TWO-DIMENSIONAL MATERIALS AND USES THEREOF

CROSS-REFERENCE TO RELATED PATENT APPLICATION

[0001] This application claims priority to U.S. Application No. 15/099,295, filed on April 14, 2016, entitled "Graphene Platelet-Based Polymers and Uses Thereof." This application also claims priority to U.S. Application No. 15/099,482, filed on April 14, 2016, entitled "METHOD FOR MAKING TWO-DIMENSIONAL MATERIALS AND COMPOSITE MEMBRANES THEREOF HAVING SIZE-SELECTIVE PERFORATIONS." This application also claims priority to U.S. Application No. 15/099,304, filed April 14, 2016, entitled "METHODS FOR IN VIVO AND IN VITRO USE OF GRAPHENE AND OTHER TWO-DIMENSIONAL

MATERIALS." This application also claims priority to U.S. Application No. 15/099,410, filed April 14, 2016, entitled "SELECTIVE INTERFACIAL MITIGATION OF GRAPHENE DEFECTS." This application also claims priority to U.S. Application No. 15/099,420, filed April 14, 2016, entitled "TWO-DFMENSIONAL MEMBRANE STRUCTURES HAVING FLOW PASSAGES." This application also claims priority to U.S. Application No. 15/099,289, filed April 14, 2016, entitled "MEMBRANES WITH TUNABLE SELECTIVITY." This application also claims priority to U.S. Application No. 15/099,276, filed April 14, 2016, entitled "BIOLOGICALLY-RELEVANT SELECTIVE ENCLOSURES FOR PROMOTING GROWTH AND VASCULARIZATION," which claims priority to U.S. Provisional Application No.

62/202,056, filed August 6, 2015. This application also claims priority to U.S. Application No. 15/099,447, filed April 14, 2016, entitled "HEALING OF THIN GRAPHITIC-BASED

MEMBRANES VIA CHARGED PARTICLE IRRADIATION." This application also claims priority to U.S. Application No. 15/099,239, filed April 14, 2016, entitled "PERFORATED SHEETS OF GRAPHENE-BASED MATERIAL," which claims priority to U.S. Provisional Application No. 62/201,527, filed August 5, 2015. This application also claims priority to U.S. Application No. 15/099,269, filed April 14, 2016, entitled "PERFORATABLE SHEETS OF GRAPHENE-BASED MATERIAL," which claims priority to U.S. Provisional Application No. 62/201,539, filed August 5, 2015. This application also claims priority to U.S. Application No. 15/099,099, filed April 14, 2016, entitled "NANOP ARTICLE MODIFICATION AND

PERFORATION OF GRAPHENE," which claims priority to U.S. Provisional Application No. 62/202,122, filed August 6, 2015 This application also claims priority to U.S. Application No. 15/099,056, filed April 14, 2016, entitled "METHODS FOR IN SITU MONITORING AND CONTROL OF DEFECT FORMATION OR HEALING." This application also claims priority to U.S. Application No. 15/099,464, filed April 14, 2016, entitled "METHOD FOR TREATING GRAPHENE SHEETS FOR LARGE-SCALE TRANSFER USING FREE-FLOAT METHOD." This application also claims priority to U.S. Application No. 15/099,193, filed April 14, 2016, entitled "IMPLANTABLE GRAPHENE MEMBRANES WITH LOW CYTOTOXICITY." All of these applications are incorporated herein by reference in their entirety.

FIELD

[0002] The present embodiments generally relate to two-dimensional materials, such as graphene as well as graphene platelet-based polymers, and methods of use and production thereof.

BACKGROUND

[0003] Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or a few layered sheets (e.g., about 20 or less) of fused six- membered rings forming an extended planar lattice. In its various forms, graphene has garnered widespread interest for use in a number of applications, primarily due to its favorable

combination of high electrical and thermal conductivity values, good in-plane mechanical strength, and unique optical and electronic properties. In many aspects, the properties of graphene parallel those of carbon nanotubes, since both nanomaterials are based upon an extended sp ² -hybridized carbon framework. Other two-dimensional materials having a thickness of a few nanometers or less and an extended planar lattice are also of interest for various applications. In an embodiment, a two dimensional material has a thickness of 0.3 to 1.2 nm. In other embodiment, a two dimensional material has a thickness of 0.3 to 3 nm.

[0004] Synthesizing graphene in a regular lattice is difficult due to the irregular occurrence of defects in as-synthesized two-dimensional materials. Such defects will also be equivalently referred to herein as "apertures," or "holes." Of particular interest for practical applications, for example, involving filtration, separation or selective containment, is the ability to make defect- free material of practical dimension. Processing and handling, which in most instances is required to use these materials, can also induce further defects in as-synthesized graphene and other two-dimensional materials.

[0005] Although pristine graphene typically displays the highest electrical conductivity values, it can sometimes be desirable to tune the electrical conductivity and modify the band structure. Tailoring of the band structure can be accomplished, for example, by introducing a plurality of defects (i.e., holes or perforations) within the graphene basal plane or increasing the number of such defects. The band structure can be influenced by both the size, type, and number of holes present. Applications that have been proposed for graphene include optical devices, mechanical structures, and electronic devices. In addition to the foregoing applications, there has been some interest in perforated graphene for filtration applications, particularly single-layer perforated graphene. Current techniques used to perforate CVD graphene include oxidation processes (e.g., UV ozone, plasma oxidation, and high temperatures), ion beams, template cutting, and direct synthesis using specialized growth substrates.

[0006] Drug and cell delivery in both immune competent and immune incompetent organisms is a problem in medical research and practice today. Recent studies use polymeric devices and hydrogels as a delivery vehicle. Some examples include polytetrafluoroethylene (e.g., expanded PTFE) with a backing of unwoven polyester mesh, silicone, hydrogels, alginate, cellulose sulfate, collagen, gelatin, agarose, chitosan and the like. Current delivery vehicles and devices are challenged by biofouling, biocompatibility issues, and a lengthy diffusion time of substances out of the vehicle. The thickness of current state devices can limit efficacy, due in part to limited diffusion of nutrients into the devices and/or impeded transfer of substances into and out of the device. Low permeability, at least in part, due to thickness and mechanical stability in view of physical stress and osmotic stress can also be problematic. Moreover, replicating the cellular walls, selective channels, and the semi-permeance that biological membranes provide has long proven to be a challenge for synthetic membranes or semipermeable walls, especially when integrating those membranes in vitro or in vivo. In addition, current membranes insufficiently achieve immunoisolation, especially in the context of xenogenic, allogenic, and autogenic transplants. SUMMARY

[0007] Some embodiments include a process comprising: exposing a multilayered material to ions provided by an ion source, the multilayered material comprising a first layer comprising a two-dimensional first material and a second layer of a second material in contact with the first layer, the ions being provided with an ion energy ranging from 1.0 keV to 10 keV, and a flux from 0.1 nA/mm ² to 100 nA/mm ² ; and producing a perforated two-dimensional material by producing a plurality of holes in the two- dimensional first material by interacting a plurality of ions provided by the ion source, neutralized ions or a combination thereof with the two-dimensional first material and with the second material. introducing one or more occluding moieties at least partially into the at least one uncovered pore to occlude the at least one uncovered pore; introducing a composite membrane comprising a porous substrate having a plurality of pores and a sheet of the perforated two-dimensional material disposed on a surface of the porous substrate and defining a top surface of the membrane, wherein the sheet of two - dimensional material covers at least a portion of the pores of the substrate and wherein at least one pore of the substrate is not covered by the two-dimensional material; incorporating the perforated two-dimensional material into a blood filtration device that

comprises two or more membranes which each comprise the perforated two-dimensional material, and further comprises a membrane a membrane comprising a cross-linked graphene platelet polymer comprising a plurality of cross-linked graphene platelets comprising a graphene portion and a cross-linking portion, the cross-linking portion contains a 4 to 10 atom link, and the cross-linked graphene platelet polymer being produced by reaction of an epoxide functionalized graphene platelet and a (meth)acrylate or (meth)acryl amide functionalized cross-linker; exposing blood from a patient to the blood filtration device having a hemodialysis membrane comprising the perforated two-dimensional material, the two-dimensional first material being disposed upon a porous support structure; removing a contaminant from the blood with the hemodialysis membrane; and recirculating purified blood to the patient.

[0008] In some embodiments, the perforated two-dimensional material is graphene-based material. In some embodiments, graphene-based material is single-layer graphene. In some embodiments, the perforated two-dimensional material is graphene oxide. In some embodiments, the one or more occluding moieties are particles sized for at least partial introduction into an uncovered pore, but which cannot exit the uncovered pore. In some embodiments, the particles are deformable or swellable. In some embodiments, the particles are deformable and pressure or energy is applied to the particles after they are introduced into the at least one uncovered pore. In some embodiments, heat or light of a selected wavelength is applied to the particles after they are introduced into the at least one uncovered pore. In some embodiments, an electron or ion beam is applied to the particles after they are introduced into the at least one uncovered pore.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 shows an illustrative scanning electron microscope (SEM) image of defects and apertures that can be present in a graphene sheet on a porous substrate. The illustrated graphene sheet has been transferred to an etched silicon nitride film in a rigid silicon support to create a composite membrane. The diameter of arrayed substrate pores is 600nm. Visible apertures in the graphene film appear black and range in size from approximately 10nm (limit of SEM resolution) to 600nm (fully uncovered substrate pore).

[0010] FIG. 2 shows an illustrative schematic demonstrating how a perforated graphene sheet or sheet of another two-dimensional material can undergo backside functionalization according to concepts described herein.

[0011] FIG. 3 shows an illustrative schematic demonstrating how a perforated graphene sheet or sheet of another two-dimensional material can undergo occlusion by flowing a catalyst there through according to concepts described herein. [0012] FIG. 4 shows an illustrative schematic demonstrating how a perforated graphene sheet or sheet of another two-dimensional material can undergo occlusion with a carbonaceous material or a non-carbonaceous material in the presence of a light ion beam, or a high temperature annealing step according to concepts described herein.

[0013] FIG. 5 shows an illustrative schematic demonstrating how apertures in multiple layered graphene sheets or sheets of other two-dimensional materials can become differentially occluded according to concepts described herein.

[0014] FIG. 6 shows an illustrative schematic demonstrating occlusion of uncovered substrate pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate according to concepts described herein.

[0015] FIG. 7 shows an illustrative schematic demonstrating occlusion of uncovered substrate pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate according to concepts described herein. In the illustrated embodiment, the substrate pores are tapered to facilitate occlusion and anchoring of the particle in the uncovered pore.

[0016] FIG. 8 shows an illustrative schematic demonstrating occlusion of uncovered substrate pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate according to concepts described herein. The substrate pores are exemplified as tapered. In the illustrated embodiment, an initial particle occludes the pore and secondary particles of size smaller than the initial particle are introduced into the uncovered occluded pores to facilitate anchoring and ensure complete occlusion.

[0017] FIG. 9 shows an illustrative schematic demonstrating occlusion of uncovered pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate according to concepts described herein. The substrate pores are exemplified as having a ledge or other form of narrowing at the pore exit to facilitate retention and anchoring of the particle in the uncovered pore. Such a ledge or other narrowing can for example be formed by deposition of a selected material to the backside of the substrate.

[0018] FIG. 10 shows an illustrative schematic demonstrating occlusion of uncovered substrate pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate according to concepts described herein. Pores in the substrate are illustrated as being interconnecting and non-uniform in diameter. Pores are shown as occluded by introduction of a plurality of particles.

[0019] FIG. 11 shows an illustrative schematic demonstrating occlusion of uncovered substrate pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate including swellable particles according to concepts described herein.

[0020] FIG. 12 shows an illustrative schematic demonstrating occlusion of uncovered substrate pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate according to concepts described herein. In the illustrated embodiment, no occluding moiety needs to be introduced into the uncovered pore. The substrate material itself is swellable, on contact for example with an absorbable fluid. Swelling of the substrate material surrounding the uncovered pore results in occlusion of the pore.

[0021] FIG. 13 shows an illustrative schematic demonstrating occlusion of uncovered substrate pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate including an appropriate chemical reagent applied to initiate reaction between reactive groups on a particle and at a pore exit according to concepts described herein.

[0022] FIGs. 14A and 14B show an illustrative schematic demonstrating occlusion of uncovered pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate wherein energy, such as light of selected wavelengths is applied to the back side of the composite membrane to facilitate anchoring of the particle in the uncovered pore according to concepts described herein. FIG. 14B illustrates resultant anchoring of the particle in the substrate pore.

[0023] FIGs. 15A and 15B show an illustrative schematic demonstrating occlusion of uncovered pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate wherein energy is applied to the back side of the composite membrane to deform and conglomerate or active chemical reactions between particles and/or between particles and the pores surfaces or edges to facilitate anchoring of particle in the uncovered pore according to concepts described herein. FIG. 15B illustrates resultant anchoring of the particle in the substrate pore [0024] FIG. 16 shows an illustrative schematic showing steps in an exemplary uncovered pore occlusion process according to concepts described herein.

[0025] FIG. 17 shows an illustrative schematic showing steps in another exemplary uncovered pore occlusion process according to concepts described herein.

[0026] FIG. 18 shows an illustrative schematic showing steps in another exemplary uncovered pore occlusion process according to concepts described herein.

[0027] FIG. 19 is an illustrative schematic demonstrating occlusion of uncovered pores by the process of Figure 18 according to concepts described herein. In the illustrated embodiment, the substrate pore is illustrated as having uniform diameter along its length. Shaped pores can also be employed. The illustrated embodiment shows the formation of a cured material or polymer within the uncovered pore to occlude the pore.

[0028] FIGs. 20A and 20B are SEM images illustrating latex bead healing (occlusion) of uncovered pores in a composite membrane according to concepts described herein.

[0029] FIGs. 21A and 21B are SEM images illustrating the progress of latex bead healing (occlusion) of uncovered pores in a composite membrane according to concepts described herein.

[0030] FIG. 22A is a graph of flow rate (μΐνπήη) (left axis, diamonds) and cumulative permeate (right axis, squares) as a function of time through a composite membrane that is being subjected to uncovered substrate pore occlusion according to concepts described herein.

[0031] FIG. 22B is a SEM image showing occlusion of 1250nm diameter pores in a silicon nitride substrate by a single graphene sheet, while FIG. 22C is a SEM image of occlusion of 1250nm diameter pores in a silicon nitride substrate after subsequent application of a second sheet of graphene.

[0032] FIG. 23 is an example reaction scheme of some embodiments.

[0033] FIG. 24 is an example configuration of a filter module of some embodiments. In these embodiments, there are four different functionalized cross-linked graphene platelet polymer composition layers, each of which is functionalized to remove or reduce the concentration of a different contaminant. [0034] FIGURE 25 shows an illustrative schematic of some embodiments of a composite structure comprising a two-dimensional material and a two fibrous layers. The fibrous layers allow for capillary ingrowth that brings the blood supply close to the two-dimensional material to facilitate exchange of molecules with the cells, proteins, tissue or the like on the opposite side of the two-dimensional material. SEM micrographs show embodiments with fibrous layers that have different pore sizes.

[0035] FIGURES 26A-F illustrate some embodiments with various configurations of enclosure configurations.

[0036] FIGURES 27A and 3B are schematic illustrations of some embodiments of an enclosure implemented for immunoisolation of living cells.

[0037] FIGURES 28A-C illustrate some embodiments for preparing an enclosure.

[0038] FIG. 29 is a top down representation of a graphene material including repaired defects.

[0039] FIG. 30 is a cross-section of the graphene material of FIG. 29 along line A-A' .

[0040] FIG. 31 is a representation of a graphene material including defects and pores when exposed to a first reactant and a second reactant to repair the defects through interfacial polymerization.

[0041] FIG. 32 is a representation of the graphene material of FIG. 31 after the reactants have contacted each other through the defect.

[0042] FIG. 33 is the graphene material of FIG. 32 after the polymerization of the reactants forming a polymer region filling the defect.

[0043] FIG. 34 is a representation of a repaired graphene material, according to one embodiment.

[0044] FIG. 35 is a representation of a repaired graphene material, according to one embodiment.

[0045] FIG. 36 is a representation of a repaired graphene material, according to one embodiment.

[0046] FIG. 37 is a representation of a graphene material including a defect when exposed to a first reactant and a second reactant to repair the defect through interfacial polymerization. [0047] FIG. 38 is a representation of the graphene material of FIG. 37 after the reactants have contacted each other through the defect.

[0048] FIG. 39 is a top down representation of a graphene material including pores, holes, and defects.

[0049] FIG. 40 is the graphene material of FIG. 39 after the holes and defects have been filled by interfacial polymerization.

[0050] FIG. 41 is a top down representation of a graphene material that has undergone interfacial polymerization to repair defects and form a polymer handling region.

[0051] FIG. 42 is a representation of a cross-section of a graphene material that has undergone interfacial polymerization to repair defects and form a polymer handling region, where the graphene material is adhered to a support.

[0052] FIG. 43 is a top down representation of an enclosure including a graphene material and a polymer handling region.

[0053] FIG. 44 is a cross-section of the enclosure of FIG. 43 along line B-B' .

[0054] FIG. 45 is a top down representation of a graphene material that includes holes formed therein arranged such that an in-situ grown support structure may be produced.

[0055] FIG. 46 is a top down representation of the graphene material of FIG. 45 after undergoing interfacial polymerization.

[0056] FIG. 47 is a representation of a cross-section of FIG. 46 along line C-C .

[0057] FIG. 48 is a representation of a cross-section of a graphene material that has undergone interfacial polymerization to repair defects and form a polymer handling region, where the graphene material is adhered to a support such that the distance between the polymer regions on the support is less than the length of the graphene between the polymer regions.

[0058] FIG. 49 is a perspective view of a two-dimensional membrane layered structure according to one embodiment.

[0059] FIG. 50 is a perspective of the two-dimensional membrane layered structure of FIG. 49 having fluid flow passages.

[0060] FIG. 51 is a cross-sectional view of the two-dimensional membrane layered structure having the flow passages of FIG. 50 according to a first embodiment. [0061] FIG. 52 is a cross-sectional view of the two-dimensional membrane layered structure having the flow passages of FIG. 50 according to a second embodiment.

[0062] FIG. 53 is a scanning electron microscope (SEM) micrograph of a support substrate layer having interlayer supports to form fluid flow passages.

[0063] FIG. 54 is a detailed SEM micrograph of the support substrate layer of FIG. 53.

[0064] FIG. 55 illustrates some embodiments having a membrane with two porous graphene- based material layers, where the membrane allows passage of both water and salt ions.

[0065] FIG. 56 illustrates some embodiments having a membrane with two porous graphene- based material layers, where applied pressure on the membrane excludes passage of salt ions.

[0066] FIG. 57 illustrates some embodiments having a membrane with three porous graphene-based material layers, where applied pressure on the membrane excludes passage of salt ions.

[0067] FIG. 58 illustrates some embodiments having a membrane with two porous graphene- based material layers, where applied voltage across the membrane excludes passage of salt ions.

[0068] FIG. 59 shows photographs of some embodiments of contamination-based spacer substances formed into various shapes or patterns. Figure 59A shows contamination-based substances formed into a single line. Figure 59B shows contamination-based substances patterned into a star. Figures 59C and 59D show contamination-based spacer substances formed into a dot array (Figure 59D provides a photograph with increased magnification as compared to Figure 59C).

[0069] FIGURE 60 is a schematic of some embodiments of graphene.

[0070] FIGURES 61 A-E show illustrative schematics of some embodiments with various configurations of enclosure configurations comprising a two-dimensional material.

[0071] FIGURES 62A and 62B are schematic illustrations of some embodiments of an enclosure implemented for immunoisolation of living cells.

[0072] FIGURES 63A-63C illustrate some embodiments for preparing an enclosure.

[0073] FIGURE 64 shows an illustrative SEM micrograph of some embodiments with a plurality of electrospun PVDF fibers deposited on graphene. [0074] FIGURE 65 shows an illustrative schematic of some embodiments of a composite structure comprising a two-dimensional material and a two fibrous layers. The fibrous layers allow for capillary ingrowth that brings the blood supply close to the two-dimensional material to facilitate exchange of molecules with the cells, proteins, tissue or the like on the opposite side of the two-dimensional material. SEM micrographs show embodiments with fibrous layers that have different pore sizes.

[0075] FIGURE 66 shows schematic illustrations of some embodiments of composite structures comprising two-dimensional materials (e.g., graphene), an optional intermediate layer (e.g., track etched polymer membrane), and a fibrous layer having a tighter fiber spacing nearer the two-dimensional material and an increasing effective pore size further from the two- dimensional material. Figure 66A also shows SEM micrographs from various locations in the composite structure. Also included are variants with direct substrate deposition (66B), hybrid thin membrane + deposition (66C), hybrid substrate deposition + thin film polymer sandwiching a graphene layer (66D) and hybrid substrate position + thin polymer film on the same side of a graphene layer.

[0076] FIGURE 67 shows an illustrative schematic of some embodiments of corrugation of graphene or graphene-based material after chemical vapor deposition on a planar growth substrate (1) by: pressing the graphene or graphene-based material and growth substrate onto a corrugated template (2); followed by application of an electrospun fibrous layer (3); removal of the graphene or graphene-based material, growth substrate and fibrous layer from the template (4); and etching of the growth substrate (5), to produce graphene or graphene-based material on electrospun material with a high surface area (6).

[0077] FIGURE 68 shows an illustrative schematic of some embodiments of a corrugated cylindrical workpiece for receiving graphene or graphene-based material on a growth substrate, as shown in Figure 65. Electrospray deposition of a fibrous layer on a non-rotated or planar surface produces a randomly distributed fibrous layer (A), whereas rotation of a cylindrical workpiece during the electrospray process produces an aligned fibrous layer (B). In the figure, entire outside of the cylinder is corrugated. [0078] FIGURE 69 shows an illustrative schematic of some embodiments of a process for manufacturing a two-dimensional material on a fibrous layer with mesh reinforcement.

[0079] FIGURE 70 shows a SEM micrograph of some embodiments of two layers of graphene or graphene-based material on a fibrous layer.

[0080] FIGURE 71 shows SEM micrographs of some embodiments of single-layer or two- layer graphene on a substrate at various magnifications and using two different electrospinning recipes, as set forth in the figure. In both recipes, 7% nylon 6,6 was electrospun and graphene was transferred to the electrospun layer. Arrows in the figure demonstrate defects and/or areas where the graphene drapes.

[0081] FIGURE 72 shows a high-magnification SEM micrograph of some embodiments of single-layer graphene on a substrate prepared according to recipe 1 in Figure 71.

[0082] FIGURE 73A shows micrographs of some embodiments of substrate layers (e.g., termed "tortuous path membrane" and "track etched membrane" in the figure); a two- dimensional membrane; and a composite structure with both a substrate layer and a two- dimensional membrane layer. FIGURE 73B illustrates exemplary embodiments of materials subjected to cytotoxicity testing and implantation testing. Figure 73 C shows photographs of some embodiments of exemplary test materials used in cytotoxicity studies.

[0083] FIGURE 74 illustrates some embodiments for using graphene in methods of immunoi sol ati on .

[0084] FIGURE 75 illustrates some embodiments for preparing and using graphene in methods of immunoisolation.

[0085] FIGURE 76 illustrates some embodiments of devices prepared with a composite structure (e.g., a substrate layer + a perforated graphene layer).

[0086] FIGURE 77 illustrates examples of substances that can be selectively excluded by two-dimensional graphene in some embodiments, based on the size of the pores in the two- dimensional graphene (graphics not to scale).

[0087] FIGURE 78 shows some embodiments of graphene disposed on various substrates, including: track-etched polyimide, track-etched polycarbonate, microporous SiN, electrospun membrane, PVDF microfiltration membrane, nanoporous SiN, carbon nanomaterial membrane, and SiN microseive. The right-most sub-figures show increased magnification views of graphene on an SiN microporous substrate.

[0088] FIGURE 79 shows micrographs of some embodiments of custom track-etched polyimide (TEPI).

[0089] FIGURE 80 shows a micrograph of some embodiments of graphene disposed on track-etched polyimide that has been nanoparticle perforated.

[0090] FIGURE 81 shows a magnified view of the micrograph shown in Figure 80. The right side of the figure is an increased magnification view that corresponds with the white box on the left side of the figure.

[0091] FIGURE 82 shows a micrograph of some embodiments of graphene disposed on electrospun nylon 6,6.

[0092] FIGURE 83 shows a magnified view of the micrograph shown in Figure 82.

[0093] FIGURE 84 shows diffusive transport of small (Allura Red AC) and large (silver nanoparticles) analytes across a substrate layer and a perforated graphene layer of some embodiments as compared to a control. In the graph, the lighter-shaded bars correspond to permeability of Allura Red AC, and the darked-shaded bars correspond to the ration of Allura Red AC permeability: silver nanoparticle permeability. The figure also includes a picture of a device that can be used to test diffusive transport.

[0094] FIGURE 85 shows data related to normalized diffusive transport of fluorescein conjugated to immunoglobulin-G (IgG) across a SiN substrate layer, a control membrane (Biopore), a perforated graphene layer, and an unperforated graphene layer. In the bar graph, the order of bars from left to right correspond with: (i) Bare Chip (left-most bar), (ii) perforated graphene, (iii) unperforated graphene (bar with lowest flux), and (iv) Biopore (right-most bar). In the line graph, the lines correspond, from highest IgG concentration to lower IgG

concentration, with: (i) Uncoated chip, (ii) perforated graphene, (iii) Biopore membrane, and (iv) unperforated graphene (represented by dots).

[0095] FIGURE 86 shows data related to permeability of fluorescein across perforated graphene (line with highest analyte concentration in the graph) and a control membrane

(Biopore) (line with second highest analyte concentration in the graph), and permeability of IgG across perforated graphene (line with 3rd highest analyte concentration in the graph) and a control membrane (line with lowest analyte concentration in the graph).

[0096] FIGURE 87 shows photographs of some embodiments where 100 nm diameter Red (580/605) FluoSpheres are restricted from traversing perforated graphene, but fluorescein is able to traverse the perforated graphene.

[0097] FIGURE 88 shows data related to permeability of fluorescein across various substrate layers, substrate layers coated with perforated graphene, and unperforated graphene. In the figure, the left-most section relates to permeability experiments conducted on a control membrane (Biopore); the middle section relates to permeability experiments conducted with uncoated substrate TEPI-400/7 (the left two bars in the middle section) and TEPI-400/7 coated with 2 layers of unperforated graphene (the right-most bar in the middle section); the right-most section relates to permeability experiments conducted with uncoated substrate TEPI-460/25 (the left five bars), TEPI-460/25 coated with 2 layers of perforated graphene (the two bars with the lowest permeability), and TEPI-460/25 coated with perforated graphene (the right six bars).

[0098] Figure 89A shows data related to permeability of fluorescein across an uncoated substrate, across a perforated graphene-coated substrate, and across an unperforated graphene- coated substrate. Figure 89B shows data related to permeability of FluoroShere across an uncoated substrate and a graphene-coated substrate.

[0099] FIGURE 90 shows TEM images of some embodiments of perforated graphene with various pore sizes. The two left-most images show perforated graphene with relatively small pores; the two right-most images show perforated graphene with relatively large pores.

[0100] FIGURE 91 shows SEM images of some embodiments showing consistent perforation of graphene over relatively large areas.

[0101] FIGURE 92 shows SEM images of some embodiments showing the ability to tune pore sizes in perforated graphene via dilation.

[0102] FIG. 93 is a flow chart illustrating a method of forming a graphenic-based membrane according to concepts disclosed herein. [0103] FIG. 94 is a scanning transmission electron microscopy (STEM) micrograph of a graphene-based material before charged particle irradiation at a level for healing according to concepts disclosed herein.

[0104] FIG. 95 is a magnified image of the STEM micrograph of FIG. 94 with arrows pointing to some identified defects.

[0105] FIG. 96 is a STEM micrograph of the region shown in FIG. 95 after charged particle irradiation for healing.

[0106] FIG. 97 is a STEM micrograph of another region of the graphene-based material shown in FIG. 94 before charged particle irradiation at a level for healing.

[0107] FIG. 98 is a STEM micrograph of the region shown in FIG. 97 after charged particle irradiation for healing.

[0108] FIG. 99 is a transmission electron microscope (TEM) image illustrating a graphene based material after conditioning treatment.

[0109] FIG. 100 is another TEM image illustrating a graphene based material after conditioning treatment.

[0110] FIGS. 101A and 101B are transmission electron microscope (TEM) images illustrating a portion of a sheet of graphene based material after perforation using UV-oxygen treatment.

[0111] FIGS. 102A and 102B are TEM images illustrating a portion of a sheet of graphene based material after perforation using Xe+ ions.

[0112] FIG. 103 and FIG. 104 are TEM images illustrating graphene based material after perforation using Ne+ ions.

[0113] FIG. 105 and FIG. 106 are TEM images illustrating graphene based material after perforation using He+ ions.

[0114] FIG. 107 is a transmission electron microscopy image demonstrating perforation through two independently stacked layers of graphene by nanoparticles.

[0115] FIG. 108 is a transmission electron microscopy image demonstrating perforation through bilayer graphene by nanoparticles. [0116] FIG. 109 is a transmission electron microscopy image demonstrating perforation by a collimated nanoparticle beam at a non-zero angle with respect to the normal of the graphene- containing sheet.

[0117] FIGS 110A and H0B show the porosity present in a graphene-containing sheet after nanoparticle perforation (FIG. 110A) and after nanoparticle perforation followed by ion beam irradiation (FIG. H0B).

[0118] FIG. 111 is a scanning electron microscopy image of two independently stacked layers of single layer graphene on a TEPI (460/25) substrate perforated by Ag P particles.

[0119] FIG. 112 shows a flowchart for a method for monitoring defect formation or healing via detection of scattered, emitted or transmitted radiation or particles, according to some embodiments.

[0120] FIG. 113 shows a flowchart for a method for monitoring defect formation or healing via detection of movement of an analyte, according to some embodiments.

[0121] FIG. 114 shows a flowchart for a method for monitoring defect formation or healing via measurement of electrical conductivity, according to some embodiments.

[0122] FIG. 115 shows a flowchart for a method for monitoring defect formation or healing via Joule heating and temperature measurement, according to some embodiments.

[0123] FIGs. 116A and 116B show schematics of exemplary systems for monitoring defect formation or healing, according to the embodiments of the present invention.

[0124] FIG. 117A is a schematic, perspective view of a growth substrate used in the formation of a graphene sheet according to some embodiments.

[0125] FIG. 117B is a schematic, perspective view of the graphene sheet formed on the growth substrate of FIG. 117 A.

[0126] FIG. 118 is a schematic view of a transfer preparation apparatus to prepare the graphene sheet of FIG. 117B for free-float transfer.

[0127] FIG. 119A is a schematic, perspective view of an etching step of the growth substrate from the prepared graphene sheet of FIG. 118 using a free-float transfer method.

[0128] FIG. 119B is a schematic, perspective view of a transfer step of the prepared graphene sheet of FIG. 118 to a functional substrate using the free-float transfer method. [0129] FIG. 120 shows a large-scale graphene sheet prepared using the transfer preparation apparatus of FIG. 118 after removal of the growth substrate.

[0130] FIG. 121 shows the large-scale graphene sheet of FIG. 120 after transfer to a functional substrate using the free-float transfer method.

[0131] FIG. 122 is a scanning electron microscope (SEM) micrograph of a graphene sheet transferred to a functional substrate using the free-float transfer method.

[0132] FIG. 123 is a detailed view of the SEM micrograph of FIG. 122.

DETAILED DESCRIPTION

METHOD FOR MAKING TWO-DIMENSIONAL MATERIALS AND COMPOSITE MEMBRANES THEREOF HAVING SIZE-SELECTIVE PERFORATIONS

[0133] A variety of two-dimensional materials useful according to concepts disclosed herein are known in the art. In various embodiments, the two-dimensional material comprises graphene, carbon nanomembranes (CNM), molybdenum disulfide, or boron nitride (specifically the hexagonal crystalline form of boron nitride). In an embodiment, the two-dimensional material is a graphene-based material. In more particular embodiments, the two-dimensional material is graphene. Graphene according to embodiments can include single-layer graphene, multi -layer graphene, or any combination thereof. Other nanomaterials having an extended two- dimensional molecular structure can also constitute the two-dimensional material in the various embodiments. For example, molybdenum sulfide is a representative chalcogenide having a two- dimensional molecular structure, and other various chalcogenides can constitute the two- dimensional material in the embodiments. Choice of a suitable two-dimensional material for a particular application can be determined by a number of factors, including the chemical and physical environment into which the graphene or other two-dimensional material is to be terminally deployed.

[0134] In an embodiment, the two dimensional material useful in membranes herein is a sheet of graphene-based material. Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. In an embodiment, graphene-based materials also include materials which have been formed by stacking independent single sheet or multilayer graphene sheets. In embodiments, multilayer graphene includes 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In embodiments, graphene is the dominant material in a graphene-based material. For example, a graphene-based material comprises at least 30% graphene by weight, or at least 40% graphene, or at least 50% graphene, or at least 60% graphene, or at least 70% graphene, or at least 80%) graphene, or at least 90% graphene, or at least 95% graphene. In embodiments, a graphene-based material comprises a range of graphene content selected from 30% to 95%, or from 40% to 80% from 50% to 70%, from 60% to 95% or from 75% to 100%. In embodiments, a graphene-based material comprises a range of up to 35% oxygen by atomic ratio.

[0135] As used herein, a "domain" refers to a region of a material where atoms are uniformly ordered into a crystal lattice. A domain is uniform within its boundaries, but different from a neighboring region. For example, a single crystalline material has a single domain of ordered atoms. In an embodiment, at least some of the graphene domains are nanocrystals, having a domain size from 1 to 100 nm or 10-100 nm and optionally up to about 1 cm. In an embodiment, at least some of the graphene domains have a domain size greater than 100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm and optionally up to about 1 cm. "Grain boundaries" formed by crystallographic defects at edges of each domain differentiate between neighboring crystal lattices. In some embodiments, a first crystal lattice may be rotated relative to a second crystal lattice, by rotation about an axis perpendicular to the plane of a sheet, such that the two lattices differ in "crystal lattice orientation".

[0136] In an embodiment, the sheet of graphene-based material comprises a sheet of single or multilayer graphene or a combination thereof. In an embodiment, the sheet of graphene-based material is a sheet of single or multilayer graphene or a combination thereof. In another embodiment, the sheet of graphene-based material is a sheet comprising a plurality of

interconnected single or multilayer graphene domains. In an embodiment, the interconnected domains are covalently bonded together to form the sheet. When the domains in a sheet differ in crystal lattice orientation, the sheet is polycrystalline.

[0137] In embodiments, the thickness of the sheet of graphene-based material is from 0.34 to 10 nm, from 0.34 to 5 nm, or from 0.34 to 3 nm. In an embodiment, a sheet of graphene-based material comprises intrinsic or native defects. Intrinsic or native defects are those resulting from preparation of the graphene-based material in contrast to perforations which are selectively introduced into a sheet of graphene-based material or a sheet of graphene. Such intrinsic or native defects include, but are not limited to, lattice anomalies, pores, tears, cracks or wrinkles. Lattice anomalies can include, but are not limited to, carbon rings with other than 6 members (e.g. 5, 7 or 9 membered rings), vacancies, interstitial defects (including incorporation of non- carbon atoms in the lattice), and grain boundaries.

[0138] In an embodiment, a sheet of graphene-based material optionally further comprises non-graphenic carbon-based material located on the surface of the sheet of graphene-based material. In an embodiment, the non-graphenic carbon-based material does not possess long range order and may be classified as amorphous. In embodiments, the non-graphenic carbon- based material further comprises elements other than carbon and/or hydrocarbons. Non-carbon elements which may be incorporated in the non-graphenic carbon include, but are not limited to, hydrogen, oxygen, silicon, nitrogen, copper and iron. In embodiments, the non-graphenic carbon-based material comprises hydrocarbons. In embodiments, carbon is the dominant material in non-graphenic carbon-based material. For example, a non-graphenic carbon-based material comprises at least 30% carbon by weight, or at least 40% carbon, or at least 50% carbon, or at least 60% carbon, or at least 70% carbon, or at least 80% carbon, or at least 90% carbon, or at least 95% carbon. In embodiments, a non-graphenic carbon-based material comprises a range of carbon selected from 30% to 95%, or from 40% to 80%, or from 50% to 70%.

[0139] Two-dimensional materials in which pores are intentionally created are referred to herein as "perforated," such as "perforated graphene-based materials," "perforated two- dimensional materials," or "perforated graphene." Two-dimensional materials are, most generally, those which have atomically thin thickness from single-layer sub-nanometer thickness to a few nanometers and which generally have a high surface area. Two-dimensional materials include metal chalogenides (e.g., transition metal dichalogenides), transition metal oxides, hexagonal boron nitride, graphene, silicene and germanene (see: Xu et al. (2013) "Graphene-like Two-Dimensional Materials) Chemical Reviews 113 :3766-3798). [0140] Two-dimensional materials include graphene, a graphene-based material, a transition metal dichalcogenide, molybdenum disulfide, a-boron nitride, silicene, germanene, or a combination thereof. Other nanomaterials having an extended two-dimensional, planar molecular structure can also constitute the two-dimensional material in the various embodiments. For example, molybdenum disulfide is a representative chalcogenide having a two-dimensional molecular structure, and other various chalcogenides can constitute the two-dimensional material in embodiments. In another example, two-dimensional boron nitride can constitute the two- dimensional material in an embodiment of the concepts disclosed herein. Choice of a suitable two-dimensional material for a particular application can be determined by a number of factors, including the chemical and physical environment into which the graphene, graphene-based or other two-dimensional material is to be deployed.

[0141] The present disclosure is directed, in part, to sheets of graphene-based material or other two-dimensional materials containing a plurality of perforations therein, where the perforations have a selected size and chemistry, as well as pore geometry. In embodiments, the perforated graphene, perforated graphene-based materials and other perforated two-dimensional materials contain a plurality of size-selected perforations ranging from about 3 to 15 angstroms in size. In a further embodiment, the perforation size ranges from 3 to 10 angstroms or from 3 to 6 angstroms in size. The present disclosure is further directed, in part, to perforated graphene, perforated graphene-based materials and other perforated two-dimensional materials containing a plurality of size-selected perforations ranging from about 3 to 15 angstroms in size and having a narrow size distribution, including but not limited to a 1-10% deviation in size or a 1-20% deviation in size. In an embodiment, the characteristic dimension of the perforations is from about 3 to 15 angstroms in size.

[0142] The present disclosure is also directed, in part, to perforated graphene, perforated graphene-based materials and other perforated two-dimensional materials containing a plurality of perforations ranging from about 5 to about 1000 angstroms in size. In further embodiments, the perforations range from 10 to 100 angstroms, 10 to 50 angstroms, 10 to 20 angstroms or 5 to 20 angstroms. In a further embodiment, the perforation size ranges from 100 nm up to 1000 nm or from 100 nm to 500 nm. The present disclosure is further directed, in part, to perforated graphene, perforated graphene-based materials and other perforated two-dimensional materials containing a plurality of perforations ranging from about 5 to 1000 angstrom in size and having a narrow size distribution, including but not limited to a 1-10% deviation in size or a 1-20% deviation in size. In an embodiment, the characteristic dimension of the perforations is from 5 to 1000 angstrom.

[0143] For circular perforations or apertures, the characteristic dimension is the diameter of the perforation or aperture. In embodiments relevant to non-circular pores, the characteristic dimension can be taken as the largest distance spanning the perforation or aperture, the smallest distance spanning the perforation or aperture, the average of the largest and smallest distance spanning the perforation or aperture, or an equivalent diameter based on the in-plane area of the perforation or aperture. As used herein, perforated graphene-based materials include materials in which non-carbon atoms have been incorporated at the edges of the pores.

[0144] The present disclosure particularly describes methods directed to occluding apertures in a sheet of a graphene-based material or other two-dimensional material that are larger than a given threshold size, thereby reducing the plurality of apertures to a desired size and optionally with a specific chemistry. In embodiments, the reduced size of the aperture falls within the perforation and aperture size ranges given above. The threshold size can be chosen at will to meet the needs of a particular application. Perforations or apertures are sized as described herein to provide desired selective permeability of a species (atom, molecule, protein, virus, cell, etc.) for a given application. Selective permeability relates to the propensity of a porous material or a perforated two-dimensional material to allow passage (or transport) of one or more species more readily or faster than other species. Selective permeability allows separation of species which exhibit different passage or transport rates through perforations or apertures. In two-dimensional materials selective permeability correlates at least in part to the dimension or size (e.g., diameter) of perforations or apertures and the relative effective size of the species. Selective permeability of the perforations or apertures in two-dimensional materials such as graphene-based materials can also depend on functionalization of the perforation or aperture (if any) and the specific species that are to be separated. For electrically conductive two dimensional materials, selective permeability can be affected by application of a voltage bias to the membrane. Selective permeability of gases can also depend upon adsorption of a gas species on the filtration material, e.g., graphene. Adsorption at least in part can affect the local concentration of the gas species at the surface of the filtration material. Separation of two or more species in a mixture includes a change in the ratio(s) (weight or molar ratio) of the two or more species in the mixture after passage of the mixture through a perforated two-dimensional material.

[0145] The chemistry of the perforated apertures can be the same or different after being occluded according to the embodiments described herein. In various embodiments, occluding the apertures can involve occluding apertures within a particular size range such that no apertures remain within the size range, thereby conferred selectivity to the "healing" of the graphene-based material or other two-dimensional material. The embodiments of the healing processes described herein are applicable to both "through-holes" (i.e., pores in a single two-dimensional sheet) and "intralayer flow" (i.e., passages existing between stacked layers of individual single layer two-dimensional sheets or multiple layer sheets of 2D material. Passages can include laterally offset pores within multiple two-dimensional sheets. Through-holes can also exist in multiple two-dimensional sheets when the pores are not substantially laterally offset from one another in the various layers

[0146] The embodiments described herein allow specific chemistries to be readily applied in a homogenous manner to graphene-based materials and other two-dimensional materials to allow for tunable activity across many applications. While the chemistries described herein can be applied homogenously to an entire surface of the sheet of graphene or other two-dimensional material, they generally provide specific activation of particular perforations or apertures of a given size using a carefully sized moiety that allows for aperture modification to take place. The described techniques can be advantageous in allowing the homogenous application of chemistry to the graphene-based material or other two-dimensional material surface while only occluding perforations or apertures of a certain desired size. The homogenous application of the various chemistries described herein can facilitate scalable production and manufacturing ease.

Perforation or aperture modification can confer a specific chemistry to the perforations or apertures (e.g., functional selectivity, hydrophobicity, and the like) and allow for at least partial occlusion of the perforations or apertures to take place in various embodiments. Such selective modification of the apertures can allow selective separations to take place using the graphene- based material, including size-based separations.

[0147] For example, perforations or apertures can be selectively modified by various known methods to contain hydrophobic moieties, hydrophilic moieties, ionic moieties, polar moieties, reactive chemical groups, for example, amine-reactive groups (chemical species that react with amines) carboxylate-reactive groups (chemical species that react with carboxylates), amines or carboxylates (among many others), polymers and various biological molecules, including for example, amino acids, peptide, polypeptides, enzymes or other proteins, carbohydrates and various nucleic acids.

[0148] Furthermore, the techniques described herein can be configured to at least partially occlude large apertures within the sheet of graphene-based material or other two-dimensional material in preference to smaller apertures, thereby allowing the smaller apertures to remain open and allow flow to be maintained therethrough. This type of selective flow can allow molecular sieving to take place using the graphene-based material or other two-dimensional material, rather than the solution-diffusion model provided by current polymeric solutions. In some aspects, apertures or defects are blocked to provide flow reduction or blockage within a range of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more. In further aspects, however, all the apertures and/or defects in a graphene-based material or other two-dimensional material can be occluded in order to substantially block passage or flow, herein 99% or more through the two-dimensional material or block passage or flow through the two-dimensional material entirely. In an embodiment, a graphene-based material or other two-dimensional material that is occluded in order to substantially or entirely block passage or flow through the material can be used as a starting material for forming a size-selected perforated two- dimensional material. Size-selected perforations can be introduced into the substantially or entirely blocked two-dimensional material employing art known methods for generating perforations of a selected size and, if any, a selected functionalization.

[0149] Although the description herein is primarily directed to graphene-based materials, it is to be recognized that other two-dimensional materials or near two-dimensional materials can be treated in a like manner. The at least partially occluded graphene-based materials prepared according to the techniques described herein can be used for occluding fluid flow, particularly liquid or gas flow for separations, including filtration membranes and filtration systems. In addition, they can be used in optical or electronic applications.

[0150] In some embodiments, the graphene-based material can be transferred from its growth substrate to a porous substrate in the course of, before or after practicing the embodiments described herein.

[0151] By way of example, a sheet of a graphene-based material or sheet of another two- dimensional material can have a plurality of perforations therein, and a direction of fluid flow therethrough can establish an "upstream" side (alternative the top surface) and a "downstream" side (alternatively the bottom surface) of the sheet. The downstream side of the sheet of the graphene-based material or sheet of another two-dimensional material can be next to or in contact with a substrate, such as a porous substrate. In some embodiments, the substrate can provide support to the sheet of the graphene-based material while practicing the various techniques described herein. The perforations in the sheet of a graphene-based material or sheet of another two-dimensional material can be intentionally placed therein, or they can occur natively during its synthesis. According to the embodiments of the present disclosure, the perforations can have a distribution of sizes, which can be known or unknown. By placing an occluding moiety within a flow contacting the graphene-based material or other two-dimensional material, apertures having a desired size profile can become occluded according to the embodiments described herein.

[0152] In some embodiments described herein, a fluid containing a sized moiety can be flowed through the sheet of graphene-based material or other two-dimensional material. The sized moiety can lodge in some of the apertures in the sheet and induce occlusion of at least the portion of the apertures in the sheet in which the sized moiety lodges. In other embodiments, a sized moiety can occlude fluid flow on the sheet of graphene-based material or other two- dimensional material from the upstream side of the graphene. Various embodiments of these various flow configurations are described below.

[0153] Occluding at least a portion of the apertures in the foregoing manner can result in reducing the size and number of apertures, possibly modifying a flow path and making the graphene-based material or other two-dimensional material suitable for use in an intended application. For example, the graphene-based material or sheet of another two-dimensional material can be processed in the foregoing manner to produce a cutoff pore size in a molecular filter. Depending on the nature of the moiety in the flow path, the moiety can be covalently or non-covalently attached to the graphene-based material, or mechanically connected to the graphene-based material.

[0154] In some embodiments, the "downstream" side of the sheet of graphene-based material can be "primed" or functionalized with oxygen via plasma oxidation or the like, such that the graphene-based material can be reactive with a moiety passing through the apertures. In some embodiments, the moiety in the flow path can bind to functional groups introduced to the graphene-based material, such that the moiety binds to the graphene-based material and the apertures become at least partially occluded. Suitable binding motifs can include, but are not limited to, addition chemistry, crosslinking, covalent bonding, condensation reactions, esterification, or polymerization. In various embodiments, the occluding moiety can be sized to reflect a particular cutoff regime, such that it only passes through apertures having a certain threshold size or shape. For example, in various embodiments, the occluding moieties can be a substantially flat molecule or spherical in shape. POSS® silicones (polyhedral oligomeric silsesquioxanes), for example, represent one particular type of occluding moiety that can be made at a very specific size and functionalized to tether to oxygenated functionalization on the apertures. Other examples, of useful occluding moieties include fullerenes, dendrites, dextran, micelles or other lipid aggregates, and micro-gel particles. Some or all of these techniques may be applied to other two-dimensional materials as well.

[0155] In various embodiments, the graphene-based material can be perforated and functionalized with oxygen, such as treating the graphene-based material with oxygen or a dilute oxygen plasma, thereby functionalizing the graphene-based material with oxygen moieties. In some embodiments, the graphene based material can be functionalized in this manner while on a copper substrate, or any other metallic/growth or polymeric substrate as would be known by those with ordinary skill in the art. Subsequently, the oxygen functionalities can be reacted via a chemistry that converts the oxygenated functionalities into a leaving group (such as a halide group, particularly a fluoro group, or sulfonic acid analogs, such as tosylates, triflates, mesylates, and the like). This chemistry results in sites on the substrate that are vulnerable to nucleophilic attack and can be used for additional chemistry, as detailed above, or allowing the graphene based material to bind to the substrate. In some embodiments, the graphene-based material can functionalized with oxygen so as to provide graphene oxide platelet membranes.

[0156] FIG. 2 shows an illustrative schematic demonstrating how a graphene sheet or sheet of another two-dimensional material can undergo backside functionalization according to the embodiments described herein to occlude undesired apertures or defects in the sheet. In the drawings, Side A refers to the side of the graphene or other two-dimensional material being exposed to the upstream flow and Side B refers to the backside of the graphene or other two- dimensional material. In certain embodiments, the flow may be across the two dimensional material instead of merely through. The backside of the sheet may be primed or activated to react with the occluding moieties. In an embodiments, the backside of the sheet is functionalized to bind or react with occluding moieties. As shown in FIG. 2, the occluding moieties do not pass through aperture or defects having a size too small for the occluding moieties to pass through, but the occluding moieties do pass through the larger undesirable apertures or defects. On passage through the undesired apertures or defects, the occluding moieties react and/or bind at the aperture or defect and occlude the aperture or defect. The flow is sufficiently high that diffusion of occluding moieties on the backside of the sheet away from the aperture that the exit is minimized to avoid occlusion of the smaller apertures or defects. While the aforementioned chemistry provides a technique for backside attack, it should also be recognized that a sheet of graphene or other two-dimensional material can also be functionalized or primed such that it can undergo front side attack, particularly in cases where transfer is less desirable during the processing of a product. Front side attack can ensure retention of configuration. Front side attack can occur similarly to the methods depicted in FIG. 2, with the exception that bonding occurs on the upstream or Side A of the graphene sheet or sheet of other two-dimensional material and there may be less selectivity in bonding holes of a desired size over larger apertures. In an embodiment, methods to permit front side attack on the surface of a graphene-based material again begin with an oxygen functional group on the surface of the graphene-based material, which are treated with an agent such as pyridine, triflate, or analogs thereof to provide a good leaving group. Subsequent chemistry can be conducted to remove the copper growth substrate and use additional chemistry to occlude holes or apertures below the diameter which the moiety may pass. Note that both front side and back side approaches allow for a

homogenous application of chemistries, which can be desirable for scalability.

[0157] In other embodiments, the downstream side of the graphene-based material or other two-dimensional material can be primed with an occluding substance and a moiety that catalyzes the reaction of the occluding substance with the graphene or other two-dimensional material can pass through the apertures. Thus, in this case, the moiety does not become bonded to the graphene or other two-dimensional material itself. Figure 3 shows an illustrative schematic demonstrating how a perforated graphene sheet or sheet of another two-dimensional material can undergo occlusion by flowing a catalyst therethrough. In more particular embodiments, the methods can include treating the graphene or graphene-based material with lithium and an appropriate charge transfer catalyst in order to create polyethylene glycol chains around the periphery of the graphene perforations. In some embodiments, such a reaction scheme can be conducted under substantially anhydrous conditions. In some embodiments, the moiety in the flow can catalyze the reaction with a polymer substrate (e.g., upon which the graphene-based material or other two-dimensional material is placed following transfer from its growth substrate), which can be considered a subset of the foregoing embodiments.

[0158] In the foregoing embodiments, also depicted in FIGs. 2 and 3, priming the graphene- based material can involve casting a porous substrate, such as a polymer substrate, onto the graphene-based material when it is on its copper growth substrate. The copper can then be etched away. Thereafter, the porous substrate can be exposed to light or some other form of electromagnetic radiation to cause a change in the substance, thereby making it no longer permeable. In alternative embodiments, the porous substrate can be exposed to a compound that binds to the porous substrate. In still other alternative embodiments, the porous substrate can maintain its porosity when practicing the embodiments described herein.

[0159] In still further embodiments, carbonaceous or non-carbonaceous materials can be flowed over the graphene based material or other two-dimensional material and become tethered to the open apertures. Suitable materials can include, for example, graphene nanoplatelets (G Ps), fullerenes of various sizes, hexagonal, boron nitride, or carbon nanotubes. In more particular embodiments, tethering of the carbonaceous or non-carbonaceous material can be accomplished by utilizing a light or gentle ion beam, a high temperature annealing step, exposing to light to generate a photo-active reaction. The high temperature annealing step could comprise isocyanate crosslink chemistry. In some embodiments, flow through the two-dimensional material can become completely blocked. In some embodiments, smaller apertures can remain open. FIGURE 4 shows an illustrative schematic demonstrating how a perforated graphene sheet or sheet of another two-dimensional material can undergo occlusion with a carbonaceous material or a non-carbonaceous material in the presence of a light ion beam or a high temperature annealing step. In the depicted embodiments, the carbonaceous or non-carbonaceous materials are flowed laterally across the sheet of graphene-based material or other two-dimensional material, rather than passing through the apertures. In alternative embodiments, the

carbonaceous materials or non-carbonaceous materials can be flowed through the sheet of graphene-based material or other two-dimensional material.

[0160] In still other embodiments, functionalization or activation of multiple, potentially different layered sheets of graphene-based materials or other two-dimensional material (e.g. complementary chemistries) can be leveraged to allow for flow not only through channels, but also via intralayer flow. Healing or partial occlusion can result from further modification of apertures. That is, layered sheets of two-dimensional material can be differentially

functionalized according to the embodiments described herein. FIG. 3 shows an illustrative schematic demonstrating how a perforated graphene sheet or sheet of another two-dimensional material can undergo occlusion by flowing a catalyst there through. FIG. 4 shows an illustrative schematic demonstrating how a perforated graphene sheet or sheet of another two-dimensional material can undergo occlusion with a carbonaceous material or a non-carbonaceous material in the presence of a light ion beam, or a high temperature annealing step. FIG. 5 shows an illustrative schematic demonstrating how apertures in multiple layered graphene sheets or sheets of other two-dimensional materials can become differentially occluded. In some embodiments, the functionalization can be chosen such that apertures of different sizes are occluded in the various graphene sheets.

[0161] The present disclosure is directed, in part, to composite membranes formed from a porous substrate having a plurality of pores with a sheet of two-dimensional material disposed on the surface of the porous substrate and defining a top surface of the composite membrane. A portion of the pores of the substrate are covered by the two-dimensional material and a portion of the pores of the substrate are not covered by the two-dimensional material due, for example, to defects formed during synthesis of the two-dimensional material, formed during handling of the two-dimensional material, or formed when the two-dimensional material is disposed on the porous substrate. Defects or apertures in the two-dimensional material can result in undesired passage of species through the composite membrane. It is desired for use in filtration

applications, that substantially all of the substrate pores are covered by the two-dimensional material so that passage through the membrane is primarily controlled by passage through the two-dimensional material. In a specific embodiment, substantially all pores of the substrate are covered by a two-dimensional material that contains perforations of a desired size range for selective passage through the membrane. In a specific embodiment, perforations in the two- dimensional material have a selected chemistry at the perforation as discussed above. The perforation in the two-dimensional material can have selected size or selected size range and discussed above. In a specific embodiment, the two-dimensional material is a graphene-based material. In a specific embodiment, the two-dimensional material is a graphene-based material which comprises single-layer graphene or multi-layer graphene.

[0162] The disclosure provides methods for occluding uncovered substrate pores in the composite membrane as described above. In an embodiment, the method includes introducing one or more occluding moieties at least partially into at least one uncovered pore to occlude the at least one uncovered pore. In specific embodiments, 50% or more of the uncovered substrate pores are occluded. In more specific embodiments, 60% or more, 75% or more, 80% or more, 90% or more, 95% or more or 99% or more of the uncovered substrate pores are occluded. In specific embodiments, occlusion of uncovered pores reduced flow through the composite membrane (compared to the non-occlude membrane) by 50% or more. In specific embodiments, occlusion of uncovered pores reduced flow through the composite membrane (compared to the non-occluded membrane) by 60% or more, 75% or more, 80% or more, 90% or more, 95% or more or 99% or more.

[0163] The extent of occlusion of uncovered pores can be assessed by various methods. Detection of uncovered pores can, for example, be assessed using a selected assay fluid, e.g., a detectible gas, such as SF ₆ , to detect the location (or approximate location) of uncovered pores by passage of the assay fluid. Uncovered pores may be detected by use of the passage of detectible chemical species, particles, electrons, UV or visible light through the pores. The presence of uncovered pores can also be detected by various imaging methods. The presence and or location (or approximate location) of pores can be assessed using various imaging methods (including scanning electron microscopy, scanning probe microscopy, scanning tunneling microscopy, atomic force microscopy, transmission electron microscopy, x-ray spectroscopy, etc.); detecting analyte, particles or ions passing through pores (using mass spectrometry, secondary mass spectrometry, Raman spectroscopy, residual gas analysis, detecting Auger electrons, detecting nanoparticles using a microbalance, detecting charged species with a Faraday cup, detecting secondary electrons, detecting movement of analyte through defects, employing an analyte detector, identifying a composition, mass, average radius, charge or size of an analyte; detecting electromagnetic radiation passing through defects;

detecting electromagnetic radiation given off by analyte; and detecting electromagnetic radiation or particles back scattered from the membrane.

[0164] Uncovered substrate pores include those pores that are only partially covered, but through which non-selective passage can occur.

[0165] The porous substrate of the composite membrane can be any porous material compatible with a disposed two-dimensional material and particularly with a graphene-based material. The porous substrate is selected to be compatible with the application for which the composite membrane is intended. For example, compatible with the gases, liquids or other components which are to be in contact with the composite membrane. The porous substrate provides mechanical support for the two-dimensional material and must maintain this support during use. The porous support should substantially retain pores that are covered with two- dimensional material. In specific embodiments, the porous material is made of a polymer, metal glass, or a ceramic

[0166] The pores in the substrate can have uniform pore diameter along the length of the pore, or they can have a diameter that varies along this length. Pores or pore openings (entrance or exit) can be shaped, as discussed below, to facilitate retention of occluding moieties in uncovered pores. Pores may be tapered, ridged or provided with one or more ledges to facilitate retention of occluding moieties in uncovered pores. In a specific embodiment, the pore entrance and/or the pore exit is narrowed compared to the rest of the pore to facilitate retention of occluding moieties. In some embodiments, pores in the substrate are preferably of uniform size and uniform density (e.g., uniformly spaced along the substrate). In some embodiments, pores may be independent or may be interconnected with other pores (tortuous or patterend). In embodiments, pores sizes (e.g. diameters) can range from 10 nm to 10 microns or more preferably from 50 nm to 500 nm. For methods and composite membranes herein a top surface of the membrane is defined as the surface upon which the two-dimensional material is disposed. One surface contains pore entrance openings and the second surface contains pore exit openings. Introduction of occluding moieties is through pore entry openings so that introduction of such moieties is selectively into pores that are not covered by two-dimension materials. Pore entrance and exits are defined by flow direction through pores.

[0167] Occluding moieties most generally include any material that can be selectively introduced into uncovered pores and retained therein to occlude the pore. A step of chemical reaction, application of energy in the form of heat, electromagnetic radiation (e.g., UV, visible or microwave irradiation), or contact with an absorbable material can be applied to deform, swell, polymerize, cross-link or otherwise facilitate retention of occluding materials in a pore. In an embodiment, the occluding materials are one or more particles sized for entrance at least in part into an uncovered pore. Particle size and pore shape may be selected to facilitate retention in the uncovered pores. Particles may be deformable, for example, by application of pressure, heat, microwave radiation or light of a selected wavelength (e.g. UV light), or by ion bombardment. Deformable particles introduced into pores are retained in pores after deformation. Particles may be swellable, where the size of a particle increases on contact with an absorbable material which induces swelling. The absorbable material can, for example, be a fluid including liquids or gases, water or an aqueous solution or a miscible mixture of water and an organic solvent, a polar organic solvent or a non-polar organic solvent. The swellable particle and the absorbable fluid are selected to achieve a desired level of swelling to achieve retention in the pore.

[0168] Occluding particles can be made of any suitable material. In specific embodiments, particles selected from metal particles, silica particles, particles of metal oxide, or polymer particles. In specific embodiments, particles are made of melamine, polystyrene or polymethyl methacrylate (PMMA). In a specific embodiment, the particles are made of latex (polystyrene). In specific embodiments, the substrate pore occluding particles are themselves permeable to provide a selective permeability through the occluded pores. In specific embodiments, the substrate pore occluding particles are permeable to fluid flow and provide for separation of components in the fluid. Permeable materials could include hydrogels, polymers, proteins, zeolites, metal-organic framework materials, or thin film solution membranes.

[0169] Particle size is generally selected based on the pores sizes present in the substrate so that the particle can enter the pore and be retained in the pore. Particles may be monodisperse if the pore entrance openings are uniform in size. A mixture of particles of different sizes can be employed when pore openings are non-uniform in size. A mixture of particles of different sizes (having a selected particle size distribution or being polydisperse in size) can be used, if pores with different (or unknown sizes) are present in the substrate. In an embodiment, the occluding particle is selected to have particle size that is approximately the same size as a pore entrance opening. The occluding particle may be slightly larger for tapered pores and slightly smaller for non-tapered pores such that the pores have a larger cross-section on the side of the substrate exposed to upstream flow. In an embodiment, particles are sized for at least partial introduction into an uncovered pore, but wherein the particle cannot exit the uncovered pore. Exit from the pore can be inhibited or prevented by providing shaped pores in which the pores are narrowed at some point along its length. Particles useful in the methods herein will in an embodiment range in size from 10 nm to 10 microns.

[0170] In an embodiment, employing deformable or swellable particles, the respective particles are deformed or swollen after introduction to an uncovered pore. [0171] In an embodiment occlusion may be facilitated through controlled fouling, where a fluid is flowed to the composite membrane surface, and material from the fluid is bonded to composite membrane pores that are defective. The fouling may be controlled such that it blocks non-selective pores.

[0172] In an embodiment occlusion may be facilitated through healing with particles in air or gas. Particles are aerosolized and/or suspended in air and then forced through the membrane, such as by having convective flow of the air through the membrane. The convective flow of the air could be facilitated by applying a pressure differential across the membrane. The particles could be those described herein, with the methods described herein for fixing the particles to the membrane.

[0173] In an embodiment, occluding particles carry one or more chemical reactive groups for reaction with compatible reactive groups in the at least one uncovered pore, on the surface of the substrate at the uncovered pore or on other particles to facilitate anchoring of at least one particle in at least one uncovered pore. The particles can carry any one or more of a reactive chemical species, for example, the reactive species may be an amine, a carboxylate acid, an activated ester, a thiol, an aldehyde or a hydroxyl group. Particles useful in the concepts disclosed herein which carry reactive groups can be prepared by known methods or may be obtained from commercial sources. Reactive groups on the particles can react with compatible reactive groups on the surface of the substrate at an uncovered pore, within the pore or at pore openings to facilitate retention in the pore. In an embodiment, particles may react with other particles in the pore to facilitate retention in the pore. One of ordinary skill in the art can employ a variety of chemically reactive groups to facilitate reaction with a pore to facilitate retention and anchoring in the pore. It will be appreciated that chemical reaction between particles, between particles and the pore surface, edges, openings or exits can be activated or induced by introduction of a reactive species, reagent or catalyst into a pore containing at least one occluding particle. It will also be appreciated that a chemical reaction between particles, between particles and the pore surface, edges, openings or exits can be activated or induced, for example, by heating, microwave irradiation, irradiation with light of selective wavelength (e.g., UV radiation) or by application of an ion beam, or by any method known in the art that is compatible with the materials employed.

[0174] In an embodiment, the occluding moieties are selected from one or more monomers, oligomers, uncured polymers or uncross-linked polymers. These occluding moieties are introduced selectively into uncovered pores and wherein the monomers, oligomers or polymers are polymerized, cured or cross-linked after they are introduced into the at least one uncovered pore. Polymerization can be effected for example by introduction of a polymerization catalyst, heating, microwave irradiation, or irradiation with light of a selected wavelength or by any method known in the art that is compatible with the materials employed. Curing of an uncured polymer or cross-linking of a polymer can be effected by any art-known method, for example by introduction of a curing or cross-linking reagent or application of heating, microwave irradiation, or irradiation with light of a selected wavelength or by any method known in the art that is compatible with the materials employed.

[0175] In an embodiment, the occlusion method further comprising a second step of introducing secondary particles into the at least one uncovered pore having a first particle therein occluding the uncovered pore, where the secondary particles are sized to be smaller than the first particle. In this embodiment, the initial particle and the secondary particles may be deformable or swellable as described above and may be deformed or swollen after introduction of the secondary particles. The initial particle and the secondary particles may carry one or more reactive groups as described above for chemical reaction of particles in the pores to facilitate retention in the pore.

[0176] In an embodiment, the composite membrane further comprises a coating layer on the top surface of the porous substrate between that surface and the sheet of two-dimensional material. Chemical reaction of a particle or other occluding moiety with reactive species on this coating at the entrance of the uncovered pore can facilitate anchoring and retention in the pore.

[0177] Occluding moieties are introduced to the top surface of the composite membrane where the occluding moieties can enter uncovered substrate pores. Introduction can be by any appropriate method and preferably is by application of a flow of fluid containing a selected concentration of occluding moieties. In a specific embodiment, the fluid is an aqueous solution carrying a selected concentration of occluding moieties. The concentration of occluding moieties in the flow introduced can be readily optimized empirically to optimize the

effectiveness or efficiency of occlusion. Effectiveness or efficiency of occlusion can be assessed by measurement of the flow rate through the composite membrane, by accumulation rate of permeate or by an assessment of the selectivity of flow. A decreasing flow rate or a leveling off of permeate accumulation indicates successful occlusion. In an embodiment, the flow of occluding moieties includes a surfactant to decrease or minimize clumping or aggregation of occluding moieties and to facilitate entry of occluding moieties into uncovered pores. The inclusion of an appropriate surfactant is particularly beneficial for the introduction of occluding particles. In a specific embodiment, the surfactant is a non-ionic surfactant, such as

(polyethylene glycol sorbitan monooleate). One of ordinary skill in the art can readily select a surfactant appropriate for the methods herein.

[0178] In a preferred embodiment, introduction of occluding moieties to the top surface of the composite membrane is by application of a cross-flow to the surface. The velocity of the cross-flow can be varied according to desired results. According to an embodiment, the pressure and flow may be varied as desired. In an embodiment, the shear velocity of the flow may be controlled. In an embodiment, the pressure across the composite membrane may be stopped while shear flowing. In an embodiment, the pressure on both sides of the membrane may be equalized. In an embodiment, the pressure may be controlled in cycles to alternately provide flow forward and then backward. The pressure on one or both sides of the membrane may be pulsed. Further, peristaltic pump rate and dimensions of the channel through the composite membrane may be controlled according to embodiments.

[0179] In an embodiment, the pore occlusion method further comprising a step of cleaning the top surface after introduction of occluding moieties into uncovered pores to remove occluding moieties that have not entered uncovered pores. This cleaning step can comprise flow of an appropriate fluid (gas or liquid) to or across the top surface of the membrane. In a specific embodiment, a flow of water or an aqueous solution is applied to or across the top surface of the membrane. In a specific embodiment, the aqueous solution contains a surfactant (as discussed above) to decrease clumping or aggregation of particles on the top surface. [0180] In an embodiment, the introduction and cleaning steps as well any intervening steps to facilitate retention of particles in uncovered pores (e.g., deformation, chemical reaction, swelling or application of energy) are repeated until additional occlusion of pores ends or until a selected level of uncovered pore occlusion is achieved. As discussed above, various methods for accessing the extent or efficiency of pore occlusion can be employed.

[0181] In an embodiment, cycles of introduction and cleaning can be repeated until at least 80% of the uncovered pores are occluded. In an embodiment, cycles of introduction and cleaning can be repeated until at least 95% of the uncovered pores are occluded. In an embodiment, cycles of introduction and cleaning can be repeated until at least 99% of the uncovered pores are occluded.

[0182] In preferred embodiments, the two-dimensional material is a graphene-based material. In preferred embodiments, the two-dimensional materials is a sheet of graphene containing single layer graphene, few layer graphene (having 2-20 layers) or multilayer graphene.

[0183] In an embodiment, the pore occlusion method can be practiced without introducing an occluding moiety into uncovered pores. In this embodiment, a composite membrane as discussed above is provided wherein a sheet of two-dimensional material covers at least a portion of the pores of the substrate; but wherein at least one pore of the substrate is not covered by the two-dimensional material. In this embodiment, the substrate material forming the pores comprises a swellable material. The substrate itself may be made of a swellable material or more preferably the substrate material surrounding the pores is formed of a swellable material. For example, a coating of swellable material can be applied to the inside surfaces of the substrate pores. Selective introduction of an absorbable material into the uncovered pores results in local swelling of the swellable material surrounding the uncovered pore and occlusion of the uncovered pore. In an embodiment, the uncovered pores are selectively contacted with an absorbable fluid.

[0184] The disclosure further provides a composite membrane comprising a porous substrate having a plurality of pores and a sheet of two-dimensional material disposed on a surface of the porous substrate and defining a top surface of the membrane, wherein the sheet of two - dimensional material covers at least a portion of the pores of the substrate, wherein at least one pore of the substrate is not covered by the two-dimensional material and wherein at least one uncovered pore of the substrate is occluded with an occluding moiety. In an embodiment, the composite membrane has at least one uncovered pore occluded with one or more particles or occluded with a polymer, cured polymer or cross-linked polymer formed in the at least one uncovered pore.

[0185] FIG. 6 schematically illustrates occlusion of uncovered pores in the substrate of a composite membrane having a graphene-based material sheet disposed upon a porous substrate forming a top surface thereof. The occluding moiety is illustrated as a particle. A particle, size- selected based on pore size, to at least partially enter an uncovered pore is illustrated. As discussed herein a plurality of particles are introduced to the top surface of the membrane and a portion of the particles enter and are retained in the uncovered pores. A particle enters at least one uncovered pore and occludes the pore preventing passage though the occluded pore. The substrate may be pre-wetted in some embodiments.

[0186] FIG. 7 schematically illustrates an exemplary occlusion method applied to a composite membrane having a graphene-based material sheet disposed upon a porous substrate where the substrate pores of the membrane are tapered such that the pores have a larger cross- section on the side of the substrate exposed to upstream flow to facilitate retention of the occluding material and anchoring of the occluding material in the uncovered pore. This embodiment is exemplified with an occluding particle which is size-selected to enter an uncovered pore and is inhibited or prevented from exiting the pore by tapering of the pore. It will be appreciated the substrate pores can be variously shaped to facilitate retention of occluding moieties, particularly particles. The direction of particle flow is illustrated as perpendicular to the membrane top surface. It will be appreciated that cross-flow parallel to the top surface can be applied to introduce occluding moieties to the top surface.

[0187] FIG. 8 schematically illustrates another exemplary occlusion method where the composite membrane has a graphene-based material sheet disposed upon a porous substrate where the substrate pores are tapered to facilitate retention of the occluding material and anchoring of the occluding material in the uncovered pore. In the illustrated embodiment, an initial particle is introduced into the uncovered pore to occlude the pore and secondary particles of size smaller than the initial particle are introduced into the uncovered occluded pores to facilitate anchoring.

[0188] FIG. 9 schematically illustrates another exemplary occlusion method applied to composite membranes having a graphene-based material sheet disposed upon a porous substrate. The substrate pores are exemplified as having a ledge or other form of narrowing along their length and specifically at the pore exit to facilitate retention and anchoring of an occluding moiety (exemplified as a particle) in the uncovered pore. In an embodiment, the particle may be inserted into the pore, and then a ridge may be formed after the particle is inserted.

[0189] FIG. 10 schematically illustrates another exemplary occlusion of uncovered substrate pores in a composite membrane having a graphene-based material sheet disposed upon the porous substrate. Pores in the substrate are illustrated as being partially interconnected and nonuniform in diameter, and may form a tortuous path through the substrate. Pores are shown as occluded by introduction of a plurality of particles. It is to be noted that if pore connections exist between covered and uncovered pores that occlusion of uncovered pores may reduce desired flow through covered pores.

[0190] FIG. 11 schematically illustrates another exemplary occlusion of uncovered pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate. The substrate pores are illustrated as having a ledge or other form of narrowing at both the entrance and exit from the pores. In the illustrated embodiment, the particle is swellable, such that the non-swollen particle is of a size that will enter the uncovered pore, but which on swelling is of a size that will not exit the uncovered pore. In specific embodiments, the swellable particle is composed at least in part of a swellable polymer. More specifically, the swellable particle is composed at least in part of a swellable amorphous polymer. In specific embodiments, the swellable particle is composed at least in part of a hydrogel. Swelling ratios of swellable polymers and hydrogel can be adjusted by variation of composition of the polymer or hydrogel as is known in the art. Swelling can for example be initiated on contact with an absorbable fluid, such as water or an organic solvent. For example, a hydrogel can be swollen employing absorption of water or aqueous solution. For example, a non-polar or hydrophobic polymer can be swollen with a hydrocarbon solvent. For example, a polar or hydrophilic polymer can be swollen with water or alcohol or mixtures thereof.

[0191] In embodiments illustrated in FIGs. 6-11, occluding particles can optionally be provided with one or more reactive groups as discussed above which can react or can be activated to react with compatible chemical moieties in the pores, at the edges of the pores, at the substrate surface at the pore opening or pore exit or disposed on ledges or other structures within the pores. Such chemical reactions facilitate anchoring of the particle in the pore. In an embodiment, occluding particles can be provided with compatible reactive chemical groups for reactions between particles in a pore.

[0192] FIG. 12 schematically illustrates another exemplary occlusion of uncovered pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate. In the illustrated embodiment, no occluding moiety is introduced into the uncovered pore. The substrate material itself is swellable, on contact for example with an absorbable fluid. Swelling of the substrate material surrounding the uncovered pore results in occlusion of the pore. In a related embodiment, the substrate is not made entirely of a swellable material, but the inside surface of the pores of the substrate are provided with a coating that is swellable.

[0193] FIG. 13 schematically illustrates another exemplary occlusion of uncovered pores in a composite membrane having a graphene-based material sheet disposed upon a surface of a porous substrate. In the illustrated embodiment, the substrate pore is shown as having a narrowing of the pore diameter at the pore exit. In the illustrated embodiment, a chemical reaction is activated to anchor the particle at the pore exit. Activation in this case is introduced to the surface of the membrane without the graphene-based material (also designated the backside of the membrane). A chemical reaction can be activated variously, by providing a reagent or catalyst or by providing activating energy, such as heat, light or activating ions or particles. The angled lines indicate at least for application of electromagnetic radiation or beams of electrons, ions or the like, that irradiation or bombardment can be applied at an angle with respect to the surface such that only a portion of the length of the pore is contacted. For example, an appropriate chemical reagent is applied as illustrated to initiate reaction between reactive groups on the particle and at the pore exit. [0194] FIGs. 14A and 14B schematically illustrates another exemplary occlusion of uncovered pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate. In the illustrated embodiment, the substrate pore is shown in FIGs. 14A and B as having a narrowing of the pore diameter at the pore exit. In the illustrated embodiment, after introduction of the particle into the pore energy is applied to the back side of the membrane as shown to cause a change to the occluding particle, such as deformation as shown in FIG. 14B. The occluding particle may be sensitive to applied energy, such as light of selected wavelengths, or contact with electron or ion beams. The applied energy facilitates deformation of the particle in the uncovered pore facilitating anchoring of the particle in the pore.

[0195] FIGs. 15A and B schematically illustrate another exemplary occlusion of uncovered pores in a composite membrane having a graphene-based material sheet disposed upon a porous substrate. In the illustrated embodiment, the substrate pore is shown as having a narrowing of the pore diameter at the pore exit. In the illustrated embodiment of FIG. 15 A, a plurality of particles is shown as introduced into the uncovered pore. In the illustrated embodiment, the particles and/or the pore surfaces or edges carry reactive chemical groups. In the illustrated embodiment of FIG. 15 A, energy, for example in the form of light of selected wavelength, an electron or ion beam, or a chemical reagent is applied to the bottom side of the composite membrane to activate chemical reactions between particles and/or between particles and the pores surfaces or edges to facilitate anchoring of particle in the uncovered pore. The result of application of energy is shown in FIG. 15B where the particles are anchored in the substrate pore [0196] FIG. 16 schematically illustrates steps in an exemplary uncovered pore occlusion process. A graphene sheet is disposed on a porous substrate, illustrated with uniform pores, to form a composite membrane. A portion of the pores are covered by the graphene and a portion of the pores are not covered by the graphene (uncovered pores). Particles sized to at least partially enter an uncovered pore are introduced to the top surface of the composite membrane where they enter uncovered pores, but not covered pores. In the illustrated embodiment, pressure, heat, or light or alternatively solvent swelling is applied to the particles in the uncovered pores to deform the particles or swell the particles to occlude the pores. Particles do not bond to the graphene. The particles are optionally subjected to an optional curing step after deformation. The curing step is for example a thermoset cure achieved by heating. An alternative exemplary cure is achieved catalytically by exposure to a catalyst or curing agent. A cleaning step is then applied to wash off and remove excess particles. The steps of introducing the particles, application of pressure, energy or solvent swelling, and cleaning are repeated until a desired level of pore occlusion is obtained.

[0197] FIG. 17 schematically illustrates steps in an exemplary uncovered pore occlusion process. A porous substrate is provided with a coating which is compatible with graphene and may enhance adhesion to graphene. The coating provided does not occlude substrate pores. A graphene sheet is then disposed on the coated porous substrate, illustrated with uniform pores to form a composite membrane. A portion of the pores are covered by the graphene and a portion of the pores are not covered by the graphene (uncovered pores). Particles sized to at least partially enter an uncovered pores are introduced to the top surface of the composite member where they enter uncovered pores, but not covered pores. In the illustrated embodiment, particles optionally bond to the substrate or to the coating on the substrate to facilitate anchoring of the particles to occlude uncovered pores. Particles do not bond to the graphene. The particles may be subjected to an optional curing step after bonding. A cleaning step is then applied to wash off and remove excess particles. The steps of introducing the particles, bonding and optionally curing of particles, and cleaning are repeated until a desired level of pore occlusion is obtained.

[0198] FIG. 18 schematically illustrates steps in an exemplary uncovered pore occlusion process. A graphene sheet is disposed on a porous substrate, illustrated with uniform pores to form a composite membrane. A portion of the pores are covered with the graphene and a portion of the pores are not covered by the graphene (uncovered pores). Polymerizable monomers or oligomers or a curable or cross-linkable polymer are introduced into the uncovered pores. These precursors do not enter graphene covered pores. The precursors are polymerized, cured or cross- linked within the uncovered pores to occlude the pores. Polymerization or curing can be facilitated by application of heat, light of selected wavelength or of chemical reagents including polymerization catalyst and/or cross-linking agents. A cleaning step is then applied to wash off and remove excess unreacted precursors and any catalysts or reagents employed. The steps of introducing precursors for polymerization, curing or cross-linking, polymerization, curing and /or cross linking and cleaning are repeated until a desired level of pore occlusion is obtained.

[0199] FIG. 19 schematically illustrates exemplary results of occlusion of uncovered pores as in the process of FIG. 18. In the illustrated embodiment, the substrate pores are shown as having uniform diameter along their length. Shaped pores can also be employed. The illustrated embodiment shows the formation of a cured material or polymer within the uncovered pore to occlude the pore. The direction of flow for introduction of occluding moieties is illustrated as flow perpendicular to the substrate top surface. It will be appreciated that flow can also be applied in the illustrated embodiments in a cross-flow configuration, where flow is parallel to the substrate top surface.

[0200] FIGs. 20A and 20B are scanning electron microscopy (SEM) images illustrating latex bead healing (occlusion) of uncovered pores in a composite membrane. The SEM images were taken while tilted at 35-degrees relative to normal of the substrate surface. Areas covered in graphene are dark gray and areas without graphene coverage are light gray. FIG. 20A is a lower magnification (x2500) SEM image showing areas on the composite membrane that are covered or not covered by graphene. Substrate pores (450 nm diameter) which are uncovered by graphene are being occluded by latex beads. Latex beads are also shown clumping on the surface of the composite membrane. Latex beads do not damage the graphene. Pores in the substrate that are uncovered by graphene are being occluded by the latex beads. FIG. 20B is a higher magnification SEM image (x8500) of the same composite membrane showing latex beads occluding uncovered pores in the substrate. Those latex beads which are occluding substrate pores are visible embedded at varying depths into the substrate pores.

[0201] FIGs.21 A and 21B are SEM images illustrating the progress of latex bead healing (occlusion) of uncovered pores in a composite membrane. Latex beads of particle size 0.46 μπι were employed to occlude substrate pores of 0.45 μπι. The graphene sheet is light gray and a large area of dark gray is a microdefect in the graphene. A number of apertures in the graphene are circled in the images. FIG. 21 A is an image taken after 5 cycles of alternating latex bead introduction and cleaning, both via cross-flow across the membrane surface. Latex beads were introduced at a lppm dilution in DI water containing 0.1% polysorbate-80 and a biocide (50ppm NaI3). Cleaning cycles were performed with the same solution sans latex beads. A three-port flow apparatus with input and output ports allowed for flow across the surface of the graphene- coated surface of composite membrane plus "permeate" flow through membrane. Most uncovered pores are occluded after 5 cycles of alternating latex bead introduction and cleaning, but the circled pores are not occluded. FIG. 2 IB is an image taken after another 4 of alternating latex bead introduction and cleaning (a total of 9 cycles). Additional apertures are occluded in this second image including all of the apertures circled in the image. The occlusion process illustrated in these images was found however not to be optimized. In particular, it was found that variation of the concentration of the occluding particles in the flow introduced to the membrane could affect efficiency of the occlusion process. It was also found that addition of a surfactant with the flow of particles reduced aggregation and clumping of particles. Clumping of particles on the membrane top surface is preferably minimized or avoided.

[0202] FIG. 22B is a SEM image showing occlusion of 1250nm diameter pores in a silicon nitride substrate by a single graphene sheet, while FIG. 22C is a SEM image of occlusion of 1250nm diameter pores in a silicon nitride substrate after subsequent application of a second sheet of graphene. As can be seen, a majority of uncovered pores resulting from defects in the first graphene sheet are subsequently occluded by the second graphene sheet. Thus, layering of individual sheets of the two-dimensional material is an effective method for occluding pores in a composite membrane that arise from intrinsic or native defects and defects generated during the processing and handling of the two-dimensional material. Occlusion of the substrate pores can be significantly improved when subsequent sheets of the two dimensional material are applied, because the intrinsic or native defects and defects generated during processing and handling are independent for each layer thus the probability of an unoccluded substrate pore is exponentially reduced with each successive layer. Such a method is most effective for fabrication of a size- selective composite membrane when the size-selected perforations are introduced to the multilayer stack of two-dimensional materials. In some embodiments, the methods described herein for occluding apertures in a sheet of a two-dimensional material may then be beneficially employed to a multi-layer stack of two dimensional materials. [0203] FIG. 22A is a graph of flow rate (μΙ7ιηίη) (left axis, diamonds) and cumulative permeate (right axis, squares) as a function of time through a graphene coated composite membrane that is being subjected to uncovered pore occlusion employing latex particles. Thus the y-axis in FIG. 22A corresponds to the flow rates through the membrane (input-to-permeate). The flow rates noted are the cross-membrane (input-to-output) rates, while the pressures noted are the pressure difference between graphene side of membrane and the permeate outlet. The flows that are cross-membrane (input-to-output) are labeled as "x-flow" in FIG. 22A, and should not be confused with the y-axis of the graph of FIG. 22A. The occlusion process employed to obtain the illustrated results differed from that illustrated in FIGs. 21 A and 21B, in that the concentration of particles applied to the top surface was optimized to improve efficiency of occlusion. The porous substrate of the composite membrane is etched silicon nitride in a rigid silicon support. The silicon nitride has a plurality of patterned 0.45 μπι pores. The composite membrane is assessed in a cross-flow arrangement with pressure applied as indicated. A three- port flow apparatus with input and output ports allowed for flow across the surface of the graphene-coated surface of composite membrane plus "permeate" flow through membrane. A baseline cycle in which an aqueous solution containing only surfactant (0.1% polysorbate-80) with 20 mL/min flow at 45 psi is initially applied. An occlusion cycle includes a step of introduction of latex beads (0.46 μπι beads) and a subsequent cleaning step. The latex beads are carried in aqueous solution at a concentration of 0.5 ppm (0.1% polysorbate-80 and biocide (50 ppm NaI3) and introduced in cross-flow to the composite membrane at 20 mL/min at 45 psi. The cleaning step is cross-flow application of an aqueous solution containing surfactant at a 20mL/min flow at 0 psi to remove latex beads remaining on surface. The occlusion step and cleaning step are applied for 7-11 minutes as indicated in FIG. 22 A. The figure follows flow rate and cumulative permeate for three full occlusion/cleaning cycles. Introduction of latex beads in the first occlusion step is shown to produce an immediate greater than 99% reduction in flow rate. Cleaning steps induce a small flow rate recovery (<5% of the flow rate before occlusion), which is then reversed by subsequent occlusion steps. Similarly, cumulative permeate levels off immediately on introduction of the latex beads. No increase in flow rate or the rate of accumulation of permeate is observed on application of additional occlusion cycles. In an embodiment, the concentration of beads may set according to the desired result. In an embodiment, small beads may be introduced, while larger beads are used to remove the small beads in the cleaning step. In an embodiment where bead agglomeration is not detrimental, the agglomerated beads may provide a filtering function.

GRAPHENE PLATELET-BASED POLYMERS AND USES THEREOF

[0204] Some embodiments provided herein are cross-linked graphene platelet polymers, compositions thereof, filtration devices comprising the cross-linked graphene platelet polymers and/or compositions thereof and methods for using and making the same.

[0205] Cross-linked graphene platelet polymers

[0206] Some of the polymers described herein comprise a graphene portion or moiety and a crosslinking portion or moiety.

[0207] The graphene portion or moiety may be a graphene platelet that may be chemically bound directly or indirectly to one or more crosslinking portions or moieties. Crosslinking may be by covalent or other bonding mechanism such as ionic, van der Waals, etc.

[0208] The graphene portion in some embodiments comprises a reacted graphene platelet.

[0209] The graphene platelet may have a very thin but wide aspect ratio. The graphene platelet may comprise several sheets of graphene, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 sheets of graphene. It is understood that the various sheets are not necessarily the same width, e.g., one or more of the sheets may be a partial sheet that covers only a portion of the sheet in which it is associated with or in contact. For example, a partial sheet may cover about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% of the portion of the sheet in which it is associated with or in contact.

[0210] The particle diameter of the graphene platelet may range from sub-micron (for example, about 10 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 1000 nm) up to about 100 microns (for example up to about 1 micron, 2 microns, 3 microns, 4 microns, 5 microns, 6 microns, 7 microns, 8 microns, 9 microns, 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns). Various ranges between the disclosed particle diameters may be utilized. It is understood that the graphene platelets will not necessarily be perfect circular particles. Thus, the particle diameter may be measured from the widest points of the graphene platelet.

[0211] The size of the graphene platelets may also be expressed as an average size or a plurality of graphene platelets. For example, in some embodiments, the average size of a plurality of graphene platelets used may be about 10 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 1000 nm, about 100 microns (for example up to about 1 micron, 2 microns, 3 microns, 4 microns, 5 microns, 6 microns, 7 microns, 8 microns, 9 microns, 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns. The coefficient of variation for the average size may be greater than zero to about 25%. For example, the coefficient of variation may be about 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0212] The graphene platelets in some of the embodiments may be functionalized. This functionalization may result in a direct or indirect chemical bond to the one or more crosslinking portions or moieties, or it may provide additional functionality to the resulting cross-linked graphene platelet polymer.

[0213] In some embodiments, the graphene platelet comprises one or more reactive moieties capable of reacting with a crosslinking molecule. In some embodiments, the graphene platelet is functionalized as disclosed in Hunt, A., et al. Adv. Funct. Mater., 22(18), pp. 3950-3957, 2012. The one or more reactive moieties, for example, may be capable of reacting with a

(meth)acrylate or (meth)acryl amide moiety, or may be capable of reacting, e.g., with a hydroxyl moiety, a carbonyl moiety, an epoxy moiety, an ether linkage, and phosphide, phosphate, sulfide and/or a sulfate. In some embodiments, the one or more reactive moieties of the graphene platelet can be an epoxy functional group or amine, and graphene/carbon nitride via reaction in a nitrogen plasma. In some embodiments, the one or more reactive moieties is a "capped" moiety that is capable of converting to a reactive moiety upon, e.g., chemical, heat or UV treatment. In various embodiments the graphene has a variable C/O ratio that maximizes the mechanical strength and the variation is C/O ratio of 2/1, 5/1, 10/1, 20/1, 30/1, 40/1, 50/1, 60/1 70/1, 80/1 90/1, and 100/1. The speciation of the graphene would be either hydroxyl or epoxy, when reactions with amines or cyanates, can form one pot epoxy or urethane networks. In some embodiments, the one or more reactive moieties are a "capped" moiety that is capable of converting to a reactive moiety upon, e.g., chemical, heat or UV treatment. In some

embodiments, the graphene platelet may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 reactive moieties. In some embodiments, a plurality of graphene platelets used in the polymer may have an average of about 2, 3, 4, 5, 6, 7, 8, 9 or 10 reactive moieties. The coefficient of variation for this average may be greater than zero to about 25%. For example, the coefficient of variation may be about 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0214] In some embodiments, the graphene platelet comprises one or more functional moieties. These moieties are different from the reactive moieties in that they do not react with the crosslinking molecule, but rather they ultimately impart some functionalization to the resulting cross-linked graphene platelet polymer. Some embodiments utilize functional moieties including thiol moieties, fluorocarbon functionalized areas of the graphene platelet and/or phosphorus, silane and siloxane functional groups.

[0215] The crosslinking portions or moieties in some embodiments may be a crosslinking portion that is chemically bound directly or indirectly to two or more graphene portions or moieties. The cross-linked graphene may form ordered layers wherein the crosslinking moiety controls the spacing between ordered layers.

[0216] In some embodiments, the crosslinking portion comprises a reacted di-, tri- or tetra- functional crosslinking compound. The functional group may be a (meth)acrylate or

(meth)acrylamide moiety, or may be capable of reacting with a hydroxyl or epoxy moiety. In some embodiments, the di-, tri- or tetra-functional crosslinking compound contains the same functional groups. In other embodiments, the functional groups on the di-, tri- or tetra-functional crosslinking compound are different. The crosslinking compound also includes a spacer portion between the functional groups. The spacer group remains in the crosslinking portions or moieties after the functional groups have reacted. For example, the spacer group may comprise 1 to 10 atoms in a linear chain, for example, carbon, oxygen or sulfur atoms, phosphide, phosphate or inorganic moieties as well, silicon and transition metals. The length of the spacer groups will determine the class of the filtered species. The spacer group between adjacent or laterally stacked graphene platelets of 1 to 6 carbons, with a carbon-carbon single bond of 1.54 A, allows for selectivity of ionic filtration, for species up to 1 nm in diameter. Longer spacers, or branching can enable selectivity for viruses and other pathogens. For example, the spacer may be longer, but still provide spacing between graphene platelets that allows for selective size exclusion of certain viruses and other pathogens of a particular size. The spacing may be determined based on the desired viruses and other pathogen that should be excluded. In some embodiments, the spacer group is a CI -CI0 linear chain, or a C3-C20 branched chain. One or more of the carbons may be replaced by an oxygen and/or sulfur atom. The CI -CI0 linear chain or C3-C20 branched chain may comprise methylene groups, which may be optionally substituted with one or more halogen of hydroxyl group thiol groups, phosphate, or phosphide.

[0217] The crosslinking portions or moieties of the present disclosure provide a spacing between two or more graphene portions or moieties. In some embodiments, the cross-linking portion or moiety provides a 4 to 10 atom link between two or more graphene portions or moieties. In other embodiments, the cross-linking portion provides a 4 to 10 atom link between two or more graphene portions or moieties provide a spacing between individual graphene platelet moieties of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or 3.5 nm. The spacing between individual graphene platelet moieties may be determined by molecular modeling of the reacted cross-linking portion, or by microscopic methods, electroacoustic spectroscopy to measure particle and spacing size in aqueous media as well as the zeta potential of the surfaces. Also, x- ray diffraction may be employed to measure inter-plate gallery spacing in lamellar structures. Methods to control the spacing between vertically stacked graphene platelets can employ flexible, e.g., polyphenylene oxide repeat units or rigid carbon spacers with, e.g., polyphenylene or polynorbornene rods to provide a consistent spacer between graphene plates.

[0218] In other embodiments, the crosslinking portions or moieties of the present disclosure can include spacer moieties. For example, the crosslinking portion may include moieties to attach to the graphene platelets (e.g., covalently, ionically, etc.) and the spacer moiety. Spacer substances can include polymers, fibers, hydrogels, molecules, nanostructures, nanoparticles and allotropes that are responsive to an environmental stimulus. In some embodiments, the spacer substance is a smart polymer, such as a hygroscopic polymer; a thin polymer that expands when hydrated; or an amorphous polymer, such as a porous amorphous polymer. In some embodiments, the spacer substance comprises electrospun fibers that can be swelled upon exposure to a solvent. In some embodiments the spacer substance comprises materials with a high thermal expansion coefficient, which expand or contract in response to a temperature stimulus. In some embodiments, the spacer substance is deliquescent. In some embodiments, the spacers are substantially inert. In some embodiments, the spacers are not inert (i.e., they can be reactive).

[0219] Exemplary spacer substances also include structural proteins, collagen, keratin, aromatic amino acids, and polyethylene glycol. Such spacer substances can be responsive to changes in tonicity of the environment surrounding the spacer substance, pi-bonding availability, and/or other environmental stimuli.

[0220] In some embodiments, the spacer substance is a piezoelectric, electrostrictive, or ferroelectric magnetic particle. In some embodiments, the magnetic particle comprises a molecular crystal with a dipole associated with the unit cell. In some embodiments, the magnetic particles can be oriented based on an external magnetic field. Exemplary magnetic particles include lithium niobate, nanocrystals of 4-dimethylamino-N-methyl-4-stilbazolium tosylate (DAST)), crystalline polytetrafluoroethylene (PTFE), electrospun PTFE, and combinations thereof.

[0221] In some embodiments, the spacer substance heats up faster or slower than its surroundings. Without being bound by theory, it is believed that such embodiments will allow the rate of passage of permeants, or a subset of permeants, across the membrane to be increased and/or decreased.

[0222] In some embodiments, spacer substances respond to electrochemical stimuli. For instance, a spacer substance can be an electrochemical material (e.g., lithium ferrophosphate), where a change in oxidation state of the spacer substance (e.g., from 2- to 3-) alters permeability of the membrane. In some embodiments, changing the oxidation state of the spacer substances alters the interaction between the spacer substance and potential permeants. In some

embodiments, the change in oxidation state results from a redox-type reaction. In some embodiments, the change in oxidation state results from a voltage applied to the membrane. [0223] In some embodiments, the spacer substance comprises contamination structures formed by utilizing a focused ion beam, e.g., to modify heavy levels of contamination on graphene-based material into more rigid structures. For instance, in some embodiments, mobilization and migration of contamination on the surface of the graphene-based material occurs— coupled in some embodiments with some slight beam induced deposition— followed by modification and induced bonding where the beam is applied. In some embodiments, combining contamination structures allows the geometry, thickness, rigidity, and composition of the spacer substance to be tuned to respond to an environmental stimulus (e.g., pressure).

[0224] Exemplary spacer substance includes particle substances such as metal nanoparticles (e.g., silver nanoparticles), oxide nanoparticles, octadecyltrichlorosilane nanoparticles, carbon nanotubes, and fullerenes. In some embodiments, the spacer substance includes nanorods, nano- dots (including decorated nano-dots), nanowires, nanostrands, and lacey carbon materials.

[0225] In some embodiments, the spacer moiety is responsive to an environmental stimulus, for example, the spacer substance may expand and/or contracts in response to the environmental stimulus. The spacer substance may reversibly expand and/or reversibly contract in response to the environmental stimulus. For instance, conformational changes between trans and cis forms of a spacer substance can alter the effective diameter of the spacer substance (by way of example, a spacer substance could be a polymer with an embedded diazo dye, where exposure to the appropriately colored light alters the volume of the dye based on cis-/trans- conformational changes). In some embodiments, the spacer substance undergoes a physical and/or chemical transformation that is pH-modulated or optically modulated. In some embodiments, the environmental stimulus degrades the spacer substance to alter the effective diameter of the spacer substance.

[0226] In some embodiments, the environmental stimulus induces a conformational change in the spacer substance that alters the effective length of the spacer substance. Environmental stimuli may include, for example, changes in temperature, pressure, pH, ionic concentration, solute concentration, tonicity, light, voltage, electric fields, magnetic fields, pi-bonding availability, and combinations thereof. [0227] The polymers described herein may include additional monomeric components, biocompatible silicone, hexamethyl tnsiloxane (D3), epoxy, both cyclohexyl epoxies, amenable to UV curing, and epichlorohydrin, amenable to substitution on the carbonyl functionality of graphene. The epichlorohydrin may be curable via thermal methods, and provides a durable, cross-linked graphene polymer. Some polymeric cross-linkers initiators may be curing agents, such as diamines. Other monomers and chain spacers may be included, such as aromatic and alkyl di-carboxylic acids curing via the hydroxyl functionality on the graphene platelets to create polyester cured graphene.

[0228] The cross-linked graphene platelet polymers described herein have a sufficient crosslink density to prevent large gaps of uncured section of graphene, which may allow, e.g., salt, to pass unimpeded through greater than about 1 nm holes (or spaces between platelets). In some embodiments, the cross-linked graphene platelet polymers have a crosslink density of 0- 0.33 (for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, or 0.33, and measured by differential scanning calorimetry. In some other embodiments, the cross-linked graphene platelet polymer compositions contain less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% holes (or spaces between platelets) greater than about 1 nm. In some other embodiments, the cross-linked graphene platelet polymer

composition is substantially free of holes (or spaces between platelets) greater than about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 nm. In some embodiments, cross-linked graphene platelet polymer composition comprises holes (or spaces between platelets) between 0.5 and 2.0 nm. The other embodiments, the space between platelets changes in response to an environmental stimulus as described herein. The space between platelets may be between 0.5 and 2.0 nm after or before the change in response to an environmental stimulus as described herein.

[0229] In other embodiments, the cross-linked graphene platelet polymer composition has a crosslink density sufficient to reduce the sodium content in a 3.5% saline solution by at least 50, 60, 70, 80, 90 or 100 fold when passed through the cross-linked graphene platelet polymer composition having a thickness of about 100 nm. Other embodiments include, e.g., about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nm, or values in between.

[0230] Figure 23 demonstrates an exemplary reaction scheme of an embodiment of the present disclosure, wherein the graphene platelet comprises a reactive epoxide moiety and the functional crosslinking compounds are di-functional crosslinking compounds containing either hydroxyl moieties or acrylate moieties.

[0231] Additional Optional Components of cross-linked graphene platelet polymers

[0232] As mentioned above, some embodiments of the graphene platelet comprise one or more functional moieties. In addition, the cross-linked graphene platelet polymer compositions may be further functionalized to remove or reduce one or more deleterious contaminant from a liquid or gas passing through the cross-linked graphene platelet polymer composition. For example, the holes (or spaces between platelets) within the cross-linked graphene platelet polymer composition may be patterned with silver nanoclusters that, e.g., deactivate the bacteria. In other embodiments, the cross-linked graphene platelet polymer composition may be further treated with quaternary alkyl-ammonium bromide compounds that, e.g., have been shown to coordinate with the phospholipid shell of viruses. In other embodiments, the cross-linked graphene platelet polymer composition may include ionically or chemisorbed ammonium compounds that are not covalently bound to the cross-linked graphene platelet polymer.

[0233] Membranes

[0234] The cross-linked graphene platelet polymer may be formed into membranes that remove or reduce one or more deleterious contaminant from a liquid or gas passing through the cross-linked graphene platelet polymer composition. In some embodiments the liquid is water. In some embodiments, the cross-linked graphene platelet polymer compositions may be mounted on a support structure.

[0235] In other embodiments, the cross-linked graphene platelet polymer may be formed into membranes that isolate or concentrate one or more desired components from a liquid or gas passing through the cross-linked graphene platelet polymer composition. For example, rare earth ions may be isolated or concentrated from, e.g., seawater by reducing water content where certain components of the seawater (e.g., water) are capable of passing through the cross-linked graphene platelet polymer composition, but the desired compounds, such as rare earth ions, are incapable of passing through the cross-linked graphene platelet polymer composition.

[0236] The membranes in some embodiments may include more than one cross-linked graphene platelet polymer composition layers. For example, the different layers may be incorporated into a membrane module, wherein the various layers each has a particular functionality. For example, the filter module comprising at least two separate filters or membranes each comprising a cross-linked graphene platelet polymer composition layer, wherein each filter or membrane is functionalized in a different manner, e.g., wherein the cross- linking moieties generate a spacing of about 1 nanometer between individual graphene platelet moieties, wherein the cross-linking portion contains a 4 to 10 atom link, wherein the cross-linked graphene platelets comprise a thiol moiety, wherein the cross-linked graphene platelets further comprise a metal nanocluster, wherein the cross-linked graphene platelets further comprise a quaternary alkyl-ammonium bromide, or wherein the graphene platelet moieties contains fluorocarbon functionalization.

[0237] In some embodiments, different layers of the composite membrane module are all incorporated into a modular container, where different modules are incorporated as required to remove/remediate various contaminants as required by the end-user. Figure 24 provides an exemplary configuration of a filter module of an embodiment of the present disclosure. In this embodiment, there are four different functionalized cross-linked graphene platelet polymer composition layers, each of which is functionalized to remove or reduce the concentration of a different contaminant.

[0238] In other embodiments, the composite membrane may be used as a separation/barrier layer or for immunoisolation of a second material that is meant to be isolated from an immune response when placed in a biological system (e.g., an animal such as a mammal). For example, it may be used to separate one environment from another within a biological system. The spacing between individual graphene platelet moieties may be such that certain biological components are excluded from passing through the composite membrane. [0239] In other embodiments, the composite membrane may be used in transdermal applications, wherein the spacing between individual graphene platelet moieties may be such that certain biological components are excluded from passing through the composite membrane.

[0240] Methods of Use

[0241] The filters and membranes of the disclosure have broad application, including in water filtration, immune-isolation (i.e., protecting substances from an immune reaction), timed drug release (e.g., sustained or delayed release), hemodialysis, and hemofiltration. Some embodiments described herein comprise a method of water filtration, water desalination, water purification, immune-isolation, timed drug release, hemodialysis, or hemofiltration, where the method comprises exposing a membrane to an environmental stimulus, and wherein the membrane comprises a cross-linked graphene platelet polymer described herein.

[0242] Some embodiments include a method of increasing the purity of a liquid or gas, comprising contacting a first portion of a liquid or gas having an impurity with a filter or membrane comprising the cross-linked graphene platelet polymer compositions to form a second portion of a liquid or gas, wherein the second portion of a liquid or gas contains a lower concentration of the impurity. In some embodiments, the liquid or gas is liquid water. In other embodiments, the liquid or gas is a liquid in a physiological environment, e.g., in an animal, such as a mammal or human. In some embodiments, the impurity is a salt that may be ionized (e.g., NaCl salt or sodium and chloride ions) or a heavy metal or bacteria (or microorganisms, such as viruses) or a hydrocarbon or a larger biological compound such as antibodies (whereas the filter or membrane can allow passage of biological compounds such as insulin, proteins and/or other biological material (e.g., RNA, DNA, and/or nucleic acids))). In some embodiments, the second portion of liquid or gas (e.g., water) is formed by passing the first portion of liquid or gas (e.g., water) through the cross-linked graphene platelet polymer compositions or filters or membranes of the present disclosure. In some embodiments, the second portion of liquid or gas (e.g., water) contains 100-fold or less of the impurity as is found in the first portion of liquid or gas (e.g., water).

[0243] Some embodiments include a method of concentrating a material of interest from a liquid or gas, comprising contacting a first portion of a liquid or gas having a composition of interest with a filter or membrane comprising the cross-linked graphene platelet polymer compositions to form a second portion of liquid or gas, wherein the second portion of liquid or gas contains a lower concentration of the composition of interest, and collecting the composition of interest that does not pass through the filter or membrane. Some embodiments include a method of concentrating a composition of interest from water by reducing the water content of a solution of that composition. In some embodiments, the composition of interest may be a rare- earth element.

[0244] In some embodiments, the cross-linked graphene platelet polymer compositions and filter or membrane may be used as a pre-filtration device. For example, some embodiments include a method of increasing the purity of water, comprising contacting a first portion of water having an impurity with a filter or membrane comprising the cross-linked graphene platelet polymer compositions to form a second portion of water, wherein the second portion of water contains a lower concentration of the impurity, followed by contacting the second portion of water with a perforated graphene filter or membrane. Some exemplary perforated graphene filters and membranes are described in the art.

[0245] Other embodiments include membranes wherein the spacing between individual graphene platelet moieties is such that is allows certain compounds to pass freely, but retards the passage of other, larger compounds. In some embodiments include membranes wherein the spacing and functionalization between individual graphene platelet moieties is such that is allows certain compounds to pass freely, but retards the passage of other compounds that interact with the graphene platelet moieties or a functional compound contained in the cross-linked graphene platelet polymer. In exemplary embodiments, a membrane that allows passage of water but excludes salt ions (e.g. Na+ and C1-) can be tuned to allow passage of both water and salt ions. In other exemplary embodiments, the membrane can be tuned to allow passage of biological compounds such as insulin, proteins and/or other biological material (e.g., RNA, DNA, and/or nucleic acids), but to exclude passage of other larger biological compounds such as antibodies. In some embodiments, the membrane can be tuned to be permeable to oxygen and nutrients, but to exclude passage of cells (such as immune cells), viruses, bacteria, antibodies, and/or complements of the immune system. In some embodiments, the membrane can be tuned from one that allows passage of antibodies to one that inhibits passage of antibodies.

[0246] Other embodiments include methods of encasing a material and selectively allowing matter of a certain size to contact the encased material. The linked graphene platelet polymer compositions and filters or membranes may be used as encapsulating materials within a biological system, wherein the spacing between individual graphene platelet moieties is such that is allows certain compounds to pass freely, but retards the passage of compounds, such as antibodies from traversing the graphene platelet polymer composition. In exemplary

embodiments, a membrane that allows passage of water but excludes salt ions (e.g. Na+ and C1-) can be tuned to allow passage of both water and salt ions. In other exemplary embodiments, the membrane can be tuned to allow passage of biological compounds such as insulin, proteins and/or other biological material (e.g., RNA, DNA, and/or nucleic acids), but to exclude passage of other larger biological compounds such as antibodies. In some embodiments, the membrane can be tuned to be permeable to oxygen and nutrients, but to exclude passage of cells (such as immune cells), viruses, bacteria, antibodies, and/or complements of the immune system. In some embodiments, the membrane can be tuned from one that allows passage of antibodies to one that inhibits passage of antibodies.

[0247] Methods of Making

[0248] The cross-linked graphene platelet polymers may be formed by reacting one or more functionalized graphene platelets with one or more functionalized crosslinking compounds of the present disclosure. In some embodiments, the functionalized graphene platelets of the present disclosure and the functionalized crosslinking compounds of the present disclosure are reacted by heat or radiation (e.g., UV) or e-beam.

METHODS FOR IN VIVO AND IN VITRO USE OF GRAPHENE AND OTHER TWO- DIMENSIONAL MATERIALS

[0249] Methods of some embodiments comprise using graphene-based materials and other two-dimensional materials to transport, deliver, and or separate substances. Some embodiments comprise to enclosures formed from graphene-based materials and other two-dimensional materials on or suspended across a suitable substrate or substrates which can be porous or non- porous, which can serve as a delivery vehicle in an environment external to the enclosure, particularly in a biological environment. Some embodiments comprise enclosures formed from graphene-based materials or other two-dimensional materials containing cells, pharmaceuticals, therapeutic agents and other medicaments.

[0250] In some embodiments, enclosures are configured for long-term in vivo implantation for the delivery of pharmaceuticals, therapeutic agents or other medicaments directly to a biological environment can improve compliance with a dosing regimen relative to traditional oral and intravenous delivery methods that require patient or medical personnel intervention. In some embodiments, enclosures may be configured as oral capsules or suppositories. In some embodiments, an enclosure may be provided in a gelatin capsule for ease of swallowing. In some embodiments, enclosures may be physically coupled with or integrated into a device that ensures contact of the enclosure with the skin of a subject for transdermal drug delivery. For example, a device for ensuring contact between an enclosure and skin may comprise a pocket for receiving the enclosure and microneedles or other relief features for penetrating the stratum cornea and anchoring the device and enclosure to the skin of a subject. In some embodiments, a sheath or vascularization device may be provided or surgically placed within a subject and enclosures may be inserted into and removed from the sheath or vascularization device. The sheath or vascularization device may, for example, be tubular and rigid, perforated or permeable, so long as it is capable of withstanding forces provided in an in vivo environment. In some embodiments, a sheath or vascularization device is biocompatible. In some embodiments, a sheath or vascularization device comprises graphene. Enclosures disposed in a sheath or vascularization device may be exchanged in a minimally invasive manner when their contents are depleted, damaged, or otherwise compromised, or when an enclosure captures an analyte for ex vivo analysis. For example, an interior of an enclosure may comprise a molecule, protein (e.g., antibody), or other substance (e.g., chelating agent) that ionically, covalently or

electrostatically binds the analyte, thereby producing a chemical complex having a diameter too large to escape from the enclosure. In some embodiments, the analyte may be bound to an interior surface of an enclosure. In some embodiments, enclosures that capture analytes for ex vivo analysis may be used without a sheath or vascularization device. For example, an enclosure for capturing an analyte may be surgically inserted into a subject at a specific site for a period of time, then surgically removed, or an enclosure for capturing an analyte may be ingested and passed through the digestive system.

[0251] In some embodiments, enclosures can be configured to deliver pharmaceuticals, therapeutic agents or other medicaments directly to a biological environment. In some embodiments, enclosure are used for treating medical conditions (including chronic medical conditions) requiring a substantially continuous release and/or slow release of a pharmaceutical, therapeutic agent, or other medicament. In some embodiments, enclosures elute drugs to a biological environment at a rate that is substantially constant, e.g., in accordance with zero-order kinetics. In some embodiments, the enclosures elute drugs with a delayed release profile. In some embodiments, implanted or ingested enclosures elute drugs with a delayed release profile.

[0252] Graphene represents an atomically thin layer of carbon in which the carbon atoms reside as closely spaced atoms at regular lattice positions, and can possess favorable mechanical and electrical properties, including optical properties, thinness, flexibility, strength, conductivity (e.g., for potential electrical stimulation), tunable porosity when perforated, and permeability. The regular lattice positions can have a plurality of defects present therein, which can occur natively or be intentionally introduced to the graphene basal plane. Such defects will also be equivalently referred to herein as "pores," "apertures," "perforations," or "holes." Aside from such apertures, graphene and other two-dimensional materials can represent an impermeable layer to many substances. Therefore, when sized properly, the apertures in the impermeable layer of such materials can be useful for ingress and egress to an enclosure formed from the impermeable layer.

[0253] Some embodiments comprise graphene-based enclosures that are capable of delivering a target to an in vivo or in vitro location while maintaining a barrier (e.g., an immunoisolation barrier) in an organism or similar biological environment. Encapsulation of molecules or cells with bi-directional transport across a semi-permeable membrane, such as perforated graphene or other two-dimensional materials, while sequestering cells or the like in a biological environment (such as in an organism) can enable treatments to overcome graft rejection, the need for repeated dosages of drugs (e.g., drugs with short half-lives), and excess surgical intervention. The foregoing can be accomplished by providing technology to allow xenogenic and allogenic tissue transplants, autogenic transplants for subjects with autoimmune disorders, long term low-dose therapeutic levels of a drug, and even sense-response paradigms to treat aliments after surgical intervention, thereby reducing complications from multiple surgeries at the same site.

[0254] Some embodiments comprise enclosures formed by two-dimensional materials configured for deployment within a tissue or organ, e.g., spanning a space between walls of a tissue or organ. For example, enclosures may be suspended inside or adjacent to an artery or an organ. In some embodiments, inlet and outlet ports of an enclosure may be aligned with fluid flow within a blood vessel such that the device is configured in-line or in parallel with the fluid flow.

[0255] Some embodiments comprise enclosures formed by two-dimensional materials where the enclosure or a compartment thereof comprises at least one opening. For example, a doughnut-shaped or toroid-shaped enclosure comprising an opening can receive vasculature, nerves or nerve bundles, heart valves, bones and the like through the opening, which may anchor or secure the enclosure at a site in need of therapeutic agents contained within the enclosure.

[0256] Some embodiments comprise enclosures formed by two-dimensional materials, where the enclosure or a component thereof comprises a lumen in the form of a tube or port for introducing or removing cells, pharmaceuticals, therapeutic agents and other substances into/from the enclosure. Such a lumen, tube, or port can be joined with the two-dimensional material of the enclosure, for example, by physical methods of clamping or crimping and/or chemical methods implementing a sealant (e.g., silicone). In some embodiments, the lumen, tube, or port can be joined with an impermeable region (which, e.g., can be non-graphene) that is connected or sealed to the two-dimensional material. In some embodiments, a lumen comprises a self-sealing end for receiving the substance via syringe.

[0257] In some embodiments, perforated graphene and other two-dimensional materials can readily facilitate the foregoing while surpassing the performance of current delivery vehicles and devices, including immune-isolating devices. Without being bound by theory, it is believed that graphene can accomplish the foregoing due to its thinness, flexibility, strength, conductivity (for potential electrical stimulation), tunable porosity, and permeability in the form of perforations therein. The thinness and subsequent transport properties across the graphene membrane surface can allow a disruptive time response to be realized compared to the lengthy diffusion seen with thicker polymeric membranes of comparable size performance.

[0258] Two-dimensional materials include those which are atomically thin, with thickness from single-layer sub-nanometer thickness to a few nanometers, and which generally have a high surface area. Two-dimensional materials include metal chalogenides (e.g., transition metal dichalogenides), transition metal oxides, hexagonal boron nitride, graphene, silicene and germanene (see: Xu et al. (2013) "Graphene-like Two-Dimensional Materials) Chemical Reviews 113 :3766-3798). Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or a few layered sheets (e.g., about 20 or less) of fused six-membered rings forming an extended sp2-hybridized carbon planar lattice. In its various forms, graphene has garnered widespread interest for use in a number of applications, primarily due to its favorable combination of high electrical and thermal conductivity values, good in-plane mechanical strength, and unique optical and electronic properties. Other two- dimensional materials having a thickness of a few nanometers or less and an extended planar lattice are also of interest for various applications. In some embodiments, a two dimensional material has a thickness of 0.3 to 1.2 nm or 0.34 to 1.2 nm. In some embodiments, a two dimensional material has a thickness of 0.3 to 3 nm or 0.34 to 3 nm.

[0259] In various embodiments, the two-dimensional material comprises a sheet of a graphene-based material. In some embodiments, the sheet of graphene-based material is a sheet of single or multilayer graphene or a sheet comprising a plurality of interconnected single or multilayer graphene domains. In some embodiments, the multilayer graphene domains have 2 to 5 layers or 2 to 10 layers. In some embodiments, the layer comprising the sheet of graphene- based material further comprises non-graphenic carbon-based material located on the surface of the sheet of graphene-based material. In some embodiments, the amount of non-graphenic carbon-based material is less than the amount of graphene. In some embodiments, the amount of graphene in the graphene-based material is from 60% to 95% or from 75% to 100%. [0260] In some embodiments, the characteristic size of the perforation is from 0.3 to 10 nm, from 1 to 10 nm, from 5 to 10 nm, from 5 to 20 nm, from 5 to 25 nm, from 5 to 30 nm, from 7 to 25 nm, from 7 to 20 nm, from 10 to 25 nm, from 15 to 25 nm, from 10 nm to 50 nm, from 50 nm to 100 nm, from 50 nm to 150 nm, from 100 nm to 200 nm, or from 100 nm to 500 nm. In some embodiments, the average pore size is within the specified range. In some embodiments, 70% to 99%, 80% to 99%, 85% to 99% or 90 to 99% of the perforations in a sheet or layer fall within a specified range, but other pores fall outside the specified range.

[0261] The technique used for forming the graphene or graphene-based material is not believed to be particularly limited, and may be used to form single-layer graphene or graphene- based materials (SLG) or few-layer graphene or graphene-based materials (FLG). For example, in some embodiments, CVD graphene or graphene-based material can be used. In various embodiments, the CVD graphene or graphene-based material can be liberated from its growth substrate (e.g., Cu) and transferred to a polymer backing. Likewise, the techniques for introducing perforations to the graphene or graphene-based material are also not believed to be particularly limited, other than being chosen to produce perforations within a desired size range. Suitable techniques are described, for example, in U.S. Patent Pub. Nos. 2013/0249147, 2014/0272286 and 2015/0221474, each of which is incorporated by reference herein in its entirety. Perforations are sized to provide desired selective permeability of a species (atom, ion, molecule, DNA, RNA, protein, virus, cell, etc.) for a given application. Selective permeability relates to the propensity of a porous material or a perforated two-dimensional material to allow passage (or transport) of one or more species more readily or faster than other species. Selective permeability allows separation of species which exhibit different passage or transport rates. In two-dimensional materials selective permeability correlates to the dimension or size (e.g., diameter) of apertures and the relative effective size of the species. Selective permeability of the perforations in two-dimensional materials such as graphene-based materials can also depend on functionalization (e.g., of perforations if any, or the surface of the graphene-based material) and the specific species that are to be separated. Selective permeability can also depend on the voltage applied across the membrane. Separation of two or more species in a mixture includes a change in the ratio(s) (weight or molar ratio) of the two or more species in the mixture after passage of the mixture through a perforated two-dimensional material.

[0262] Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. In some embodiments, graphene-based materials also include materials which have been formed by stacking single or multilayer graphene sheets. In some embodiments, multilayer graphene includes 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In some embodiments, graphene is the dominant material in a graphene-based material. For example, a graphene-based material comprises at least 20% graphene, at least 30%> graphene, or at least 40% graphene, or at least 50%) graphene, or at least 60%> graphene, or at least 70% graphene, or at least 80%> graphene, or at least 90% graphene, or at least 95% graphene. In some embodiments, a graphene-based material comprises a range of graphene selected from 30% to 95%, or from 40% to 80% from 50% to 70%, from 60% to 95% or from 75% to 100%.

[0263] As used herein, a "domain" refers to a region of a material where atoms are uniformly ordered into a crystal lattice. A domain is uniform within its boundaries, but different from a neighboring region. For example, a single crystalline material has a single domain of ordered atoms. In some embodiments, at least some of the graphene domains are nanocrystals, having domain size from 1 to 100 nm or 10-100 nm. In some embodiments, at least some of the graphene domains have a domain size greater than 100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm. Some embodiments comprise a domain size up to about 1 mm. "Grain boundaries" formed by cry stall ographic defects at edges of each domain differentiate between neighboring crystal lattices. In some embodiments, a first crystal lattice may be rotated relative to a second crystal lattice, by rotation about an axis perpendicular to the plane of a sheet, such that the two lattices differ in "crystal lattice orientation".

[0264] In some embodiments, the sheet of graphene-based material comprises a sheet of single or multilayer graphene or a combination thereof. In some embodiments, the sheet of graphene-based material is a sheet of single or multilayer graphene or a combination thereof. In some embodiments, the sheet of graphene-based material is a sheet comprising a plurality of interconnected single or multilayer graphene domains. In some embodiments, the interconnected domains are covalently bonded together to form the sheet. When the domains in a sheet differ in crystal lattice orientation, the sheet is polycrystalline.

[0265] In some embodiments, the thickness of the sheet of graphene-based material is from 0.34 to 10 nm, from 0.34 to 5 nm, or from 0.34 to 3 nm. In some embodiments, a sheet of graphene-based material comprises intrinsic or native defects. Intrinsic or native defects are those resulting from preparation of the graphene-based material in contrast to perforations which are selectively introduced into a sheet of graphene-based material or a sheet of graphene. Such intrinsic or native defects include, but are not limited to, lattice anomalies, pores, tears, cracks or wrinkles. Lattice anomalies can include, but are not limited to, carbon rings with other than 6 members (e.g. 5, 7 or 9 membered rings), vacancies, interstitial defects (including incorporation of non-carbon atoms in the lattice), and grain boundaries.

[0266] In some embodiments, the layer comprising the sheet of graphene-based material further comprises non-graphenic carbon-based material located on the surface of the sheet of graphene-based material. In some embodiments, the non-graphenic carbon-based material does not possess long range order and may be classified as amorphous. In some embodiments, the non-graphenic carbon-based material further comprises elements other than carbon and/or hydrocarbons. Non-carbon elements which may be incorporated in the non-graphenic carbon include, but are not limited to, hydrogen, oxygen, silicon, copper and iron. In some

embodiments, the non-graphenic carbon-based material comprises hydrocarbons. In some embodiments, carbon is the dominant material in non-graphenic carbon-based material. For example, a non-graphenic carbon-based material comprises at least 30% carbon, or at least 40% carbon, or at least 50% carbon, or at least 60% carbon, or at least 70% carbon, or at least 80% carbon, or at least 90% carbon, or at least 95% carbon. In some embodiments, a non-graphenic carbon-based material comprises a range of carbon selected from 30% to 95%, or from 40% to 80%, or from 50% to 70%.

[0267] Nanomaterials that contain pores in its basal plane, regardless of whether they are intrinsically or natively present or intentionally created, will be referred to herein as "perforated two-dimensional materials." Exemplary perforated two-dimensional materials include perforated graphene-based materials and/or other perforated graphene. The term "perforated graphene-based materials" is used herein to denote a two-dimensional material comprising a graphene sheet with defects in its basal plane, regardless of whether the defects are intrinsically or natively present or intentionally produced. Perforated graphene-based materials include perforated graphene.

[0268] In some embodiments, the perforated two-dimensional material contains a plurality of holes of size (or size range) appropriate for a given enclosure application. The size distribution of holes may be narrow, e.g., limited to a 1-10% + 3% deviation in size, or a 1-20% + 5% deviation in size, or a 1-30% + 5% deviation in size. In some embodiments, the characteristic dimension of the holes is selected for the application. For circular holes, the characteristic dimension is the diameter of the hole. In some embodiments relevant to non-circular pores, the characteristic dimension can be taken as the largest distance spanning the hole, the smallest distance spanning the hole, the average of the largest and smallest distance spanning the hole, or an equivalent diameter based on the in-plane area of the pore. These examples illustrate that various pore geometries or shapes may be implemented in a two-dimensional membrane, such as circular, oval, diamond, slits and the like. As used herein, perforated graphene-based materials include materials in which non-carbon atoms have been incorporated at the edges of the pores.

[0269] In various embodiments, the two-dimensional material comprises graphene, molybdenum disulfide, or hexagonal boron nitride. In more particular embodiments, the two- dimensional material can be graphene. Graphene can includes single-layer graphene, multi-layer graphene, or any combination thereof. Other nanomaterials having an extended two-dimensional molecular structure can also constitute the two-dimensional material in the some embodiments. For example, molybdenum disulfide is a representative chalcogenide having a two-dimensional molecular structure, and other various chalcogenides can constitute the two-dimensional material in some embodiments. Choice of a suitable two-dimensional material for a particular application can be determined by a number of factors, including the chemical and physical environment into which the graphene or other two-dimensional material is to be terminally deployed. In some embodiments, materials employed in making an enclosure are biocompatible or can be made biocompatible. In some embodiments, combinations of two-dimensional materials may be used in a multilayer or multi-sheet configuration to make an enclosure. For example, a first two- dimensional material in the multilayer or multi-sheet configuration, nearer an interior of an enclosure, could provide structural support while a second two-dimensional material of the multilayer or multi-sheet configuration, nearer the external environment, could impart biocompatibility.

[0270] The process of forming holes in graphene and other two-dimensional materials will be referred to herein as "perforation," and such nanomaterials will be referred to herein as being "perforated." In a graphene sheet an interstitial aperture is formed by each six carbon atom ring structure in the sheet and this interstitial aperture is less than one nanometer across. In particular, this interstitial aperture is believed to be about 0.3 nanometers across its longest dimension (the center to center distance between carbon atoms being about 0.28 nm and the aperture being somewhat smaller than this distance). Perforation of sheets comprising two-dimensional network structures typically refers to formation of holes larger than the interstitial apertures in the network structure.

[0271] Due to the atomic-level thinness of graphene and other two-dimensional materials, it can be possible to achieve high liquid throughput fluxes during separation or filtration processes, even with holes that are in the ranges of from 0.3 to 10 nm, from 1 to 10 nm, from 5 to 10 nm, from 5 to 20 nm, from 10 nm to 50 nm, from 50 nm to 100 nm, from 50 nm to 150 nm, from 100 nm to 200 nm, or from 100 nm to 500 nm.

[0272] Chemical techniques can be used to create holes in graphene and other two- dimensional materials. Exposure of graphene or another two-dimensional material to ozone or atmospheric pressure plasma (e.g., an oxygen/argon or nitrogen/argon plasma) can effect perforation.

[0273] In some embodiments, holes can be created using focused ion beam drilling, ion bombardment, nanoparticle bombardment, and combinations thereof. In some embodiments, lithographic techniques can be used to remove matter from the planar structure of two- dimensional materials to create holes.

[0274] In various embodiments, the holes produced in the graphene or other two-dimensional material can range from about 0.3 nm to about 50 nm in size. In some embodiments, hole sizes can range from 1 nm to 50 nm. In some embodiments, hole sizes can range from 1 nm to 10 nm. In some embodiments, hole sizes can range from 5 nm to 10 nm. In some embodiments, hole sizes can range from 1 nm to 5 nm. In some embodiments, the holes can range from about 0.5 nm to about 2.5 nm in size. In some embodiments, the hole size is from 0.3 to 0.5 nm. In some embodiments, the hole size is from 0.5 to 10 nm. In some embodiments, the hole size is from 5 nm to 20 nm. In some embodiments, the hole size is from 0.7 nm to 1.2 nm. In some embodiments, the hole size is from 10 nm to 50 nm. In some embodiments where larger hole sizes are preferred, the hole size is from 50 nm to 100 nm, from 50 nm to 150 nm, or from 100 nm to 200 nm.

[0275] The term substance is used genetically herein to refer to atoms, ions, molecules, macromolecules, viruses, cells, particles and aggregates thereof. Substances of particular interest are molecules of various size, including biological molecules, such as DNA, RNA, proteins and nucleic acids. Substances can include pharmaceuticals, drugs, medicaments and therapeutics, which include biologies and small molecule drugs.

[0276] Figure 25 shows an illustrative schematic demonstrating the thickness of graphene in comparison to conventional drug delivery vehicles and devices. The biocompatibility of graphene can further promote this application, particularly by functionalizing the graphene to be compatible with a particular biological environment (e.g., via available edge bonds, bulk surface functionalization, pi-bonding, and the like). Functionalization can provide membranes having added complexity for use in treating local and systemic disease. Figure 25 illustrates a wall of an enclosure formed with perforated two-dimensional material having hole sizes in the range of 400-700 nm which will retain active cells. The external biological environment abutting the enclosure (the full enclosure is not shown) is illustrated with an optional porous substrate layer adjacent and external to the perforated two-dimensional material and an optional woven support material external to the perforated two-dimensional material. As illustrated, implantation of such an enclosure contemplates vascularization into any such substrate layer materials. In some embodiments intended to provide immunoisolation, hole sizes can be tailored to prevent entrance of antibodies into the enclosure.

[0277] In various embodiments, sealed enclosures, primarily formed from a two-dimensional material, such as graphene, that remain capable of bi-directional transportation of materials. In various embodiments, at least one section or panel of the enclosure contains appropriately sized perforations in the two-dimensional material to allow ingress and egress, respectively, of materials of a desired size to and from the interior of the enclosure.

[0278] In some embodiments, the two-dimensional material, such as graphene, can be affixed to a suitable porous substrate. Suitable porous substrates can include, for example, thin film polymers; ceramics and inorganic materials, such as Si ₃ N ₄ , SiO ₂ , Si; thin metal films (e.g., Ti, Au); track-etched polyimide; polycarbonate; PET; and combinations thereof.

[0279] In some embodiments, the enclosure comprises two or more two-dimensional material layers. In some embodiments, an intermediate layer is positioned between two separate two-dimensional layers. In some embodiments, the intermediate layer is porous. In some embodiments, the intermediate layer comprises carbon nanotubes, lacey carbon, nanoparticles, lithographically patterned low-dimensional materials, silicon and silicon nitride micromachined material, a fine mesh, such as a transmission electron microscopy grid, or combinations of these.

[0280] In some embodiments, the intermediate layer is functionalized. In some

embodiments, functionalization comprises surface charges (e.g., sulfonates) attached to or embedded in the intermediate layer. Without being bound by theory, it is believed that surface charges can impact molecules and/or ions that can traverse the membrane. In some

embodiments, functionalization comprises specific binding sites attached to or embedded in the intermediate layer. In some embodiments, functionalization comprises proteins or peptides attached to or embedded in the intermediate layer. In some embodiments, functionalization comprises antibodies and/or antigens (e.g., IgG-binding antigens) or an antibody-binding fragment thereof attached to or embedded in the intermediate layer. In some embodiments, functionalization comprises adsorptive substances attached to or embedded in the intermediate layer. In some embodiments, functionalization involves catalytic and/or regenerative substances or groups. In some embodiments, functionalization comprise a negatively or partially negatively charged group (e.g., oxygen) attached to or embedded in the intermediate layer. In some embodiments, functionalization comprises a positively or partially positively charged group attached to or embedded in the intermediate layer. In some embodiments, the functionalization moieties are free to diffuse within the intermediate layer. In some embodiments, the functionalization moieties are trapped between two two-dimensional material layers, but are not restricted to a single position in the channel (i.e., they are mobile within the intermediate layer, but are inhibited from traversing the two-dimensional material layers, e.g., based the size of the pores in the two-dimensional material layers). In some embodiments, functionalization of the intermediate layer functions as an entrainment layer, and inhibits substances from traversing the membrane that would be able to traverse the membrane absent the functionalization. Thus, in some embodiments functionalization imparts a selective permeability upon the membrane based on properties of potential permeants such as charge, hydrophobicity, structure, etc.

[0281] In some embodiments, a substrate layer is disposed on one or both surfaces of the graphene-based material layer. Without being bound by theory, it is believed that the substrate layer can improve biocompatibility of membranes, for instance by reducing biofouling;

preventing protein adsorption-related problems; enhancing vascularization and/or tissue ingrowth or distribution; supporting cells; and/or separating cells to prevent or inhibit clumping or agglomerating. In some embodiments, the substrate layer can increase vascularization near the enclosure, thus prompting the formation of blood vessels and/or tissue ingrowth in close proximity to the enclosure.

[0282] In some embodiments, the substrate is disposed directly on the graphene-based material layer. In some embodiments, the substrate is disposed indirectly on the graphene-based material layer; for instance, an intermediate layer can be positioned between the substrate layer and the graphene-based material layer. In some embodiments, the graphene-based material layer is suspended on a substrate layer. In some embodiments, the substrate layer is affixed to the graphene-based material layer.

[0283] The substrate layer can be porous and/or nonporous. In some embodiments, the substrate layer contains porous and nonporous sections. In some embodiments the substrate layer comprises a porous or permeable fibrous layer. Porous substrates include, for example, one or more of ceramics and thin film polymers. Exemplary ceramics include nanoporous silica (silicon dioxide), silicon, SiN, and combinations thereof. In some embodiments, the substrate layer comprises track-etched polymers, expanded polymers, patterned polymers, woven polymers, and/or non-woven polymers. In some embodiments, the substrate layer comprises a plurality of polymer filaments. In some embodiments, the polymer filaments can comprise a thermopolymer, thermoplastic or melt polymer, e.g., that can be molded or set in an optional annealing step. In some embodiments, the polymer filaments are hydrophobic. In some embodiments, the polymer filaments are hydrophilic. In some embodiments, the substrate layer comprises a polymer selected from the group consisting of polysulfones, polyurethane, polymethylmethacrylate (PMMA), polyglycolid acid (PGA), polylactic acid (PLA), polyethylene glycol (PEG), polylactic-co-glycolic acid (PLGA), polyamides (such as nylon-6,6, supramid and nylamid), polyimides, polypropylene, polyethersulfones (PES), polyvinylidine fluoride (PVDF), cellulose acetate, polyethylene, polypropylene, polycarbonate, polytetrafluoroethylene (PTFE) (such as Teflon), polyvinylchloride (PVC), polyether ether ketone (PEEK), mixtures and block co-polymers of any of these, and combinations and/or mixtures thereof. In some embodiments, the polymers are biocompatible, bioinert and/or medical grade materials.

[0284] In some embodiments, the substrate layer comprises a biodegradable polymer. In some embodiments, a substrate layer forms a shell around the enclosure. In some embodiments, the substrate layer shell, or a portion thereof, can be dissolved or degraded, e.g., in vitro.

[0285] Suitable techniques for depositing or forming a porous or permeable polymer on the graphene-based material layer include casting or depositing a polymer solution onto the graphene-based material layer or intermediate layer using a method such as spin-coating, curtain coating, doctor-blading, immersion coating, electrospinning, or other similar techniques.

Electrospinning technique are described, e.g., in US 2009/0020921 and/or U.S. Application No. 14/609,325, both of which are hereby incorporated by reference in their entirety.

[0286] In some embodiments, the process for forming a substrate layer includes an electrospinning process in which a plurality of polymer filaments are laid down to form a porous mat, e.g., on the graphene-based material layer. In some embodiments, the mat has pores or voids located between deposited filaments of the fibrous layer. Figure 25 shows an illustrative SEM micrograph of a graphene or graphene-based film deposited upon a plurality of electrospun PVDF fibers. In some embodiments, the electrospinning process comprises a melt

electrospinning process or a solution electrospinning process, such as a wet electrospinning process or a dry electrospinning process. (See, e.g., Sinha-Ray et al. J. Membrane Sci. 485, 1 July 2015, 132-150.) In some embodiments, the polymer can be present in a spin dope at a concentration of 2 wt.% to 15 wt.%, or 5 wt.% to 10 wt.%, or about 7 wt.%. Suitable solvents for the spin dope include any solvent that dissolves the polymer to be deposited and which rapidly evaporates, such as m-cresol, formic acid, dimethyl sulfoxide (DMSO), ethanol, acetone, dimethylacetamide (DMAC), dimethylformamide (DMF), water, and combinations thereof. In some embodiments, the spin dope solvent is biocompatible and/or bioinert. In some

embodiments, the amount of solvent used can influence the morphology of the substrate layer. In dry electrospinning processes, the spun fibers of the fibrous layer can remain as essentially discrete entities once deposited. In some embodiments, wet electrospinning processes deposit the spun fibers such that they are at least partially fused together when deposited. In some embodiments, the size and morphology of the deposited fiber mat (e.g., degree of porosity, effective pore size, thickness of fibrous layer, gradient porosity) can be tailor based on the electrospinning process used.

[0287] The porosity of the fibrous layer can include effective porosity values— i.e., void space values— (e.g. measured via imagery or porometry methods) of up to about 95% (i.e., the layer is 95% open), about 90%, about 80%, or about 60% with a broad range of pore sizes. In some embodiments, a single spinneret can be moved to lay down a mat of the fibrous layer. In other embodiments, multiple spinnerets can be used for this purpose. In some embodiments, the spun fibers in an electrospun fibrous layer can have a fiber diameter ranging from about 1 nm to about 100 μm, or about 10 nm to about 1 μm, or about 10 nm to about 500 nm, or about 100 nm to about 200 nm, or about 50 nm to about 120 nm, or about 1 μπι to about 5 μm, or about 1 μπι to about 6 μm, or about 5 μπι to about 10 μπι. In some embodiments, the fiber diameter is directly correlated with a depth (Z-axis) of a pore abutting the graphene-based material layer (disposed in the X-Y plane), and large diameter fibers can lead to large unsupported spans of material.

[0288] In some embodiments, the substrate layer can have pores with an effective pore size of from about 1 nm to about 100 μm, or about 10 nm to about 1 μm, or about 10 nm to about 500 nm, or about 100 nm to about 200 nm, or about 50 nm to about 120 nm, or about 1 μπι to about 5 μηι, or about 1 μιη to about 6 μηι, or about 5 μιη to about 10 μιη. Pore diameters in the substrate layer can be measure, for example, via a bubble point method.

[0289] In some embodiments, the substrate layer can have an average pore size gradient throughout its thickness. "Pore size gradient," describes a layer with a plurality of pores, where the average diameter of the pores increases or decreases based on the proximity of the pore to the graphene-based material layer. For example, a fibrous layer can have an average pore size gradient that decreases nearer the surface of a graphene-based material. In some embodiments, an average pore size of the fibrous layer is smaller nearer the surface of the graphene-based material than at an opposite surface of the fibrous layer. For example, the fibrous layer can have effective pore diameters of from about 1 μπι to about 6 μπι close to the intermediate layer or the graphene-based material layer which can increase to greater than 100 μπι at the maximum distance away from the intermediate layer or graphene-based material layer.

[0290] In some embodiments, the fibrous layer can have a "porosity gradient" throughout its thickness, which can be measured for instance using imagery. "Porosity gradient," as used herein, describes a change, along a dimension of the fibrous layer, in the porosity or total pore volume as a function of distance from the graphene-based material layer. For example, throughout the thickness of the porous fibrous layer, the porosity can change in a regular or irregular manner. A porosity gradient can decrease from one face of the fibrous layer to the other. For example, the lowest porosity in the fibrous layer can be located spatially closest to the graphene-based material layer, and the highest porosity can be located farther away (e.g., spatially closer to an external environment). A porosity gradient of this type can be achieved by electrospinning fibers onto a graphene-based material layer such that a fiber mat is denser near the surface of the graphene-based material layer and less dense further from the surface of the graphene-based material layer. In some embodiments, a substrate layer can have a relatively low porosity close to the graphene-based material layer, a higher porosity at a mid-point of the fibrous layer thickness (which can, for example, contain a supporting mesh for reinforcement or other particles), and return to a relatively low porosity at an external surface distal to the graphene-based material layer. [0291] In some embodiments, the substrate layer can have a "permeability gradient" throughout its thickness. "Permeability gradient," as used herein, describes a change, along a dimension of the fibrous layer, in the "permeability" or rate of flow of a liquid or gas through a porous material. For example, throughout the thickness of the fibrous layer, the permeability can change in a regular or irregular manner. A permeability gradient can decrease from one face of the fibrous layer to the other. For example, the lowest permeability in the fibrous layer can be located spatially closest to the graphene-based material layer, and the highest permeability can be located farther away. Those of skill in the art will understand that permeability of a layer can increase or decrease without pore diameter or porosity changing, e.g., in response to chemical functionalization, applied pressure, voltage, or other factors.

[0292] It should also be noted that in some embodiments, the enclosure can be supported by one or more support structures. In some embodiments, the support structure can itself have a porous structure wherein the pores are larger than those of the graphene-based material layer. In some embodiments, the support structure is entirely porous (i.e., the support structure is formed as a frame at a perimeter of a graphene-based material layer). In some embodiments, the support structure is at least in part non-porous comprising some structure interior to a perimeter of a graphene-based material layer.

[0293] In some embodiments, the thickness and structure of the substrate layer can be chosen to convey a desired degree of structural support (e.g., to prevent tearing and/or buckling) to the graphene-based material layer. In various embodiments, the substrate layer can have a thickness of about 1 mm or less, or about 1 μm or less. In some embodiments, a thickness of the substrate layer can range from about 100 nm to about 100 μm, or about 1 μm to about 50 μm, or about 10 μπι to about 20 μm, or about 15 μπι to about 25 μm. In some embodiments, the substrate layer has a thickness greater than about 5 μm, or greater than about 10 μm, or greater than about 15 μm. In some embodiments, the substrate layer has a thickness of less than 1 μm.

[0294] In some embodiments, both the graphene-based material layer and the substrate layer include a plurality of pores therein. In some embodiments, both the graphene-based material layer and the substrate layer contain pores, and the pores in the graphene-based material layer are smaller, on average, than the pores in the substrate layer. In some embodiments, the median pore size in the graphene-based material layer are smaller than the median pore size in the substrate layer. For example, in some embodiments, the substrate layer can contain pores with an average and/or median diameter of about 1 μιη or larger and the graphene-based material layer can contain pores with an average and/or median diameter of about 10 nm or smaller. Accordingly, in various embodiments, the average and/or median diameter of pores in the graphene-based material layer are at least about 10-fold smaller than are the average and/or median diameter of pores in the substrate layer. In some embodiments, the average and/or median diameter of pores in the graphene-based material layer are at least about 100-fold smaller than are the average and/or media diameter of pores in the substrate layer.

[0295] In some embodiments, the substrate layer can provide a scaffold for tissue growth, cell growth, support, and/or vascularization. In some embodiments, the substrate layer or wall comprises additives, such as pharmaceuticals, cells, growth factors (e.g., VEGF), signaling molecules, cytokines, clotting factors, blood thinners, immunosuppressants, antimicrobial agents, hormones, antibodies, minerals, nutrients or combinations thereof. In some embodiments, additives such as pharmaceuticals, cells, growth factors, clotting factors, blood thinners, immunosuppressants, antimicrobial agents, hormones, antibodies, antigens (e.g., IgG-binding antigens) or an antibody-binding fragment thereof, minerals, nutrients or combinations thereof are positioned on the inside of the disclosure. In some embodiments, the substrate layer or wall comprises materials toxic to bacteria or cells (without being bound by theory, it is believed that incorporating toxic materials into the wall will prevent passage of potentially dangerous cells across the wall).

[0296] In some embodiments, additives beneficially promote cell or tissue viability or growth, reduce or prevent infection, improve vascularization to or near the enclosure, improve biocompatibility, reduce biofouling, and/or reduce the risk of adverse reactions. In some embodiments, additives can modulate properties, such as hydrophobicity or hydrophilicity, of the substrate layer. In some embodiments, additives can be used to modulate elution of a substance from a compartment in the enclosure. For instance, additives can confer shell-like properties to a substrate layer, such that degradation or removal of the additives allows substances in the compartment to escape the enclosure (and, by extension, substances from the external environment to enter to enclosure).

[0297] In some embodiments, the enclosures have a single compartment without sub- compartments. In some embodiments, the enclosures can have a plurality of sub-compartments within the main enclosure each sub-compartment comprises perforated two-dimensional material to allow passage of one or more substance into or out of the sub-compartment. In such embodiments, sub-compartment can have any useful shape or size. In some embodiments, 2 or 3 sub-compartments are present. Several examples of enclosure sub -compartments are illustrated in Figures 26A-26F. In Figure 26A, a nested configuration is illustrated, the main enclosure B completely contains a smaller enclosure A, such that substances in the centermost enclosure A can pass into the main enclosure B, and potentially react with or within the main compartment during ingress and egress therefrom. In this embodiment, one or more substance in A can pass into B and one or more substance in A can be retained in A and not to B. Two sub compartments in which one or more substance can pass directly between the sub-compartments are in direct fluid communication. Passage between sub-compartments and between the enclosure and the external environment is via passage through the holes of a perforated two-dimensional material. The barrier (membrane, i.e. perforated two-dimensional material) between compartment A and B can be permeable to all substances in A or selectively permeable to certain substances in A. The barrier (membrane) between B and the external environment can be permeable to all substances in B or selectively permeable to certain substances in B. In Figure 26A, sub-compartment A is in direct fluid communication with sub-compartment B which in turn is in direct fluid

communication with the external environment. Compartment A in this nested configuration is only in indirect fluid communication with the external environment via intermediate passage into sub-compartment B. The two-dimensional materials employed in different sub-compartments of a given enclosure may be the same or different materials and the perforations or hole sizes in the two-dimensional material of different sub-compartments may be the same or different dependent upon the substances involved and the application.

[0298] In Figure 26B the enclosure is bisected with an impermeable wall (e.g., formed of non-porous or non-permeable sealant) forming sub-compartments A and B, such that both sections have access to the egress location independently, but there is no direct or indirect passage of substances from A to B. (It will be appreciated, however, that substances exiting A or B may enter the other sub-compartment indirectly via the external environment.)

[0299] In Figure 26C the main enclosure is again bisected into sub-compartments A and B, but with a perforated material forming the barrier between the sub-compartments. Both sub- compartments not only have access to the egress location independently, but in some

embodiments also can interact with one another, i.e. the sub-compartments are in direct fluid communication. In some embodiments, the barrier (membrane) between compartments A and B is selectively permeable, for example allowing at least one substance in A to pass into B, but not allowing the substances originating in B to pass to A.

[0300] Figure 26D illustrates an enclosure having three compartments. The enclosure is illustrated with sub-compartment A having egress into sub-compartment B, which in turn has egress into sub -compartment C, which in turn has egress to the external environment.

Compartments A and B have no egress to the external environment, i.e. they are not in direct fluid communication with the external environment. Adjacent sub-compartments A and B and adjacent sub-compartments B and C are each separated by a perforated two-dimensional material and are thus in direct fluid communication with each other. Sub-compartment A is only in indirect fluid communication with compartment C and the external environment via sub- compartment B or B and C, respectively. Various other combinations of semi-permeable barrier (membranes) or non-permeable barriers can be employed to separate compartments in the enclosures. Various perforation size constraints can change depending on how the enclosure is ultimately configured (e.g., if one enclosure is within another versus side-by-side). Regardless of the chosen configuration, the boundaries or at least a portion thereof, of the enclosure can be constructed from a two-dimensional material in order to realize the benefits thereof, specifically such that the thickness of the active membrane is less than the diameter of the target to be passed selectively across the membrane. In some embodiments, the pore size of the two-dimensional material can range between about 0.3 nm to about 10 nm in size. Larger pore sizes are also possible. [0301] Figure 26E illustrates an enclosure having multiple compartments in a radial array around a central compartment. In the embodiment shown, central compartment E is surrounded by four compartments A-D. Top and bottom surfaces of compartment E may also be joined to compartments that are not shown. In addition to a square or rectangular shape for compartment E, the central compartment of a radial array may have a hexagonal, octahedral, decahedral, dodecahedral or circular shape to increase the number of connection points for the radially arranged compartments.

[0302] The enclosure of Figure 26E is illustrated with compartment E having egress into compartments A-D, which in turn have egress to the external environment. Compartment E does not have egress to the external environment, i.e. it is not in direct fluid communication with the external environment. Compartments A-D have egress to the external environment through at least one section of permeable two-dimensional membrane, but in some embodiments compartments A-D may be formed entirely by a permeable two-dimensional membrane.

[0303] Figure 26F illustrates an enclosure having a single compartment (A) and no sub- compartments. In the Figure, the compartment is in direct fluid communication with an environment external to the enclosure.

[0304] In an example of the operation of an enclosure configured as a radial array, compartment E may independently transfer molecules to, receive molecules from, or exchange molecules with compartments A, B, C and/or D. In some embodiments, compartment E may contain a biological organism producing a molecule that is transferred to one or more of compartments A-D, which may contain different molecules capable of reacting with the molecule produced in the central compartment. In some embodiments, central compartment E may receive one or more molecules from one or more of the radial compartments A-D, such that compartment E acts as a reaction chamber. In such an embodiment, it may be useful for compartments A-D to only have egress to an external environment through central compartment E. The perforated two-dimensional material separating the central compartment from each of the radially arranged compartments may be the same or different in terms of composition and hole size. [0305] In some embodiments, the sub-compartments are connected by microfluidic channels. In some embodiments, the microfluidic channels comprise valves. In some embodiments, substances can diffuse between sub-compartments. In some embodiments, substances can pass between sub-compartments via a tortuous path membrane. In some embodiments, reaction rates between substances in two sub-compartments can be controlled by modulating the ability of the substances to pass from the first sub-compartment to the second sub-compartment, and vice versa.

[0306] Some embodiments comprise a device comprising more than one enclosure (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 enclosures), where at least a portion of the enclosures are connected such that a reservoir is formed between the enclosures. In some embodiments, at least a portion of the enclosures are connected by microfluidic channels. In some embodiments, the microfluidic channels comprise valves. In some embodiments, substances can diffuse between at least a portion of the enclosures. In some embodiments, substances can pass between at least a portion of the enclosures via a tortuous path membrane. In some embodiments, reaction rates between substances in different enclosures can be controlled by modulating the ability of the substances to pass from one enclosure to the other enclosure, and vice versa.

[0307] Some embodiments comprise two or more enclosures configured in a similar manner to the sub-compartments described above. For instance, two enclosures can be positioned in a nested configuration, where only the outer enclosure is in fluid communication with an environment external to the enclosure. In some embodiments with a nested enclosure

configuration, the outermost enclosure comprises a substance that is released over a period of days, weeks, months, or years. In some embodiments, the innermost enclosure comprises a substance that is released after the substance in the outermost enclosure is substantially depleted, at which point the substance from the innermost enclosure can pass through the outermost enclosure and into the external environment. In some embodiments, a polymer protective shell (e.g., a polymer coating) surrounding the inner enclosure is degraded after a certain time period, for instance after the substance in the outermost enclosure is depleted. In some embodiments, devices with more than two nested enclosures can be used. Without being bound by theory, it is believed that a nested enclosure configuration can be used for sustained substance release and/or weaning a subject off a pharmaceutical product.

[0308] Some embodiments comprise a means for moving substances and/or fluid between sub-compartments. Some embodiments comprise a means for moving substances and/or fluid between enclosures and/or reservoirs positioned between the enclosures. For instance, passage of substances and/or fluids can be in response to a concentration gradient, electric potential, or pressure difference. In some embodiments, passage of substances and/or fluids can be in response to activating or deactivating electrically gated pores.

[0309] In some embodiments, passage of substances and/or fluid is via osmosis. In some embodiments, an osmotic engine is used to influence passage of substances and/or fluids. In some embodiments, osmosis is triggered based on a change in basal cell chemistry. For instance, the presence of antibodies or an immune-mediated response can trigger the release of substances from the enclosure (for example, an immune response could trigger release of antibiotics from the enclosure device).

[0310] In some embodiments, a piston is used to influence passage of substances and/or fluids (e.g., the piston can be used to push out or draw in substances/fluids from an enclosure and/or reservoir). In some embodiments, passage of substances and/or fluid between enclosures and/or reservoirs is via an automated or triggered release of the substances and/or fluid. In some embodiments, the passage is triggered by a microchip positioned in or on the device. In some embodiments, the microchip is triggered by a triggering device located external to the enclosure device.

[0311] It should also be noted that in some embodiments, the enclosure can be supported by one or more support structures. In some embodiments, the support structure can itself have a porous structure wherein the pores are larger than those of the two-dimensional material. In some embodiments, the support structure is entirely porous. In some embodiments, the support structure is at least in part non-porous.

[0312] The multiple physical embodiments for the enclosures and their uses can allow for various levels of interaction and scaled complexity of problems to be solved. For example, a single enclosure can provide drug elution for a given time period, or there can be multiple sizes of perforations to restrict or allow movement of distinct targets, each having a particular size.

[0313] In some embodiments, added complexity with multiple sub-compartments can allow for interaction between target compounds to catalyze or activate a secondary response (i.e., a "sense-response" paradigm). For example, if there are two sections of an enclosure that have access to egress independently, exemplary compound A may undergo a constant diffusion into the body, or either after time or only in the presence of a stimulus from the body. In such embodiments, exemplary compound A can activate exemplary compound B, or inactivate functionalization blocking exemplary compound B from escaping. The bindings to produce the foregoing effects can be reversible or irreversible. In some embodiments, exemplary compound A can interact with chemical cascades produced outside the enclosure, and a metabolite subsequent to the interaction can release exemplary compound B (by inactivating

functionalization). Further examples utilizing effects that take place in a similar manner include using source cells (non-host, allogenic) contained in an enclosure, within which secretions from the cell can produce a "sense-response" paradigm.

[0314] In some embodiments, growth factors and/or hormones can be loaded in the enclosure to encourage vascularization (see Figure 25). In the foregoing embodiments, cell survival can be far superior as a result of bi-directional transport of nutrients and waste.

[0315] In some embodiments, the relative thinness of graphene can enable bi-directional transport across the membrane enclosure in close proximity to blood vessels, particularly capillary blood vessels, and other target cells. In some embodiments, using a graphene-based enclosure can provide differentiation over other solutions accomplishing the same effect because the graphene membrane is not appreciably limiting the permeability. Thus, in some embodiments the diffusion of molecules through the medium or interstitial connections can limit the movement of a target.

[0316] In some embodiments, a "sense-response" paradigm with graphene is enabled by a superior time response. The biocompatibility of graphene can further enhance this application. Further, due to its extreme thinness, graphene is less susceptible to biofouling and clogging than traditional permeable materials and adsorbed species may be removed by electrification of graphene. Expansion to functionalized graphene membranes for added complexity in treating local and systemic disease is also predicted to lower the degree of biofouling, due to electrostatic repulsion by the functional moieties. Additionally, the mechanical stability of graphene can make it suitable to withstand physical stresses and osmotic stresses within the body.

[0317] Figures 27A and 27B provide a schematic illustration of enclosures with a single compartment for immunoisolation (it will be appreciated that the enclosure can having a plurality of sub-compartments, for example, two or three sub-compartments). The enclosure (2730) of Figure 27 A is shown as a cross-section formed by an inner sheet or layer (2731) comprising perforated two-dimensional material, such as a graphene-based material, and an outer sheet or layer (2732) of a substrate material (though in some embodiments, the inner layer comprises the substrate material, and the outer layer comprises the perforate two-dimensional material). The substrate material can be porous, selectively permeable or non-porous, and/or and non- permeable. However at least a portion of the support material is porous or selectively permeable. The enclosures in Figure 27 contain selected living cells (2733). Figure 27B provides an alternative cross-section of the enclosure of Figure 27 A, showing the space or cavity formed between a first composite layer (2732/2731) and a second composite layer (2732/2731) (in the figure, the cavity is depicted to contain roughly circular symbols, which can be cells or any other substance) where a sealant 2734 is illustrated as sealing the edges of the composite layers. It will be appreciated that seals at the edges of the composite layers can be formed employing physical methods, such as clamping, crimping, or with adhesives. Methods and materials for forming the seals at the edges are not particularly limiting. In some embodiments, the sealing material provides a non-porous and non-permeable seal or closure. In some embodiments, a portion of the enclosure is formed from a sealant, such as a silicone, epoxy, polyurethane or similar material. In some embodiments, the sealant is biocompatible. For instance, in some

embodiments the seal does not span the entire length or width of the device. In some

embodiments, the seal forms a complete loop around the cavity. In some embodiments, the seal is formed as a frame at a perimeter of a two-dimensional material. In some embodiments, the seal is positioned, at least in in part, interior to a perimeter of a two-dimensional material. [0318] If cells are placed within the enclosure, at least a portion of the enclosure can be permeable to oxygen and nutrients sufficient for cell growth and maintenance and permeable to waste products. In some embodiments, the enclosure is not permeable to cells (such as immune cells), viruses, bacteria, antibodies, and/or complements of the immune system. Thus, in some embodiments, cells from the external environment cannot enter the enclosure and cells in the enclosure are retained. In some embodiments, the enclosure is permeable to desirable products, such as growth factors produced by the cells. The cells within the enclosure are immune- isolated. In some embodiments, hole sizes in perforated two-dimensional materials useful for immunoisolation range in size from about 1-20 nm, about 1-10 nm, about 3-10 nm, or about 3-5 nm. In some embodiments, the holes are from about 1 nm to about 30 nm in size, such as about 30 nm, about 20 nm, about 18 nm, about 15 nm, about 10 nm, about 5 nm, or about 3 nm. See, e.g., Song et al., Scientific Reports, 6: 23679, doi: 10.1038/srep23679 (2016), which is incorporated herein by reference in its entirety.

[0319] Figures 28A-28C illustrate an exemplary method for forming an enclosure and introducing selected substances, for example cells therein. The method is illustrated with use of a sealant for forming the enclosure. The exemplary enclosure has no sub-compartment.

Enclosures with sub-compartments, for example nested or adjacent sub-compartments can be readily prepared employing the illustrated method. As illustrated in Figure 28A, a first composite layer or sheet is formed by placing a sheet or layer of two-dimensional material, particularly a sheet of graphene-based material or a sheet of graphene (2841), in contact with a support layer (2842). At least a portion of the support layer (2842) of the first composite is porous or permeable. Pore size of the support layer is generally larger than the holes or apertures in the two-dimensional material employed and may be tuned for the environment (e.g. body cavity). A layer of sealant (2844), e.g. silicone, is applied on the sheet or layer of perforated two-dimensional material outlining a compartment of the enclosure wherein the sealant will form a non-permeable seal around a perimeter of the enclosure. Formation of a single compartment is illustrate in Figures 28A-28C, however, it will be appreciated that multiple independent compartments within an enclosure can be formed by an analogous process. A second composite layer formed in the same way as the first can be prepared and positioned with the sheet or layer of perforated two dimensional materials in contact with the sealant. (Alternatively, a sealant can be applied to a portion of composite layer and the layer can be folded over in contact with the sealant to form an enclosure. A seal is then formed between the two composite layers.

Appropriate pressure may be applied to facilitate sealing without damaging the two-dimensional material or its support. It will be appreciated that an alternative enclosure can be formed by applying a sheet or layer of non-porous and non-permeable support material in contact with the sealant. In this case only a portion of the enclosure is porous and permeable. Sealed composite layers are illustrated in Figure 284B where it is shown that the sealed layers can be trimmed to size around the sealant to form the enclosure.) The enclosure formed is shown to have an external porous support layer 2842, the sheet or layer of perforated two-dimensional material (2841) being positioned as an internal layer, with sealant 2844 around the perimeter of the enclosure. As illustrated in Figure 28C, cells or other substances that would be excluded from passage through the perforated to-dimensional sheet or layer can be introduced into the enclosure after it formed by injection through the sealant layer. Any perforation formed by such injection can be sealed as needed. It will be appreciated that substances and cells can be introduced into the enclosure prior to formation of the seal. Those in the art will appreciate that sterilization methods appropriate for the application envisioned may be employed during or after the preparation of the enclosure.

[0320] In some embodiments an enclosure comprises perforated two-dimensional material encapsulating a substance, such that the substance is released to an environment external to the enclosure by passage through the holes in the perforated two-dimensional material. In some embodiments, the enclosure encapsulates more than one different substance. In some

embodiments, not all of the different substances are released to an environment external to the enclosure. In some embodiments, all of the different substances are released into an environment external to the enclosure. In some embodiments, different substances are released into an environment external to the enclosure at different rates. In some embodiments, different substances are released into an environment external to the enclosure at the same rates.

[0321] In some embodiments, the enclosure comprises two or more sub-compartments, wherein at least one sub-compartment is in direct fluid communication with an environment external to the enclosure through holes in a two-dimensional material of the sub-compartment. In some embodiments, each sub-compartment comprises a perforated two-dimensional material and each sub-compartment is in direct fluid communication with an environment external to the enclosure, through holes in the two-dimensional material of each sub-compartment.

[0322] In some embodiments, an enclosure is subdivided into two sub-compartments separated from each other at least in part by perforated two-dimensional material, such that the two-sub-compartments are in direct fluid communication with each other through holes in two- dimensional material. In some embodiments, the enclosure is subdivided into two-sub- compartments each comprising two-dimensional material which sub-compartments are in direct fluid communication with each other through holes in two-dimensional material and only one of the sub-compartments is in direct fluid communication with an environment external to the enclosure. In some embodiments, the enclosure is subdivided into two-sub-compartments each comprising two-dimensional material which sub-compartments are in direct fluid communication with each other through holes in two-dimensional material and both of the sub-compartments are also in direct fluid communication with an environment external to the enclosure.

[0323] In some embodiments, the enclosure has an inner sub-compartment and an outer sub- compartment each comprising a perforated two-dimensional material, wherein the inner sub- compartment is entirely enclosed within the outer sub-compartment, the inner and outer compartments are in direct fluid communication with each other through holes in two- dimensional material and the inner sub-compartment is not in direct fluid communication with an environment external to the enclosure.

[0324] In some embodiments, where an enclosure has a plurality of sub-compartments each comprising a two-dimensional material, the sub-compartments are nested one within the other, each of which sub-compartments is in direct fluid communication through holes in two- dimensional material with the sub-compartment(s) to which it is adjacent, the outermost sub- compartment in direct fluid communication with an environment external to the enclosure, the remaining plurality of sub-compartments not in direct fluid communication with an environment external to the enclosure. [0325] In some embodiments, where the enclosure is subdivided into a plurality of sub- compartments, each comprising a two-dimensional material, each sub-compartment is in direct fluid communication with one or more adjacent sub-compartments, and only one sub- compartment is in direct fluid communication with an environment external to the enclosure.

[0326] In some embodiments, the enclosure comprises two sub-compartments, where (i) the first sub-compartment is in fluid communication with an environment external to the enclosure and comprises a substance such as a pharmaceutical, a drug, a medicament, a therapeutic, a biologic, a small molecule, and combinations thereof and (ii) the second compartment comprises a semi-permeable membrane not abutting the first sub-compartment. In some embodiments, osmosis occurs across the semi-permeable membrane in the second sub-compartment, thereby increasing pressure on the first sub-compartment (e.g., using a piston-like driving force). In some embodiments, this increased pressure increases the diffusion rate of the substance in the first sub-compartment into the environment external to the enclosure.

[0327] In some embodiments, the at least one substance within the enclosure that is released to an environment external to the enclosure through holes in two-dimensional material is a pharmaceutical, therapeutic or drug. In some embodiments, e.g., when the released substance is a pharmaceutical, therapeutic or drug, the two-dimensional material of the enclosure for release of the substance comprises holes ranging in size from 1-50 nm. In some embodiments, e.g., when the released substance is a pharmaceutical, therapeutic or drug, the two-dimensional material of the enclosure for release of the substance comprises holes ranging in size from 1-10 nm.

[0328] In some embodiments, the substance within the enclosure is cells and the size of the holes in the two-dimensional material is selected to retain the cells within the enclosure and to exclude immune cells and antibodies from entering the enclosure from an environment external to the enclosure. In some embodiments, useful for cells, the enclosure is divided into a plurality of sub-compartments and one or more sub-compartments contain cells. An enclosure can contain different cells with a sub-compartment or different cells within different sub- compartments of the same enclosure. In some embodiments useful for cells, the enclosure is a nested enclosure wherein the cells are within the inner sub-compartment. [0329] In some embodiments, an enclosure has an inner sub-compartment and an outer sub- compartment each comprising a perforated two-dimensional material wherein the inner sub- compartment is entirely enclosed within the outer sub-compartment, the inner and outer compartments are in direct fluid communication through holes in two-dimensional material of the inner sub-compartment, the inner sub-compartment is not in direct fluid communication with an environment external to the enclosure and the outer compartment is in direct fluid communication with an environment external to the enclosure.

[0330] In some embodiments useful with cells, an enclosure has a plurality of sub- compartments each of which comprises perforated two-dimensional material and each of which sub -compartments is in direct fluid communication with one or more adjacent sub- compartments, the cells being within one or more cell-containing sub-compartments each of which are not in direct fluid communication with an environment external to the enclosure.

[0331] In some embodiments of enclosures containing cells, the cells are yeast cells or bacterial cells. In some embodiments of enclosures containing cells, the cells are mammalian cells. In some embodiments of enclosures containing cells, the size of the holes, in the two- dimensional material of the enclosure or sub-compartment, ranges from 1-10 nm, 3-10 nm, or from 3-5 nm.

[0332] In some embodiments, two-dimensional material in the enclosure is supported on a porous substrate. In some embodiments, the porous substrate can be polymer or ceramic.

[0333] In some embodiments the two-dimensional material is a graphene-based material. In some embodiments, the two-dimensional material is graphene.

[0334] In some embodiments, at least a portion of the holes, or a portion thereof, in the two- dimensional materials of the enclosure are functionalized. In some embodiments, the external surface of the enclosure is functionalized. In some embodiments, functionalization comprises surface charges (e.g., sulfonates) attached to the pores and/or surface of the enclosure. Without being bound by theory, it is believed that surface charges can impact molecules and/or ions that can traverse the membrane. In some embodiments, functionalization comprises specific binding sites attached to the pores and/or the surface of the enclosure. In some embodiments, functionalization comprises proteins or peptides attached to the pores and/or the surface of the enclosure. In some embodiments, functionalization comprises adsorptive substances attached to the pores and/or the surface of the enclosure. In some embodiments, functionalization involves catalytic and/or regenerative substances or groups. In some embodiments, functionalization comprise a negatively or partially negatively charged group (e.g., oxygen) attached to the pores and/or the surface of the enclosure. In some embodiments, functionalization comprises a positively or partially positively charged group attached to the pores and/or the surface of the enclosure.

[0335] In some embodiments, functionalizing the pores and/or the surface of the enclosure functions: to restrict contaminants from traversing the membrane; to act as a disposable filter, capture, or diagnostic tool; increase biocompatibility (e.g., when polyethylene glycol is used for functionalization); increase filtration efficiency; and/or to increase selectivity at or near the pores or in asymmetric membranes.

[0336] In some embodiments, at least a portion of the two-dimensional material is conductive and a voltage can be applied to at least a portion of the conductive two-dimensional material. The voltage can be an AC or DC voltage. The voltage can be applied from a source external to the enclosure. In some embodiments, a device comprising a two-dimensional material (such as an enclosure device) further comprises connectors and leads for application of a voltage from an external source to the two-dimensional material.

[0337] Some embodiments comprise methods of employing an enclosure in a selected environment for delivery of one or more substance to the environment. In some embodiments, the environment is a biological environment. In some embodiments, the enclosure is implanted into biological tissue. In some embodiments, the enclosure device is positioned such that the device or enclosure is positioned partially inside a subject's body and partially outside a subject's body (e.g., an enclosure can be used as a port or wound covering to allow drugs or biologies to be introduced without cells or other contaminants entering the body). In some embodiments, the enclosure is injected (e.g., through a needle). In some embodiments, the enclosure is ingested. In some embodiments, the enclosure is employed for delivery of a pharmaceutical, a drug or a therapeutic. [0338] In some embodiments, a method comprises introducing an enclosure comprising perforated two-dimensional material into a an environment, the enclosure containing at least one substance; and releasing at least a portion of at least one substance through the holes of the two- dimensional material to the environment external to the enclosure. In some embodiments, the enclosure contains cells which are not released from the enclosure and the at least one substance a portion of which is released is a substance generated by the cells in the enclosure.

[0339] In some embodiments, a method comprises introducing an enclosure comprising perforated two-dimensional material to an environment, the enclosure containing at least one first substance; and receiving a second substance from the environment into the enclosure. In some embodiments, the first substance is cells, a second substance is nutrients and another second substance is oxygen.

[0340] In some embodiments, the support layer can be a polymer or a ceramic material. Useful exemplary ceramics include nanoporous silica, silicon or silicon nitride. Useful porous polymer supports include solution-diffusion membranes, track-etched polymers, expanded polymers or non-woven polymers. The support material can be porous or permeable. A portion, e.g., a wall, side or portion thereof, of an enclosure or a sub-compartment can be non-porous polymer or ceramic. Biocompatible polymers and ceramics are preferred. A portion of the enclosure can be formed from a sealant, such as a silicone, epoxy, polyurethane or similar material. Biocompatible sealants are preferred.

[0341] In some embodiments, a non-perforated wall or portion thereof of an enclosure is a metallic, polymeric or ceramic material. Biocompatible metals, polymers and ceramics are preferred, such as medical grade materials. In some embodiments, a non-perforated wall of an enclosure may be treated, e.g., on a surface interfacing with an external environment, to provide or improve biocompatibility.

[0342] Additionally, the conductive properties of graphene-based or other two-dimensional membranes can allow for electrification to take place from an external source. In exemplary embodiments, an AC or DC voltage can be applied to conductive two-dimensional materials (e.g., in a device such as an enclosure device). The conductivity properties of graphene can provide additional gating to charged molecules or substances. Electrification can occur permanently or only a portion of the time to affect gating. Directional gating of charged molecules can be directed not only through the pores (or restrict travel through pores), but also to the surface of the graphene to adsorb or bind and encourage growth, promote formation of a protective layer, or provide the basis or mechanism for other biochemical effects (e.g., on the body).

[0343] In some embodiments, the membranes allow for electrostatic control of charged species, for instance in nanofluidic or microfluidic systems. In some embodiments, the membranes allow for control of charged species by varying the applied voltage, for instance in nanofluidic or microfluidic systems. In some embodiments, the membrane can be tuned to manipulate ion transport at low and/or high ion concentrations. In some embodiments, the membrane is an ion-selective membrane. In some embodiments, the membrane comprises one or more voltage-gated ion channels, such as voltage-gated pores. In some embodiments, the membranes mimic biological voltage-gated ion channels. Inn some embodiments, the gated graphene functions as an artificial membrane, e.g., when used in an artificial organ or organelle. In some embodiments, the membrane is a solid-state membrane. In some embodiments, nanochannel or nanopore transistors can be used to manipulate ion transport.

[0344] In some embodiments, the membrane can be tuned using low or high applied voltages. In some embodiments, the membrane allows high ionic flux. In some embodiments, the membrane allows low ion flux. In some embodiments, pores in the membrane modulate current of ions at low gate voltages and/or display high selectivity. In some embodiments, ion flux across the membrane can be turned on or off at low applied voltages, such as < 500 mV. In some embodiments, ion flux across the membrane can be turned on or off at biologically relevant ion concentrations, such as up to 1 M. In some embodiments, the applied voltage can modulate on species selectivity, e.g., cation or anion selectivity.

[0345] In some embodiments, nanopores can be electrostatically controlled at low voltages and biologically relevant ion concentrations. In some embodiments, membranes are used in separation and sensing technologies. In some embodiments, membranes are used in water filtration, energy storage, microfluidic devices, nanofluidic devices, and/or therapeutic methods. Thus, some embodiments relate to methods for separating ions or other substances; methods for sensing ions; methods for storing energy; methods for filtering water; and/or methods of treating a disease or condition. Some embodiments relate to methods of nanofiltration and/or microfiltration. Some embodiments comprise using gating to control release of substances. Some embodiments comprise using gating to allow for different substances to be release at different times. Some embodiments comprise allowing different substances to pass through the membrane at different times, thus modulating when and how substances mix and interact with other substances in a specific order.

[0346] Both permanent and temporary binding to the graphene is possible in such embodiments. In addition to the foregoing advantages, some embodiments can also be advantageous in that they not only represent a disruptive technology for state of the art vehicle and other devices, but they can also permit these vehicles and devices to be used in new ways. For example, cell line developments, therapeutic releasing agents, and/or sensing paradigms (e.g., MRSw's, MR-based magnetic relaxation switches, see; Koh et al. (2008) Ang. Chem. Int'l Ed. Engl, 47(22)4119-4121) can be used to mitigate biofouling and bioreactivity, conveying superior permeability and less delay in response, and providing mechanical stability. That is, the enclosures can allow existing technologies to be implemented in ways not previously possible.

[0347] Some embodiments comprise enclosures where graphene allows implementation of a sense-response system. For instance, graphene can be used to sense a variety of biomolecules, such as insulin. In some embodiments, the biomolecules are "sensed" based on an interaction between compounds with the graphene or with functional groups attached to the graphene.

Without being bound by theory, it is believed that the sense-response paradigm provides a feedback mechanism for monitoring the state of encapsulated materials.

[0348] Some embodiments comprise bioartificial liver configurations comprising an enclosure. For instance, hepatocytes or liver cells can be encapsulated by the enclosure. In some embodiments, enclosures comprising encapsulated hepatocytes is implanted into a subject in need thereof, such as a subject with impaired liver function. In some embodiments, enclosures comprising encapsulated hepatocytes are used in an extracorporeal medical procedure. In some embodiments, the enclosure is loadable or reloadable, such that a metabolite can be injected into the enclosure to elicit a reaction, or the number or type of cells inside the enclosure can be modified (e.g., the cells inside the enclosure can be replaced).

[0349] Some embodiments comprise artificial kidney configurations comprising an enclosure. For instance, kidney cells can be encapsulated by the enclosure. In some

embodiments, enclosures comprising encapsulated kidney cells can be implanted into a subject in need thereof. In some embodiments, the enclosure is loadable or reloadable, such that a metabolite can be injected into the enclosure to elicit a reaction, or the number or type of cells inside the enclosure can be modified (e.g., the cells inside the enclosure can be replaced).

[0350] Some embodiments comprise artificial lungs comprising an enclosure. In some embodiments, the compartment in the enclosure is in gaseous communication with an environment external to the compartment.

[0351] In addition to the in vivo and in vitro uses described above, some embodiments can be utilized in other areas as well. Some embodiments can be used in non-therapeutic applications such as, for example, the dosage of probiotics in dairy products (as opposed to the presently used microencapsulation techniques to increase viability during processing for delivery to the GI tract). In this regard and others, it should be noted that the enclosures and devices formed therefrom can span several orders of magnitude in size, depending on manufacturing techniques and various end use requirements. Nevertheless, the enclosures are believed to be able to be made small enough to circulate through the bloodstream. On the opposite end of the spectrum, the enclosures can be made large enough to implant (on the order of a few inches or greater). These properties can result from the two-dimensional characteristics of the graphene and its growth over large surface areas.

SELECTIVE INTERFACIAL MITIGATION OF GRAPHENE DEFECTS

[0352] Graphene based materials and other two-dimensional materials may have undesirable defects present therein. Defects, as utilized herein, are undesired openings formed in the graphene material. The presence of defects may render the graphene material unsuitable for filtration-type applications, as the defects may allow undesired molecules to pass through the material. In such applications, the presence of defects above a cutoff size or outside of a selected size range can be undesirable. On the other hand, defects below a critical size required for application-specific separation may be useful from a permeability perspective, as long as such defects do not negatively impact the integrity of the graphene. In some embodiments, defects may include holes, tears, slits, or any other shape or structure. Defects may be the result of manufacturing or handling the graphene material.

[0353] A process for repairing or mitigating the presence of defects in the graphene materials increases the utility of the materials as filtration or permeable membranes. The repair process may selectively produce a polymer material within the defects of the graphene material, preventing flow through the defects. The repair process may produce a graphene material 2900 with polymer regions 2910 that have filled defects in the graphene material, as shown in FIGS. 29 and 30. The polymer regions 2910 are thin and may be a single layer of polymer molecules. As shown in FIG. 30, while the polymer regions 2910 are thin, they are thicker than the graphene material 2900. For example, a single layer of graphene may have a thickness of about 3.5 angstroms, while a polymer region including a single layer of polymer molecules may have a thickness of a few nanometers or more, depending on the polymer.

[0354] Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or a few layered sheets (e.g., about 20 or less) of fused six- membered rings forming an extended sp2-hybridized carbon planar lattice. Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. As utilized herein, graphene material may refer to graphene or a graphene-based material. In some embodiments, graphene-based materials also include materials which have been formed by stacking single or multilayer graphene sheets. In some embodiments, multilayer graphene includes 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In some embodiments, layers of multilayered graphene are stacked, but are less ordered in the z direction (perpendicular to the basal plane) than a thin graphite crystal.

[0355] In some embodiments, a sheet of graphene-based material is a sheet of single or multilayer graphene or a sheet comprising a plurality of interconnected single or multilayer graphene domains. In some embodiments, the multilayer graphene domains have 2 to 5 layers or 2 to 10 layers. As used herein, a "domain" refers to a region of a material where atoms are uniformly ordered into a crystal lattice. A domain is uniform within its boundaries, but different from a neighboring region. For example, a single crystalline material has a single domain of ordered atoms. In some embodiments, at least some of the graphene domains are nanocrystals, having a domain size from 1 nm to 100 nm, such as 10 nm to 100 nm. In some embodiments, at least some of the graphene domains have a domain size greater than 100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm. In some embodiments, a domain of multilayer graphene may overlap a neighboring domain. "Grain boundaries" formed by crystallographic defects at edges of each domain differentiate between neighboring crystal lattices. In some embodiments, a first crystal lattice may be rotated relative to a second crystal lattice, by a rotation about an axis perpendicular to the plane of a sheet, such that the two lattices differ in "crystal lattice orientation."

[0356] In some embodiments, the sheet of graphene-based material is a sheet of single or multilayer graphene or a combination thereof. In some other embodiments, the sheet of graphene-based material is a sheet comprising a plurality of interconnected single or multilayer graphene domains. The interconnected domains may be covalently bonded together to form the sheet. When the domains in a sheet differ in crystal lattice orientation, the sheet may be considered polycrystalline.

[0357] In some embodiments, the thickness of the sheet of graphene-based material is from 0.3 nm to 10 nm, such as from 0.34 nm to 10 nm, from 0.34 nm to 5 nm, or from 0.34 nm to 3 nm. In some embodiments, the thickness may include both single layer graphene and non- graphenic carbon.

[0358] In some embodiments, graphene is the dominant material in a graphene-based material. For example, a graphene-based material may comprise at least 20% graphene, such as at least 30% graphene, at least 40% graphene, at least 50% graphene, at least 60% graphene, at least 70%) graphene, at least 80% graphene, at least 90% graphene, at least 95% graphene, or more. In some embodiments, a graphene-based material may comprise a graphene content range selected from 30% to 100%, such as from 30% to 95%, such as from 40% to 80%, from 50% to 70%, from 60% to 95%, or from 75% to 100%. In some embodiments, the amount of graphene in the graphene-based material is measured as an atomic percentage. The amount of graphene in the graphene-based material is measured as an atomic percentage utilizing known methods including transmission electron microscope examination or, alternatively, if TEM is ineffective, another similar measurement technique.

[0359] In some embodiments, a sheet of graphene-based material may further comprise non- graphenic carbon-based material located on at least one surface of the sheet of graphene-based material. In an embodiment, the sheet is defined by two base surfaces (e.g. top and bottom faces of the sheet) and side faces (e.g. the side faces of the sheet). In some embodiments, non- graphenic carbon-based material is located on one or both base surfaces of the sheet. In some embodiments, the sheet of graphene-based material includes a small amount of one or more other materials on the surface, such as, but not limited to, one or more dust particles or similar contaminants.

[0360] In some embodiments, the amount of non-graphenic carbon-based material is less than the amount of graphene. In some other embodiments, the amount of non-graphenic carbon material is three to five times the amount of graphene; this may be measured in terms of mass. In additional embodiments, the non-graphenic carbon material is characterized by a percentage by mass of said graphene-based material selected from the range of 0% to 80%. In some

embodiments, the surface coverage of the sheet of non-graphenic carbon-based material is greater than zero and less than 80%, such as from 5% to 80%, from 10% to 80%, from 5% to 50%), or from 10%> to 50%. This surface coverage may be measured with transmission electron microscopy. In some embodiments, the amount of graphene in the graphene-based material is from 60%) to 95% or from 75% to 100%. The amount of graphene in the graphene-based material is measured as mass percentage utilizing known methods, preferentially using transmission electron microscopy (TEM) examination or, alternatively, if TEM is ineffective, using other similar techniques.

[0361] In some embodiments, the non-graphenic carbon-based material does not possess long range order and may be classified as amorphous. The non-graphenic carbon-based material may further comprise elements other than carbon and/or hydrocarbons. In some embodiments, non-carbon elements which may be incorporated in the non-graphenic carbon include hydrogen, oxygen, silicon, copper, and iron. In further embodiments, the non-graphenic carbon-based material comprises hydrocarbons. In some embodiments, carbon is the dominant material in non- graphenic carbon-based material. For example, a non-graphenic carbon-based material may comprise at least 30% carbon, such as at least 40% carbon, at least 50% carbon, at least 60% carbon, at least 70% carbon, at least 80% carbon, at least 90% carbon, or at least 95% carbon. In some embodiments, a non-graphenic carbon-based material comprises a range of carbon selected from 30% to 95%, such as from 40% to 80%, or from 50% to 70%. The amount of carbon in the non-graphenic carbon-based material may be measured as an atomic percentage utilizing known methods preferentially using transmission electron microscope examination or, alternatively, if TEM is ineffective, using other similar techniques.

[0362] In some embodiments, the graphene material may be in the form of a macroscale sheet. As used herein, a macroscale sheet may be observable by the naked eye. In some embodiments, at least one lateral dimension of the macroscopic sheet may be greater than 1 mm, such as greater than 5 mm, greater than 1 cm, or greater than 3 cm. In some embodiments, the macroscopic sheet may be larger than a flake obtained by exfoliation. For example, the macroscopic sheet may have a lateral dimension greater than about 1 micrometer. In some embodiments, the lateral dimension of the macroscopic sheet may be less than 10 cm. In some embodiments, the macroscopic sheet may have a lateral dimension of from 10 nm to 10 cm, such as from 1 mm to 10 cm. As used herein, a lateral dimension is generally perpendicular to the thickness of the sheet.

[0363] As used herein, the term "two-dimensional material" may refer to any extended planar structure of atomic thickness, including both single- and multi-layer variants thereof. Multi-layer two-dimensional materials may include up to about 20 stacked layers. In some embodiments, a two-dimensional material suitable for the present structures and methods can include any material having an extended planar molecular structure and an atomic level thickness. Particular examples of two-dimensional materials include graphene films, graphene- based material, transition metal dichalcogenides, metal oxides, metal hydroxides, graphene oxide, hexagonalboron nitride, silicone, germanene, or other materials having a similar planar structure. Specific examples of transition metal dichalcogenides include molybdenum disulfide and niobium diselenide. Specific examples of metal oxides include vanadium pentoxide. Graphene or graphene-based films according to the embodiments herein can include single-layer or multi-layer films, or any combination thereof. Choice of a suitable two-dimensional material can be determined by a number of factors, including the chemical and physical environment into which the graphene, graphene-based material or other two-dimensional material is to be terminally deployed, ease of perforating the two-dimensional material, and the like. The processes and structures disclosed herein with respect to graphene materials are also applicable to two-dimensional materials.

[0364] Pores as described herein may be sized to provide desired selective permeability of a species (atom, molecule, protein, virus, cell, etc.) for a given application. Selective permeability relates to the propensity of a porous material or a perforated two-dimensional material to allow passage (or transport) of one or more species more readily or faster than other species. Selective permeability allows separation of species which exhibit different passage or transport rates. In two-dimensional materials selective permeability correlates to the dimension or size (e.g., diameter) of apertures and the relative effective size of the species. Selective permeability of the perforations in two-dimensional materials such as graphene-based materials can also depend on functionalization of perforations (if any) and the specific species that are to be separated.

Separation of two or more species in a mixture includes a change in the ratio(s) (weight or molar ratio) of the two or more species in the mixture after passage of the mixture through a perforated two-dimensional material.

[0365] In some embodiments, a characteristic size of the pores may be from 0.3 nm to 500 nm, such as from 0.3 nm to 10 nm, from 1 nm to 10 nm, from 5 nm to 10 nm, from 5 nm to 20 nm, from 10 nm to 50 nm, from 50 nm to 100 nm, from 50 nm to 150 nm, from 100 nm to 200 nm, or from 100 nm to 500 nm. The characteristics size may refer to the average pore size. In some embodiments, from 70% to 99%, such as from 80% to 99%, from 85% to 99%, or from 90% to 99%), of the pores in a sheet or layer fall within a specified range, and the remaining pores fall outside the specified range.

[0366] The size distribution of the pores may be narrow, e.g., limited to 0.1 to 0.5 coefficient of variation. For circular pores, the characteristic dimension may be the diameter of the hole. For non-circular pores, the characteristic dimension may be the largest distance spanning the hole, the smallest distance spanning the hole, the average of the largest and smallest distance spanning the hole, or an equivalent diameter based on the in-plane area of the pore.

[0367] Quantitative image analysis of pore features may include measurement of the number, area, size and/or perimeter of pores. In some embodiments, the equivalent diameter of each pore is calculated from the equation A= π d2/4, where d is the equivalent diameter of the pore and A is the area of the pore. When the pore area is plotted as a function of equivalent pore diameter, a pore size distribution may be obtained. The coefficient of variation of the pore size may be calculated herein as the ratio of the standard deviation of the pore size to the mean of the pore size.

[0368] In some embodiments, the ratio of the area of the pores to the area of the sheet is used to characterize the sheet. The area of the sheet may be taken as the planar area spanned by the sheet. In some embodiments, characterization may be based on the ratio of the area of the perforations to the sheet area excluding features such as surface debris. In some additional embodiments, characterization may be based on the ratio of the area of the pores to a suspended area of the sheet. In some embodiments, the pore area may comprises 0.1% or greater, such as 1%) or greater, or 5% or greater of the sheet area. In some embodiments, the pore are may comprise less than 15% of the sheet area, such as less than 10% of the sheet area. In some embodiments, the pore area may comprise from 0.1% to 15% of the sheet area, such as from 1% to 15%) of the sheet area, from 5%> to 15%> of the sheet area, or from 1%> to 10%> of the sheet area. In some embodiments, the pores may be located over greater than 10%>, such as greater than 15%> of the area, of a sheet of graphene-based material. In some embodiments, the pore density may be from 2 pores pre nm ² to 1 pore per μηι ² .

[0369] The defect repair process includes the application of a first reactant to a first side of the graphene material and a second reactant to a second side of the graphene material. As shown in FIG. 37, molecules of the first reactant material 3110 are disposed on a first side of the graphene material 2900 and molecules of the second reactant material 3120 are disposed on a second side of the graphene material 2900. A defect 2912 in the graphene material 2900 allows the first reactant 3110 to contact the second reactant 3120 as shown in FIG. 38. The first reactant 3110 and the second reactant 3120 may pass through any defect 2912 with a size larger than the reactant molecules. The interaction between the first reactant 3110 and the second reactant 3120 produces a polymerization reaction and forms a polymer 3110 in the defect. As shown in FIG. 34, the polymerization reaction may continue until the polymer 3110 fills the defect and the first reactant 3110 and second reactant 3120 are no longer able to pass through the defect and interact. The thickness of the polymer region may depend on the type of polymer employed and the reaction conditions. In some embodiments the thickness of the polymer region may be greater than a few nanometers, such as greater than 3 nm, greater than 10 nm, greater than 25 nm, greater than 50 nm, greater than 100 nm, greater than 1 μm, greater than 10 μm, greater than 100 μm, or more. In some embodiments, the polymer regions may have a thickness in the range of 3 nm to 100 μm, such as from 10 nm to 50 μm, or from 10 nm to 500 nm.

[0370] The first reactant may be any reactant capable of producing a polymer when in contact with the second reactant. The first reactant may be provided in the form of a liquid solution or suspension. In some embodiments the first reactant may be a monomer or oligomer. The monomer or oligomer may include a diamine, such as hexamethylene diamine, or a polystyrene monomer. The first reactant may be biocompatible or bio-inert.

[0371] The second reactant may be any reactant capable of producing a polymer when in contact with the first reactant. The second reactant may be provided in the form of a liquid solution, liquid suspension, gas, or plasma. The second reactant may be a monomer, an oligomer, or a catalyst that initiates polymerization. In some embodiments the second reactant may be a dicarboxylic acid, such as hexanedioic acid. In some embodiments the second reactant may be a polymerization catalyst, such as azobisisobutyronitrile (AIBN). The second reactant may be provided in an aqueous solution or an oil based solution. The second reactant may be

biocompatible or bio-inert.

[0372] In some embodiments, the reactants may be selected from monomers or oligomers that include any of the following functional groups: hydroxyl, ether, ketone, carboxyl, aldehyde, amine, or combinations thereof. The monomers or oligomers may be selected from any appropriate species that includes a functional group capable of reacting with a counterpart reactant to produce a polymer. [0373] In some embodiments, the reactants may be selected to produce a step or condensation polymerization. A step or condensation polymerization reaction is self-limiting, as once the defects are filled such that the reactants can no longer pass through the defect the polymerization reaction will cease due to a lack of reactants. The self-limiting nature of the step or condensation polymerization reaction allows the defects in the graphene material to be fully repaired without concern that polymer formation will continue until pores and desired fluid flow channels are blocked.

[0374] In some embodiments, the reactants may be selected to produce an addition or chain polymerization reaction. To produce an addition or chain polymerization one of the reactants may be a monomer, oligomer, or polymer and the second reactant may be an initiator. The addition or chain polymerization reaction may continue until the reaction is quenched or the reactant supply is exhausted. In practice, the extent of the addition or chain polymerization may be controlled by quenching the reaction after a predetermined time that is selected to ensure that sufficient repair of the defects in the graphene material has occurred. In some embodiments, the quenching of the reaction may be achieved by introducing a quenching reagent, such as oxygen, to the reaction system. An addition or chain polymerization reaction may be useful in applications where it is desirable for the polymer to be formed in areas beyond the immediate defects of the graphene material. The ability to form more extensive polymer regions allows the interfacial polymerization process to produce polymer regions with additional functionality, such as providing adhesion enhancements, mechanical reinforcement, or chemical functionalization. An exemplary reactant pair for an addition or chain polymerization may be an AIBN aqueous solution and a vapor phase polystyrene.

[0375] The polymer formed during the repair process may be any appropriate polymer. In some embodiments, the polymers formed utilizing a step or condensation polymerization reaction may include polyamide, polyimide, polyester, polyurethane, polysiloxane, phenolic resin, epoxy, melamine, polyacetal, polycarbonate, and co-polymers thereof. The polymers formed utilizing an addition or chain polymerization reaction may include polyacrylonitrile, polystyrene, poly(methyl methacrylate), poly(vinyl acetate), or co-polymers thereof. In some embodiments, the polymer formed during the repair process may be a biocompatible or bio-inert polymer. In some embodiments, the polymer formed during the repair process may be semipermeable, such that some materials or molecules may diffuse through the polymer regions that fill the defects. In some embodiments, the polymer may be porous or non-porous.

[0376] In some embodiments the first reactant and the second reactant may have a size larger than a desired pore size of the graphene material. The use of reactants with such a size allows for the selective repair of only those defects that have a size greater than the desired pore size, as the reactants are unable to pass through the defects and pores with a size less than the desired pore size of the graphene material. The size of a defect, as utilized herein, may refer to the effective diameter of the defect. The effective diameter of a defect is the diameter of the largest spherical particle that will pass through the defect. The effective diameter may be measured by any appropriate method, such as imaging with a scanning electron microscope and then calculating the effective diameter of the defect. The size of a reactant, as utilized herein, may refer to the effective diameter of the reactant. In some embodiments, the effective diameter of the reactant may be the diameter of a sphere that is capable of passing through the same openings that the reactant can pass through. In some embodiments, the effective diameter of polymeric materials may refer to the diameter of gyration, with the diameter of gyration being twice the radius of gyration.

[0377] In some embodiments, a reactant with a large size may be a dendrimer. In some embodiments, the dendrimers may include a surface containing any of the functional groups described herein for the reactants. For example, the dendrimers may include hydroxyl, amine, sulfonic acid, carboxylic acid, or quaternary ammonium functional groups on the surface thereof. The large reactants may have a size of at least about 15 nm, such as at least about 20 nm, about 25 nm, about 30 nm, about 40 nm, about 50 nm, about 75 nm, about 100 nm, about 125 nm, or more. In some embodiments, a reactant with a large size may be a reactant with a diameter of gyration that is equivalent to the effective diameter of the smallest defect targeted for repair. Exemplary reactants of this type may include high molecular weight polymers with end groups including the functional groups described above for the reactants. In some embodiments, a large reactant may be an ionic polymer, where the first and second reactants are selected to have opposite charges. [0378] As shown in FIGS. 31 and 32, the graphene material 2900 may include defects 2912 and pores 2914. The use of a first reactant material 3110 and a second reactant material 3120 with a size greater than the size of the pores 2914 prevents the reactant molecules from passing through and polymerizing in the pores 2914. The first reactant material 3110 and the second reactant material 3120 pass through and polymerize in the defects 2912. In this manner, the interfacial polymerization repair process is capable of selectively repairing only those defects having a size that is greater than a desired pore size. As shown in FIG. 33, after the repair process the defects are filled by a polymer region 110, while the pores 2914 are open and allow the passage of fluid there through.

[0379] In some embodiments the first reactant material and the second reactant material are provided in forms that allow the manner in which the reactants diffuse into each other to be controlled. The way in which the reactants interact influences the location of the polymer produced by the polymerization. In some embodiments the reactants are provided in a form that does not allow significant amounts of diffusion of either reactant in to the other, which produces a polymer region that has a midpoint that substantially aligns with the graphene material, as shown in FIG. 34. The reactants may be provided in solutions that are immiscible with each other, such that the interface between the solutions is maintained along the plane of the graphite material. The immiscible solutions may be any appropriate combination, such as an aqueous solution and an oil-based solution. In some embodiments the diffusion of the reactants in to the other reactant solution may be prevented by selecting reactants that are not soluble in the solvent forming the other solution. For example, a first reactant that is not soluble in oil may be provided in an aqueous solution and a second reactant that is not soluble in water may be provided in an oil solution, producing limited or no diffusion of the reactants to the counterpart solution. In some embodiments, the first reactant and second reactant may be selected such that one of the reactants is cationic and the second reactant is anionic.

[0380] In some embodiments the reactants may be selected such that one of the reactants is capable of diffusing readily into the other reactant. As shown in FIG. 35, the first reactant material 3110 may be selected such that the second reactant material 3120 diffuses therein, producing a polymer 3110 that is located substantially on the side of the graphene material 2900 that the first reactant is disposed on. A similar effect may be produced when the first reactant is a liquid solution and the second reactant is a gas, such that the first reactant does not diffuse in to the gas of the second reactant.

[0381] In some embodiments the second reactant material 3120 may be selected such that the first reactant material 3110 diffuses in the second reactant material 3120. A reactant system of this type produces a polymer 3110 that is located substantially on the side of the graphene material 2900 on which the second reactant material 3120 is disposed, as shown in FIG. 36.

[0382] In some embodiments, the interaction of the reactants through the defects in the graphene material may be the result of diffusion. In some embodiments, the reactants may be heated to increase the diffusion thereof and the likelihood that the reactants will interact. In some other embodiments, the reactants may be ionic, with the first and second reactants having opposite charges. The opposite charges of the ionic polymers produces an attraction between the reactants, ensuring that the reactants interact across the defects of the graphene material to produce a polymer. In some embodiments, electrophoresis may be employed to facilitate interaction between ionic and polar reactants. In some embodiments, the reactants may have a dipole, such that an electric or magnetic field may be applied to the reactants to drive motion of the reactants in the system and produce interaction between the reactants. In some embodiments, an electrical potential may be applied across the graphene material, attracting the reactants to the surface thereof and enhancing interaction between the reactants.

[0383] The polymer regions formed in the defects may be attached to the graphene material by any suitable interaction. In some embodiments, the polymer regions may be attached to the graphene material through mechanical interaction. One example of mechanical interaction occurs includes a polymer region formed such that the portion of the polymer region in plane with the graphene material is has a smaller dimension than the portions of the polymer region formed on either side of the graphene material. The larger ends of the polymer region mechanically interact with the graphene material to prevent the polymer region from being pulled out of the defect. In some embodiments, the graphene material and the polymer region may be attached by van der Waals attraction. [0384] In some embodiments, the graphene material may be functionalized to produce covalent or non-covalent interactions between the graphene material and the polymer regions. In some embodiments, the graphene material may be rendered hydrophobic or hydrophilic by treating the graphene material before forming the polymer regions, such that the interaction between the graphene material and the polymer region is strengthened. In some embodiments, the graphene material may be treated to form functional groups, such as hydroxyl, carbonyl, carboxylic, or amine groups. The functionalization may be achieved through any appropriate process, such as oxidation of the graphene material. In some embodiments, the graphene material may be oxidized by thermal treatment, ultraviolet oxidation, plasma treatment, sulfuric acid treatment, nitric acid treatment, or permanganate treatment. In some embodiments, the graphene material may be aminated by ammonia treatment. The oxidation may be limited to the area of the graphene material containing defects, as the chemical bonds of the graphene material are generally more reactive in the areas adjacent to defects than in the basal plane. The functional groups produced by the treatment of the graphene material may form covalent bonds with the polymer regions, such that the polymer regions are attached to the graphene material by the covalent bonds.

[0385] The reactants may be selected such that the produced polymer is capable of adhering to a support over which the graphene material may be disposed. In some embodiments, graphene materials do not covalently bond to support materials, thus by selecting a polymer material to repair defects in the graphene material that will adhere to a support structure the adhesion of the repaired graphene material to the support structure may be improved. The increased adhesion may be demonstrated by immersing the sample in a solvent that does not attack the polymer regions or the support structure and agitating the sample. In some embodiments, the increased adhesion may be demonstrated by applying a back pressure to the support structure side of the graphene material, and measuring the delamination/rupture pressure. The materials exhibiting improved adhesion have a higher delamination/rupture pressure than graphene materials that lack the polymer regions.

[0386] The support structure may be any appropriate structure that supports the graphene material without hindering the desired applications of the graphene material, such as filtration or selective permeability. The support structure may be a polymer material, such as a polycarbonate material. In the case that the support is a polycarbonate material, the polymer may be an epoxy. The support may be a porous material, such that the graphene material is supported while also allowing fluid to flow to and through the graphene material.

[0387] A porous material that may be useful as a support structure for the graphene material may include one or more selected from ceramics and thin film polymers. In some embodiments, ceramic porous materials may include silica, silicon, silicon nitride, and combinations thereof. In some embodiments, the porous material may include track-etched polymers, expanded polymers, patterned polymers, non-woven polymers, woven polymers, and combinations thereof.

[0388] The support structure may include a polymer selected from the group consisting of polysulfones, polyurethane, polymethylmethacrylate (PMMA), polyethylene glycol (PEG), polylactic-co-glycolic acid (PLGA), PLA, PGA, polyamides (such as nylon-6,6, supramid and nylamid), polyimides, polypropylene, polyethersulfones (PES), polyvinylidine fluoride (PVDF), cellulose acetate, polyethylene, polypropylene, polycarbonate, polytetrafluoroethylene (PTFE) (such as Teflon), polyvinylchloride (PVC), polyether ether ketone (PEEK), mixtures and block co-polymers of any of these, and combinations and/or mixtures thereof. In some embodiments, the polymers are biocompatible, bioinert and/or medical grade materials.

[0389] The repaired graphene material may be adhered to the support structure material by placing the repaired graphene material in contact with the support structure material. In some embodiments, the support structure may be treated to promote adhesion to the polymer regions of the repaired graphene material. The adhesion promoting treatment may include any appropriate process, such as subjecting the surface of the support structure to ultra violet oxidation. As shown in FIG. 42, the graphene material 4000 may be separated from the support structure material 4050 by a gap as a result of the thickness and location of the polymer regions 4012 repairing the defects and polymer handling region 4040. The size of the gap may be controlled by altering the location of the polymer regions formed during the repair process, as described above.

[0390] The increase in adhesion of the repaired graphene material to the support structure is a function of the proportion of polymer regions in the repaired graphene material. In some embodiments, a minimum amount of polymer regions, and thereby adhesion, may be ensured by forming holes in the graphene layer before the repair process. As utilized herein, holes refer to openings purposefully formed in the graphene material that will be plugged by the polymer material during the repair process. In some embodiments, the holes may fall within the defect classification, as they are undesired in the repaired membrane material. In some embodiments, the holes may have any appropriate size, such as any of the sizes of the pores described herein. In some embodiments, the holes may have a size that is greater than the desired pore size, such that the holes may be filled during the repair process and the pores may remain open.

[0391] The holes may be formed in the graphene material by any appropriate process, such as ion bombardment, chemical reaction, nanoparticle impacting or mechanical cutting. In some embodiments, the holes may be formed by any of the processes described herein for the formation of pores in the material. The holes may be arranged in a periodic array with a predetermined pattern and spacing across the surface of the graphene material. As shown in FIG. 39, the holes may be arranged in a plurality of rows, with a defined spacing between holes within each row, and a defined spacing between rows. The spacing of the holes in the rows may be the same in multiple rows, or different in each row. The spacing between the rows may be uniform, such that the spacing between each adjacent pair of rows is the same, or varied, such that the spacing between pairs of adjacent rows may be different. In some embodiments, the spacing of the holes in adjacent rows may be in phase, such that the holes in adjacent rows are aligned, as shown in FIG. 39. In some other embodiments, the spacing of the holes in the rows may be out of phase, such that the holes in adjacent rows are not aligned. In some embodiments, the holes may be arranged in a repeating pattern. In some embodiments, the holes may have a random distribution across the surface of the graphene material.

[0392] In some embodiments, the holes may be formed such that the holes account for at least about 5% of the area of the graphene material before repair, such as at least about 10%, about 15%), about 20%, about 25%, about 30%>, about 40%, or more. In some embodiments, the holes may have an area of less than about 50% of the area of the graphene material before repair, such as less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, or less. The minimum area of the holes of the graphene material may be selected such that the polymer regions of the repaired graphene material produce at least a desired degree of adhesion between the repaired graphene material and the support structure.

[0393] The adhesion between the repaired graphene material and the support structure produces a graphene membrane assembly. The adhesion between the polymer regions of the repaired graphene material and the support structure may include van der Waals forces, chemical bonds, molecular entanglement, or combinations thereof. In some embodiments, the polymer of the polymer regions may include polar a group, such as a hydroxyl, carbonyl, amine, epoxide, or combinations thereof, that exhibits stronger van der Waals attraction to the support structure than the graphene material. In some embodiments, the polymer of the polymer regions may include functional groups or side chains that readily react with the support structure to form chemical bonds. The chemical bonding between the polymer regions and the support structure may be initiated by any appropriate process, such as exposure to ultraviolet (UV) radiation, thermal treatment, or combinations thereof. In some embodiments, the polymer molecules of the polymer regions may be entangled with polymer molecules of the support structure. The molecular entanglement may be produced by thermal treatment of the graphene membrane assembly, such that the polymer of the polymer region and the support structure are softened without degrading. In such applications, the polymer of the polymer region and the polymer of the support structure may be selected to have similar thermal properties, such that both polymers are softened sufficiently at the treatment temperature to produce entanglement of the polymer molecules. The graphene membrane assembly exhibits improved performance and service life when compared to a graphene membrane without polymer adhesion regions disposed on support structures.

[0394] In some embodiments, the polymer regions may be adhered to a support structure in a manner that increases the surface area of the graphene material provided on the support structure. As shown in FIG. 48, polymer regions 4812 of a repaired graphene material may be adhered to a support structure 4850 such that the graphene material 4800 forms folds, drapes, or bends that increase the available surface area of the graphene material. The increased surface area may be produced by adhering the polymer regions 4812 to the support structure 4850 such that the distance between the polymer regions on the support structure is less than the length of the graphene material between the polymer regions. The graphene material may then fold or bend to accommodate the shorter distance between the polymer regions, and produce an increased graphene surface area for a given support structure area. In some embodiments, the folds, drapes, or bends may be formed by performing the repair process in a solvent that induces swelling in the polymer, and then exchanging the solvent for another solvent that does not swell the polymer. The resulting shrinkage of the polymer regions may relax the graphene and create folds, drapes, or bends therein. In some embodiments, the polymer regions may be softened by heating to relieve stress in the polymer material, creating folds, drapes, or bends in the graphene material. In some embodiments, movements of the repaired graphene material that includes the polymer regions may produce the folds, drapes, or bends in the graphene material.

[0395] In some embodiments, the support structure may be formed in situ during the repair process. To create the in situ support structure, holes may be formed in the graphene material in a pattern and spacing that will result in an interconnected polymer layer, while still maintaining an area of the graphene material sufficient to allow the desired performance of the graphene material. The holes may be produced utilizing any of the procedures described herein, and with any of the shapes and sizes described herein. The holes may be formed in any appropriate pattern, and with any appropriate size. In some embodiments, the holes may be formed in linear arrangements such that the distance between the holes is significantly smaller than the size of the holes. The holes may be arranged in lines, circles, squares, or any other appropriate pattern. As shown in FIG. 45, the holes 4510 may be formed in the graphene material 4480 in a series of lines, where the spacing between the holes within the lines is small in comparison to the size of the holes. The polymer may grow beyond the beyond the borders of the holes, such that the formed polymer regions associated with each hole fuse or merge together, producing a substantially continuous support structure. As shown in FIG. 46, the in situ formed polymer support structure may include a polymer handling region 4540 fused with the polymer regions 4412 formed in the holes. The extent to which the polymer regions extend beyond the borders of the holes is illustrated in FIG. 47, which shows that portions of the polymer regions filling the holes 4412 and/or polymer handling regions 4540 may extend over portions of the graphene material 4480 to fuse and form a substantially continuous in situ support structure. [0396] The in situ support structure may resemble a porous polymer layer, with the graphene material extending across the pores in the support structure. In some embodiments, the in situ support structure may be produced by disposing a porous layer over the graphene membrane prior to forming the in situ support structure, and removing the porous layer after the formation of the in situ support to form fluid flow channels in the in situ support. The porous layer may be a mesh, such as a polymer mesh. The removal of the porous layer may be achieved by any appropriate process, such as dissolving the porous layer. In some embodiments, the graphene material employed in the formation of the in situ support structure may be free of defects other than the holes produced for the purpose of forming the in situ support structure.

[0397] In some embodiments, the repair process may be extended to produce a polymer handling region attached to the graphene material. The polymer handling region 4040 may form a frame around the graphene material 4000 as shown in FIG. 31. The polymer handling region 4040 may be formed in the same process and at the same time as the polymer regions 4012 that repair defects in the graphene material 4000. The polymer handling region may be produced by extending the first reactant and the second reactant beyond the edges of the graphene material, such that the first and second reactants form a polymer extending from the edge of the graphene material. The polymer handling region allows the repaired graphene material to be handled more easily, as the polymer handling region may be more damage resistant than the graphene material. Additionally, the polymer handling region allows the repaired graphene material to be manipulated without directly contacting the graphene material, reducing the opportunity for defects to form in the graphene material after the repair process. In some embodiments, the repair process may be utilized to form a polymer handling region on a graphene material that is free of defects.

[0398] The polymer handling region may have any appropriate size and geometry. As shown in FIG. 41, the polymer handling region 4040 may be in the form of a substantially continuous border that extends along the circumference of the graphene material 4000. The polymer handling region may extend for a distance of at least about 1 mm from the edge of the graphene material, such as at least about 2 mm, about 5 mm, about 1 cm, about 2 cm, about 5 cm, or more. The polymer handling region may have a thickness on the same scale as the polymer regions that plug defects in the graphene material described herein. In some embodiments, the polymer handling region has the same thickness as the polymer regions that plug defects in the graphene material. In some embodiments, the polymer handling region may extend along at least a portion of an edge of the graphene region, such as along one or more edges of the graphene material.

[0399] In some embodiments, the polymer handling region 4040 may also function as a sealing region that prevents fluid from flowing around the edges of the graphene material. The polymer handling region may be adhered and sealed to a support structure 4050, as shown in FIG. 42. In some embodiments, the polymer handling region may be utilized to mount the graphene material in a device or test fixture. The polymer handling region 4342 of a first repaired graphene material 4302 may be sealed to the polymer handling region 4344 of a second repaired graphene material 4304 to form a graphene enclosure or envelope, as shown in FIGS. 43 and 44. The graphene enclosure or envelope forms an interior volume 4370 that is defined by the first graphene material 4302 and the second graphene material 4304.

[0400] The polymer repair process may be conducted before or after forming pores in the graphene material. In cases where the pores are formed in the graphene material before the repair process, the repair process may employ reactants with a size selected to repair only defects greater in size than the desired pores, as described above. In this manner the desired pores are maintained in the repaired graphene material, while defects larger than the desired pore size are repaired with a polymer region. Performing the repair process after forming the pores allows for pore forming procedure that results in a less controlled pore size to be employed, as pores formed that are larger than the desired size will be repaired. Additionally, defects may be formed in the graphene material during the pore forming process and repairing the graphene material after the pore forming process prevent defects formed in the pore forming process from being present in the finished material. This produces a graphene material with more uniform pore sizes.

[0401] In some embodiments, the process of producing a perforated graphene material may include forming pores in a graphene material, forming holes in the graphene material to increase adhesion of the graphene material to a substrate, and repairing the graphene material utilizing an interfacial polymerization process. After forming the pores and holes and before repairing the graphene material, the graphene material 3900 includes pores 3930, holes 3910 and defects 3920, as shown in FIG. 39. After repairing the graphene material, the graphene material 3900 includes pores 3930, polymer regions filling the defects 3922, and polymer regions filling the holes 3912, as shown in FIG. 40. The repaired graphene material may be free of defects and holes that are larger than the desired pore size. The forming of the holes in the graphene material is an optional step, and may not be performed where an increase in adhesion between the graphene material and a substrate is not desired. For example, a process without the formation of holes may produce a graphene material 4000 that includes pores 3930, a polymer region filling defects 4012, and a polymer handling region 4040.

[0402] In some embodiments the graphene material may be produced by repairing defects in the graphene material with interfacial polymerization and forming pores in the material by any appropriate process. In some embodiments, pores may be formed in the graphene material by ultraviolet oxidation, plasma treatment, ion irradiation, or nanoparticle bombardment. The pore formation may occur before or after the repair of the graphene material.

[0403] Ion-based perforation processes may include methods in which the graphene-based material is irradiated with a directional ion source. In some embodiments, the ion source is collimated. The ion source may be a broad field or flood ion source. A broad field or flood ion source can provide an ion flux which is significantly reduced compared to a focused ion beam. The ion source inducing perforation of the graphene or other two-dimensional material in embodiments of the present disclosure is considered to provide a broad ion field, also commonly referred to as an ion flood source. In some embodiments, the ion flood source does not include focusing lenses. In some embodiments, the ion source may be operated at less than atmospheric pressure, such as at 10 ^-3 to 10 ^-5 torr or 10 ^-4 to 10 ^-6 torr. The environment may also contain background amounts (e.g. on the order of 10 ^-5 torr) of oxygen (02), nitrogen (N2) or carbon dioxide (C02). The ion beam may be perpendicular to the surface of the layer(s) of the material (incidence angle of 0 degrees) or the incidence angle may be from 0 to 45 degrees, 0 to 20 degrees, 0 to 15 degrees or 0 to 10 degrees. In some embodiments, exposure to ions does not include exposure to a plasma.

[0404] Ultraviolet oxidation based perforation processes may include methods in which the graphene-based material is simultaneously exposed to ultraviolet (UV) light and an oxygen containing gas. Ozone may be generated by exposure of an oxygen containing gas such as oxygen or air to the UV light. Ozone may also be supplied by an ozone generator device. In some embodiments, the UV oxidation based perforation method further includes exposure of the graphene-based material to atomic oxygen. Suitable wavelengths of UV light may include, but are not limited to, wavelengths below 300 nm, such as from 150 nm to 300 nm. In some embodiments, the intensity of the UV light may be from 10 to 100 mW/cm ² at 6 mm distance or 100 to 1000 mW/cm ² at 6 mm distance. For example, suitable UV light may be emitted by mercury discharge lamps (e.g. a wavelength of about 185 nm to 254 nm). In some embodiments, UV oxidation is performed at room temperature or at a temperature greater than room

temperature. In some embodiments, UV oxidation may be performed at atmospheric pressure (e.g. 1 atm) or under vacuum.

[0405] In some embodiments, the pores may be formed by nanoparticle bombardment.

Nanoparticle bombardment may employ a nanoparticle beam or a cluster beam. In some embodiments, the beam is collimated or is not collimated. Furthermore, the beam need not be highly focused. In some embodiments, a plurality of the nanoparticles or clusters is singly charged. In additional embodiments, the nanoparticles comprise from 500 to 250,000 atoms, such as from 500 to 5,000 atoms.

[0406] A variety of metal particles are suitable for use in the methods of the present disclosure. For example, nanoparticles of Al, Ag, Au, Ti, Cu and nanoparticles comprising Al, Ag, Au, Ti, Cu are suitable. Metal Ps can be generated in a number of ways including magnetron sputtering and liquid metal ion sources (LMIS). Methods for generation of nanoparticles are further described in Cassidy, Cathal, et al. "Inoculation of silicon nanoparticles with silver atoms." Scientific reports 3 (2013), Haberland, Hellmut, et al. "Filling of micron- sized contact holes with copper by energetic cluster impact." Journal of Vacuum Science & Technology A 12.5 (1994): 2925-2930, Bromann, Karsten, et al. "Controlled deposition of size- selected silver nanoclusters." Science 274.5289 (1996): 956-958, Palmer, R. E., S. Pratontep, and H-G. Boyen. "Nanostructured surfaces from size-selected clusters." Nature Materials 2.7 (2003): 443-448, Shyjumon, L, et al. "Structural deformation, melting point and lattice parameter studies of size selected silver clusters." The European Physical Journal D-Atomic, Molecular, Optical and Plasma Physics 37.3 (2006): 409-415, Allen, L. P., et al. "Craters on silicon surfaces created by gas cluster ion impacts." Journal of applied physics 92.7 (2002): 3671-3678, Wucher, Andreas, Hua Tian, and Nicholas Winograd. "A Mixed Cluster Ion Beam to Enhance the Ionization Efficiency in Molecular Secondary Ion Mass Spectrometry." Rapid communications in mass spectrometry : RCM 28.4 (2014): 396-400. PMC. Web. 6 Aug. 2015 and Pratontep, S., et al. "Size-selected cluster beam source based on radio frequency magnetron plasma sputtering and gas condensation." Review of scientific instruments 76.4 (2005): 045103, each of which is hereby incorporated by reference for its description of nanoparticle generation techniques.

[0407] Gas cluster beams can be made when high pressure gas adiabatically expands in a vacuum and cools such that it condenses into clusters. Clusters can also be made ex situ such as C60 and then accelerated towards the graphene.

[0408] In some embodiments, the nanoparticles are specially selected to introduce moieties into the graphene. In some embodiments, the nanoparticles function as catalysts. The moieties may be introduced at elevated temperatures, optionally in the presence of a gas. In other embodiments, the nanoparticles introduce" chiseling" moieties, which are moieties that help reduce the amount of energy needed to remove an atom during irradiation.

[0409] In some embodiments, the size of the produced pores is controlled by controlling both the nanoparticle size and the nanoparticle energy. Without wishing to be bound by any particular belief, if all the nanoparticles have sufficient energy to perforate, then the resulting pores are believed to correlate with the nanoparticle sizes selected. However, the size of the pore is believed to be influenced by deformation of the nanoparticle during the perforation process. This deformation is believed to be influenced by both the energy and size of the nanoparticle and the stiffness of the graphene layer(s). A grazing angle of incidence of the nanoparticles can also produce deformation of the nanoparticles. In addition, if the nanoparticle energy is controlled, it is believed that nanoparticles can be deposited with a large mass and size distribution, but that a sharp cutoff can still be achieved.

[0410] Without wishing to be bound by any particular belief, the mechanism of perforation is believed to be a continuum bound by sputtering on one end (where enough energy is delivered to the graphene sheet to atomize the carbon in and around the NP impact site) and ripping or fracturing (where strain induced failure opens a torn hole but leaves the graphene carbons as part of the original sheet). Part of the graphene layer may fold over at the site of the rip or fracture. In an embodiment the cluster can be reactive so as to aid in the removal of carbon (e.g. an oxygen cluster or having trace amounts of a molecule known to etch carbon in a gas cluster beam i.e. a mixed gas cluster beam). Without wishing to be bound by any particular belief, the stiffness of a graphene layer is believed to be influenced by both the elastic modulus of graphene and the tautness of the graphene. Factors influencing the elastic modulus of a graphene layer are believed to include temperature, defects (either small defects or larger defects from P irradiation), physisorption, chemisorption and doping. Tautness is believed to be influenced by coefficient of thermal expansion mismatches (e.g. between substrate and graphene layer) during deposition, strain in the graphene layer, wrinkling of the graphene layer. It is believed that strain in a graphene layer can be influenced by a number of factors including application of gas pressure to the backside (substrate side) of a graphene layer, straining of an elastic substrate prior to deposition of graphene, flexing of the substrate during deposition, and defecting the graphene layer in controlled regions to cause the layer to locally contract and increase the local strain.

[0411] In some embodiments, nanoparticle perforation can be further controlled by straining the graphene layers during perforation to induce fracture, thereby "ripping" or "tearing" one or more graphene layers. In some embodiments, the stress is directional and used to preferentially fracture in a specific orientation. For example, ripping of one or more graphene sheets can be used to create "slit" shaped apertures; such apertures can be substantially larger than the nanoparticle used to initiate the aperture. In additional embodiments, the stress is not oriented in a particular direction.

[0412] In some embodiments, the pores may be functionalized. In some embodiments, the pores are functionalized by exposure to gas during and/or following the perforation process. The exposure to gas may occur at temperatures above room temperature. In some embodiments, the pores can have more than one effective functionalization. For example, when the top and the bottom layers of a graphite stack are exposed to different functionalizing gases, more than one effective functionalization can be produced. In further embodiments, a thin layer of a

functionalizing moiety is applied to the surface before NP perforation, during NP perforation and after P perforation. As compatible with the P process, the thin layer may be formed by applying a fluid to the surface. In embodiments, the gas pressure is 10 ^-4 Torr to atmospheric pressure. In embodiments, functionalizing moieties include, but are not limited to water, water vapor, PEG, oxygen, nitrogen, amines, and carboxylic acid.

[0413] The preferred gasses for before and during functionalization depend on the reaction of graphene and the gas within the high energy environment of the particle impact. This would be within about 100 nm of the edge of the particle impact. This fits into two general classes, and the gases would be added at a partial pressure of from lx10 ^-6 Torr to lx10 ^-3 Torr. The first class would be species that reacts with radicals, carbanions (negative charge centered on a carbon) and carbocations (positive charge centered on a carbon). Representative molecules include carbon dioxide, ethylene oxide and isoprene. The second class would be species that fragment to create species that react with graphene and defective graphene. Representative molecules would be polyethylene glycol, diacytylperoxide, azobisisobutyronitrile, and phenyl diazonium iodide.

[0414] In some embodiments, it is desirable and advantageous to perforate multiple graphene sheets at one time rather than perforating single graphene sheets individually, since multi-layer graphene is more robust and less prone to the presence of intrinsic or defects that align through all the layers than is single-layer graphene. In addition, the process is stepwise efficient, since perforated single-layer graphene can optionally be produced by exfoliating the multi-layer graphene after the pore definition process is completed. The pore size is also tailorable in the processes described herein. Thus, the nanoparticle perforation processes described herein are desirable in terms of the number, size and size distribution of pores produced.

[0415] The multi -layer graphene subjected to nanoparticle perforation may contain between about 2 stacked graphene sheets and about 20 stacked graphene sheets. Too few graphene sheets may lead to difficulties in handling the graphene as well as an increased incidence of intrinsic or native graphene defects. Having more than about 20 stacked graphene sheets, in contrast, may make it difficult to perforate all of the graphene sheets. In some embodiments, the multilayer sheets may be made by individually growing sheets and making multiple transfers to the same substrate. In some embodiments, the multi-layer graphene perforated by the techniques described herein can have 2 graphene sheets, or 3 graphene sheets, or 4 graphene sheets, or 5 graphene sheets, or 6 graphene sheets, or 7 graphene sheets, or 8 graphene sheets, or 9 graphene sheets, or 10 graphene sheets, or 11 graphene sheets, or 12 graphene sheets, or 13 graphene sheets, or 14 graphene sheets, or 15 graphene sheets, or 16 graphene sheets, or 17 graphene sheets, or 18 graphene sheets, or 19 graphene sheets, or 20 graphene sheets.

[0416] The reactants may be applied to the graphene material by any appropriate process. In some embodiments the graphene material may be disposed between liquid solutions or suspensions containing the reactants, and the liquid solutions and suspensions may or may not be flowing past the surfaces of the graphene material. The liquid solutions or suspensions of reactants may be applied to the graphene material by rollers, brushes, spray nozzles, or doctor blades. In some embodiments, the reactants may be applied to the graphene material in droplet form, such as through the use of an inkjet apparatus. In some embodiments a liquid solution or suspension containing a reactant may be disposed on one side of the graphene material and the other side of the graphene material may be exposed to a gas phase reactant.

[0417] In some embodiments, the graphene material may be floated on the surface of a liquid suspension or solution containing one of the reactants. The graphene material may be free of a support structure when it is floated on the liquid. In some embodiments, the graphene material may be disposed on a support structure when floated on the liquid, the support structure may include support structures that function to maintain the position of the graphene material on the surface of the liquid and support structures that may be utilized to handle the graphene material after repair. In some embodiments, a mesh material may be employed as a support structure to maintain the graphene material on the surface of the liquid. In some embodiments, a porous polymer may be employed as a support structure that may also be used to handle or manipulate the graphene material after the repair process. In some other embodiments, a support structure including a sacrificial layer that is removed during or after the repair process may be employed.

[0418] In some embodiments, the reactants may be applied to an enclosure or envelope including the graphene material. As shown in FIG. 43, an enclosure or envelope including the graphene material may include a lumen 4360 that allows access to the interior volume 4370 of the enclosure. In a repair process of the graphene material included in the enclosure, the first reactant may be supplied to the interior volume of the enclosure, and the second reactant may be applied to the exterior of the enclosure. The manner of exposing the exterior of the enclosure to the second reactant may include any of the application processes described herein. After the completion of the repair process, the first reactant may be removed from the enclosure and a desired component may be loaded in to the interior space of the enclosure.

[0419] The repaired graphene material described herein may be employed in any appropriate process or device. In some embodiments the graphene material may be utilized in filtration devices, such as devices utilized in deionization, reverse osmosis, forward osmosis, contaminant removal, and wastewater treatment processes. The graphene material may also be employed in a biomedical device as a selectively permeable membrane. In some embodiments, the graphene material may be employed in a viral clearance or protein separation process.

[0420] The graphene materials described herein may be employed as membranes in water filtration, immune-isolation (i.e., protecting substances from an immune reaction), timed drug release (e.g., sustained or delayed release), hemodialysis, and hemofiltration. The graphene materials described herein may be employed in a method of water filtration, water desalination, water purification, immune-isolation, timed drug release, hemodialysis, or hemofiltration, where the method comprises exposing a membrane to an environmental stimulus.

[0421] In some embodiments, methods of filtering water may include passing water through a membrane including the graphene materials described herein. Some embodiments include desalinating or purifying water comprising passing water through a membrane including the graphene materials described herein. The water can be passed through the membrane by any known means, such as by diffusion or gravity filtration, or with applied pressure.

[0422] Some embodiments include methods of selectively separating or isolating substances in a biological environment, wherein a membrane including the graphene materials described herein separates or isolates biological substances based on characteristics of the substance, such as size. Such methods can be useful in the context of disease treatment, such as in the treatment of diabetes. In some embodiments, biological substances below a certain size threshold can migrate across the membrane. In some embodiments, even biological substances below the size threshold are excluded from migrating across the membrane due to functionalization of membrane pores and/or channels. TWO-DIMENSIONAL MEMBRANE STRUCTURES HAVING FLOW PASSAGES

[0423] Some embodiments relate to a two-dimensional membrane layered structure having a plurality of flow passages formed between a two-dimensional membrane layer and a support substrate layer. The flow passages provide an increase in pore utilization by allowing fluid to flow through pores in the two-dimensional membrane layer that do not overlap with passages present in the support substrate. With the flow passages, fluid may flow from the non- overlapping pores through the flow passages to a nearby support substrate passage. In some embodiments, the flow passages may be formed by interlayer supports laterally disposed on an upper surface of the support substrate layer and/or by grooves laterally formed on the upper surface of the support substrate layer. In addition to increasing overall pore utilization of the membrane layer and total flow through the membrane structure, the flow passages may also provide sufficient support to the two-dimensional membrane without adding undesirable strain to the membrane layer.

[0424] FIG. 49 shows a perspective view of a two-dimensional membrane layered structure 4900. The two-dimensional membrane layered structure 4900 may include a two-dimensional membrane layer 4910 having a plurality of pores 4915, and a support substrate layer 4920 having a plurality of substrate passages 4925 that extend along a thickness of the support substrate layer 4920.

[0425] The two-dimensional membrane layer 4910 may be a single-layer two-dimensional material or a stacked two-dimensional material. Most generally, a single-layer two dimensional material is atomically thin, having an extended planar structure and a thickness on the nanometer scale. Single-layer two-dimensional materials generally exhibit strong in-plane chemical bonding relative to the weak coupling present between layers when such layers are stacked. Examples of single-layer two-dimensional materials include metal chalcogenides (e.g., transition metal dichalcogenides), transition metal oxides, boron nitrate (e.g., hexagonal boron nitride), graphene, silicone, and germanene, carbon nanomembranes (CNM), and molybdenum disulfide. Stacked two-dimensional materials may include a few layers (e.g., about 20 or less) of a single- layer two-dimensional material or various combinations of single-layer two-dimensional materials. In some embodiments, the two-dimensional membrane layer 4910 may be a graphene or graphene-based two-dimensional material as a single-layer two-dimensional material or a stacked two-dimensional material.

[0426] The support substrate layer 4920 may include any appropriate planar-type substrate. In some embodiments, the support substrate layer 4920 may be made from ceramic porous materials, such as silica, silicon, silicon nitride, and combinations thereof. In some embodiments, he support substrate layer 4920 may be made from a polymer material, such as track-etched polymers, expanded polymers, patterned polymers, non-woven polymers, and combinations thereof. The support substrate layer 4920 may include a polymer selected from the group consisting of polysulfones, polyurethane, polymethylmethacrylate (PMMA), polyethylene glycol (PEG), polylactic-co-glycolic acid (PLGA), PLA, PGA, polyamides (such as nylon-6,6, supramid and nylamid), polyimides, polypropylene, polyethersulfones (PES), polyvinylidine fluoride (PVDF), cellulose acetate, polyethylene, polypropylene, polycarbonate,

polytetrafluoroethylene (PTFE) (such as Teflon), polyvinylchloride (PVC), polyether ether ketone (PEEK), mixtures and block co-polymers of any of these, and combinations and/or mixtures thereof. In some embodiments, the polymers may be biocompatible, bioinert and/or medical grade materials. In some embodiments, the support substrate layer 4920 may be a track- etched polycarbonate (TEPC) membrane. In other embodiments, the support substrate layer 4920 may be a membrane formed by silicon nitride, track-etched polyimide, track-etched polyester, track-etched SiN, nanoporous silicon, nanoporous silicon nitride, an electrospun membrane, and/or a PVDF membrane. In some embodiments, the substrate passages 4925 may be formed randomly or in a patterned manner.

[0427] As shown in FIG. 49, when disposing the two-dimensional membrane layer 4910 on the support substrate 4920, only a certain percentage of pores 4915 overlap with the substrate passages 4925. For example, a first portion of pores 4915a overlap with, or are in fluid communication with, the support substrate passages 4925. The first portion of pores 4915a contributes to overall pore utilization because they allow fluid to flow through to the substrate passages 4925. On the other hand, a second portion of pores 4915b do not overlap with, or fail to be in fluid communication with, the support substrate passages 4925. Thus, the second portion of pores 4915b does not contribute to overall pore utilization because fluid cannot flow from these pores into the support substrate passages 4925. Moreover, as further shown in FIG. 49, in some cases, a support substrate passage 4925a may fail to be in fluid communication with any of the pores 4915. Thus, this blocked support substrate passageg 4925a receives no fluid flow, thereby decreasing the overall utilization of the support substrate passages 4925.

[0428] FIG. 50 illustrates a perspective view of the two-dimensional membrane layered structure 4900 having a plurality of flow passages 4955 disposed between the two-dimensional membrane layer 4910 and the support substrate layer 4920. As shown in FIG. 50, the flow passages 4955 provide a means for the second portion of pores 4915b to be utilized by providing a flow path for fluid to flow from a respective pore 4915b to a nearby substrate passage 4925. In some embodiments, the flow passages 4955 may also provide a flow path from the second portion of pores 4915b to the blocked support substrate passage 4925a such that fluid may flow through the blocked support substrate passage 4925a. Thus, the integration of the flow passages 4955 provide for an increase in the overall pore utilization of the two-dimensional membrane layer 4910 and a means for increased fluid flow through the support substrate layer 4920.

[0429] FIG. 51 shows a cross-sectional view of the two-dimensional membrane layered structure 4900 having flow passages 4955 formed therein in accordance with a first embodiment. As shown in FIG. 51, a plurality of interlayer supports 4950 may be disposed laterally on an upper surface 4926 of the support substrate layer 4920. The two-dimensional membrane layer 4910 is then disposed onto the upper surface 4926 of the support substrate layer 4920 having the interlayer supports 4950 disposed thereon. When layered, the interlayer supports 4950 support the two-dimensional membrane layer 4910 while pushing the two-dimensional membrane layer 4910 upward from the support substrate layer 4920 to form the flow passages 4955 that allow fluid to flow through the pores 4915b.

[0430] The interlayer supports 4950 may take numerous forms. For example, in some embodiments, the interlayer supports 4950 may be carbon nanotubes. For example, FIGS. 53 and 54 show interlayer supports in the form of carbon nanotubes spray-coated onto an upper surface of a support substrate layer in the form of a track-etched polyimide layer before the two- dimensional membrane layer is disposed onto the support substrate layer. As shown the figures, the carbon nanotubes are disposed onto the upper surface of the support substrate layer so as to extend laterally across the upper surface to provide access for fluid flow to the substrate passages. In some embodiments, the carbon nanotubes may be single-walled or multi-walled.

[0431] In other embodiments, the interlayer supports 4950 may be electrospun fibers. In yet other embodiments, the interlayer supports 4950 may be nanorods, nanoparticles, (e.g., oxide nanoparticles, octadecyltrichlorosilane nanoparticles), fullerenes, collagen, keratin, aromatic amino acids, polyethylene glycol, lithium niobate particles,, decorated nano-dots, nanowires, nanostrands, lacey carbon material, proteins polymers (e.g., hygroscopic polymer, thin polymer, amorphous polymer), hydrogels, self-assembled monolayers, allotropes, nanocrystals of 4- dimethylamino-N-methyl-4-stilbazolium tosylate, crystalline polytetrafluoroethylene, or combinations thereof. In some embodiments, the density of the interlayer supports 4950 may comprise about 5% to about 50% of the total area of the upper surface 4926 of the substrate support layer 4920 to provide a sufficient pore utilization increase to the composite 4900, while at the same time minimizing a decrease in mechanical support of the two-dimensional membrane layer 4910. In some embodiments, the density of the interlayer supports 4950 comprises 40 to 45 % of the total area of the upper surface 4926 of the support substrate layer 4920.

[0432] The interlayer supports 4950 may be applied to the upper surface 4926 of the support substrate layer 4920 in any appropriate manner. For example, the some embodiments, the interlayer supports 4950 may be carbon nanotubes that may be spray-coated in random orientations onto the upper surface 4926 of the support substrate layer 4920. The spray-coating may be controlled such that the density of the carbon nanotubes applied to the upper surface 4926 may be fine-tuned. Other methods of disposing the interlayer supports 4950 onto the upper surface 4926 of the support substrate layer 4920 may include, but are not limited to, electrostatic deposition, drop casting, spin-coating, sputtering, lithography, ion beam induced deposition, atomic layer deposition, or electron beam induced deposition. Additional methods include the application of electrospun fibers by an electric field, the acceleration of nanoparticles by a potential, or the use of an ion beam to irradiate material present on the upper surface 4926 to form raised structures on the upper surface 4926 of the support substrate layer 4920.

[0433] FIG. 52 shows a cross-sectional view of the two-dimensional membrane layered structure 4900 having flow passages 4955 formed therein in accordance with a second embodiment. As shown in FIG. 52, before transferring the two-dimensional membrane layer 4910 onto the support substrate layer 4920, a plurality of grooves 4950' are laterally formed onto the upper surface 4926 of the support substrate layer 4920 to form the flow channels 4955. As such, fluid may flow through the pores 4915b to the support substrate channels 4925, thereby increasing the overall pore utilization of the two-dimensional membrane layer 4910.

[0434] The plurality of grooves 4950' may be formed into the upper surface 4926 of the support substrate layer 4925 by any appropriate means. For example, in some embodiments, the grooves 4950' may be formed by etching the upper surface 4926. In other embodiments, the grooves 4950' may be formed by focused ion beam milling, lithography methods (e.g., optical, electron beam lithography, extreme UV lithography) on resists followed by suitable etching (e.g., reactive ion etching), block copolymer mask focused on columnar and aligned structures followed by suitable etching, laser ablation, nanoimprint, scanning probe lithography, shadowmask deposition followed by suitable etching, or sparse aperture masking methods.

[0435] In some embodiments, the grooves 4950' may be formed in a regular, structured pattern, such as a lattice-like pattern, or in a random pattern. In addition, the grooves 4950' may be formed to have a depth and a width of about 1 nm to about 5μιη. In some embodiments, the depth and width may range from about 10 nm to about 1000 nm. In other embodiments, the depth and width of the grooves 4950' may range from about 50 nm to about 250 nm. In certain embodiments, the density of the grooves 4950' may comprise up to 50 to 75 % of the total area of the upper surface 4926 of the support substrate layer 4920.

[0436] Some embodiments, such as described above, allow for the formation of fluid flow passages in a two-dimensional membrane layered structure. In some embodiments, the flow passages may be formed by interlayer supports disposed on an upper surface of the support substrate layer. In other embodiments, the flow passages may be formed by grooves provided on the upper surface of the support substrate layer. In yet other embodiments, the flow passages may be formed both by interlayer supports disposed on the upper surface of the support substrate layer and grooves provided on the upper surface of the support substrate layer. The fluid flow passages may be formed between the two-dimensional support layer and the support substrate layer such that an increase in the amount of pores that allow fluid to flow to passages in the support substrate layer may be realized. Thus, the flow passages may result in an increase in overall pore utilization in the layered structure without resulting in a loss of mechanical support provided to the two-dimensional membrane.

[0437] The two-dimensional membrane layered structures described herein have broad application, including in water filtration, immune-isolation, (i.e., protecting substances from an immune reaction), timed drug release (e.g., sustained or delayed release), hemodialysis, and hemofiltration. Some embodiments described herein comprise a method of water filtration, water desalination, water purification, immune-isolation, timed drug release, hemodialysis, or hemofiltration, where the method comprises exposing a two-dimensional membrane layered structure to an environmental stimulus, and wherein the two-dimensional membrane layered structure comprises a two-dimensional membrane layer having a plurality of pores (e.g., a porous graphene-based material) and a support substrate layer having a plurality of substrate passages.

[0438] Some embodiments include methods of filtering water comprising passing water through a two-dimensional membrane layered structure. Some embodiments include

desalinating or purifying water comprising passing water through a two-dimensional membrane layered structure. The water can be passed through the two-dimensional membrane layered structure by any known means, such as by diffusion or gravity filtration, or with applied pressure (e.g., applied with a pump or via osmotic pressure).

[0439] Some embodiments include methods of selectively separating or isolating substances in a biological environment, wherein the two-dimensional membrane layered structure separates or isolates biological substances based on characteristics of the substance, such as size. Such methods can be useful in the context of disease treatment, such as in the treatment of diabetes. In some embodiments, biological substances below a certain size threshold can migrate across the two-dimensional membrane layered structure. In some embodiments, even biological substances below the size threshold are excluded from migrating across the two-dimensional membrane layered structure due to functionalization of the plurality of pores, the plurality of substrate passages, the plurality of interlayer supports and/or the plurality of grooves.

[0440] In some embodiments, the plurality of pores, or at least a portion thereof, is functionalized. In some embodiments, the plurality of substrate passages, or at least a portion thereof, is functionalized, for instance by attaching or embedding a functional group. In some embodiments, the plurality of interlayer supports and/or the plurality of grooves, or at least a portion thereof, is functionalized, for instance by attaching or embedding a functional group. In some embodiments, the functionalization moieties are trapped between two layers, but are not restricted to a single position in the flow passages (i.e., they are mobile within the flow passages, but are inhibited from traversing the layers, e.g., based the size of the pores in the two- dimensional membrane layer). In some embodiments, functionalization comprises surface charges (e.g., sulfonates) attached to the pores, substrate passages, interlayer supports, and/or grooves. Without being bound by theory, it is believed that surface charges can impact which molecules and/or ions can traverse the two-dimensional membrane layered structure. In some embodiments, functionalization comprises specific binding sites attached to the pores, substrate passages, interlayer supports, and/or grooves. In some embodiments, functionalization comprises proteins or peptides attached to the pores, substrate passages, interlayer supports, and/or grooves. In some embodiments, functionalization comprises antibodies and/or antigens (e.g., IgG-binding antigens) attached to the pores, substrate passages, interlayer supports, and/or grooves. In some embodiments, functionalization comprises adsorptive substances attached to the pores, substrate passages, interlayer supports, and/or grooves. In some embodiments, functionalization involves catalytic and/or regenerative substances or groups. In some embodiments, functionalization comprises a negatively or partially negatively charged group (e.g., oxygen) attached to the pores, substrate passages, interlayer supports, and/or grooves. In some embodiments, functionalization comprises a positively or partially positively charged group attached to the pores, substrate passages, interlayer supports, and/or grooves.

[0441] In some embodiments, functionalizing the pores, substrate passages, interlayer supports, and/or grooves functions to: restrict contaminants from traversing the two-dimensional membrane layered structure; act as a disposable filter, capture, or diagnostic tool; increase biocompatibility (e.g., when polyethylene glycol is used for functionalization); increase filtration efficiency; position the interlayer supports (e.g., interlayer supports can be positioned near the pores via affinity -based functionalization in the pores; additional spacers can be positioned in interlaminar areas); increase selectivity at or near the pores or in asymmetric two-dimensional membrane layered structure; and/or protect interlayer supports (e.g., from the external environment or from a particular vulnerability such as degradation).

[0442] Some embodiments have been described in detail with particular reference to preferred embodiments thereof, but it will be understood by those skilled in the art that variations and modifications can be effected within the spirit and scope of the claims.

MEMBRANES WITH TUNABLE SELECTIVITY

[0443] Some embodiments include membranes, and methods of making membranes, with tunable selectivity, e.g., where the membrane can adapt to environmental conditions. In some embodiments, the membrane can be tuned as a result of being adjusted to alter selectivity. Some other embodiments include methods of altering membrane permeability and methods of using membranes with tunable selectivity.

[0444] Tunable Membranes

[0445] Membranes of some embodiments are formed with multiple layers of porous graphene-based material, where the layers are positioned or stacked such that a space between the layers can function as a channel or conduit. In some embodiments, the membrane comprises at least two layers of porous graphene-based material, such as from about 2 to about 10 layers, or from about 2 to about 5 layers of porous graphene-based material. In some embodiments, the membrane comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 layers of porous graphene-based material. The number of channels in the membrane depends in part on the number of graphene-based material layers in the membrane. Thus, two graphene-based material layers can form one channel; three graphene-based material layers can form two channels. In some embodiments, the walls of the channel comprise the graphene-based material layers.

[0446] In some embodiments, a graphene-based material layer comprises a single sheet of graphene-based material. In some other embodiments, a graphene-based material layer comprises multiple sheets of graphene-based material, such as from about 2 to about 5 sheets of graphene-based material. When a layer comprises multiple sheets of graphene-based material, the sheets of can be combined in the layer via, e.g., covalent bonding and/or van der Waals forces. Graphene-based materials are discussed in greater detail later in this application. [0447] The porous graphene-based material layers in the membrane can be structurally similar, structurally identical, or structurally different from other porous graphene-based material layers in the membrane. For instance, in some embodiments, all graphene-based material layers have the same number of graphene sheets. In some embodiments, the number of graphene sheets in a layer is different from the number of graphene sheets in a different layer. The porous graphene-based material layers in the membrane can be chemically similar, chemically identical, or chemically different from other porous graphene-based material layers in the membrane. In some embodiments, graphene-based material layers can be functionalized with similar, identical, or different functional groups from other graphene-based material layers.

[0448] The thickness of the membrane depends in part on the number of layers present in the membrane and/or on the number of graphene-based material sheets in the membrane. In some embodiments, the membrane is at least 5 nm thick, such as from about 5 nm to about 250 nm thick, from about 5 to about 20 nm thick, or from about 20 to about 50 nm thick.

[0449] Membranes of some of the embodiments provide a means for increasing and/or decreasing the diameter of the channel. For example, at least one spacer substance can be positioned in the channel between the graphene-based material layers. In some embodiments, the spacer substance is responsive to an environmental stimulus. Exemplary environmental stimuli include changes in temperature, pressure, pH, ionic concentration, solute concentration, tonicity, light, voltage, electric fields, magnetic fields, pi-bonding availability, and combinations thereof. In some embodiments, the spacer substance is responsive to a single environmental stimulus. In some embodiments, the spacer substance is responsive to two or more

environmental stimuli.

[0450] The properties of the responsive spacer substances can be altered upon exposure to an environmental stimulus. For instance, in some embodiments the spacer substance can expand and/or contract in response to an environmental stimulus. By way of example, the effective diameter of the spacer substance can be reduced in response to an increase in applied pressure. This is demonstrated in Figures 60-62, which shows a membrane channel with altered selectivity following application of pressure (initially, both water and ions, electrolytes, and/or salts in solution can traverse the membrane - Figure 60; increased pressure compresses the spacer substance and prevents the salt from traversing the membrane - Figure 61; Figure 62 demonstrates that pressure can be used to compress a spacer substance in a membrane with three graphene layers). The term "effective diameter" as it relates to spacer substances refers to the distance between two points on the spacer substance, where the points interact with different graphene-based material layers that form a channel of the membrane (i.e., the height of the spacer substance in the membrane). In this way, the effective diameter of the spacer substance influences the diameter of the channel. An initial effective diameter of the spacer substances can be determined prior to incorporating the spacer substance into the membrane, for instance via transmission electron microscopy (TEM) tomography. For some spacer substances, such as nanoparticles and other particle-based spacer substances, the effective diameter can be determined via scanning electron microscopy (SEM).

[0451] In some embodiments, the effective diameter of the spacer substance can be increased upon removal of or reduction in applied pressure. In some embodiments, the effective diameter of the spacer substance increases upon hydration and/or decreases upon dehydration. In some embodiments, the spacer substance is capable of undergoing a physical and/or chemical transformation in the membrane based on an interaction with an activating substance, such as an affinity-based interaction or a chemical reduction. In some embodiments, the environmental stimulus induces a conformational change in the spacer substance that alters the effective diameter of the spacer substance. For instance, conformational changes between trans and cis forms of a spacer substance can alter the effective diameter of the spacer substance (by way of example, a spacer substance could be a polymer with an embedded diazo dye, where exposure to the appropriately colored light alters the volume of the dye based on cis-/trans- conformational changes). In some embodiments, the spacer substance undergoes a physical and/or chemical transformation that is pH-modulated or optically modulated. In some embodiments, the environmental stimulus degrades the spacer substance to alter the effective diameter of the spacer substance.

[0452] In some embodiments, the effective diameter of the spacer substance can be altered by applying a voltage to the membrane or via electrowetting. See, for instance, Figure 63, showing that the spacing between layers can be altered via voltage-sensitive spacer substances. In some embodiments, a voltage or field source is applied across the membrane. In some embodiments, the voltage assists in moving the permeant across the membrane. In some embodiments, the voltage is applied across the membrane using a power source, such as a battery, a wall outlet, or an applied RF field (or other beam). In some embodiments, a voltage of about 1 mV to about 900 mV is applied across the membrane. In some embodiments, the voltage is applied to a graphene-based material layer in-plane. In some embodiments, the graphene can be biased, for instance with the use of an insulating material. In some

embodiments, the voltage applied across the membrane is low enough that the graphene does not delaminate and/or low enough that the voltage does not induce electrolysis.

[0453] The responsive change in spacer substance properties can be reversible or

irreversible. In some embodiments, the spacer substances reversibly expands and/or reversibly contracts in response to the environmental stimulus. Therefore, in some embodiments, the size of the spacer substance can be repeatedly increased and then decreased in succession. In some embodiments, the size of spacer substance can be increased or decreased, but not both. In some embodiments, the size of the spacer substance can be increased or decreased irreversibly.

[0454] Spacer substances can include polymers, fibers, hydrogels, molecules, nanostructures, nanoparticles, self-assembled monolayers, and allotropes that are responsive to an environmental stimulus. In some embodiments, the spacer substance is a smart polymer, such as a hygroscopic polymer; a thin polymer that expands when hydrated; or an amorphous polymer, such as a porous amorphous polymer. In some embodiments, the spacer substance comprises electrospun fibers that can be swelled upon exposure to a solvent. In some embodiments the spacer substance comprises materials with a high thermal expansion coefficient, which expand or contract in response to a temperature stimulus. In some embodiments, the spacer substance is deliquescent. In some embodiments, the spacers are substantially inert. In some embodiments, the spacers are not inert (i.e., they can be reactive).

[0455] Exemplary spacer substance includes particle substances such as metal nanoparticles (e.g., silver nanoparticles), oxide nanoparticles, octadecyltrichlorosilane nanoparticles, carbon nanotubes, and fullerenes. In some embodiments, the spacer substance includes nanorods, nano- dots (including decorated nano-dots), nanowires, nanostrands, and lacey carbon materials. [0456] Exemplary spacer substances also include structural proteins, collagen, keratin, aromatic amino acids, and polyethylene glycol. Such spacer substances can be responsive to changes in tonicity of the environment surrounding the spacer substance, pi-bonding availability, and/or other environmental stimuli.

[0457] In some embodiments, the spacer substance is a piezoelectric, electrostrictive, or ferroelectric magnetic particle. In some embodiments, the magnetic particle comprises a molecular crystal with a dipole associated with the unit cell. In some embodiments, the magnetic particles can be oriented based on an external magnetic field. Exemplary magnetic particles include lithium niobate, nanocrystals of 4-dimethylamino-N-methyl-4-stilbazolium tosylate (DAST)), crystalline polytetrafluoroethylene (PTFE), electrospun PTFE, and combinations thereof.

[0458] In some embodiments, the spacer substance heats up faster or slower than its surroundings. Without being bound by theory, it is believed that such embodiments will allow the rate of passage of permeants, or a subset of permeants, across the membrane to be increased and/or decreased.

[0459] In some embodiments, spacer substances respond to electrochemical stimuli. For instance, a spacer substance can be an electrochemical material (e.g., lithium ferrophosphate), where a change in oxidation state of the spacer substance (e.g., from 2- to 3-) alters permeability of the membrane. In some embodiments, changing the oxidation state of the spacer substances alters the interaction between the spacer substance and potential permeants. In some

embodiments, the change in oxidation state results from a redox-type reaction. In some embodiments, the change in oxidation state results from a voltage applied to the membrane.

[0460] In some embodiments, the spacer substance comprises contamination structures formed by utilizing a focused ion beam, e.g., to modify heavy levels of contamination on graphene-based material into more rigid structures. For instance, in some embodiments, mobilization and migration of contamination on the surface of the graphene-based material occurs - coupled in some embodiments with some slight beam induced deposition - followed by modification and induced bonding where the beam is applied. In some embodiments, combining contamination structures allows the geometry, thickness, rigidity, and composition of the spacer substance to be tuned to respond to an environmental stimulus (e.g., pressure). Exemplary contamination-based spacer substances are shown in Figures 64A-D.

[0461] In some embodiments, spacer substances have an affinity for graphene. In some embodiments, spacer substances have a higher affinity for graphene than for substances or solutions that can permeate the membrane.

[0462] In some embodiments, the spacer substance is chemically modified to have a functional group or a desired physiochemical property. For example, in some embodiments the spacer substance is modified to be hydrophobic. In some embodiments, the spacer substance is modified to be hydrophilic. In some embodiments, the spacer substance is modified by addition of hydroxyl groups. In some embodiments, the spacer substance is attached to antibody receptors. In some embodiments, the spacer substance is attached to proteins, enzymes, and/or catalysts. For example, in some embodiments a metallic, organometallic, and/or zeolite-based functional group can act as a catalyst for precursors that enter the membrane. In some embodiments, spacer substances are functionalized to preferentially orient a permeant (e.g., water or a solvent). In some embodiments, a permeant that is in the preferential orientation can traverses the membrane in that preferential orientation, whereas a permeant that is not the preferential orientation does not.

[0463] In some embodiments, membranes include a plurality of a single type of spacer substance (e.g., a plurality of nanoparticles). In some embodiments, membranes include a multiple types of spacer substances (e.g., nanoparticles and polymers). In some embodiments, the spacer substance is a porous layer, such as a porous amorphous polymer layer. In some embodiments, the spacer substance is a self-assembled co-polymer that leaves channels between graphene-based material layers (e.g., the channels can be a sub-nm in diameter to about 40 nm in diameter). In some embodiments, the spacer substance or substances between two graphene- based material layers can be the same as or different from the spacer substance or substances between two other graphene-based material layers. That is, the spacer substance or substances in one membrane channel can be the same as or different from the spacer substance or substances in a different membrane channel. [0464] The diameter of the channel can be tailored based on the density and/or size of the spacer substance incorporated into the membrane. For instance, without being bound by theory, an increase in spacer substance density is believed to be associated with an increase in channel diameter as compared to a channel comprising the same spacer substance, but at a lower density. Indeed, an increased distance between spacer substances (i.e., a low density) allows flexible graphene-based material layers to attain stable configurations in which portions of different layers are in close proximity, thereby lowering the diameter of the channel.

[0465] In some embodiments, the spacer substances are incorporated at a sufficiently low density to allow inter-layer interactions (e.g., interactions between graphene in different layers). In some embodiments, the spacer substances are incorporated at a sufficiently high density to allow chemical interactions (e.g., covalent or van der Waals interactions) between the layers and the spacer substances, but to prevent inter-layer chemical interactions. In some embodiments, both layer-spacer substance and inter-layer chemical interactions are present in the membrane.

[0466] In some embodiments, the spacer substances are positioned in the channel with an average distance between spacer substances of from 10 nm to about 150 nm. In some embodiments, the spacer density is such that spacer substances cover up to about 50% of the surface area of center of the channel - i.e., in a 2D-plane along the center of the channel, spacer substances cover up to about 50% of the area of that plane. In some embodiments, the spacer density is such that the spacer substances cover up to about 40%, up to about 30%, up to about 20%), or up to about 10%> of the surface area of the center of the channel. Spacer density can be calculated, for instance, based on the amount of spacer substance used, the dimensions of the membrane, and the dimensions of the spacer.

[0467] As mentioned above, the size of the spacer substances can also impact properties of the membrane. For instance, spacer substances with a relatively large effective diameter can be used to prepare channels with a relatively high maximum diameter. The term "maximum diameter" as it relates to channel width is defined by the diameter of the channel at a point of interaction between a layer and the spacer substance in the channel with the largest effective diameter. Notably, because of the flexibility/conformity of graphene-based materials, the diameter of a channel at any given location can be higher or lower than the maximum diameter. In some embodiments, spacer substances with relatively small effective diameter can be used to prepare channels with a relatively low maximum diameter. In some embodiments, the spacer substances have an effective diameter of from about 0.3 nm to about 100 nm, such from about 0.3 nm to about 0.5 nm, from about 0.5 nm to about 2 nm, or from about 20 nm to about 50 nm.

[0468] In some embodiments, the spacer substance is restricted from traversing the graphene-based material layers. For example, in some embodiments, the spacer substance is larger than the size of the pores in the graphene-based material layers, or larger than a portion of the pores in the graphene-based material layers. In some embodiments the spacer substance is larger in one dimension than the size of the pores, or a portion thereof, in the graphene-based material layers. In some embodiments, the spacer substance interacts with the graphene-based material layer (e.g., via covalent bonding or van der Waals interactions). In some embodiments, the interactions between the spacer substance and the graphene-based material layer is stronger than an interaction between the spacer substance and permeants that pass through the membrane.

[0469] The effective diameter of the spacer substance can be tunable, i.e., it can be altered upon exposure to an environmental stimulus. In this regard, the effective diameter of spacer substances can be altered, upon exposure to an environmental stimulus, by about 0.3 nm to about 50 nm, such as from about 0.3 nm to about 0.5 nm, from about 0.5 nm to about 2 nm, from about 10 nm to about 20 nm, or from about 20 nm to about 50 nm. In some embodiments, the effective diameter of the spacer substance can be reduced by about 0.3 nm to about 50 nm, such as from about 0.3 nm to about 0.5 nm, from about 0.5 nm to about 2 nm, from about 10 nm to about 20 nm, or from about 20 nm to about 50 nm. In some embodiments, the effective diameter of the spacer substance can be increased by about 0.3 nm to about 50 nm, such as from about 0.3 nm to about 0.5 nm, from about 0.5 nm to about 2 nm, from about 10 nm to about 20 nm, or from about 20 nm to about 50 nm.

[0470] In some embodiments, the membrane comprises one or more channels that are impermeable (i.e., the channel diameter is about 0, and the channel is referred to as being in a closed position) before and/or after exposure to an environmental stimulus. In some

embodiments, the diameter of the channel is from about 0.3 nm to about 100 nm, such from about 0.3 nm to about 0.5 nm, from about 0.5 nm to about 2 nm, or from about 20 nm to about 50 nm. In some embodiments the channel diameter is about 0.5 nm, about 1 nm, about 2 nm, about 5 nm, about 10 nm, about 15, nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm. In some embodiments, the channel diameter is smaller than the diameter of pores, or a portion of pores, in the graphene-based material layers. The phrases "channel diameter" and "diameter of the channel" are defined by the diameter of substances that can traverse the membrane. For example, substances with a diameter of more than 10 nm are inhibited from traversing a membrane with a channel diameter of 10 nm or less; substances with a diameter of 50 nm or more are inhibited from traversing a membrane with a channel diameter of 50 nm or less. Channel diameter can be assessed, for example, using a flow test to determine the size cutoff for substances that can traverse the membrane. In some embodiments, particles smaller than the diameter of the channel are also inhibited from traversing the channel, for instance due to interactions with the graphene-based material layer or due to a solvation shell around the particle.

[0471] The channel diameter can be the larger than, about the same as, or smaller than the diameter of pores on the graphene-based material layer. In some embodiments, the channel diameter is smaller than the average diameter of pores in the graphene-based material layer, such as about 5% smaller, about 10% smaller, about 20% smaller, or about 50% smaller. In some embodiments, the channel diameter is about the same as the average diameter of pores in the graphene-based material layer. In some embodiments, the channel diameter is larger than the average diameter of pores in the graphene-based material layer, and the channel is

functionalized; in such embodiments, the channel diameter can be estimated based on the diameter of the spacer substances.

[0472] In some embodiments, the channel diameter is tunable, i.e., it can be altered upon exposure to an environmental stimulus, by from about 0.3 nm to about 50 nm, such as from about 0.3 nm to about 0.5 nm, from about 0.5 nm to about 2 nm, or from about 20 nm to about 50 nm. In some embodiments, the channel diameter can be reduced by about 0.3 nm to about 50 nm, such as from about 0.3 nm to about 0.5 nm, from about 0.5 nm to about 2 nm, or from about 20 nm to about 50 nm. In some embodiments, the channel diameter can be increased by about 0.3 nm to about 50 nm, such as from about 0.3 nm to about 0.5 nm, from about 0.5 nm to about 2 nm, or from about 20 nm to about 50 nm. By altering the properties of the spacer substances with an environmental stimulus, permeability and/or selectivity of the membrane (e.g., as measured by a flow test) can also be altered. Thus, membranes of some of the embodiments are responsive to one or more environmental stimuli. For instance, channels located between membrane layers can be increased or decreased in diameter as a result of changes in the size of the spacer substances. In exemplary embodiments, a membrane that allows passage of water but excludes salt ions (e.g. Na+ and C1-) can be tuned to allow passage of both water and salt ions. In other exemplary embodiments, the membrane can be tuned to allow passage of biological compounds such as insulin, proteins and/or other biological material (e.g., RNA, DNA, and/or nucleic acids), but to exclude passage of other larger biological compounds such as antibodies. In some embodiments, the membrane can be tuned to be permeable to oxygen and nutrients, but to exclude passage of cells (such as immune cells), viruses, bacteria, antibodies, and/or complements of the immune system. In some embodiments, the membrane can be tuned from one that allows passage of antibodies to one that inhibits passage of antibodies.

[0473] Tunable membranes have broad application, including in water filtration, immune- isolation (i.e., protecting substances from an immune reaction), timed drug release (e.g., sustained or delayed release), hemodialysis, and hemofiltration. Some embodiments described herein comprise a method of water filtration, water desalination, water purification, immune- isolation, timed drug release, hemodialysis, or hemofiltration, where the method comprises exposing a membrane to an environmental stimulus, and wherein the membrane comprises a first layer with a porous graphene-based material and a second layer with a porous graphene-based material.

[0474] Some embodiments include methods of filtering water comprising passing water through a membrane. Some embodiments include desalinating or purifying water comprising passing water through a membrane. The water can be passed through the membrane by any known means, such as by diffusion or gravity filtration, or with applied pressure (e.g. applied with a pump or via osmotic pressure).

[0475] Some embodiments include methods of selectively separating or isolating substances in a biological environment, wherein the membrane separates or isolates biological substances based on characteristics of the substance, such as size. Such methods can be useful in the context of disease treatment, such as in the treatment of diabetes. In some embodiments, biological substances below a certain size threshold can migrate across the membrane. In some embodiments, even biological substances below the size threshold are excluded from migrating across the membrane due to functionalization of membrane pores and/or channels.

[0476] In some embodiments, the pores, or at least a portion thereof, are functionalized. In some embodiments, the channels, or at least a portion thereof, are functionalized, for instance by attaching or embedding a functional group. In some embodiments, the functionalization moieties are trapped between two graphene-based material layers, but are not restricted to a single position in the channel (i.e., they are mobile within the channel, but are inhibited from traversing the two-dimensional material layers, e.g., based the size of the pores in the graphene-based material layers). In some embodiments, functionalization comprises surface charges (e.g., sulfonates) attached to the pores and/or channels. Without being bound by theory, it is believed that surface charges can impact which molecules and/or ions can traverse the membrane. In some embodiments, functionalization comprises specific binding sites attached to the pores and/or channels. In some embodiments, functionalization comprises proteins or peptides attached to the pores and/or the channel. In some embodiments, functionalization comprises antibodies and/or antigens (e.g., IgG-binding antigens) attached to the pores and/or channels. In some embodiments, functionalization comprises adsorptive substances attached to the pores and/or channels. In some embodiments, functionalization involves catalytic and/or regenerative substances or groups. In some embodiments, functionalization comprises a negatively or partially negatively charged group (e.g., oxygen) attached to the pores and/or channels. In some embodiments, functionalization comprises a positively or partially positively charged group attached to the pores and/or channels.

[0477] In some embodiments, functionalizing the pores and/or channels functions to: restrict contaminants from traversing the membrane; act as a disposable filter, capture, or diagnostic tool; increase biocompatibility (e.g., when polyethylene glycol is used for functionalization); increase filtration efficiency; position the spacer substances in the channels (e.g., spacers can be positioned near the pores via affinity -based functionalization in the pores; additional spacers can be positioned in interlaminar areas); increase selectivity at or near the pores or in asymmetric membranes; and/or protect spacer substances (e.g., from the external environment or from a particular vulnerability such as degradation).

[0478] Substrate Layer

[0479] In some embodiments, a substrate layer is disposed on one or both surfaces of the membrane. Without being bound by theory, it is believed that the substrate layer can improve biocompatibility of membranes, for instance by reducing biofouling, preventing protein adsorption-related problems, and/or enhancing vascularization. In some embodiments, the substrate layer can increase vascularization and/or tissue ingrowth near the membrane, thus prompting the formation of blood vessels and/or tissue ingrowth in close proximity to the membrane.

[0480] In some embodiments, the substrate layer has a thickness of about 1 mm or less, about 1 μπι or less, or about 100 nm or less. In some embodiments, a thickness of the substrate layer can range from about 100 nm to about 100 ?m, or about 1 μιη to about 50 μm, or about 10 μπι to about 20 μm, or about 15 μιη to about 25 μιη. In some embodiments, the substrate layer has a thickness about 10 μιη or greater, or about 15 μιη or greater. In some embodiments, the substrate layer has a thickness of less than 1 μιη. In some embodiments, the substrate layer has a thickness of about 10 nm to about 100 nm, or about 20 nm to about 50 nm.

[0481] In some embodiments, the enclosure can be supported by one or more support structures. In some embodiments, the support structure can itself have a porous structure wherein the pores are larger than those of the two-dimensional material. In some embodiments, the support structure is formed as a frame at a perimeter of a two-dimensional material. In some embodiments, the support structure is positioned, at least in part, interior to a perimeter of a two- dimensional material. In some embodiments, the substrate layer can convey a desired degree of structural support (e.g., to prevent tearing and/or buckling) to the two-dimensional material layer.

[0482] In some embodiments, two or more substrate layers are positioned on the same side of the membrane (e.g., two or more substrate layers can be positioned on the outside of an enclosure comprising the membrane). In some embodiments, the substrate is disposed directly on (or affixed directly to) a graphene-based material layer. In some embodiments, the substrate is disposed on or affixed to the graphene-based material layer with high conformance (e.g., by disposing a slightly wet substrate on the graphene-based material layer). In some embodiments, the substrate is disclosed with low conformance. In some embodiments, the substrate is disposed indirectly on (or affixed indirectly to) the graphene-based material; for instance, an intermediate layer can be positioned between the substrate layer and the graphene-based material layer. In some embodiments, the substrate layer is disposed or directly or indirectly on (or affixed directly or indirectly to) another substrate layer. In some embodiments, the graphene-based material layer is suspended on a substrate layer. In some embodiments, the substrate layer is affixed to the graphene-based material layer.

[0483] In some embodiments, the substrate layer can increase vascularization near the membrane, thus prompting the formation of blood vessels and/or tissue ingrowth in close proximity to the membrane. In some embodiments, the increased vascularization contributes to decreasing the effective distance between the blood stream and substances being eluted through the membrane. In some embodiments, the increased vascularization contributes to viability of substances, such as cells, enclosed within an enclosure comprising the membrane.

[0484] The substrate layer can be porous and/or nonporous. In some embodiments, the substrate layer contains porous and nonporous sections. In some embodiments the substrate layer comprises a porous or permeable fibrous layer. Porous substrates include, for example, one or more of ceramics and thin film polymers. Exemplary ceramics include nanoporous silica (silicon dioxide), silicon, SiN, and combinations thereof. In some embodiments, the substrate layer comprises track-etched polymers, expanded polymers, patterned polymers, woven polymers, and/or non-woven polymers. In some embodiments, the substrate layer comprises a plurality of polymer filaments. In some embodiments, the polymer filaments can comprise a thermopolymer, thermoplastic polymer, or melt polymer, e.g., that can be molded or set in an optional annealing step. In some embodiments, the polymer filaments are hydrophobic. In some embodiments, the polymer filaments are hydrophilic. In some embodiments, the substrate layer comprises a polymer selected from the group consisting of polysulfones, polyurethane, polymethylmethacrylate (PMMA), polyglycolid acid (PGA), polylactic acid (PLA), polyethylene glycol (PEG), polylactic-co-glycolic acid (PLGA), polyamides (such as nylon-6,6, supramid and nylamid), polyimides, polypropylene, polyethersulfones (PES), polyvinylidine fluoride (PVDF), cellulose acetate, polyethylene, polypropylene, polycarbonate, polytetrafluoroethylene (PTFE) (such as Teflon), polyvinylchloride (PVC), polyether ether ketone (PEEK), mixtures and block co-polymers of any of these, and combinations and/or mixtures thereof. In some embodiments, the polymers are biocompatible, bioinert and/or medical grade materials.

[0485] In some embodiments, the substrate layer comprises a biodegradable polymer. In some embodiments, a substrate layer forms a shell around an enclosure comprising the membrane (e.g., it completely engulfs the enclosure). In some embodiments, the substrate layer shell can be dissolved or degraded, e.g., in vitro. In some embodiments, the shell can be loaded with additives, including additives that protect substances inside the enclosure from air or prevent the need for a stabilizing agent.

[0486] Suitable techniques for depositing or forming a porous or permeable polymer on the graphene-based material layer include casting or depositing a polymer solution onto the graphene-based material layer or intermediate layer using a method such as spin-coating, spray coating, curtain coating, doctor-blading, immersion coating, electrospinning, or other similar techniques. Electrospinning techniques are described, e.g., in US 2009/0020921 and/or U.S. Application No. 14/609,325, both of which are hereby incorporated by reference in their entirety.

[0487] In some embodiments, the process for forming a substrate layer includes an electrospinning process in which a plurality of polymer filaments are laid down to form a porous mat, e.g., on the graphene-based material layer. In some embodiments, the mat has pores or voids located between deposited filaments of the fibrous layer. Figure 64 shows an illustrative SEM micrograph of a graphene or graphene-based film deposited upon a plurality of electrospun PVDF fibers. In some embodiments, the electrospinning process comprises a melt

electrospinning process or a solution electrospinning process, such as a wet electrospinning process or a dry electrospinning process. (See, e.g., Sinha-Ray et al. J. Membrane Sci. 485, 1 July 2015, 132-150.) In some embodiments, the polymer can be present in a spin dope at a concentration of 2 wt.% to 15 wt.%, or 5 wt.% to 10 wt.%, or about 7 wt.%. Suitable solvents for the spin dope include any solvent that dissolves the polymer to be deposited and which rapidly evaporates, such as m-cresol, formic acid, dimethyl sulfoxide (DMSO), ethanol, acetone, dimethylacetamide (DMAC), dimethylformamide (DMF), water, and combinations thereof. In some embodiments, the spin dope solvent is biocompatible and/or bioinert. In some

embodiments, the amount of solvent used can influence the morphology of the substrate layer. In dry electrospinning processes, the spun fibers of the fibrous layer can remain as essentially discrete entities once deposited. In some embodiments, wet electrospinning processes deposit the spun fibers such that they are at least partially fused together when deposited. In some embodiments, the size and morphology of the deposited fiber mat (e.g., degree of porosity, effective pore size, thickness of fibrous layer, gradient porosity) can be tailored based on the electrospinning process used.

[0488] The porosity of the fibrous layer can include effective void space values (e.g.

measured via imagery) up to about 95% (i.e., the layer is 95% open), about 90%, about 80%, or about 60%), with a broad range of void space sizes. In some embodiments, a single spinneret can be moved to lay down a mat of the fibrous layer. In some embodiments, multiple spinnerets can be used for this purpose. In some embodiments, the spun fibers in an electrospun fibrous layer can have a fiber diameter ranging from about 1 nm to about 100 μm, or about 10 nm to about 1 μm, or about 10 nm to about 500 nm, or about 100 nm to about 200 nm, or about 50 nm to about 120 nm, or about 1 μm to about 5 μm, or about 1 μπι to about 6 μm, or about 5 μm to about 10 μπι. In some embodiments, the fiber diameter is directly correlated with a depth (Z-axis) of a pore abutting the graphene-based material layer (disposed in the X-Y plane), and large diameter fibers can lead to large unsupported spans of material.

[0489] In some embodiments, the substrate layer can have pores (e.g., void spaces) with an effective pore size of from about 1 nm to about 100 μm, or about 10 nm to about 1 μm, or about 10 nm to about 500 nm, or about 100 nm to about 200 nm, or about 50 nm to about 120 nm, or about 1 μπι to about 5 μm, or about 1 μπι to about 6 μm, or about 5 μπι to about 10 μπι. Pore diameters in the substrate layer can be measured, for example, via porometry methods (e.g., capillary flow porometry) or extrapolated via imagery.

[0490] In some embodiments, the substrate layer can have an average pore size gradient throughout its thickness. "Pore size gradient" describes a layer with a plurality of pores, where the average diameter of the pores increases or decreases based on the proximity of the pore to the graphene-based material layer. For example, a fibrous layer can have an average pore size gradient that decreases nearer the surface of a graphene-based material. In some embodiments, an average pore size of the fibrous layer is smaller nearer the surface of the graphene-based material than at an opposite surface of the fibrous layer. For example, the fibrous layer can have effective pore diameters of less than about 200 nm close to the intermediate layer or the graphene-based material layer which can increase to greater than 100 μπι at the maximum distance away from the intermediate layer or graphene-based material layer.

[0491] In some embodiments, the fibrous layer can have a "porosity gradient" throughout its thickness, which can be measured for instance using imagery. "Porosity gradient" describes a change, along a dimension of the fibrous layer, in the porosity or total pore volume as a function of distance from the graphene-based material layer. For example, throughout the thickness of the porous fibrous layer, the porosity can change in a regular or irregular manner. A porosity gradient can decrease from one face of the fibrous layer to the other. For example, the lowest porosity in the fibrous layer can be located spatially closest to the graphene-based material layer, and the highest porosity can be located farther away (e.g., spatially closer to an external environment). A porosity gradient of this type can be achieved by electrospinning fibers onto a graphene-based material layer such that a fiber mat is denser near the surface of the graphene- based material layer and less dense further from the surface of the graphene-based material layer. In some embodiments, a substrate layer can have a relatively low porosity close to the graphene- based material layer, a higher porosity at a mid-point of the fibrous layer thickness (which can, for example, contain a supporting mesh for reinforcement or other particles), and return to a relatively low porosity at an external surface distal to the graphene-based material layer.

[0492] In some embodiments, the substrate layer can have a "permeability gradient" throughout its thickness. "Permeability gradient," as used herein, describes a change, along a dimension of the fibrous layer, in the "permeability" or rate of flow of a liquid or gas through a porous material. For example, throughout the thickness of the fibrous layer, the permeability can change in a regular or irregular manner. A permeability gradient can decrease from one face of the fibrous layer to the other. For example, the lowest permeability in the fibrous layer can be located spatially closest to the graphene-based material layer, and the highest permeability can be located farther away. Those of skill in the art will understand that permeability of a layer can increase or decrease without pore diameter or porosity changing, e.g., in response to chemical functionalization, applied pressure, voltage, or other factors.

[0493] In some embodiments, both the graphene-based material layer and the substrate layer include a plurality of pores therein. In some embodiments, both the graphene-based material layer and the substrate layer contain pores, and the pores in the graphene-based material layer are smaller, on average, than the pores in the substrate layer. In some embodiments, the median pore size in the graphene-based material layer is smaller than the median pore size in the substrate layer. For example, in some embodiments, the substrate layer can contain pores with an average and/or median diameter of about 1 μιη or larger and the graphene-based material layer can contain pores with an average and/or median diameter of about 10 nm or smaller. Accordingly, in some embodiments, the average and/or median diameter of pores in the graphene-based material layer is at least about 10-fold smaller than the average and/or median diameter of pores in the substrate layer. In some embodiments, the average and/or median diameter of pores in the graphene-based material layer is at least about 100-fold smaller than are the average and/or media diameter of pores in the substrate layer.

[0494] In some embodiments, the substrate layer can provide a scaffold for tissue growth, cell growth and/or vascularization. In some embodiments, the substrate layer or wall comprises additives, such as pharmaceuticals, cells, growth factors (e.g., VEGF), signaling molecules, cytokines, clotting factors, blood thinners, immunosuppressants, antimicrobial agents, hormones, antibodies, minerals, nutrients or combinations thereof. In some embodiments, additives such as pharmaceuticals, cells, growth factors, clotting factors, blood thinners, immunosuppressants, antimicrobial agents, hormones, antibodies, antigens (e.g., IgG-binding antigens) or an antibody- binding fragment thereof, minerals, nutrients or combinations thereof are positioned on the inside of the disclosure. In some embodiments, the substrate layer or membrane comprises materials toxic to bacteria or cells (without being bound by theory, it is believed that incorporating toxic materials into the wall will prevent passage of potentially dangerous or detrimental cells across the membrane). [0495] In some embodiments, additives beneficially promote cell or tissue viability or growth, reduce or prevent infection, improve vascularization to or near the membrane, improve biocompatibility, reduce biofouling, and/or reduce the risk of adverse reactions. In some embodiments, additives can modulate properties, such as hydrophobicity or hydrophilicity, of the substrate layer. In some embodiments, additives can be used to modulate elution of a substance from a compartment in the enclosure. For instance, additives can confer shell-like properties to a substrate layer, such that degradation or removal of the additives allows substances to traverse the membrane.

[0496] In some embodiments, an intermediate layer promotes adhesion between the graphene-based material layer and the substrate layer. Thus, in some embodiments, the enclosure comprises an intermediate layer disposed between the graphene-based material layer and the substrate layer. In some embodiments, the enclosure comprises an intermediate layer positioned between two substrate layers on the same side of the graphene-based material layer.

[0497] In some embodiments, the intermediate layer comprises carbon nanotubes, lacey carbon, nanoparticles, lithographically patterned low-dimensional materials, silicon and silicon nitride micromachined material, a fine mesh, such as a transmission electron microscopy grid, or combinations of these. In some embodiments, the intermediate layer can be a thin, smooth, porous polymer layer, such as a track etched polymer. In some embodiments, the intermediate layer has a thickness of from 3 nm to 10 μm, 10 nm to 10 μm, 50 nm to 10 μm, 100 nm to 10 μm, 500 nm to 10 μm, 1 μm to 10 μm, or 2 μπι to 6 μπι.

[0498] Graphene-Based Materials

[0499] As discussed above, membranes of some of the embodiments comprise graphene- based materials.

[0500] Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or a few layered sheets (e.g., about 20 or less) of fused six- membered rings forming an extended sp2-hybridized carbon planar lattice. Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. In some embodiments, graphene-based materials also include materials which have been formed by stacking single or multilayer graphene sheets. In some embodiments, multilayer graphene includes 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In some embodiments, layers of multilayered graphene are stacked, but are less ordered in the z direction (perpendicular to the basal plane) than a thin graphite crystal.

[0501] In some embodiments, a sheet of graphene-based material may be a sheet of single or multilayer graphene or a sheet comprising a plurality of interconnected single or multilayer graphene domains, which may be observed in any known manner such as using for example small angle electron diffraction, transmission electron microscopy, etc.. In some embodiments, the multilayer graphene domains have 2 to 5 layers or 2 to 10 layers. As used herein, a domain refers to a region of a material where atoms are substantially uniformly ordered into a crystal lattice. A domain is uniform within its boundaries, but may be different from a neighboring region. For example, a single crystalline material has a single domain of ordered atoms. In some embodiments, at least some of the graphene domains are nanocrystals, having domain size from 1 to 100 nm or 10-100 nm. In some embodiments, at least some of the graphene domains have a domain size greater than from 100 nm to 1 cm, or from 100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm. In some embodiments, a domain of multilayer graphene may overlap a neighboring domain. Grain boundaries formed by crystallographic defects at edges of each domain may differentiate between neighboring crystal lattices. In some embodiments, a first crystal lattice may be rotated relative to a second crystal lattice, by rotation about an axis perpendicular to the plane of a sheet, such that the two lattices differ in crystal lattice orientation.

[0502] In some embodiments, the sheet of graphene-based material is a sheet of single or multilayer graphene or a combination thereof. In some other embodiments, the sheet of graphene-based material is a sheet comprising a plurality of interconnected single or multilayer graphene domains. In some embodiments, the interconnected domains are covalently bonded together to form the sheet. When the domains in a sheet differ in crystal lattice orientation, the sheet is polycrystalline. [0503] In some embodiments, the thickness of the sheet of graphene-based material is from 0.3 to 10 nm, 0.34 to 10 nm, from 0.34 to 5 nm, or from 0.34 to 3 nm. In some embodiments, the thickness includes both single layer graphene and the non-graphenic carbon.

[0504] In some embodiments, a sheet of graphene-based material comprises intrinsic or native defects. Intrinsic or native defects may result from preparation of the graphene-based material in contrast to perforations which are selectively introduced into a sheet of graphene- based material or a sheet of graphene. Such intrinsic or native defects may include, but are not limited to, lattice anomalies, pores, tears, cracks or wrinkles. Lattice anomalies can include, but are not limited to, carbon rings with other than 6 members (e.g. 5, 7 or 9 membered rings), vacancies, interstitial defects (including incorporation of non-carbon atoms in the lattice), and grain boundaries. Perforations are distinct from openings in the graphene lattice due to intrinsic or native defects or grain boundaries, but testing and characterization of the final membrane such as mean pore size and the like encompasses all openings regardless of origin since they are all present.

[0505] In some embodiments, graphene is the dominant material in a graphene-based material. For example, a graphene-based material may comprise at least 20% graphene, 30% graphene, or at least 40% graphene, or at least 50% graphene, or at least 60% graphene, or at least 70%) graphene, or at least 80% graphene, or at least 90% graphene, or at least 95% graphene. In some embodiments, a graphene-based material comprises a range of graphene selected from 30% to 95%, or from 40% to 80% from 50% to 70%, from 60% to 95% or from 75%) to 100%). The amount of graphene in the graphene-based material is quantified as an atomic percentage utilizing known methods including scanning transmission electron microscope examination, or alternatively if STEM or TEM is ineffective another similar measurement technique.

[0506] In some embodiments, a sheet of graphene-based material further comprises non- graphenic carbon-based material located on at least one surface of the sheet of graphene-based material. In some embodiments, the sheet is exemplified by two base surfaces (e.g. top and bottom faces of the sheet, opposing faces) and side faces (e.g. the side faces of the sheet). In some further embodiments, the "bottom" face of the sheet is that face which contacted the substrate during growth of the sheet and the "free" face of the sheet opposite the "bottom" face. In some embodiments, non-graphenic carbon-based material may be located on one or both base surfaces of the sheet (e.g. the substrate side of the sheet and/or the free surface of the sheet). In some further embodiments, the sheet of graphene-based material includes a small amount of one or more other materials on the surface, such as, but not limited to, one or more dust particles or similar contaminants.

[0507] In some embodiments, the amount of non-graphenic carbon-based material is less than the amount of graphene. In some further embodiments, the amount of non-graphenic carbon material is three to five times the amount of graphene; this is measured in terms of mass. In some additional embodiments, the non-graphenic carbon material is characterized by a percentage by mass of said graphene-based material selected from the range of 0% to 80%. In some embodiments, the surface coverage of the sheet of non-graphenic carbon-based material is greater than zero and less than 80%, from 5% to 80%, from 10% to 80%, from 5% to 50% or from 10%) to 50%. This surface coverage may be measured with transmission electron microscopy, which gives a projection. In some embodiments, the amount of graphene in the graphene-based material is from 60%> to 95% or from 75% to 100%. The amount of graphene in the graphene-based material is quantified as an atomic percentage utilizing known methods preferentially using transmission electron microscope examination, or alternatively if STEM is ineffective using an atomic force microscope.

[0508] In some embodiments, the non-graphenic carbon-based material does not possess long range order and is classified as amorphous. In some embodiments, the non-graphenic carbon-based material further comprises elements other than carbon and/or hydrocarbons. In some embodiments, non-carbon elements which may be incorporated in the non-graphenic carbon include hydrogen, oxygen, silicon, copper, and iron. In some further embodiments, the non-graphenic carbon-based material comprises hydrocarbons. In some embodiments, carbon is the dominant material in non-graphenic carbon-based material. For example, a non-graphenic carbon-based material in some embodiments comprises at least 30% carbon, or at least 40% carbon, or at least 50% carbon, or at least 60% carbon, or at least 70% carbon, or at least 80% carbon, or at least 90% carbon, or at least 95% carbon. In some embodiments, a non-graphenic carbon-based material comprises a range of carbon selected from 30% to 95%, or from 40% to 80%), or from 50%> to 70%. The amount of carbon in the non-graphenic carbon-based material is quantified as an atomic percentage utilizing known methods preferentially using transmission electron microscope examination, or alternatively if STEM is ineffective using atomic force microscope.

[0509] Perforation techniques suitable for use in perforating the graphene-based materials may include described herein ion-based perforation methods and UV-oxygen based methods.

[0510] Ion-based perforation methods include methods in which the graphene-based material is irradiated with a directional source of ions. In some further embodiments, the ion source is collimated. In some embodiments, the ion source is a broad beam or flood source. A broad field or flood ion source can provide an ion flux which is significantly reduced compared to a focused ion beam. The ion source inducing perforation of the graphene or other two- dimensional material is considered to provide a broad ion field, also commonly referred to as an ion flood source. In some embodiments, the ion flood source does not include focusing lenses. In some embodiments, the ion source is operated at less than atmospheric pressure, such as at 10- ³ to 10 ^-5 torr or 10 ^-4 to 10 ^-6 torr. In some embodiments, the environment also contains background amounts (e.g. on the order of 10 ^-5 torr) of oxygen (02), nitrogen (N2) or carbon dioxide (CO ₂ ). In some embodiments, the ion beam may be perpendicular to the surface of the layer(s) of the material (incidence angle of 0 degrees) or the incidence angle may be from 0 to 45 degrees, 0 to 20 degrees, 0 to 15 degrees or 0 to 10 degrees. In some further embodiments, exposure to ions does not include exposure to plasma.

[0511] In some embodiments, UV-oxygen based perforation methods include methods in which the graphene-based material is simultaneously exposed to ultraviolet (UV) light and an oxygen containing gas Ozone may be generated by exposure of an oxygen containing gas such as oxygen or air to the UV light. Ozone may also be supplied by an ozone generator device. In some embodiments, the UV-oxygen based perforation method further includes exposure of the graphene-based material to atomic oxygen. Suitable wavelengths of UV light include, but are not limited to wavelengths below 300 nm or from 150 nm to 300 nm. In some embodiments, the intensity from 10 to 100 mW/cm ² at 6mm distance or 100 to 1000 mW/cm ² at 6mm distance. For example, suitable light is emitted by mercury discharge lamps (e.g. about 185 nm and 254 nm). In some embodiments, UV/oxygen cleaning is performed at room temperature or at a temperature greater than room temperature. In some further embodiments, UV/oxygen cleaning is performed at atmospheric pressure (e.g. 1 atm) or under vacuum.

[0512] Perforations are sized as described herein to provide desired selective permeability of a species (atom, molecule, protein, virus, cell, etc.) for a given application. Selective

permeability relates to the propensity of a porous material or a perforated two-dimensional material to allow passage (or transport) of one or more species more readily or faster than other species. Selective permeability allows separation of species which exhibit different passage or transport rates. In two-dimensional materials selective permeability correlates to the dimension or size (e.g., diameter) of apertures and the relative effective size of the species. Selective permeability of the perforations in two-dimensional materials such as graphene-based materials can also depend on functionalization of perforations (if any) and the specific species. Separation or passage of two or more species in a mixture includes a change in the ratio(s) (weight or molar ratio) of the two or more species in the mixture during and after passage of the mixture through a perforated two-dimensional material.

[0513] In some embodiments, the characteristic size of the perforation is from 0.3 to 10 nm, from 1 to 10 nm, from 5 to 10 nm, from 5 to 20 nm, from 10 nm to 50 nm, from 50 nm to 100 nm, from 50 nm to 150 nm, from 100 nm to 200 nm, or from 100 nm to 500 nm. In some embodiments, the average pore size is within the specified range. In some embodiments, 70% to 99%, 80% to 99%, 85% to 99% or 90 to 99% of the perforations in a sheet or layer fall within a specified range, but other pores fall outside the specified range.

[0514] Nanomaterials in which pores are intentionally created may be referred to as perforated graphene, perforated graphene-based materials or perforated two-dimensional materials, and the like. Perforated graphene-based materials include materials in which non- carbon atoms have been incorporated at the edges of the pores. Pore features and other material features may be characterized in a variety of manners including in relation to size, area, domains, periodicity, coefficient of variation, etc. For instance, the size of a pore may be assessed through quantitative image analysis utilizing images preferentially obtained through transmission electron microscopy, and if TEM is ineffective, through atomic force microscopy, and if AFM is ineffective, through scanning electron microscopy, as for example presented in Figs. 60 and 61. The boundary of the presence and absence of material identifies the contour of a pore. The size of a pore may be determined by shape fitting of an expected species against the imaged pore contour where the size measurement is characterized by smallest dimension unless otherwise specified. For example, in some instances, the shape may be round or oval. The round shape exhibits a constant and smallest dimension equal to its diameter. The width of an oval is its smallest dimension. The diameter and width measurements of the shape fitting in these instances provide the size measurement, unless specified otherwise.

[0515] Each pore size of a test sample may be measured to determine a distribution of pore sizes within the test sample. Other parameters may also be measured such as area, domain, periodicity, coefficient of variation, etc. Multiple test samples may be taken of a larger membrane to determine that the consistency of the results properly characterizes the whole membrane. In such instance, the results may be confirmed by testing the performance of the membrane with test species. For example, if measurements indicate that certain sizes of species should be restrained from transport across the membrane, a performance test provides verification with test species. Alternatively, the performance test may be utilized as an indicator that the pore measurements will determine a concordant pore size, area, domains, periodicity, coefficient of variation, etc.

[0516] The size distribution of holes may be narrow, e.g., limited to 0.1-0.5 coefficient of variation. In some embodiments, the characteristic dimension of the holes is selected for the application.

[0517] In some embodiments involving circular shape fitting the equivalent diameter of each pore is calculated from the equation A= π d2/4. Otherwise, the area is a function of the shape fitting. When the pore area is plotted as a function of equivalent pore diameter, a pore size distribution may be obtained. The coefficient of variation of the pore size may be calculated herein as the ratio of the standard deviation of the pore size to the mean of the pore size as measured across the test samples. The average area of perforations is an averaged measured area of pores as measured across the test samples. [0518] In some embodiments, the ratio of the area of the perforations to the ratio of the area of the sheet may be used to characterize the sheet as a density of perforations. The area of a test sample may be taken as the planar area spanned by the test sample. Additional sheet surface area may be excluded due to wrinkles other non-planar features. Characterization may be based on the ratio of the area of the perforations to the test sample area as density of perforations excluding features such as surface debris. Characterization may be based on the ratio of the area of the perforations to the suspended area of the sheet. As with other testing, multiple test samples may be taken to confirm consistency across tests and verification may be obtained by performance testing. The density of perforations may be, for example, 2 per nm ² (2/ nm ² ) to 1 per μm ² (1/ μm ² ).

[0519] In some embodiments, the perforated area comprises 0.1% or greater, 1% or greater or 5% or greater of the sheet area, less than 10% of the sheet area, less than 15% of the sheet area, from 0.1% to 15% of the sheet area, from 1% to 15% of the sheet area, from 5% to 15% of the sheet area or from 1% to 10% of the sheet area. In some further embodiments, the

perforations are located over greater than 10% or greater than 15% of said area of said sheet of graphene-based material. A macroscale sheet is macroscopic and observable by the naked eye. In some embodiments, at least one lateral dimension of the sheet is greater than 3 cm, greater than 1 cm, greater than 1 mm or greater than 5 mm. In some further embodiments, the sheet is larger than a graphene flake which would be obtained by exfoliation of graphite in known processes used to make graphene flakes. For example, the sheet has a lateral dimension greater than about 1 micrometer. In an additional embodiment, the lateral dimension of the sheet is less than 10 cm. In some further embodiments, the sheet has a lateral dimension (e.g., perpendicular to the thickness of the sheet) from 10 nm to 10 cm or greater than 1 mm and less than 10 cm.

[0520] Chemical vapor deposition growth of graphene-based material typically involves use of a carbon containing precursor material, such as methane and a growth substrate. In some embodiments, the growth substrate is a metal growth substrate. In some embodiments, the metal growth substrate is a substantially continuous layer of metal rather than a grid or mesh. Metal growth substrates compatible with growth of graphene and graphene-based materials include transition metals and their alloys. In some embodiments, the metal growth substrate is copper based or nickel based. In some embodiments, the metal growth substrate is copper or nickel. In some embodiments, the graphene-based material is removed from the growth substrate by dissolution of the growth substrate.

[0521] In some embodiments, the sheet of graphene-based material is formed by chemical vapor deposition (CVD) followed by at least one additional conditioning or treatment step. In some embodiments, the conditioning step is selected from thermal treatment, UV-oxygen treatment, ion beam treatment, and combinations thereof. In some embodiments, thermal treatment may include heating to a temperature from 200° C to 800° C at a pressure of 10 ^-7 torr to atmospheric pressure for a time of 2 hours to 8 hours. In some embodiments, UV-oxygen treatment may involve exposure to light from 150 nm to 300 nm and an intensity from 10 to 100 mW/cm ² at 6mm distance for a time from 60 to 1200 seconds. In some embodiments, UV- oxygen treatment may be performed at room temperature or at a temperature greater than room temperature. In some further embodiments, UV-oxygen treatment may be performed at atmospheric pressure (e.g. 1 atm) or under vacuum. In some embodiments, ion beam treatment may involve exposure of the graphene-based material to ions having an ion energy from 50 eV to 1000 eV (for pretreatment) and the fluence is from 3 x 10 ¹⁰ ions/cm ² to 8 x 10 ¹¹ ions/cm ² or 3 x 10 ¹⁰ ions/cm ² to 8 x 10 ¹³ ions/cm2 (for pretreatment). In some further embodiments, the source of ions may be collimated, such as a broad beam or flood source. In some embodiments, the ions may be noble gas ions such as Xe ⁺ . In some embodiments, one or more conditioning steps are performed while the graphene-based material is attached to a substrate, such as a growth substrate.

[0522] In some embodiments, the conditioning treatment affects the mobility and/or volatility of the non-graphitic carbon-based material. In some embodiments, the surface mobility of the non-graphenic carbon-based material is such that when irradiated with perforation parameters such as described herein, the non-graphenic carbon-based material, may have a surface mobility such that the perforation process results ultimately in perforation. Without wishing to be bound by any particular belief, hole formation is believed to be related to beam induced carbon removal from the graphene sheet and thermal replenishment of carbon in the hole region by non grapheme carbon. The replenishment process may be dependent upon energy entering the system during perforation and the resulting surface mobility of the non-graphenic carbon based material. To form holes, the rate of graphene removal may be higher than the non- graphenic carbon hole filling rate. These competing rates depend on the non-graphenic carbon flux (e.g., mobility [viscosity and temperature] and quantity) and the graphene removal rate (e.g., particle mass, energy, flux).

[0523] In some embodiments, the volatility of the non-graphenic carbon-based material may be less than that which is obtained by heating the sheet of graphene-based material to 500°C for 4 hours in vacuum or at atmospheric pressure with an inert gas.

[0524] In various embodiments, CVD graphene or graphene-based material can be liberated from its growth substrate (e.g., Cu) and transferred to a supporting grid, mesh or other supporting structure. In some embodiments, the supporting structure may be configured so that at least some portions of the sheet of graphene-based material are suspended from the supporting structure. For example, at least some portions of the sheet of graphene-based material may not be in contact with the supporting structure.

[0525] In some embodiments, the sheet of graphene-based material following chemical vapor deposition comprises a single layer of graphene having at least two surfaces and non- graphenic carbon based material may be provided on said surfaces of the single layer graphene. In some embodiments, the non-graphenic carbon based material may be located on one of the two surfaces or on both. In some further embodiments, additional graphenic carbon may also present on the surface(s) of the single layer graphene.

[0526] Methods of Making Tunable Membranes

[0527] Tunable membranes can be made by a variety of methods. For instance, a perforated graphene layer can be combined with spacer substances in solution, such that the spacer substances self-assemble to the perforated graphene layer. Then, the solution can be reduced to induce bonding between the spacer substance and the graphene layer. After that, an additional graphene layer can be added to the solution, which can bond to the graphene layer-spacer substance complex. Attachment of the additional graphene layer can be via van der Waals forces or induced covalent bonding (e.g., as a result of an applied energy such as ion radiation). [0528] In some embodiments, spacer substances are covalently bonded to at least one graphene-based material layer. Without being bound by theory, it is believed that covalent bonding between a spacer substance and a graphene-based material layer can be induced via ion- beam induced bonding, electron-beam induced bonding, heating, chemical reactions (e.g., via reactants on - i.e., attached to - the spacer substance and the graphene-based material layer), and combinations thereof.

[0529] In some embodiments, functional moieties are attached to the spacer molecules to facilitate self-assembly on or bonding to the graphene layers. In some embodiments, the functional moieties are removed in the process of making the membrane.

[0530] In some embodiments, the spacer substances are trapped between two graphene-based material layers. In some embodiments the spacer substances are trapped between two graphene- based material layers, but are not restricted to a single position in the channel (i.e., they are mobile within the channel).

BIOLOGICALLY-RELEVANT SELECTIVE ENCLOSURES FOR PROMOTING GROWTH

AND VASCULARIZATION

[0531] Some embodiments relate to the selective passage of substances through an enclosure that encourages nearby vascularization (i.e., angiogenesis) and/or tissue ingrowth in a biological environment. Some embodiments include methods and devices for selectively separating or isolating substances in a biological environment, e.g., using a composite structure that comprises a two-dimensional material. Some embodiments include an enclosure comprising a

compartment and a wall separating the compartment from an environment external to the compartment. In some embodiments, the wall comprises a two-dimensional material layer and a substrate layer. Two-dimensional materials, such as graphene-based materials, are discussed below.

[0532] Enclosures can be in any shape. Thus, the cross-section of an enclosure can be, for example, circular, ovular, rectangular, square, or irregular-shaped. The size of the enclosure also is not limited, and can be small enough to circulate in the bloodstream (e.g., on the order of nanometers or larger) or large enough for implantation (e.g. on the order of inches or smaller). In some embodiments, the enclosure is from 100 nm to 6 inches long in its longest dimension, such as from about 100 nm to about 500 nm, about 500 nm to about 1 μm, about 1 μιη to about 500 μm, about 500 μιη to about 1 mm, about 1 mm to about 500 mm, about 500 mm to about 1 cm, about 1 cm to about 10 cm, or about 1 cm to about 6 inches long. In some embodiments, the enclosure is longer than 6 inches in its longest dimension, such as about 10 inches or about 15 inches long.

[0533] The thickness of the wall depends, in part, on the two-dimensional material layer and/or substrate layers used in the wall. Thus, in some embodiments a wall, or a portion thereof, comprising both a two-dimensional material layer and a substrate layer is at least 5 nm thick, such as from about 5 nm to about 1 μιη thick, from about 5 nm to about 250 nm thick, from about 5 to about 50 nm thick, from about 5 to about 20 nm thick, or from about 20 to about 50 nm thick. In some embodiments, the thickness of the wall is about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm thick, about 25 nm thick, about 30 nm thick, about 35 nm thick, about 40 nm thick, about 45 nm thick, about 50 nm thick, about 100 nm thick, about 150 nm thick, about 200 nm thick, about 250 nm thick, about 300 nm thick, about 400 nm thick, about 500 nm thick, about 600 nm thick, about 700 nm thick, about 800, nm thick, about 900 nm thick, or about 1 μιη thick. In some embodiments, the thickness of the wall is up to about 1 μιη thick or up to about 1 mm thick. In some embodiments, the thickness of the wall is tailored to allow bidirectional passage of oxygen and nutrients into and out of the enclosure. In some embodiments, the thickness of the wall is tailored to allow entry of oxygen and nutrients into the enclosure at sufficient concentrations to maintain viability of cells within the enclosure.

[0534] In some embodiments, the substrate layer has a thickness of about 1 mm or less, about 1 μπι or less, or about 100 nm or less. In some embodiments, a thickness of the substrate layer can range from about 100 nm to about 100 μm, or about 1 μπι to about 50 μm, or about 10 μπι to about 20 μm, or about 15 μπι to about 25 μπι. In some embodiments, the substrate layer has a thickness about 10 μπι or greater, or about 15 μπι or greater. In some embodiments, the substrate layer has a thickness of less than 1 μπι. In some embodiments, the substrate layer has a thickness of about 10 nm to about 100 nm, or about 20 nm to about 50 nm. [0535] In some embodiments, the enclosure can be supported by one or more support structures. In some embodiments, the support structure can itself have a porous structure wherein the pores are larger than those of the two-dimensional material. In some embodiments, the support structure is formed as a frame at a perimeter of a two-dimensional material. In some embodiments, the support structure is positioned in part interior to a perimeter of a two- dimensional material. In some embodiments, the substrate layer can convey a desired degree of structural support (e.g., to prevent tearing and/or buckling) to the two-dimensional material layer.

[0536] In some embodiments, a substrate layer is positioned on one or both sides or surfaces of the two-dimensional material. Thus, in some embodiments the substrate is positioned on the outside of the enclosure and in some cases is exposed to the external environment (see, e.g., Figure 76, showing some embodiments with a device with a substrate positioned on the outside of an enclosure). In some embodiments, the substrate is positioned on the inside of the enclosure, and can be separated from an environment external to the enclosure (even though the substrate can be separated from the environment external to the enclosure, it can still be exposed to components from the external environment due to pores in the two-dimensional material layer and/or substrate layer). In some embodiments, the substrate is positioned on both the outside and the inside of the enclosure. In that case, the substrate on the outside of the enclosure can contain materials that are the same as or different from the substrate on the inside of the enclosure. In some embodiments, two or more substrate layers are positioned on the same side of the two- dimensional material layer (e.g., two or more substrate layers can be positioned on the outside of the enclosure). In some embodiments, the substrate is disposed directly on the two-dimensional material. In some embodiments, the substrate is disposed on the two-dimensional material with high conformance (e.g., by disposing a slightly wet substrate on the two-dimensional material). In some embodiments, the substrate is disclosed with low conformance. In some embodiments, the substrate is disposed indirectly on the two-dimensional material; for instance, an intermediate layer can be positioned between the substrate layer and the two-dimensional material layer. In some embodiments, the substrate layer is disposed directly or indirectly on another substrate layer. In some embodiments, the two-dimensional material is suspended on a substrate layer. In some embodiments, the substrate layer is affixed to the two-dimensional material layer (see, e.g., Figure 73 A, showing exemplary substrate layers, two-dimensional material layers, and substrate affixed to a two-dimensional material; see also Figure 73B, showing some embodiments that were determined to be not cytotoxic based on cytotoxicity testing and implantation testing).

[0537] In some embodiments, the substrate layer can increase vascularization near the enclosure, thus prompting the formation of blood vessels and/or tissue ingrowth in close proximity to the enclosure. In some embodiments, the increased vascularization contributes to decreasing the effective distance between the blood stream and substances being eluted from the enclosure. In some embodiments, the increased vascularization contributes to viability of substances, such as cells, enclosed within the enclosure.

[0538] The substrate layer can be porous and/or nonporous. In some embodiments, the substrate layer contains porous and nonporous sections. In some embodiments the substrate layer comprises a porous or permeable fibrous layer. Porous substrates include, for example, one or more of ceramics and thin film polymers. Exemplary ceramics include nanoporous silica (silicon dioxide), silicon, SiN, and combinations thereof. In some embodiments, the substrate layer comprises track-etched polymers, expanded polymers, patterned polymers, woven polymers, and/or non-woven polymers. In some embodiments, the substrate layer comprises a plurality of polymer filaments. In some embodiments, the polymer filaments can comprise a thermopolymer, thermoplastic polymer, or melt polymer, e.g., that can be molded or set in an optional annealing step. In some embodiments, the polymer filaments are hydrophobic. In some embodiments, the polymer filaments are hydrophilic. In some embodiments, the substrate layer comprises a polymer selected from the group consisting of polysulfones, polyurethane, polymethylmethacrylate (PMMA), polyglycolid acid (PGA), polylactic acid (PLA), polyethylene glycol (PEG), polylactic-co-glycolic acid (PLGA), polyamides (such as nylon-6,6, supramid and nylamid), polyimides, polypropylene, polyethersulfones (PES), polyvinylidine fluoride (PVDF), cellulose acetate, polyethylene, polypropylene, polycarbonate, polytetrafluoroethylene (PTFE) (such as Teflon), polyvinylchloride (PVC), polyether ether ketone (PEEK), mixtures and block co-polymers of any of these, and combinations and/or mixtures thereof. In some embodiments, the polymers are biocompatible, bioinert and/or medical grade materials. By way of example, Figure 78 shows some embodiments of graphene disposed on various different substrates. Figures 79 shows micrographs some embodiments of custom track-etched polyimide. Figures 80 and 81 show micrographs of some embodiments of graphene disposed on track-etched polyimide. Figure 82 and 83 show micrographs of some embodiments of graphene disposed on electrospun nylon 6,6.

[0539] In some embodiments, the substrate layer comprises a biodegradable polymer. In some embodiments, a substrate layer forms a shell around the enclosure (e.g., completely engulfs the enclosure). In some embodiments, the substrate layer shell, or a portion thereof, can be dissolved or degraded, e.g., in vitro. In some embodiments, the shell can be loaded with additives, including additives that protect substances inside the enclosure from air or prevent the need for a stabilizing agent.

[0540] Suitable techniques for depositing or forming a porous or permeable polymer on the two-dimensional material include casting or depositing a polymer solution onto the two- dimensional material or intermediate layer using a method such as spin-coating, spray coating, curtain coating, doctor-blading, immersion coating, electrospinning, or other similar techniques. Electrospinning techniques are described, e.g., in US 2009/0020921 and/or U.S. Application No. 14/609,325, both of which are hereby incorporated by reference in their entirety.

[0541] In some embodiments, the process for forming a substrate layer includes an electrospinning process in which a plurality of polymer filaments are laid down to form a porous mat, e.g., on the two-dimensional material layer. In some embodiments, the mat has pores or voids located between deposited filaments of the fibrous layer. Figure 64 shows an illustrative SEM micrograph of a graphene or graphene-based film deposited upon a plurality of electrospun PVDF fibers. In some embodiments, the electrospinning process comprises a melt

electrospinning process or a solution electrospinning process, such as a wet electrospinning process or a dry electrospinning process. (See, e.g., Sinha-Ray et al. J. Membrane Sci. 485, 1 July 2015, 132-150.) In some embodiments, the polymer can be present in a spin dope at a concentration of 2 wt.% to 15 wt.%, or 5 wt.% to 10 wt.%, or about 7 wt.%. Suitable solvents for the spin dope include any solvent that dissolves the polymer to be deposited and which rapidly evaporates, such as m-cresol, formic acid, dimethyl sulfoxide (DMSO), ethanol, acetone, dimethylacetamide (DMAC), dimethylformamide (DMF), water, and combinations thereof. In some embodiments, the spin dope solvent is biocompatible and/or bioinert. In some embodiments, the amount of solvent used can influence the morphology of the substrate layer. In dry electrospinning processes, the spun fibers of the fibrous layer can remain as essentially discrete entities once deposited. In some embodiments, wet electrospinning processes deposit the spun fibers such that they are at least partially fused together when deposited. In some embodiments, the size and morphology of the deposited fiber mat (e.g., degree of porosity, effective pore size, thickness of fibrous layer, gradient porosity) can be tailored based on the electrospinning process used.

[0542] The porosity of the fibrous layer can include effective void space values (e.g.

measured via imagery) up to about 95% (i.e., the layer is 95% open), about 90%, about 80%, or about 60%, with a broad range of void space sizes. In some embodiments, a single spinneret can be moved to lay down a mat of the fibrous layer. In some embodiments, multiple spinnerets can be used for this purpose. In some embodiments, the spun fibers in an electrospun fibrous layer can have a fiber diameter ranging from about 1 nm to about 100 μm, or about 10 nm to about 1 μm, or about 10 nm to about 500 nm, or about 100 nm to about 200 nm, or about 50 nm to about 120 nm, or about 1 μm to about 5 μm, or about 1 μm to about 6 μm, or about 5 μm to about 10 μm. In some embodiments, the fiber diameter is directly correlated with a depth (Z-axis) of a pore abutting the two-dimensional material (disposed in the X-Y plane), and large diameter fibers can lead to large unsupported spans of material.

[0543] In some embodiments, the substrate layer can have pores (e.g., void spaces) with an effective pore size of from about 1 nm to about 100 μm, or about 10 nm to about 1 μm, or about 10 nm to about 500 nm, or about 100 nm to about 200 nm, or about 50 nm to about 120 nm, or about 1 μm to about 5 μm, or about 1 μm to about 6 μm, or about 5 μm to about 10 μm. Pore diameters in the substrate layer can be measured, for example, via porometry methods (e.g., capillary flow porometry) or extrapolated via imagery.

[0544] In some embodiments, the substrate layer can have an average pore size gradient throughout its thickness. "Pore size gradient" describes a layer with a plurality of pores, where the average diameter of the pores increases or decreases based on the proximity of the pore to the two-dimensional material. For example, a fibrous layer can have an average pore size gradient that decreases nearer the surface of a graphene-based material. In some embodiments, an average pore size of the fibrous layer is smaller nearer the surface of the graphene-based material than at an opposite surface of the fibrous layer. For example, the fibrous layer can have effective pore diameters of less than about 200 nm close to the intermediate layer or the two-dimensional material layer which can increase to greater than 100 μπι at the maximum distance away from the intermediate layer or two-dimensional material layer.

[0545] In some embodiments, the fibrous layer can have a "porosity gradient" throughout its thickness, which can be measured for instance using imagery. "Porosity gradient" describes a change, along a dimension of the fibrous layer, in the porosity or total pore volume as a function of distance from the two-dimensional material layer. For example, throughout the thickness of the porous fibrous layer, the porosity can change in a regular or irregular manner. A porosity gradient can decrease from one face of the fibrous layer to the other. For example, the lowest porosity in the fibrous layer can be located spatially closest to the two-dimensional material, and the highest porosity can be located farther away (e.g., spatially closer to an external

environment). A porosity gradient of this type can be achieved by electrospinning fibers onto a two-dimensional material such that a fiber mat is denser near the surface of the two-dimensional material and less dense further from the surface of the two-dimensional material. In some embodiments, a substrate layer can have a relatively low porosity close to the two-dimensional material, a higher porosity at a mid-point of the fibrous layer thickness (which can, for example, contain a supporting mesh for reinforcement or other particles), and return to a relatively low porosity at an external surface distal to the two-dimensional material.

[0546] In some embodiments, the substrate layer can have a "permeability gradient" throughout its thickness. "Permeability gradient," as used herein, describes a change, along a dimension of the fibrous layer, in the "permeability" or rate of flow of a liquid or gas through a porous material. For example, throughout the thickness of the fibrous layer, the permeability can change in a regular or irregular manner. A permeability gradient can decrease from one face of the fibrous layer to the other. For example, the lowest permeability in the fibrous layer can be located spatially closest to the graphene or graphene-based film or other two-dimensional material, and the highest permeability can be located farther away. Those of skill in the art will understand that permeability of a layer can increase or decrease without pore diameter or porosity changing, e.g., in response to chemical functionalization, applied pressure, voltage, or other factors.

[0547] In some embodiments, both the two-dimensional material layer and the substrate layer include a plurality of pores therein. In some embodiments, both the two-dimensional material and the substrate layer contain pores, and the pores in the two-dimensional material layer are smaller, on average, than the pores in the substrate layer. In some embodiments, the median pore size in the two dimensional material layer is smaller than the median pore size in the substrate layer. For example, in some embodiments, the substrate layer can contain pores with an average and/or median diameter of about 1 μιη or larger and the two-dimensional material layer can contain pores with an average and/or median diameter of about 10 nm or smaller. Accordingly, in some embodiments, the average and/or median diameter of pores in the two-dimensional material layer is at least about 10-fold smaller than the average and/or median diameter of pores in the substrate layer. In some embodiments, the average and/or median diameter of pores in the two-dimensional material layer is at least about 100-fold smaller than are the average and/or media diameter of pores in the substrate layer.

[0548] Figure 65 illustrates a portion of an enclosure in a biological environment in contact with biological tissue in which an enclosure comprises one or more substrate layers, such as fibrous layers positioned on the outside of the perforated two-dimensional material. Figure 65 also shows capillary vascularization into the substrate layer. Without being bound by theory, it is believed that the biocompatibility of graphene can further promote this application, particularly by functionalizing the graphene to improve compatibility with a particular biological environment (e.g., via available edge bonds, bulk surface functionalization, pi-bonding, and the like). Functionalization can provide enclosures having added complexity for use in treating local and systemic disease. Figure 65 also shows a wall of an enclosure with a perforated two- dimensional material having hole sizes in a range that will retain cells. The external biological environment abutting the enclosure (the full enclosure is not shown) in Figure 65 is separated from cells, proteins, etc., positioned inside the enclosure. As illustrated, in some embodiments implantation of such an enclosure contemplates vascularization into a substrate layer positioned on the outside of the enclosure.

[0549] In some embodiments, the substrate layer can provide a scaffold for tissue growth, cell growth and/or vascularization. In some embodiments, the substrate layer or wall comprises additives, such as pharmaceuticals, cells, growth factors (e.g., VEGF), signaling molecules, cytokines, clotting factors, blood thinners, immunosuppressants, antimicrobial agents, hormones, antibodies, minerals, nutrients or combinations thereof. In some embodiments, additives such as pharmaceuticals, cells, growth factors, clotting factors, blood thinners, immunosuppressants, antimicrobial agents, hormones, antibodies, antigens (e.g., IgG-binding antigens) or an antibody- binding fragment thereof, minerals, nutrients or combinations thereof are positioned on the inside of the disclosure. In some embodiments, the substrate layer or wall comprises materials toxic to bacteria or cells (without being bound by theory, it is believed that incorporating toxic materials into the wall will prevent passage of potentially dangerous or detrimental cells across the wall).

[0550] In some embodiments, additives beneficially promote cell or tissue viability or growth, reduce or prevent infection, improve vascularization to or near the enclosure, improve biocompatibility, reduce biofouling, and/or reduce the risk of adverse reactions. In some embodiments, additives can modulate properties, such as hydrophobicity or hydrophilicity, of the substrate layer. In some embodiments, additives can be used to modulate elution of a substance from a compartment in the enclosure. For instance, additives can confer shell-like properties to a substrate layer, such that degradation or removal of the additives allows substances in the compartment to escape the enclosure (and, by extension, substances from the external environment to enter to enclosure).

[0551] Some embodiments comprise a composited structure that include a two-dimensional material layer and a substrate layer. In some embodiments, a composite structure includes a support material (see, e.g., Figure 66D) disposed on an opposite side of the two-dimensional material from the substrate layer. In some embodiments, a composite structure comprises an intermediate layer between the two-dimensional material and the substrate layer, e.g., as shown in Figures 66A, 66C, and 66E. Figure 66 shows schematic illustrations of composite structures comprising two-dimensional materials (e.g., graphene), an optional intermediate layer (e.g., track etched polymer membrane), and a fibrous layer having a tighter fiber spacing nearer the two- dimensional material and an increasing effective pore size further from the two-dimensional material. Figure 66 A shows SEM micrographs of the fibrous material with (bottom two expanded micrographs) and without (top two expanded micrographs) the two-dimensional material on the surface of the fibrous material. Figure 66A also shows SEM micrographs of high fiber density (bottom), medium fiber density (middle) and low fiber density (top) substrates.

[0552] In some embodiments, the intermediate layer promotes adhesion between the two- dimensional material layer and the substrate layer. Thus, in some embodiments, the enclosure comprises an intermediate layer disposed between the two-dimensional material layer and the substrate layer. In some embodiments, the enclosure comprises an intermediate layer positioned between two substrate layers on the same side of the two-dimensional material layer.

[0553] In some embodiments, the intermediate layer comprises carbon nanotubes, lacey carbon, nanoparticles, lithographically patterned low-dimensional materials, silicon and silicon nitride micromachined material, a fine mesh, such as a transmission electron microscopy grid, or combinations of these. Figure 69 shows an illustrative schematic of a process for manufacturing a two-dimensional material on a fibrous layer with mesh reinforcement. In some embodiments, the intermediate layer can be a thin, smooth, porous polymer layer, such as a track etched polymer. In some embodiments, the intermediate layer has a thickness of from 3 nm to 10 μm, 10 nm to 10 μm, 50 nm to 10 μm, 100 nm to 10 μm, 500 nm to 10 μm, 1 μπι to 10 μm, or 2 μπι to 6 μm. In some embodiments, the composite structure has a thickness of from 1 μπι to 100 μm, 2 μm, to 75 μm, 3 μm, to 50 μm, 4 μm, to 40 μm, 5 μm to 30 μm, 6 μm, to 25 μm, or 6 μπι to 20 μm, or 6 μπι to 16 μm.

[0554] In some embodiments, an enclosure or composite structure includes a fibrous layer affixed to multiple sheets of graphene or graphene-based material. In some embodiments, the sheets of graphene or graphene-based materials are stacked upon one another with one of the sheets affixed directly or indirectly to the fibrous layer. Figure 70 shows an illustrative SEM micrograph of two layers of graphene or graphene-based material on a fibrous layer. In some embodiments, one or more sheets of graphene or graphene-based material can be affixed to a first surface of a fibrous layer and one or more sheets of graphene or graphene-based material can be affixed to a second surface of the fibrous layer. In some embodiments, the graphene- based material is applied to a fully-formed substrate layer, such as a fully-formed electrospun substrate layer. Some embodiments comprise putting multiple layers of the graphene-based material onto the substrate layer (e.g., the fully-formed substrate layer). Without being bound by theory, it is believed that adding multiple layers of graphene-based material onto the substrate layer allows complete coverage of the substrate layer with the graphene-based material.

[0555] In some embodiments, the enclosure comprises a single compartment that does not contain sub-compartments. In some embodiments, the single compartment is in fluid

communication with an external environment separated from the compartment, e.g., by a wall. In some embodiments, the enclosure has a plurality of sub-compartments. In some

embodiments, the sub-compartments are in fluid communication with an environment outside the sub-compartment. In some embodiments, each sub-compartment comprises a wall that allows passage of one or more substances into and/or out of the sub-compartment. In some

embodiments, the wall or a portion thereof comprises a perforated two-dimensional material, a polymer, a hydrogel, or some other means of allowing passage of one or more substance into and/or out of the sub-compartment. In some embodiments, an enclosure is subdivided into two sub-compartments separated from each other at least in part by perforated two-dimensional material, such that the two sub-compartments are in direct fluid communication with each other through holes in the two-dimensional material. In some embodiments, the enclosure is subdivided into two sub-compartments each comprising two-dimensional material which sub- compartments are in direct fluid communication with each other through holes in the two- dimensional material and only one of the sub-compartments is in direct fluid communication with an environment external to the enclosure. In some embodiments, the enclosure is subdivided into two sub-compartments each comprising two-dimensional material which sub- compartments are in direct fluid communication with each other through holes in the two- dimensional material and both of the sub-compartments are also in direct fluid communication with an environment external to the enclosure.

[0556] In some embodiments, the enclosure has an inner sub-compartment and an outer sub- compartment each comprising a perforated two-dimensional material, wherein the inner sub- compartment is entirely enclosed within the outer sub-compartment, the inner and outer compartments are in direct fluid communication with each other through holes in the two- dimensional material and the inner sub-compartment is not in direct fluid communication with an environment external to the enclosure.

[0557] In some embodiments, where an enclosure has a plurality of sub-compartments each comprising a two-dimensional material, the sub-compartments are nested one within the other, each of which sub-compartments is in direct fluid communication through holes in two- dimensional material with the sub-compartment(s) to which it is adjacent, the outermost sub- compartment in direct fluid communication with an environment external to the enclosure, the remaining plurality of sub-compartments not in direct fluid communication with an environment external to the enclosure.

[0558] In some embodiments, a sub-compartment can have any shape or size. In some embodiments, 2 or 3 sub-compartments are present. Several examples of enclosure sub- compartments are illustrated in Figures 61 A-61E. In Figure 61 A, a nested configuration is illustrated, such that sub-compartment B completely contains a smaller sub-compartment A, and substances in the centermost enclosure A can pass into the main enclosure B, and potentially react with or within the main compartment during ingress and egress therefrom. In this embodiment, one or more substances in A can pass into B and one or more substances in A can be retained in A and not enter B. Two sub-compartments in which one or more substances can pass directly between the sub-compartments are said to be in direct fluid communication.

Passage between sub-compartments and between the enclosure and the external environment can be via holes of a perforated two-dimensional material. In some embodiments, the barrier (e.g., a membrane) between compartment A and B can be permeable to all substances in A or to certain substances in A (i.e., selective permeability). In some embodiments, the barrier between B and the external environment can be permeable to all substances in B or selectively permeable to certain substances in B. In Figure 61 A, sub-compartment A is in direct fluid communication with sub-compartment B which in turn is in direct fluid communication with the external environment. Compartment A in this nested configuration is in indirect fluid communication with the external environment via intermediate passage into sub-compartment B. The two- dimensional materials employed in different sub-compartments of an enclosure can be the same or different materials and the perforations or hole sizes in the two-dimensional material of different sub-compartments can be the same or different.

[0559] In Figure 61B, the enclosure is bisected with an impermeable wall (e.g., formed of non-porous or non-permeable sealant) forming sub-compartments A and B, such that both sections have access to the egress location independently, but there is no direct or indirect passage of substances from A to B. (It will be appreciated, however, that substances exiting A or B can enter the other sub-compartment indirectly via the external environment.)

[0560] In Figure 61C, the main enclosure is again bisected into sub-compartments A and B, but with a perforated material forming the barrier between the sub-compartments. Both sub- compartments not only have access to the egress location independently, but also can interact with one another, i.e. the sub-compartments are in direct fluid communication. In some embodiments, the barrier between sub-compartments A and B is selectively permeable, for example allowing at least one substance in A to pass into B, but not allowing the substances originating in B to pass to A. The porosity of the barrier between sub-compartments (e.g., sub- compartments A and B) can be the same as or different than the porosity of the sub-compartment walls in direct fluid communication with an environment external to the enclosure.

[0561] Figure 6 ID illustrates an enclosure having three compartments. The enclosure is illustrated with sub-compartment A being in fluid communication with sub-compartment B, which in turn is in fluid communication with sub-compartment C, which in turn is in fluid communication with the external environment. Compartments A and B are not in fluid communication with the external environment, i.e. they are not in direct fluid communication with the external environment. Adjacent sub-compartments A and B and adjacent sub- compartments B and C are each separated by a perforated two-dimensional material and are thus in direct fluid communication with each other. Sub-compartment A is only in indirect fluid communication with compartment C and the external environment via sub-compartment B or B and C, respectively. Various other combinations of semi-permeable barrier or non-permeable barriers can be employed to separate sub-compartments. Various perforation size constraints can change depending on how the enclosure is ultimately configured. Regardless of the chosen configuration, in some embodiments the boundaries, or at least a portion thereof, of the enclosure can be constructed from a two-dimensional material such that the thickness of the two- dimensional material is less than the diameter of the substance to be passed selectively across the two-dimensional material.

[0562] Figure 6 IE illustrates an enclosure having a single compartment (A) and no sub- compartments. In the Figure, the compartment is in direct fluid communication with an environment external to the enclosure.

[0563] In some embodiments, the presence of two or more sub-compartments containing the same substance(s) provides redundancy in function so that an enclosure can remain at least partially operable so long as at least one sub-compartment is not compromised.

[0564] The multiple physical embodiments for the enclosures and their uses can allow for various levels of interaction and scaled complexity of problems to be solved. For example, a single enclosure can provide drug elution for a given time period, or there can be multiple sizes of perforations to restrict or allow movement of distinct substances, each having a particular size.

[0565] Added complexity of the embodiments described herein with multiple sub- compartments can allow for interaction between compounds to catalyze or activate a secondary response (i.e., a "sense-response" paradigm). For example, if there are two sections of an enclosure that function independently, exemplary compound A can undergo a constant diffusion into the body, or either after a given time or in the presence of a stimulus from the body. In some embodiments, exemplary compound A can activate exemplary compound B, or inactivate functionalization that otherwise blocks exemplary compound B from escaping. In some embodiments, binding interactions to produce the foregoing effects can be reversible or irreversible. In some embodiments, exemplary compound A can interact with chemical cascades produced outside the enclosure, and a metabolite subsequent to the interaction can release exemplary compound B (e.g., by inactivating functionalization). Further examples utilizing effects that take place in a similar manner include using source cells (e.g., non-host; allogenic; xenogenic; autogenic; cadeaveric; stem cells, such as fully or partially differentiated stem cells) contained in an enclosure, within which secretions from the cell can produce a "sense-response" paradigm. In some embodiments, the presence of graphene in the "sense-response" paradigm does not hinder diffusion, thus allowing a fast time response as compared to enclosures that to not contain graphene.

[0566] In some embodiments, growth factors or hormones can be loaded in the enclosure to encourage vascularization (see Figure 65). In some embodiments, survival of cells within the enclosure can be improved as a result of bi-directional passage of nutrients and waste into and out of the enclosure.

[0567] In some embodiments, the relative thinness of graphene can enable bi-directional passage across a wall (or portion thereof) of the enclosure in close proximity to blood vessels, particularly capillary blood vessels, and other cells. In some embodiments, using a graphene- based enclosure can provide differentiation over other solutions accomplishing the same effect because the graphene does not appreciably limit permeability; instead, the diffusion of molecules through the graphene apertures can limit the movement of a substance across the wall.

[0568] In some embodiments, the perforations allow for zeroth order diffusion through the wall. In some embodiments, osmotic pumps can be used to transport substances across the wall. In some embodiments, natural delta pressures in the body influence passage of substances across the wall. In some embodiments, convective pressure influences passage of substances across the wall. In some embodiments, it is possible to achieve high throughput flux through the wall of the enclosure.

[0569] Figures 62A and 62B provide a schematic illustration of enclosures with a single compartment for immunoisolation (it will be appreciated that the enclosure can having a plurality of sub-compartments, for example, two or three sub-compartments). The enclosure (6030) of Figure 62 A is shown as a cross-section formed by an inner sheet or layer (6031) comprising perforated two-dimensional material, such as a graphene-based material, and an outer sheet or layer (6032) of a substrate material (though in some embodiments, the inner layer comprises the substrate material, and the outer layer comprises the perforate two-dimensional material). The substrate material can be porous, selectively permeable or non-porous, and/or and non- permeable. However at least a portion of the support material is porous or selectively permeable. The enclosures in Figure 62 contain selected living cells (6033). Figure 62B provides an alternative cross-section of the enclosure of Figure 62 A, showing the space or cavity formed between a first composite layer (6032/6031) and a second composite layer (6032/6031) (in the figure, the cavity is depicted to contain roughly circular symbols, which can be, e.g., cells or any other substance) where a sealant 6034 is illustrated as sealing the edges of the composite layers. It will be appreciated that seals at the edges of the composite layers can be formed employing physical methods, such as clamping, crimping, or with adhesives. Methods and materials for forming the seals at the edges are not particularly limiting. In some embodiments, the sealing material provides a non-porous and non-permeable seal or closure. In some embodiments, a portion of the enclosure is formed from a sealant, such as a silicone, epoxy, polyurethane or similar material. In some embodiments, the sealant is biocompatible. For instance, in some embodiments the seal does not span the entire length or width of the device. In some

embodiments, the seal forms a complete loop around the cavity. In some embodiments, the seal is formed as a frame at a perimeter of a two-dimensional material. In some embodiments, the seal is positioned, at least in in part, interior to a perimeter of a two-dimensional material.

[0570] Some embodiments include methods for using graphene-based materials and/or other two-dimensional materials to transport, transfer, deliver, and/or allow passage of substances in or to a biological environment. Some embodiments comprise delivering substances to an environment external to the enclosure (e.g., a biological environment). In some embodiments, the substance positioned on the inside of the enclosure comprises one or more of atoms, molecules, viruses, bacteria, cells, particles and aggregates thereof. For example, the substance can include biological molecules, such as proteins (e.g., insulin), nucleic acids, DNA, and/or RNA; pharmaceuticals; drugs; medicaments; therapeutics, including biologies and small molecule drugs; and combinations thereof.

[0571] If cells are placed within the enclosure, at least a portion of the enclosure can be permeable to oxygen and nutrients sufficient for cell growth and maintenance, to waste produced by the cell (e.g., CO ₂ ), and/or to metabolites produced by the cell (e.g., insulin). In some embodiments, at least a portion of the enclosure is permeable to signaling molecules, such as glucose. In some embodiments, at least a portion of the enclosure is permeable to growth factors produced by cells, such as VEGF. [0572] In some embodiments, the enclosure is not permeable to cells (such as immune cells), viruses, bacteria, antibodies, and/or complements of the immune system. Thus, in some embodiments, cells from the external environment cannot enter the enclosure and cells in the enclosure are retained. In some embodiments, the enclosure is permeable to desirable products, such as growth factors or hormones produced by the cells (see, e.g., Figures 74 and 75, illustrating some embodiments related to immunoisolation). The cells within the enclosure can be immunoisolated (i.e., protected from an immune reaction). In some embodiments of enclosures containing cells, the cells are yeast cells, bacterial cells, stem cells, mammalian cells, human cells, porcine cells, or a combination thereof. In some embodiments useful with cells, an enclosure comprises a plurality of sub-compartments, with the cells being positioned within one or more sub-compartments. In some embodiments useful with cells, the enclosure comprises a single compartment. In some embodiments, hole sizes in perforated two-dimensional materials useful for immunoisolation range in size from 1-50 nm, 1-40 nm, 1-30 nm, 1-25 nm, 1-17 nm, 1- 15 nm, 1-12 nm, 1-10 nm, 3-50 nm, 3-30 nm, 3-20 nm, 3-10 nm, or 3-5 nm. In some

embodiments, the size of the holes is about 1 nm, about 3 nm about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 30 nm, or about 50 nm.

[0573] Figures 63A-63C illustrate an exemplary method for forming an enclosure and introducing selected substances, for example cells, therein. The method is illustrated with use of a sealant for forming the enclosure. As illustrated in Figure 63 A, a first composite layer or sheet can be formed by placing a sheet or layer of two-dimensional material, such as a sheet of graphene-based material or a sheet of graphene (6141), in contact with a substrate layer (6142). At least a portion of the substrate layer (6142) of the first composite can be porous or permeable. Pore size of the substrate layer can be larger than the holes or apertures in the two-dimensional material employed and can be tuned for the environment (e.g., body cavity). A layer of sealant (6144), e.g., silicone, is applied on the sheet or layer of perforated two-dimensional material outlining a compartment of the enclosure wherein the sealant will form a non-permeable seal around a perimeter of the enclosure. Formation of a single compartment is illustrated in Figures 63 A-63C, however, it will be appreciated that multiple independent compartments within an enclosure can be formed by an analogous process. A second composite layer formed in the same way as the first is then prepared and positioned with the sheet or layer of perforated two dimensional materials in contact with the sealant. Alternatively, a sealant can be applied to a portion of a composite layer and the layer can be folded over in contact with the sealant to form an enclosure. A seal is then formed between the two composite layers. Appropriate pressure can be applied to facilitate sealing without damaging the two-dimensional material or its support. It will be appreciated that an alternative enclosure can be formed by applying a sheet or layer of non-porous and non-permeable support material in contact with the sealant. In this case only a portion of the enclosure is porous and permeable. Other methods for sealing the enclosure include ultrasonic welding. Sealed composite layers are illustrated in Figure 63B where it is shown that the sealed layers can be trimmed to size around the sealant to form the enclosure. The enclosure formed is shown to have an external porous substrate layer 6142 with the sheet or layer of perforated two-dimensional material (6141) being positioned as an internal layer, with sealant 6144 around the perimeter of the enclosure. As illustrated in Figure 63C, cells or other substances that would be excluded from passage through the perforated two-dimensional sheet or layer can be introduced into the enclosure after it is formed by injection through the sealant layer. Any perforation formed by such injection can be sealed as needed.

[0574] In some embodiments, substances (e.g., cells) can be introduced into the enclosure prior to formation of the seal. In some embodiments, one or more ports can be provided for introducing substances into the enclosure. For example, a loading port can be provided within the sealed perimeter of the enclosure, and the loading port can be permanently or semipermanently sealed after introduction of one or more substances through the loading port. Those in the art will appreciate that sterilization methods appropriate for the application envisioned can be employed during or after the preparation of the enclosure.

[0575] In some embodiments, an enclosure comprises perforated two-dimensional material encapsulating a substance, such that the substance is released to an environment external to the enclosure by passage through the holes in the perforated two-dimensional material. In some embodiments, the enclosure encapsulates two or more different substances. In some

embodiments, not all of the different substances are released to an environment external to the enclosure. In some embodiments, all of the different substances are released into an environment external to the enclosure. In some embodiments, different substances are released into an environment external to the enclosure at different rates. In some embodiments, different substances are released into an environment external to the enclosure at the same rates.

[0576] In some embodiments of any enclosure herein at least a portion of the holes in the two-dimensional material of the enclosure are functionalized.

[0577] In some embodiments at least a portion of the two-dimensional material is conductive and a voltage can be applied to at least a portion of the conductive two-dimensional material. The voltage can be an AC or DC voltage. The voltage can be applied from a source external to the enclosure. In some embodiments, an enclosure device further comprises connectors and leads for application of a voltage from an external source to the two-dimensional material.

[0578] Additionally, the conductive properties of graphene-based or other two-dimensional materials can allow for electrification to take place from an external source. In exemplary embodiments, an AC or DC voltage can be applied to conductive two-dimensional materials (e.g., in a device such as an enclosure device). The conductivity properties of graphene can provide additional gating to charged molecules or substances. Electrification can occur permanently or only a portion of the time to affect gating. Directional gating of charged molecules can be directed not only through the pores (or restrict travel through pores), but also to the surface of the graphene to adsorb or bind and encourage growth, promote formation of a protective layer, or provide the basis or mechanism for other biochemical effects (e.g., on the body).

[0579] In some embodiments, at least once wall, or portion thereof, of the enclosure allows for electrostatic control of charged species, for instance in nanofluidic or microfluidic systems. In some embodiments, the wall allows for control of charged species by varying the applied voltage, for instance in nanofluidic or microfluidic systems. In some embodiments, the wall can be tuned to manipulate ion passage at low and/or high ion concentrations. In some

embodiments, the wall is an ion-selective membrane. In some embodiments, the wall comprises one or more voltage-gated ion channels, such as voltage-gated pores. In some embodiments, the wall mimics biological voltage-gated ion channels. In some embodiments, the wall is a solid- state membrane. In some embodiments, nanochannel or nanopore transistors can be used to manipulate ion passage.

[0580] In some embodiments, the wall can be tuned using low or high applied voltages. In some embodiments, the wall allows high ionic flux. In some embodiments, the wall allows low ion flux. In some embodiments, pores in the wall modulates current of ions at low gate voltages and/or display high selectivity. In some embodiments, ion flux across the wall can be turned on or off at low applied voltages, such as < 500 mV. In some embodiments, ion flux across the wall can be turned on or off at biologically relevant ion concentrations, such as up to 1 M. In some embodiments, the applied voltage can modulate on species selectivity, e.g., cation or anion selectivity.

[0581] In some embodiments, nanopores can be electrostatically controlled at low voltages and biologically relevant ion concentrations. In some embodiments, walls are used in separation and sensing technologies. In some embodiments, walls are used in water filtration, water desalination, water purification, osmosis, energy storage, microfluidic devices, nanofluidic devices, and/or therapeutic methods. In some embodiments, walls are used in immune-isolation (i.e., protecting substances from an immune reaction), timed drug release (e.g., sustained or delayed release), hemodialysis, and hemofiltration. Some embodiments relate to methods for separating ions or other substances; methods for sensing ions; methods for storing energy;

methods for filtering water; and/or methods of treating a disease or condition (e.g., diabetes). Some embodiments relate to methods of ultrafiltration, nanofiltration and/or microfiltration. Some embodiments comprise using gating to control release of substances. Some embodiments comprise using gating to allow for different substances to be released at different times. Some embodiments comprise allowing different substances to pass through the wall at different times, thus modulating when and how substances mix and interact with other substances in a specific order.

[0582] Some embodiments comprise a method comprising introducing an enclosure comprising perforated two-dimensional material into an environment, the enclosure containing at least one substance; and releasing at least a portion of the at least one substance through the holes of the two-dimensional material to the environment external to the enclosure. In some embodiments, the enclosure contains cells which are not released from the enclosure and the at least one substance, a portion of which is released, is a substance generated by the cells in the enclosure.

[0583] Some embodiments comprise a method comprising introducing an enclosure comprising perforated two-dimensional material to an environment, the enclosure containing at least one first substance; and allowing migration of other substances from the environment into the enclosure. In some embodiments, the first substance is cells, and other substances include nutrients and/or oxygen.

[0584] In some embodiments, a composite structure comprises perforated two-dimensional material and a first fibrous layer comprising a plurality of polymer filaments affixed to a surface of the two-dimensional material; wherein the composite structure is substantially planar. In some embodiments, the perforated two-dimensional material has a second fibrous layer affixed to a surface of the two-dimensional material opposite the first fibrous layer. In some

embodiments, the average pore size of the first fibrous layer is different from the average pore size of the second fibrous layer. In some embodiments, the first and/or second fibrous layer comprises an additive selected from the group consisting of pharmaceuticals, cells, growth factors, clotting factors, blood thinners, immunosuppressants, antimicrobial agents, hormones, antibodies, minerals, nutrients and combinations thereof. In some embodiments, the

substantially planar composite structure is flexible. In some embodiments, the substantially planar composite structure is rigid. In some embodiments, multiple composite structures are combined to form a pouch-like enclosure. Such planar composite structures can be useful, for example, as appliques for wound healing. The composite structures can also be used, for example, as a component of an adhesive bandage.

[0585] Both permanent and temporary binding of substances to the enclosure are possible. In some embodiments, enclosures represent a disruptive technology for state of the art vehicle and other devices, such that these vehicles and devices to be used in new ways. For example, cell line developments, therapeutic releasing agents, and sensing paradigms (e.g., MRSw's, MR-based magnetic relaxation switches, see; Koh et al. (2008) Ang. Chem. Int'l Ed. Engl., 47(22) 4119-4121) can be used. Moreover, some embodiments mitigate biofouling and bioreactivity, convey superior permeability and less delay in response, and provide mechanical stability.

[0586] In some embodiments, enclosures can be used in non-therapeutic applications, such as in dosing probiotics in dairy products.

[0587] In some embodiments, two-dimensional materials are atomically thin, with thickness ranging from single-layer sub-nanometer thickness to a few nanometers. Two-dimensional materials include metal chalogenides (e.g., transition metal dichalogenides), transition metal oxides, hexagonal boron nitride, graphene, silicene and germanene (see: Xu et al. (2013)

"Graphene-like Two-Dimensional Materials) Chemical Reviews 113 :3766-3798).

[0588] In some embodiments, the two-dimensional material comprises a graphene-based material.

[0589] Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or a few layered sheets (e.g., about 20 or less) of fused six- membered rings forming an extended sp ² -hybridized carbon planar lattice. Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. In some embodiments, graphene-based materials also include materials which have been formed by stacking single or multilayer graphene sheets. In some embodiments, multilayer graphene includes 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In some embodiments, layers of multilayered graphene are stacked, but are less ordered in the z direction (perpendicular to the basal plane) than a thin graphite crystal.

[0590] In some embodiments, a sheet of graphene-based material may be a sheet of single or multilayer graphene or a sheet comprising a plurality of interconnected single or multilayer graphene domains, which may be observed in any known manner such as using for example small angle electron diffraction, transmission electron microscopy, etc.. In some embodiments, the multilayer graphene domains have 2 to 5 layers or 2 to 10 layers. As used herein, a domain refers to a region of a material where atoms are substantially uniformly ordered into a crystal lattice. A domain is uniform within its boundaries, but may be different from a neighboring region. For example, a single crystalline material has a single domain of ordered atoms. In some embodiments, at least some of the graphene domains are nanocrystals, having domain size from 1 to 100 nm or 10-100 nm. In some embodiments, at least some of the graphene domains have a domain size greater than from 100 nm to 1 cm, or from 100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm. In some embodiments, a domain of multilayer graphene may overlap a neighboring domain. Grain boundaries formed by crystallographic defects at edges of each domain may differentiate between neighboring crystal lattices. In some embodiments, a first crystal lattice may be rotated relative to a second crystal lattice, by rotation about an axis perpendicular to the plane of a sheet, such that the two lattices differ in crystal lattice orientation.

[0591] In some embodiments, the sheet of graphene-based material is a sheet of single or multilayer graphene or a combination thereof. In some other embodiments, the sheet of graphene-based material is a sheet comprising a plurality of interconnected single or multilayer graphene domains. In some embodiments, the interconnected domains are covalently bonded together to form the sheet. When the domains in a sheet differ in crystal lattice orientation, the sheet is polycrystalline.

[0592] In some embodiments, the thickness of the sheet of graphene-based material is from 0.3 to 10 nm, 0.34 to 10 nm, from 0.34 to 5 nm, or from 0.34 to 3 nm. In some embodiments, the thickness includes both single layer graphene and the non-graphenic carbon.

[0593] In some embodiments, a sheet of graphene-based material comprises intrinsic or native defects. Intrinsic defects may result from preparation of the graphene-based material in contrast to perforations which are selectively introduced into a sheet of graphene-based material or a sheet of graphene. Such intrinsic or native defects may include, but are not limited to, lattice anomalies, pores, tears, cracks or wrinkles. Lattice anomalies can include, but are not limited to, carbon rings with other than 6 members (e.g. 5, 7 or 9 membered rings), vacancies, interstitial defects (including incorporation of non-carbon atoms in the lattice), and grain boundaries.

Perforations are distinct from openings in the graphene lattice due to intrinsic or native defects or grain boundaries, but testing and characterization of the final membrane such as mean pore size and the like encompasses all openings regardless of origin since they are all present. [0594] In some embodiments, graphene is the dominant material in a graphene-based material. For example, a graphene-based material may comprise at least 20% graphene, at least 30%) graphene, or at least 40% graphene, or at least 50% graphene, or at least 60%> graphene, or at least 70% graphene, or at least 80% graphene, or at least 90% graphene, or at least 95% graphene. In some embodiments, a graphene-based material comprises a range of graphene selected from 30% to 95%, or from 40% to 80% from 50% to 70%, from 60% to 95% or from 75%) to 100%). The amount of graphene in the graphene-based material is quantified as an atomic percentage utilizing known methods including scanning transmission electron microscope examination, or alternatively if STEM or TEM is ineffective another similar measurement technique.

[0595] In some embodiments, a sheet of graphene-based material further comprises non- graphenic carbon-based material located on at least one surface of the sheet of graphene-based material. In some embodiments, the sheet is exemplified by two base surfaces (e.g. top and bottom faces of the sheet, opposing faces) and side faces (e.g. the side faces of the sheet). In some further embodiments, the "bottom" face of the sheet is that face which contacted the substrate during growth of the sheet and the "free" face of the sheet opposite the "bottom" face. In some embodiments, non-graphenic carbon-based material may be located on one or both base surfaces of the sheet (e.g. the substrate side of the sheet and/or the free surface of the sheet). In some further embodiments, the sheet of graphene-based material includes a small amount of one or more other materials on the surface, such as, but not limited to, one or more dust particles or similar contaminants.

[0596] In some embodiments, the amount of non-graphenic carbon-based material is less than the amount of graphene. In some further embodiments, the amount of non-graphenic carbon material is three to five times the amount of graphene; this is measured in terms of mass. In some additional embodiments, the non-graphenic carbon material is characterized by a percentage by mass of said graphene-based material selected from the range of 0% to 80%. In some embodiments, the surface coverage of the sheet of non-graphenic carbon-based material is greater than zero and less than 80%, from 5% to 80%, from 10% to 80%, from 5% to 50% or from 10%) to 50%. This surface coverage may be measured with transmission electron microscopy, which gives a projection. In some embodiments, the amount of graphene in the graphene-based material is from 60% to 95% or from 75% to 100%. The amount of graphene in the graphene-based material is quantified as an atomic percentage utilizing known methods preferentially using transmission electron microscope examination, or alternatively if STEM is ineffective using an atomic force microscope.

[0597] In some embodiments, the non-graphenic carbon-based material does not possess long range order and is classified as amorphous. In some embodiments, the non-graphenic carbon-based material further comprises elements other than carbon and/or hydrocarbons. In some embodiments, non-carbon elements which may be incorporated in the non-graphenic carbon include hydrogen, oxygen, silicon, copper, and iron. In some further embodiments, the non-graphenic carbon-based material comprises hydrocarbons. In some embodiments, carbon is the dominant material in non-graphenic carbon-based material. For example, a non-graphenic carbon-based material in some embodiments comprises at least 30% carbon, or at least 40% carbon, or at least 50% carbon, or at least 60% carbon, or at least 70% carbon, or at least 80% carbon, or at least 90% carbon, or at least 95% carbon. In some embodiments, a non-graphenic carbon-based material comprises a range of carbon selected from 30% to 95%, or from 40% to 80%), or from 50% to 70%. The amount of carbon in the non-graphenic carbon-based material is quantified as an atomic percentage utilizing known methods preferentially using transmission electron microscope examination, or alternatively if STEM is ineffective using atomic force microscope.

[0598] Perforation techniques suitable for use in perforating the graphene-based materials may include described herein ion-based perforation methods and UV-oxygen based methods.

[0599] Ion-based perforation methods include methods in which the graphene-based material is irradiated with a directional source of ions. In some further embodiments, the ion source is collimated. In some embodiments, the ion source is a broad beam or flood source. A broad field or flood ion source can provide an ion flux which is significantly reduced compared to a focused ion beam. The ion source inducing perforation of the graphene or other two- dimensional material is considered to provide a broad ion field, also commonly referred to as an ion flood source. In some embodiments, the ion flood source does not include focusing lenses. In some embodiments, the ion source is operated at less than atmospheric pressure, such as at 10- ³ to 10 ^-5 torr or 10 ^-4 to 10 ^-6 torr. In some embodiments, the environment also contains background amounts (e.g. on the order of 10 ^-5 torr) of oxygen (O ₂ ), nitrogen (N ₂ ) or carbon dioxide (CO ₂ ). In some embodiments, the ion beam may be perpendicular to the surface of the layer(s) of the material (incidence angle of 0 degrees) or the incidence angle may be from 0 to 45 degrees, 0 to 20 degrees, 0 to 15 degrees or 0 to 10 degrees. In some further embodiments, exposure to ions does not include exposure to plasma.

[0600] In some embodiments, UV-oxygen based perforation methods include methods in which the graphene-based material is simultaneously exposed to ultraviolet (UV) light and an oxygen containing gas Ozone may be generated by exposure of an oxygen containing gas such as oxygen or air to the UV light. Ozone may also be supplied by an ozone generator device. In some embodiments, the UV-oxygen based perforation method further includes exposure of the graphene-based material to atomic oxygen. Suitable wavelengths of UV light include, but are not limited to wavelengths below 300 nm or from 150 nm to 300 nm. In some embodiments, the intensity from 10 to 100 mW/cm ² at 6mm distance or 100 to 1000 mW/cm ² at 6mm distance. For example, suitable light is emitted by mercury discharge lamps (e.g. about 185 nm and 254 nm). In some embodiments, UV/oxygen cleaning is performed at room temperature or at a temperature greater than room temperature. In some further embodiments, UV/oxygen cleaning is performed at atmospheric pressure (e.g. 1 atm) or under vacuum.

[0601] Perforations are sized as described herein to provide desired selective permeability of a species (atom, molecule, protein, virus, cell, etc.) for a given application. Selective

permeability relates to the propensity of a porous material or a perforated two-dimensional material to allow passage (or transport) of one or more species more readily or faster than other species. Selective permeability allows separation of species which exhibit different passage or transport rates. In two-dimensional materials selective permeability correlates to the dimension or size (e.g., diameter) of apertures and the relative effective size of the species. Selective permeability of the perforations in two-dimensional materials such as graphene-based materials can also depend on functionalization of perforations (if any) and the specific species. Separation or passage of two or more species in a mixture includes a change in the ratio(s) (weight or molar ratio) of the two or more species in the mixture during and after passage of the mixture through a perforated two-dimensional material.

[0602] In some embodiments, the characteristic size of the perforation is from 0.3 to 10 nm, from 1 to 10 nm, from 5 to 10 nm, from 5 to 20 nm, from 10 nm to 50 nm, from 50 nm to 100 nm, from 50 nm to 150 nm, from 100 nm to 200 nm, or from 100 nm to 500 nm. In some embodiments, the average pore size is within the specified range. In some embodiments, 70% to 99%, 80% to 99%, 85% to 99% or 90 to 99% of the perforations in a sheet or layer fall within a specified range, but other pores fall outside the specified range.

[0603] Nanomaterials in which pores are intentionally created may be referred to as perforated graphene, perforated graphene-based materials or perforated two-dimensional materials, and the like. Perforated graphene-based materials include materials in which non- carbon atoms have been incorporated at the edges of the pores. Pore features and other material features may be characterized in a variety of manners including in relation to size, area, domains, periodicity, coefficient of variation, etc. For instance, the size of a pore may be assessed through quantitative image analysis utilizing images preferentially obtained through transmission electron microscopy, and if TEM is ineffective, through atomic force microscopy, and if AFM is ineffective, through scanning electron microscopy, as for example presented in Figs. 60 and 61. The boundary of the presence and absence of material identifies the contour of a pore. The size of a pore may be determined by shape fitting of an expected species against the imaged pore contour where the size measurement is characterized by smallest dimension unless otherwise specified. For example, in some instances, the shape may be round or oval. The round shape exhibits a constant and smallest dimension equal to its diameter. The width of an oval is its smallest dimension. The diameter and width measurements of the shape fitting in these instances provide the size measurement, unless specified otherwise.

[0604] Each pore size of a test sample may be measured to determine a distribution of pore sizes within the test sample. Other parameters may also be measured such as area, domain, periodicity, coefficient of variation, etc. Multiple test samples may be taken of a larger membrane to determine that the consistency of the results properly characterizes the whole membrane. In such instance, the results may be confirmed by testing the performance of the membrane with test species. For example, if measurements indicate that certain sizes of species should be restrained from transport across the membrane, a performance test provides verification with test species. Alternatively, the performance test may be utilized as an indicator that the pore measurements will determine a concordant pore size, area, domains, periodicity, coefficient of variation, etc.

[0605] The size distribution of holes may be narrow, e.g., limited to 0.1-0.5 coefficient of variation. In some embodiments, the characteristic dimension of the holes is selected for the application.

[0606] In some embodiments involving circular shape fitting the equivalent diameter of each pore is calculated from the equation A= π d ² /4. Otherwise, the area is a function of the shape fitting. When the pore area is plotted as a function of equivalent pore diameter, a pore size distribution may be obtained. The coefficient of variation of the pore size may be calculated herein as the ratio of the standard deviation of the pore size to the mean of the pore size as measured across the test samples. The average area of perforations is an averaged measured area of pores as measured across the test samples.

[0607] In some embodiments, the ratio of the area of the perforations to the ratio of the area of the sheet may be used to characterize the sheet as a density of perforations. The area of a test sample may be taken as the planar area spanned by the test sample. Additional sheet surface area may be excluded due to wrinkles other non-planar features. Characterization may be based on the ratio of the area of the perforations to the test sample area as density of perforations excluding features such as surface debris. Characterization may be based on the ratio of the area of the perforations to the suspended area of the sheet. As with other testing, multiple test samples may be taken to confirm consistency across tests and verification may be obtained by performance testing. The density of perforations may be, for example, 2 per nm ² (2/ nm ² to 1 per μm ² (1/ μm ² )).

[0608] In some embodiments, the perforated area comprises 0.1% or greater, 1% or greater or 5%) or greater of the sheet area, less than 10% of the sheet area, less than 15% of the sheet area, from 0.1% to 15% of the sheet area, from 1% to 15% of the sheet area, from 5% to 15% of the sheet area or from 1% to 10% of the sheet area. In some further embodiments, the perforations are located over greater than 10% or greater than 15% of said area of said sheet of graphene-based material. A macroscale sheet is macroscopic and observable by the naked eye. In some embodiments, at least one lateral dimension of the sheet is greater than 3 cm, greater than 1 cm, greater than 1 mm or greater than 5 mm. In some further embodiments, the sheet is larger than a graphene flake which would be obtained by exfoliation of graphite in known processes used to make graphene flakes. For example, the sheet has a lateral dimension greater than about 1 micrometer. In an additional embodiment, the lateral dimension of the sheet is less than 10 cm. In some further embodiments, the sheet has a lateral dimension (e.g., perpendicular to the thickness of the sheet) from 10 nm to 10 cm or greater than 1 mm and less than 10 cm.

[0609] Chemical vapor deposition growth of graphene-based material typically involves use of a carbon containing precursor material, such as methane and a growth substrate. In some embodiments, the growth substrate is a metal growth substrate. In some embodiments, the metal growth substrate is a substantially continuous layer of metal rather than a grid or mesh. Metal growth substrates compatible with growth of graphene and graphene-based materials include transition metals and their alloys. In some embodiments, the metal growth substrate is copper based or nickel based. In some embodiments, the metal growth substrate is copper or nickel. In some embodiments, the graphene-based material is removed from the growth substrate by dissolution of the growth substrate.

[0610] In some embodiments, the sheet of graphene-based material is formed by chemical vapor deposition (CVD) followed by at least one additional conditioning or treatment step. In some embodiments, the conditioning step is selected from thermal treatment, UV-oxygen treatment, ion beam treatment, and combinations thereof. In some embodiments, thermal treatment may include heating to a temperature from 200° C to 800° C at a pressure of 10 ^-7 torr to atmospheric pressure for a time of 2 hours to 8 hours. In some embodiments, UV-oxygen treatment may involve exposure to light from 150 nm to 300 nm and an intensity from 10 to 100 mW/cm ² at 6mm distance for a time from 60 to 1200 seconds. In some embodiments, UV- oxygen treatment may be performed at room temperature or at a temperature greater than room temperature. In some further embodiments, UV-oxygen treatment may be performed at atmospheric pressure (e.g. 1 atm) or under vacuum. In some embodiments, ion beam treatment may involve exposure of the graphene-based material to ions having an ion energy from 50 eV to 1000 eV (for pretreatment) and the fluence is from 3 x 10 ¹⁰ ions/cm ² to 8 x 10 ¹¹ ions/cm ² or 3 x

10 10 ions/cm 2 to 8 x 1013 ions/cm 2 (for pretreatment). In some further embodiments, the source of ions may be collimated, such as a broad beam or flood source. In some embodiments, the ions may be noble gas ions such as Xe ⁺ . In some embodiments, one or more conditioning steps are performed while the graphene-based material is attached to a substrate, such as a growth substrate.

[0611] In some embodiments, the conditioning treatment affects the mobility and/or volatility of the non-graphitic carbon-based material. In some embodiments, the surface mobility of the non-graphenic carbon-based material is such that when irradiated with perforation parameters such as described herein, the non-graphenic carbon-based material, may have a surface mobility such that the perforation process results ultimately in perforation. Without wishing to be bound by any particular belief, hole formation is believed to be related to beam induced carbon removal from the graphene sheet and thermal replenishment of carbon in the hole region by non grapheme carbon. The replenishment process may be dependent upon energy entering the system during perforation and the resulting surface mobility of the non-graphenic carbon based material. To form holes, the rate of graphene removal may be higher than the non- graphenic carbon hole filling rate. These competing rates depend on the non-graphenic carbon flux (e.g., mobility [viscosity and temperature] and quantity) and the graphene removal rate (e.g., particle mass, energy, flux).

[0612] In some embodiments, the volatility of the non-graphenic carbon-based material may be less than that which is obtained by heating the sheet of graphene-based material to 500°C for 4 hours in vacuum or at atmospheric pressure with an inert gas.

[0613] In various embodiments, CVD graphene or graphene-based material can be liberated from its growth substrate (e.g., Cu) and transferred to a supporting grid, mesh or other supporting structure. In some embodiments, the supporting structure may be configured so that at least some portions of the sheet of graphene-based material are suspended from the supporting structure. For example, at least some portions of the sheet of graphene-based material may not be in contact with the supporting structure. [0614] In some embodiments, the sheet of graphene-based material following chemical vapor deposition comprises a single layer of graphene having at least two surfaces and non- graphenic carbon based material may be provided on said surfaces of the single layer graphene. In some embodiments, the non-graphenic carbon based material may be located on one of the two surfaces or on both. In some further embodiments, additional graphenic carbon may also present on the surface(s) of the single layer graphene.

[0615] In some embodiments, enclosures can be further modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the spirit and scope of the disclosure. Accordingly, the enclosures and methods are not limited by the foregoing description.

[0616] Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently. When a compound is described herein such that a particular isomer or enantiomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomer and enantiomer of the compound described individually or in any combination. One of ordinary skill in the art will appreciate that methods, device elements, starting materials and synthetic methods other than those specifically exemplified can be employed in the practice herein without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, starting materials and synthetic methods are intended to be included herein. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure.

[0617] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements. Embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0618] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope. Thus, it should be understood that although some embodiments have been specifically disclosed, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of embodiments as defined by the appended claims.

[0619] In general the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The preceding definitions are provided to clarify their specific use in the context herein.

[0620] All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).

[0621] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which embodiments pertain. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art, in some cases as of their filing date, and it is intended that this information can be employed herein, if needed, to exclude (for example, to disclaim) specific embodiments that are in the prior art. For example, when a compound is claimed, it should be understood that compounds known in the prior art, including certain compounds disclosed in the references disclosed herein (particularly in referenced patent documents), are not intended to be included in the claims.

[0622] Working Examples

[0623] Some embodiments are further illustrated by the following examples. The examples are provided for illustrative purposes only, and are not to be construed as limiting the scope or content of embodiments any way.

[0624] Example 1 - Permeability Study - Allura Red AC and Silver Nanopar tides

[0625] Permeability of small (Allura Red AC) and large (silver nanoparticles) across a SiN substrate layer and a perforated graphene layer were assessed via diffusion cell experiments. Permeability was compared to a control membrane with selectivity on the order of nanometers. Results are displayed in Figure 84, where "Bare AM Chip" is the SiN substrate, "Biopore" is the control membrane, and "Nanoporous graphene" is perforated graphene. In the Figure, results were normalized to permeability of substances through the control membrane based on area of the tested substances. Also, permeability results for Allura Red AC and silver nanoparticles through the control membrane were also normalized to each other (based on the raw data, permeability of Allura Red AC through the control membrane high than permeability of silver nanoparticles through the control membrane). The data shows that less Allura Red AC permeated the perforated graphene layer than the SiN layer. Moreover, to an even larger extent, the perforated graphene layer restricted silver nanoparticles from traversing the layer, as compared to the SiN layer.

[0626] Example 2 - Diffusive Transport of Fluorescein and IgG

[0627] Diffusive transport of fluorescein conjugated to IgG was assessed via diffusion cell experiments with respect to the following materials: a SiN substrate layer (termed "Bare Chip" in Figure 85), perforated graphene, unperforated graphene, and a control membrane (termed "Biopore" in the Figure 85). The results showed that normalized flux of IgG through the SiN substrate layer was higher than through perforated graphene. The results also showed minimum flux of IgG through unperforated graphene. Without being bound by theory, it is believed that flux observed through unperforated graphene resulted from intrinsic or native defects in the graphene.

[0628] Figure 86 compares the permeability results in Figure 85 with permeability data obtained on fluorescein alone (i.e., not conjugated to IgG). The Figure demonstrates that fluorescein permeability was higher than IgG permeability.

[0629] Example 3 - Permeability Study - FluoSpheres and Fluorescein

[0630] Permeability of 100 nm diameter Red (580/605) FluoSpheres and fluorescein was assessed via diffusion cell experiments with perforated graphene. As shown in Figure 87,

FluoSpheres added to the left side of diffusion cell did not traverse the perforated graphene.

However, fluorescein added to the left side of the diffusion cell traversed the perforated graphene, and then was detected on the right side of the diffusion cell.

[0631] Example 4 - Permeability Study - Fluorescein Across Various Substrates

[0632] Permeability of fluorescein across various substrates was measured via Permegear cells. The experiments were conducted with 7 mm diameter test materials, and with 5 μΜ fluorescein in PBSA buffer at room temperature. As shown in Figure 88, the following test materials were used: (i) a control membrane ("Biopore"); (ii) uncoated substrate TEPI-400/7

(i.e., 400 nm pores, 7 μιη thick substrate); (ii) TEPI-400/7 coated with two layers of unperforated graphene ("SLG2 Unperf); (iii) uncoated substrate TEPI-460/25; (iv) TEPI-460/25 coated with two layers of unperforated graphene; and (v) TEPI-460/25 coated with two layers of perforated graphene, where the graphene was perforated with silver nanoparticles.

[0633] The data showed that unperforated graphene substantially reduced the amount of fluorescein that traversed the substrate layers. The data also showed the coating the substrate with perforated graphene did not substantially alter permeability of the substrate. That is, the permeability of fluorescein across uncoated TEPI-460/25 was similar to that of fluorescein across TEPI-460/25 coated with perforate graphene. This was the case even if only a small percentage of graphene suspended across the substrate was perforated. For instance, the data show similar results when 12-15%, 8-10%, 5-6%, 4-5%. 3-4% or 2-3% of the graphene suspended across the substrate was porous. [0634] Additional data (not shown) further demonstrated that permeability of fluorescein across the substrate was enhanced by etching the substrate with NaOCl.

[0635] Figure 89A and 89B show diffusion for small fluorescent dye molecules (fluorescein, Figure 89A) and large 100nm FluoSpheres (Figure 89B) through uncoated substrate,

unperforated graphene-coated substrate, and perforated graphene-coated substrate. The plots show the relative fluorescence intensity for the low concentration solution side of the membrane over time. The uncoated substrate was highly permeable to both fluorescent analytes, whereas the unperforated graphene-coated membrane had low permeablility. Size selectivity of the nanoperforated graphene membrane was demonstrated by its relatively high permeability to the smaller analyte (fluorescein, Figure 89A) and low permeability to the larger analyte

(FluoSpheres, Figure 89B). Both analytes were diluted in phosphate-buffered saline

solution. Donor concentrations were 5μΜ for fluorescein and 200ppm for FluoSpheres. The substrate was a track-etched polymeric membrane with pore diameters ranging between approximately 350-450 nm. Sample area available for diffusion was 49 mm ^A 2. Testing was performed at room temperature.

FMPLANTABLE GRAPHENE MEMBRANES WITH LOW CYTOTOXICITY

[0636] Some embodiments relate to the selective passage of substances through an enclosure that encourages nearby vascularization (i.e., angiogenesis) and/or tissue ingrowth in a biological environment. Some embodiments include methods and devices for selectively separating or isolating substances in a biological environment, e.g., using a composite structure that comprises a two-dimensional material. Some embodiments include an enclosure comprising a

compartment and a wall separating the compartment from an environment external to the compartment. In some embodiments, the wall comprises a two-dimensional material layer and a substrate layer. Two-dimensional materials, such as graphene-based materials, are discussed below.

[0637] Enclosures can be in any shape. Thus, the cross-section of an enclosure can be, for example, circular, ovular, rectangular, square, or irregular-shaped. The size of the enclosure also is not limited, and can be small enough to circulate in the bloodstream (e.g., on the order of nanometers or larger) or large enough for implantation (e.g. on the order of inches or smaller). In some embodiments, the enclosure is from 100 nm to 6 inches long in its longest dimension, such as from about 100 nm to about 500 nm, about 500 nm to about 1 μm, about 1 μιη to about 500 μm, about 500 μιη to about 1 mm, about 1 mm to about 500 mm, about 500 mm to about 1 cm, about 1 cm to about 10 cm, or about 1 cm to about 6 inches long. In some embodiments, the enclosure is longer than 6 inches in its longest dimension, such as about 10 inches or about 15 inches long.

[0638] The thickness of the wall depends, in part, on the two-dimensional material layer and/or substrate layers used in the wall. Thus, in some embodiments a wall, or a portion thereof, comprising both a two-dimensional material layer and a substrate layer is at least 5 nm thick, such as from about 5 nm to about 1 μιη thick, from about 5 nm to about 250 nm thick, from about 5 to about 50 nm thick, from about 5 to about 20 nm thick, or from about 20 to about 50 nm thick. In some embodiments, the thickness of the wall is about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm thick, about 25 nm thick, about 30 nm thick, about 35 nm thick, about 40 nm thick, about 45 nm thick, about 50 nm thick, about 100 nm thick, about 150 nm thick, about 200 nm thick, about 250 nm thick, about 300 nm thick, about 400 nm thick, about 500 nm thick, about 600 nm thick, about 700 nm thick, about 800, nm thick, about 900 nm thick, or about 1 μιη thick. In some embodiments, the thickness of the wall is up to about 1 μιη thick or up to about 1 mm thick. In some embodiments, the thickness of the wall is tailored to allow bidirectional passage of oxygen and nutrients into and out of the enclosure. In some embodiments, the thickness of the wall is tailored to allow entry of oxygen and nutrients into the enclosure at sufficient concentrations to maintain viability of cells within the enclosure.

[0639] In some embodiments, the substrate layer has a thickness of about 1 mm or less, about 1 μπι or less, or about 100 nm or less. In some embodiments, a thickness of the substrate layer can range from about 100 nm to about 100 μm, or about 1 μπι to about 50 μm, or about 10 μπι to about 20 μm, or about 15 μπι to about 25 μπι. In some embodiments, the substrate layer has a thickness about 10 μπι or greater, or about 15 μπι or greater. In some embodiments, the substrate layer has a thickness of less than 1 μιη. In some embodiments, the substrate layer has a thickness of about 10 nm to about 100 nm, or about 20 nm to about 50 nm.

[0640] In some embodiments, the enclosure can be supported by one or more support structures. In some embodiments, the support structure can itself have a porous structure wherein the pores are larger than those of the two-dimensional material. In some embodiments, the support structure is formed as a frame at a perimeter of a two-dimensional material. In some embodiments, the support structure is positioned in part interior to a perimeter of a two- dimensional material. In some embodiments, the substrate layer can convey a desired degree of structural support (e.g., to prevent tearing and/or buckling) to the two-dimensional material layer.

[0641] In some embodiments, a substrate layer is positioned on one or both sides or surfaces of the two-dimensional material. Thus, in some embodiments the substrate is positioned on the outside of the enclosure and in some cases is exposed to the external environment (see, e.g., Figure 76, showing some embodiments with a device with a substrate positioned on the outside of an enclosure). In some embodiments, the substrate is positioned on the inside of the enclosure, and can be separated from an environment external to the enclosure (even though the substrate can be separated from the environment external to the enclosure, it can still be exposed to components from the external environment due to pores in the two-dimensional material layer and/or substrate layer). In some embodiments, the substrate is positioned on both the outside and the inside of the enclosure. In that case, the substrate on the outside of the enclosure can contain materials that are the same as or different from the substrate on the inside of the enclosure. In some embodiments, two or more substrate layers are positioned on the same side of the two- dimensional material layer (e.g., two or more substrate layers can be positioned on the outside of the enclosure). In some embodiments, the substrate is disposed directly on the two-dimensional material. In some embodiments, the substrate is disposed on the two-dimensional material with high conformance (e.g., by disposing a slightly wet substrate on the two-dimensional material). In some embodiments, the substrate is disclosed with low conformance. In some embodiments, the substrate is disposed indirectly on the two-dimensional material; for instance, an intermediate layer can be positioned between the substrate layer and the two-dimensional material layer. In some embodiments, the substrate layer is disposed directly or indirectly on another substrate layer. In some embodiments, the two-dimensional material is suspended on a substrate layer. In some embodiments, the substrate layer is affixed to the two-dimensional material layer (see, e.g., Figure 73 A, showing exemplary substrate layers, two-dimensional material layers, and substrate affixed to a two-dimensional material; see also Figure 73B, showing some embodiments that were determined to be not cytotoxic based on cytotoxicity testing and implantation testing).

[0642] In some embodiments, the substrate layer can increase vascularization near the enclosure, thus prompting the formation of blood vessels and/or tissue ingrowth in close proximity to the enclosure. In some embodiments, the increased vascularization contributes to decreasing the effective distance between the blood stream and substances being eluted from the enclosure. In some embodiments, the increased vascularization contributes to viability of substances, such as cells, enclosed within the enclosure.

[0643] The substrate layer can be porous and/or nonporous. In some embodiments, the substrate layer contains porous and nonporous sections. In some embodiments the substrate layer comprises a porous or permeable fibrous layer. Porous substrates include, for example, one or more of ceramics and thin film polymers. Exemplary ceramics include nanoporous silica (silicon dioxide), silicon, SiN, and combinations thereof. In some embodiments, the substrate layer comprises track-etched polymers, expanded polymers, patterned polymers, woven polymers, and/or non-woven polymers. In some embodiments, the substrate layer comprises a plurality of polymer filaments. In some embodiments, the polymer filaments can comprise a thermopolymer, thermoplastic polymer, or melt polymer, e.g., that can be molded or set in an optional annealing step. In some embodiments, the polymer filaments are hydrophobic. In some embodiments, the polymer filaments are hydrophilic. In some embodiments, the substrate layer comprises a polymer selected from the group consisting of polysulfones, polyurethane, polymethylmethacrylate (PMMA), polyglycolid acid (PGA), polylactic acid (PLA), polyethylene glycol (PEG), polylactic-co-glycolic acid (PLGA), polyamides (such as nylon-6,6, supramid and nylamid), polyimides, polypropylene, polyethersulfones (PES), polyvinylidine fluoride (PVDF), cellulose acetate, polyethylene, polypropylene, polycarbonate, polytetrafluoroethylene (PTFE) (such as Teflon), polyvinylchloride (PVC), polyether ether ketone (PEEK), mixtures and block co-polymers of any of these, and combinations and/or mixtures thereof. In some embodiments, the polymers are biocompatible, bioinert and/or medical grade materials. By way of example, Figure 78 shows some embodiments of graphene disposed on various different substrates.

Figures 79 shows micrographs some embodiments of custom track-etched polyimide. Figures 80 and 81 show micrographs of some embodiments of graphene disposed on track-etched polyimide. Figure 82 and 83 show micrographs of some embodiments of graphene disposed on electrospun nylon 6,6.

[0644] In some embodiments, the substrate layer comprises a biodegradable polymer. In some embodiments, a substrate layer forms a shell around the enclosure (e.g., it completely engulfs the enclosure). In some embodiments, the substrate layer shell, or a portion thereof, can be dissolved or degraded, e.g., in vitro. In some embodiments, the shell can be loaded with additives, including additives that protect substances inside the enclosure from air or prevent the need for a stabilizing agent.

[0645] Suitable techniques for depositing or forming a porous or permeable polymer on the two-dimensional material include casting or depositing a polymer solution onto the two- dimensional material or intermediate layer using a method such as spin-coating, spray coating, curtain coating, doctor-blading, immersion coating, electrospinning, or other similar techniques. Electrospinning techniques are described, e.g., in US 2009/0020921 and/or U.S. Application No. 14/609,325, both of which are hereby incorporated by reference in their entirety.

[0646] In some embodiments, the process for forming a substrate layer includes an electrospinning process in which a plurality of polymer filaments are laid down to form a porous mat, e.g., on the two-dimensional material layer. In some embodiments, the mat has pores or voids located between deposited filaments of the fibrous layer. Figure 64 shows an illustrative SEM micrograph of a graphene or graphene-based film deposited upon a plurality of electrospun PVDF fibers. In some embodiments, the electrospinning process comprises a melt

electrospinning process or a solution electrospinning process, such as a wet electrospinning process or a dry electrospinning process. (See, e.g., Sinha-Ray et al. J. Membrane Sci. 485, 1 July 2015, 132-150.) In some embodiments, the polymer can be present in a spin dope at a concentration of 2 wt.% to 15 wt.%, or 5 wt.% to 10 wt.%, or about 7 wt.%. Suitable solvents for the spin dope include any solvent that dissolves the polymer to be deposited and which rapidly evaporates, such as m-cresol, formic acid, dimethyl sulfoxide (DMSO), ethanol, acetone, dimethylacetamide (DMAC), dimethylformamide (DMF), water, and combinations thereof. In some embodiments, the spin dope solvent is biocompatible and/or bioinert. In some

embodiments, the amount of solvent used can influence the morphology of the substrate layer. In dry electrospinning processes, the spun fibers of the fibrous layer can remain as essentially discrete entities once deposited. In some embodiments, wet electrospinning processes deposit the spun fibers such that they are at least partially fused together when deposited. In some embodiments, the size and morphology of the deposited fiber mat (e.g., degree of porosity, effective pore size, thickness of fibrous layer, gradient porosity) can be tailored based on the electrospinning process used.

[0647] The porosity of the fibrous layer can include effective void space values (e.g.

measured via imagery) up to about 95% (i.e., the layer is 95% open), about 90%, about 80%, or about 60%, with a broad range of void space sizes. In some embodiments, a single spinneret can be moved to lay down a mat of the fibrous layer. In some embodiments, multiple spinnerets can be used for this purpose. In some embodiments, the spun fibers in an electrospun fibrous layer can have a fiber diameter ranging from about 1 nm to about 100 μm, or about 10 nm to about 1 μm, or about 10 nm to about 500 nm, or about 100 nm to about 200 nm, or about 50 nm to about 120 nm, or about 1 μπι to about 5 μm, or about 1 μπι to about 6 μm, or about 5 μπι to about 10 μπι. In some embodiments, the fiber diameter is directly correlated with a depth (Z-axis) of a pore abutting the two-dimensional material (disposed in the X-Y plane), and large diameter fibers can lead to large unsupported spans of material.

[0648] In some embodiments, the substrate layer can have pores (e.g., void spaces) with an effective pore size of from about 1 nm to about 100 μm, or about 10 nm to about 1 μm, or about 10 nm to about 500 nm, or about 100 nm to about 200 nm, or about 50 nm to about 120 nm, or about 1 μπι to about 5 μm, or about 1 μπι to about 6 μm, or about 5 μπι to about 10 μπι. Pore diameters in the substrate layer can be measured, for example, via porometry methods (e.g., capillary flow porometry) or extrapolated via imagery.

[0649] In some embodiments, the substrate layer can have an average pore size gradient throughout its thickness. "Pore size gradient" describes a layer with a plurality of pores, where the average diameter of the pores increases or decreases based on the proximity of the pore to the two-dimensional material. For example, a fibrous layer can have an average pore size gradient that decreases nearer the surface of a graphene-based material. In some embodiments, an average pore size of the fibrous layer is smaller nearer the surface of the graphene-based material than at an opposite surface of the fibrous layer. For example, the fibrous layer can have effective pore diameters of less than about 200 nm close to the intermediate layer or the two-dimensional material layer which can increase to greater than 100 μπι at the maximum distance away from the intermediate layer or two-dimensional material layer.

[0650] In some embodiments, the fibrous layer can have a "porosity gradient" throughout its thickness, which can be measured for instance using imagery. "Porosity gradient" describes a change, along a dimension of the fibrous layer, in the porosity or total pore volume as a function of distance from the two-dimensional material layer. For example, throughout the thickness of the porous fibrous layer, the porosity can change in a regular or irregular manner. A porosity gradient can decrease from one face of the fibrous layer to the other. For example, the lowest porosity in the fibrous layer can be located spatially closest to the two-dimensional material, and the highest porosity can be located farther away (e.g., spatially closer to an external

environment). A porosity gradient of this type can be achieved by electrospinning fibers onto a two-dimensional material such that a fiber mat is denser near the surface of the two-dimensional material and less dense further from the surface of the two-dimensional material. In some embodiments, a substrate layer can have a relatively low porosity close to the two-dimensional material, a higher porosity at a mid-point of the fibrous layer thickness (which can, for example, contain a supporting mesh for reinforcement or other particles), and return to a relatively low porosity at an external surface distal to the two-dimensional material.

[0651] In some embodiments, the substrate layer can have a "permeability gradient" throughout its thickness. "Permeability gradient," as used herein, describes a change, along a dimension of the fibrous layer, in the "permeability" or rate of flow of a liquid or gas through a porous material. For example, throughout the thickness of the fibrous layer, the permeability can change in a regular or irregular manner. A permeability gradient can decrease from one face of the fibrous layer to the other. For example, the lowest permeability in the fibrous layer can be located spatially closest to the graphene or graphene-based film or other two-dimensional material, and the highest permeability can be located farther away. Those of skill in the art will understand that permeability of a layer can increase or decrease without pore diameter or porosity changing, e.g., in response to chemical functionalization, applied pressure, voltage, or other factors.

[0652] In some embodiments, both the two-dimensional material layer and the substrate layer include a plurality of pores therein. In some embodiments, both the two-dimensional material and the substrate layer contain pores, and the pores in the two-dimensional material layer are smaller, on average, than the pores in the substrate layer. In some embodiments, the median pore size in the two dimensional material layer is smaller than the median pore size in the substrate layer. For example, in some embodiments, the substrate layer can contain pores with an average and/or median diameter of about 1 μιη or larger and the two-dimensional material layer can contain pores with an average and/or median diameter of about 10 nm or smaller. Accordingly, in some embodiments, the average and/or median diameter of pores in the two-dimensional material layer is at least about 10-fold smaller than the average and/or median diameter of pores in the substrate layer. In some embodiments, the average and/or median diameter of pores in the two-dimensional material layer is at least about 100-fold smaller than are the average and/or media diameter of pores in the substrate layer.

[0653] Some embodiments comprise an enclosure with low or no toxicity, such as cytotoxicity. In some embodiments, the enclosure is not cytotoxic when implanted into a subject. In some embodiments, the enclosure is not cytotoxic to cells, skin, blood, bodily fluids, or muscle. In some embodiments, the enclosure is not cytotoxic when injected into a subject. In some embodiments, the enclosure is not cytotoxic when ingested by a subject. In some embodiments, the enclosure is not cytotoxic when used in vitro.

[0654] Some embodiments comprise a two-dimensional material (e.g., a graphene based material), such as a porous two-dimensional material, with low or no toxicity, such as cytotoxicity. In some embodiments, the two-dimensional material is not cytotoxic to cells, skin, blood, bodily fluids, or muscle. In some embodiments, the two-dimensional material is not cytotoxic when implanted into a subject. In some embodiments, the two-dimensional material is not cytotoxic when injected into a subject. In some embodiments, the two-dimensional material is not cytotoxic when ingested by a subject. In some embodiments, the two-dimensional material is not cytotoxic when used in vitro. In some embodiments, a two-dimensional material can be affixed to or disposed on a second material {e.g., a substrate) without substantially affecting the cytotoxicity of the second material. In some embodiments, affixing the two-dimensional material to (or disposing it on) the second material can reduce cytotoxicity of the second material.

[0655] Some embodiments comprise a composite structure with low or no toxicity, such as cytotoxicity. In some embodiments, the composite structure is not cytotoxic to cells, skin, blood, bodily fluids, or muscle. In some embodiments, the composite structure is not cytotoxic when implanted into a subject. In some embodiments, the composite structure is not cytotoxic when injected into a subject. In some embodiments, the composite structure is not cytotoxic when ingested by a subject. In some composite structure is not cytotoxic when used in vitro.

[0656] Cytotoxicity can be measured, for instance, using cell viability assays or implantation testing. In some embodiments, greater than about 70% of cells exposed to the enclosure and/or composite structure remain viable at least 24 hours after exposure. In some embodiments, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of cells exposed to the enclosure and/or composite structure remain viable at least 24 hours after exposure.

[0657] In some embodiments, the device, enclosure and/or composite material has a bioreactivity rating of about 8.9 or less, such as from about 3.0 to about 8.9, or about 0.0 to about 2.9. In some embodiments, the device, enclosure and/or composite material has a bioreactivity rating of about 0.0, about 0.5, about 0.7, about 1.0, about 1.5, about 2.0, about 2.2, about 2.5, or about 2.9.

[0658] In some embodiments, tissue surrounding an implanted enclosure and/or composite structure do not exhibit substantial signs of cytotoxicity. Thus, in some embodiments, the enclosure and/or composite structure causes no, mild, or moderate signs of inflammation, encapsulation, hemorrhage, necrosis, discoloration, polymorphonuclear cells, lymphocytes, plasma cells, macrophages, giant cells, necrosis, neovascularization, fibrosis, fatty infiltrate, or combinations thereof in tissues exposed to the enclosure and/or composite structure. In some embodiments, macroscopic evaluation of tissue exposed to the enclosure and/or composite structure reveals no signs of inflammation, encapsulation, hemorrhage, necrosis, discoloration, or combinations thereof. In some embodiments, macroscopic evaluation of tissue exposed to the enclosure and/or composite structure reveals mild or moderate signs of inflammation, encapsulation, hemorrhage, necrosis, discoloration, or combinations thereof.

[0659] In some embodiments, microscopic evaluation of tissue exposed to the enclosure and/or composite structure reveals no signs an inflammatory response, such as signs of polymorphonuclear cells, lymphocytes, plasma cells, macrophages, giant cells, necrosis, or combinations thereof. In some embodiments, microscopic evaluation of tissue exposed to the enclosure and/or composite structure reveals minimal or mild signs an inflammatory response, such as signs of polymorphonuclear cells, lymphocytes, plasma cells, macrophages, giant cells, necrosis, or combinations thereof. In some embodiments, microscopic evaluation of tissue exposed to the enclosure and/or composite structure reveals no signs a healing response, such as neovascularization, fibrosis, fatty infiltrate, or combinations thereof. In some embodiments, microscopic evaluation of tissue exposed to the enclosure and/or composite structure reveals minimal or mild signs a healing response, such as neovascularization, fibrosis, fatty infiltrate, or combinations thereof.

[0660] In some embodiments, extent of cytotoxicity is classified based on macroscopic or microscopic evaluation, and classification can be relative to cytotoxicity of a control enclosure and/or structure. Thus, in some embodiments no, mild, or moderate signs of inflammation, encapsulation, hemorrhage, necrosis, discoloration, polymorphonuclear cells, lymphocytes, plasma cells, macrophages, giant cells, necrosis, neovascularization, fibrosis, fatty infiltrate, or combinations thereof are as compared to a control (e.g., in some embodiments, the enclosure and/or composite structure has no signs of inflammation if observed inflammation is less than is observed using a control).

[0661] Some embodiments comprise methods of releasing a substance into an environment from an enclosure with low or no toxicity (e.g., cytotoxicity) to the environment. Some embodiments comprise treating a condition or disease, such as diabetes, by an enclosure with low or no cytotoxicity into the subject. Some embodiments comprise using the non-cytotoxic or low-cytotoxic enclosure in methods of immunoisolation (i.e., protecting substances from an immune reaction), timed drug release (e.g., sustained or delayed release), hemodialysis, or hemofiltration.

[0662] Some embodiments comprise encapsulating a device with a composite structure comprising (a) a perforated graphene-based material layer and (b) a substrate layer affixed directly or indirectly to at least one surface of the perforated graphene-based material. In some embodiments, the encapsulated device has a reduced cytotoxicity as compared to a comparable device without a perforated graphene-based material layer.

[0663] Some embodiments comprise methods of coating a therapeutic device with the composite structure. In some embodiments, the composite structure is applied to the exterior of the therapeutic device. Some embodiments comprise the coated therapeutic device. In some embodiments, the coated therapeutic device has a lower toxicity (e.g., cytotoxicity) than a comparable therapeutic device that is not coated with the composite structure.

[0664] Figure 65 illustrates a portion of an enclosure in a biological environment in contact with biological tissue in which an enclosure comprises one or more substrate layers, such as fibrous layers positioned on the outside of the perforated two-dimensional material. Figure 65 also shows capillary vascularization into the substrate layer. Without being bound by theory, it is believed that the biocompatibility of graphene can further promote this application, particularly by functionalizing the graphene to improve compatibility with a particular biological environment (e.g., via available edge bonds, bulk surface functionalization, pi-bonding, and the like). Functionalization can provide enclosures having added complexity for use in treating local and systemic disease. Figure 65 also shows a wall of an enclosure with a perforated two- dimensional material having hole sizes in a range that will retain cells. The external biological environment abutting the enclosure (the full enclosure is not shown) in Figure 65 is separated from cells, proteins, etc., positioned inside the enclosure. As illustrated, in some embodiments implantation of such an enclosure contemplates vascularization into a substrate layer positioned on the outside of the enclosure. [0665] In some embodiments, the substrate layer can provide a scaffold for tissue growth, cell growth and/or vascularization. In some embodiments, the substrate layer or wall comprises additives, such as pharmaceuticals, cells, growth factors (e.g., VEGF), signaling molecules, cytokines, clotting factors, blood thinners, immunosuppressants, antimicrobial agents, hormones, antibodies, minerals, nutrients or combinations thereof. In some embodiments, additives such as pharmaceuticals, cells, growth factors, clotting factors, blood thinners, immunosuppressants, antimicrobial agents, hormones, antibodies, antigens (e.g., IgG-binding antigens) or an antibody- binding fragment thereof, minerals, nutrients or combinations thereof are positioned on the inside of the disclosure. In some embodiments, the substrate layer or wall comprises materials toxic to bacteria or cells (without being bound by theory, it is believed that incorporating toxic materials into the wall will prevent passage of potentially dangerous or detrimental cells across the wall).

[0666] In some embodiments, additives beneficially promote cell or tissue viability or growth, reduce or prevent infection, improve vascularization to or near the enclosure, improve biocompatibility, reduce biofouling, and/or reduce the risk of adverse reactions. In some embodiments, additives can modulate properties, such as hydrophobicity or hydrophilicity, of the substrate layer. In some embodiments, additives can be used to modulate elution of a substance from a compartment in the enclosure. For instance, additives can confer shell-like properties to a substrate layer, such that degradation or removal of the additives allows substances in the compartment to escape the enclosure (and, by extension, substances from the external environment to enter to enclosure).

[0667] Some embodiments comprise a composited structure that include a two-dimensional material layer and a substrate layer. In some embodiments, a composite structure includes a support material (see, e.g., Figure 66D) disposed on an opposite side of the two-dimensional material from the substrate layer. In some embodiments, a composite structure comprises an intermediate layer between the two-dimensional material and the substrate layer, e.g., as shown in Figures 66A, 66C and 66E. Figure 66 shows schematic illustrations of composite structures comprising two-dimensional materials (e.g., graphene), an optional intermediate layer (e.g., track etched polymer membrane), and a fibrous layer having a tighter fiber spacing nearer the two- dimensional material and an increasing effective pore size further from the two-dimensional material. Figure 66 A shows SEM micrographs of the fibrous material with (bottom two expanded micrographs) and without (top two expanded micrographs) the two-dimensional material on the surface of the fibrous material. Figure 66A also shows SEM micrographs of high fiber density (bottom), medium fiber density (middle) and low fiber density (top) substrates.

[0668] In some embodiments, the intermediate layer promotes adhesion between the two- dimensional material layer and the substrate layer. Thus, in some embodiments, the enclosure comprises an intermediate layer disposed between the two-dimensional material layer and the substrate layer. In some embodiments, the enclosure comprises an intermediate layer positioned between two substrate layers on the same side of the two-dimensional material layer.

[0669] In some embodiments, the intermediate layer comprises carbon nanotubes, lacey carbon, nanoparticles, lithographically patterned low-dimensional materials, silicon and silicon nitride micromachined material, a fine mesh, such as a transmission electron microscopy grid, or combinations of these. Figure 69 shows an illustrative schematic of a process for manufacturing a two-dimensional material on a fibrous layer with mesh reinforcement. In some embodiments, the intermediate layer can be a thin, smooth, porous polymer layer, such as a track etched polymer. In some embodiments, the intermediate layer has a thickness of from 3 nm to 10 μm, 10 nm to 10 μm, 50 nm to 10 μm, 100 nm to 10 μm, 500 nm to 10 μm, 1 μπι to 10 μm, or 2 μπι to 6 μπι. In some embodiments, the composite structure has a thickness of from 1 μπι to 100 μm, 2 μπι to 75 μm, 3 μπι to 50 μm, 4 μπι to 40 μm, 5 μπι to 30 μm, 6 μπι to 25 μm, or 6 μπι to 20 μm, or 6 μπι to 16 μπι.

[0670] In some embodiments, an enclosure or composite structure includes a fibrous layer affixed to multiple sheets of graphene or graphene-based material. In some embodiments, the sheets of graphene or graphene-based materials are stacked upon one another with one of the sheets affixed directly or indirectly to the fibrous layer. Figure 70 shows an illustrative SEM micrograph of two layers of graphene or graphene-based material on a fibrous layer. In some embodiments, one or more sheets of graphene or graphene-based material can be affixed to a first surface of a fibrous layer and one or more sheets of graphene or graphene-based material can be affixed to a second surface of the fibrous layer. In some embodiments, the graphene- based material is applied to a fully-formed substrate layer, such as a fully-formed electrospun substrate layer. Some embodiments comprise putting multiple layers of the graphene-based material onto the substrate layer (e.g., the fully-formed substrate layer). Without being bound by theory, it is believed that adding multiple layers of graphene-based material onto the substrate layer allows complete coverage of the substrate layer with the graphene-based material.

[0671] In some embodiments, the enclosure comprises a single compartment that does not contain sub-compartments. In some embodiments, the single compartment is in fluid

communication with an external environment separated from the compartment, e.g., by a wall. In some embodiments, the enclosure has a plurality of sub-compartments. In some

embodiments, the sub-compartments are in fluid communication with an environment outside the sub-compartment. In some embodiments, each sub-compartment comprises a wall that allows passage of one or more substances into and/or out of the sub-compartment. In some

embodiments, the wall or a portion thereof comprises a perforated two-dimensional material, a polymer, a hydrogel, or some other means of allowing passage of one or more substance into and/or out of the sub-compartment. In some embodiments, an enclosure is subdivided into two sub-compartments separated from each other at least in part by perforated two-dimensional material, such that the two sub-compartments are in direct fluid communication with each other through holes in the two-dimensional material. In some embodiments, the enclosure is subdivided into two sub-compartments each comprising two-dimensional material which sub- compartments are in direct fluid communication with each other through holes in the two- dimensional material and only one of the sub-compartments is in direct fluid communication with an environment external to the enclosure. In some embodiments, the enclosure is subdivided into two sub-compartments each comprising two-dimensional material which sub- compartments are in direct fluid communication with each other through holes in the two- dimensional material and both of the sub-compartments are also in direct fluid communication with an environment external to the enclosure.

[0672] In some embodiments, the enclosure has an inner sub-compartment and an outer sub- compartment each comprising a perforated two-dimensional material, wherein the inner sub- compartment is entirely enclosed within the outer sub-compartment, the inner and outer compartments are in direct fluid communication with each other through holes in the two- dimensional material and the inner sub-compartment is not in direct fluid communication with an environment external to the enclosure.

[0673] In some embodiments, where an enclosure has a plurality of sub-compartments each comprising a two-dimensional material, the sub-compartments are nested one within the other, each of which sub-compartments is in direct fluid communication through holes in two- dimensional material with the sub-compartment(s) to which it is adjacent, the outermost sub- compartment in direct fluid communication with an environment external to the enclosure, the remaining plurality of sub-compartments not in direct fluid communication with an environment external to the enclosure.

[0674] In some embodiments, a sub-compartment can have any shape or size. In some embodiments, 2 or 3 sub-compartments are present. Several examples of enclosure sub- compartments are illustrated in Figures 61 A-61E. In Figure 61 A, a nested configuration is illustrated, such that sub-compartment B completely contains a smaller sub-compartment A, and substances in the centermost enclosure A can pass into the main enclosure B, and potentially react with or within the main compartment during ingress and egress therefrom. In this embodiment, one or more substances in A can pass into B and one or more substances in A can be retained in A and not enter B. Two sub-compartments in which one or more substances can pass directly between the sub-compartments are said to be in direct fluid communication.

Passage between sub-compartments and between the enclosure and the external environment can be via holes of a perforated two-dimensional material. In some embodiments, the barrier (e.g., a membrane) between compartment A and B can be permeable to all substances in A or to certain substances in A (i.e., selective permeability). In some embodiments, the barrier between B and the external environment can be permeable to all substances in B or selectively permeable to certain substances in B. In Figure 61 A, sub-compartment A is in direct fluid communication with sub-compartment B which in turn is in direct fluid communication with the external environment. Compartment A in this nested configuration is in indirect fluid communication with the external environment via intermediate passage into sub-compartment B. The two- dimensional materials employed in different sub-compartments of an enclosure can be the same or different materials and the perforations or hole sizes in the two-dimensional material of different sub-compartments can be the same or different.

[0675] In Figure 61B, the enclosure is bisected with an impermeable wall (e.g., formed of non-porous or non-permeable sealant) forming sub-compartments A and B, such that both sections have access to the egress location independently, but there is no direct or indirect passage of substances from A to B. (It will be appreciated, however, that substances exiting A or B can enter the other sub-compartment indirectly via the external environment.)

[0676] In Figure 61C, the main enclosure is again bisected into sub-compartments A and B, but with a perforated material forming the barrier between the sub-compartments. Both sub- compartments not only have access to the egress location independently, but also can interact with one another, i.e. the sub-compartments are in direct fluid communication. In some embodiments, the barrier between sub-compartments A and B is selectively permeable, for example allowing at least one substance in A to pass into B, but not allowing the substances originating in B to pass to A. The porosity of the barrier between sub-compartments (e.g., sub- compartments A and B) can be the same as or different than the porosity of the sub-compartment walls in direct fluid communication with an environment external to the enclosure.

[0677] Figure 6 ID illustrates an enclosure having three compartments. The enclosure is illustrated with sub-compartment A being in fluid communication with sub-compartment B, which in turn is in fluid communication with sub-compartment C, which in turn is in fluid communication with the external environment. Compartments A and B are not in fluid communication with the external environment, i.e. they are not in direct fluid communication with the external environment. Adjacent sub-compartments A and B and adjacent sub- compartments B and C are each separated by a perforated two-dimensional material and are thus in direct fluid communication with each other. Sub-compartment A is only in indirect fluid communication with compartment C and the external environment via sub-compartment B or B and C, respectively. Various other combinations of semi-permeable barrier or non-permeable barriers can be employed to separate sub-compartments. Various perforation size constraints can change depending on how the enclosure is ultimately configured. Regardless of the chosen configuration, in some embodiments the boundaries, or at least a portion thereof, of the enclosure can be constructed from a two-dimensional material such that the thickness of the two- dimensional material is less than the diameter of the substance to be passed selectively across the two-dimensional material.

[0678] Figure 6 IE illustrates an enclosure having a single compartment (A) and no sub- compartments. In the Figure, the compartment is in direct fluid communication with an environment external to the enclosure.

[0679] In some embodiments, the presence of two or more sub-compartments containing the same substance(s) provides redundancy in function so that an enclosure can remain at least partially operable so long as at least one sub-compartment is not compromised.

[0680] The multiple physical embodiments for the enclosures and their uses can allow for various levels of interaction and scaled complexity of problems to be solved. For example, a single enclosure can provide drug elution for a given time period, or there can be multiple sizes of perforations to restrict or allow movement of distinct substances, each having a particular size.

[0681] Added complexity of the embodiments described herein with multiple sub- compartments can allow for interaction between compounds to catalyze or activate a secondary response (i.e., a "sense-response" paradigm). For example, if there are two sections of an enclosure that function independently, exemplary compound A can undergo a constant diffusion into the body, or either after a given time or in the presence of a stimulus from the body. In some embodiments, exemplary compound A can activate exemplary compound B, or inactivate functionalization that otherwise blocks exemplary compound B from escaping. In some embodiments, binding interactions to produce the foregoing effects can be reversible or irreversible. In some embodiments, exemplary compound A can interact with chemical cascades produced outside the enclosure, and a metabolite subsequent to the interaction can release exemplary compound B (e.g., by inactivating functionalization). Further examples utilizing effects that take place in a similar manner include using source cells (e.g., non-host; allogenic; xenogenic; autogenic; cadeaveric; stem cells, such as fully or partially differentiated stem cells) contained in an enclosure, within which secretions from the cell can produce a "sense-response" paradigm. In some embodiments, the presence of graphene in the "sense-response" paradigm does not hinder diffusion, thus allowing a fast time response as compared to enclosures that to not contain graphene.

[0682] In some embodiments, growth factors or hormones can be loaded in the enclosure to encourage vascularization (see Figure 65). In some embodiments, survival of cells within the enclosure can be improved as a result of bi-directional passage of nutrients and waste into and out of the enclosure.

[0683] In some embodiments, the relative thinness of graphene can enable bi-directional passage across a wall (or portion thereof) of the enclosure in close proximity to blood vessels, particularly capillary blood vessels, and other cells. In some embodiments, using a graphene- based enclosure can provide differentiation over other solutions accomplishing the same effect because the graphene does not appreciably limit permeability; instead, the diffusion of molecules through the graphene apertures can limit the movement of a substance across the wall.

[0684] In some embodiments, the perforations allow for zeroth order diffusion through the wall. In some embodiments, osmotic pumps can be used to transport substances across the wall. In some embodiments, natural delta pressures in the body influence passage of substances across the wall. In some embodiments, convective pressure influences passage of substances across the wall. In some embodiments, it is possible to achieve high throughput flux through the wall of the enclosure.

[0685] Figures 62A and 62B provide a schematic illustration of enclosures with a single compartment for immunoisolation (it will be appreciated that the enclosure can having a plurality of sub-compartments, for example, two or three sub-compartments). The enclosure (6030) of Figure 62 A is shown as a cross-section formed by an inner sheet or layer (6031) comprising perforated two-dimensional material, such as a graphene-based material, and an outer sheet or layer (6032) of a substrate material (though in some embodiments, the inner layer comprises the substrate material, and the outer layer comprises the perforate two-dimensional material). The substrate material can be porous, selectively permeable or non-porous, and/or and non- permeable. However at least a portion of the support material is porous or selectively permeable. The enclosures in Figure 62 contain selected living cells (6033). Figure 62B provides an alternative cross-section of the enclosure of Figure 62 A, showing the space or cavity formed between a first composite layer (6032/6031) and a second composite layer (6032/6031) (in the figure, the cavity is depicted to contain roughly circular symbols, which can be, e.g., cells or any other substance) where a sealant 6034 is illustrated as sealing the edges of the composite layers. It will be appreciated that seals at the edges of the composite layers can be formed employing physical methods, such as clamping, crimping, or with adhesives. Methods and materials for forming the seals at the edges are not particularly limiting. In some embodiments, the sealing material provides a non-porous and non-permeable seal or closure. In some embodiments, a portion of the enclosure is formed from a sealant, such as a silicone, epoxy, polyurethane or similar material. In some embodiments, the sealant is biocompatible. For instance, in some embodiments the seal does not span the entire length or width of the device. In some

embodiments, the seal forms a complete loop around the cavity. In some embodiments, the seal is formed as a frame at a perimeter of a two-dimensional material. In some embodiments, the seal is positioned, at least in in part, interior to a perimeter of a two-dimensional material.

[0686] Some embodiments include methods for using graphene-based materials and/or other two-dimensional materials to transport, transfer, deliver, and/or allow passage of substances in or to a biological environment. Some embodiments comprise delivering substances to an environment external to the enclosure (e.g., a biological environment). In some embodiments, the substance positioned on the inside of the enclosure comprises one or more of atoms, molecules, viruses, bacteria, cells, particles and aggregates thereof. For example, the substance can include biological molecules, such as proteins, peptides, (e.g., insulin), nucleic acids, DNA, and/or RNA; pharmaceuticals; drugs; medicaments; therapeutics, including biologies and small molecule drugs; and combinations thereof.

[0687] If cells are placed within the enclosure, at least a portion of the enclosure can be permeable to oxygen and nutrients sufficient for cell growth and maintenance, to waste produced by the cell (e.g., CO ₂ ), and/or to metabolites produced by the cell (e.g., insulin). In some embodiments, at least a portion of the enclosure is permeable to signaling molecules, such as glucose. In some embodiments, at least a portion of the enclosure is permeable to growth factors produced by cells, such as VEGF. [0688] In some embodiments, the enclosure is not permeable to cells (such as immune cells), viruses, bacteria, antibodies, and/or complements of the immune system. Thus, in some embodiments, cells from the external environment cannot enter the enclosure and cells in the enclosure are retained. In some embodiments, the enclosure is permeable to desirable products, such as growth factors or hormones produced by the cells (see, e.g., Figures 74 and 75, illustrating some embodiments related to immunoisolation). The cells within the enclosure can be immunoisolated (i.e., protected from an immune reaction). In some embodiments of enclosures containing cells, the cells are yeast cells, bacterial cells, stem cells, mammalian cells, human cells, porcine cells, or a combination thereof. In some embodiments useful with cells, an enclosure comprises a plurality of sub-compartments, with the cells being positioned within one or more sub-compartments. In some embodiments useful with cells, the enclosure comprises a single compartment. In some embodiments, hole sizes in perforated two-dimensional materials useful for immunoisolation range in size from 1-50 nm, 1-40 nm, 1-30 nm, 1-25 nm, 1-17 nm, 1- 15 nm, 1-12 nm, 1-10 nm, 3-50 nm, 3-30 nm, 3-20 nm, 3-10 nm, or 3-5 nm. In some

embodiments, the size of the holes is about 1 nm, about 3 nm about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 30 nm, or about 50 nm.

[0689] Figures 63A-63C illustrate an exemplary method for forming an enclosure and introducing selected substances, for example cells, therein. The method is illustrated with use of a sealant for forming the enclosure. As illustrated in Figure 63 A, a first composite layer or sheet can be formed by placing a sheet or layer of two-dimensional material, such as a sheet of graphene-based material or a sheet of graphene (6141), in contact with a substrate layer (6142). At least a portion of the substrate layer (6142) of the first composite can be porous or permeable. Pore size of the substrate layer can be larger than the holes or apertures in the two-dimensional material employed and can be tuned for the environment (e.g., body cavity). A layer of sealant (6144), e.g., silicone, is applied on the sheet or layer of perforated two-dimensional material outlining a compartment of the enclosure wherein the sealant will form a non-permeable seal around a perimeter of the enclosure. Formation of a single compartment is illustrated in Figures 63 A-63C, however, it will be appreciated that multiple independent compartments within an enclosure can be formed by an analogous process. A second composite layer formed in the same way as the first is then prepared and positioned with the sheet or layer of perforated two dimensional materials in contact with the sealant. Alternatively, a sealant can be applied to a portion of a composite layer and the layer can be folded over in contact with the sealant to form an enclosure. A seal is then formed between the two composite layers. Appropriate pressure can be applied to facilitate sealing without damaging the two-dimensional material or its support. It will be appreciated that an alternative enclosure can be formed by applying a sheet or layer of non-porous and non-permeable support material in contact with the sealant. In this case only a portion of the enclosure is porous and permeable. Other methods for sealing the enclosure include ultrasonic welding. Sealed composite layers are illustrated in Figure 63B where it is shown that the sealed layers can be trimmed to size around the sealant to form the enclosure. The enclosure formed is shown to have an external porous substrate layer 6142 with the sheet or layer of perforated two-dimensional material (6141) being positioned as an internal layer, with sealant 6144 around the perimeter of the enclosure. As illustrated in Figure 63C, cells or other substances that would be excluded from passage through the perforated two-dimensional sheet or layer can be introduced into the enclosure after it is formed by injection through the sealant layer. Any perforation formed by such injection can be sealed as needed.

[0690] In some embodiments, substances (e.g., cells) can be introduced into the enclosure prior to formation of the seal. In some embodiments, one or more ports can be provided for introducing substances into the enclosure. For example, a loading port can be provided within the sealed perimeter of the enclosure, and the loading port can be permanently or semipermanently sealed after introduction of one or more substances through the loading port. Those in the art will appreciate that sterilization methods appropriate for the application envisioned can be employed during or after the preparation of the enclosure.

[0691] In some embodiments, an enclosure comprises perforated two-dimensional material encapsulating a substance, such that the substance is released to an environment external to the enclosure by passage through the holes in the perforated two-dimensional material. In some embodiments, the enclosure encapsulates two or more different substances. In some

embodiments, not all of the different substances are released to an environment external to the enclosure. In some embodiments, all of the different substances are released into an environment external to the enclosure. In some embodiments, different substances are released into an environment external to the enclosure at different rates. In some embodiments, different substances are released into an environment external to the enclosure at the same rates.

[0692] In some embodiments of any enclosure herein at least a portion of the holes in the two-dimensional material of the enclosure are functionalized.

[0693] In some embodiments at least a portion of the two-dimensional material is conductive and a voltage can be applied to at least a portion of the conductive two-dimensional material. The voltage can be an AC or DC voltage. The voltage can be applied from a source external to the enclosure. In some embodiments, an enclosure device further comprises connectors and leads for application of a voltage from an external source to the two-dimensional material.

[0694] Additionally, the conductive properties of graphene-based or other two-dimensional materials can allow for electrification to take place from an external source. In exemplary embodiments, an AC or DC voltage can be applied to conductive two-dimensional materials (e.g., in a device such as an enclosure device). The conductivity properties of graphene can provide additional gating to charged molecules or substances. Electrification can occur permanently or only a portion of the time to affect gating. Directional gating of charged molecules can be directed not only through the pores (or restrict travel through pores), but also to the surface of the graphene to adsorb or bind and encourage growth, promote formation of a protective layer, or provide the basis or mechanism for other biochemical effects (e.g., on the body).

[0695] In some embodiments, at least once wall, or portion thereof, of the enclosure allows for electrostatic control of charged species, for instance in nanofluidic or microfluidic systems. In some embodiments, the wall allows for control of charged species by varying the applied voltage, for instance in nanofluidic or microfluidic systems. In some embodiments, the wall can be tuned to manipulate ion passage at low and/or high ion concentrations. In some

embodiments, the wall is an ion-selective membrane. In some embodiments, the wall comprises one or more voltage-gated ion channels, such as voltage-gated pores. In some embodiments, the wall mimics biological voltage-gated ion channels. In some embodiments, the wall is a solid- state membrane. In some embodiments, nanochannel or nanopore transistors can be used to manipulate ion passage.

[0696] In some embodiments, the wall can be tuned using low or high applied voltages. In some embodiments, the wall allows high ionic flux. In some embodiments, the wall allows low ion flux. In some embodiments, pores in the wall modulates current of ions at low gate voltages and/or display high selectivity. In some embodiments, ion flux across the wall can be turned on or off at low applied voltages, such as < 500 mV. In some embodiments, ion flux across the wall can be turned on or off at biologically relevant ion concentrations, such as up to 1 M. In some embodiments, the applied voltage can modulate on species selectivity, e.g., cation or anion selectivity.

[0697] In some embodiments, nanopores can be electrostatically controlled at low voltages and biologically relevant ion concentrations. In some embodiments, walls are used in separation and sensing technologies. In some embodiments, walls are used in water filtration, water desalination, water purification, osmosis, energy storage, microfluidic devices, nanofluidic devices, and/or therapeutic methods. In some embodiments, walls are used in immune-isolation (i.e., protecting substances from an immune reaction), timed drug release (e.g., sustained or delayed release), hemodialysis, and hemofiltration. Some embodiments relate to methods for separating ions or other substances; methods for sensing ions; methods for storing energy;

methods for filtering water; and/or methods of treating a disease or condition (e.g., diabetes). Some embodiments relate to methods of ultrafiltration, nanofiltration and/or microfiltration. Some embodiments comprise using gating to control release of substances. Some embodiments comprise using gating to allow for different substances to be released at different times. Some embodiments comprise allowing different substances to pass through the wall at different times, thus modulating when and how substances mix and interact with other substances in a specific order.

[0698] Some embodiments comprise a method comprising introducing an enclosure comprising perforated two-dimensional material into an environment, the enclosure containing at least one substance; and releasing at least a portion of the at least one substance through the holes of the two-dimensional material to the environment external to the enclosure. In some embodiments, the enclosure contains cells which are not released from the enclosure and the at least one substance, a portion of which is released, is a substance generated by the cells in the enclosure.

[0699] Some embodiments comprise a method comprising introducing an enclosure comprising perforated two-dimensional material to an environment, the enclosure containing at least one first substance; and allowing migration of other substances from the environment into the enclosure. In some embodiments, the first substance is cells, and other substances include nutrients and/or oxygen.

[0700] In some embodiments, a composite structure comprises perforated two-dimensional material and a first fibrous layer comprising a plurality of polymer filaments affixed to a surface of the two-dimensional material; wherein the composite structure is substantially planar. In some embodiments, the perforated two-dimensional material has a second fibrous layer affixed to a surface of the two-dimensional material opposite the first fibrous layer. In some

embodiments, the average pore size of the first fibrous layer is different from the average pore size of the second fibrous layer. In some embodiments, the first and/or second fibrous layer comprises an additive selected from the group consisting of pharmaceuticals, cells, growth factors, clotting factors, blood thinners, immunosuppressants, antimicrobial agents, hormones, antibodies, minerals, nutrients and combinations thereof. In some embodiments, the

substantially planar composite structure is flexible. In some embodiments, the substantially planar composite structure is rigid. In some embodiments, multiple composite structures are combined to form a pouch-like enclosure. Such planar composite structures can be useful, for example, as appliques for wound healing. The composite structures can also be used, for example, as a component of an adhesive bandage.

[0701] Both permanent and temporary binding of substances to the enclosure are possible. In some embodiments, enclosures represent a disruptive technology for state of the art vehicle and other devices, such that these vehicles and devices to be used in new ways. For example, cell line developments, therapeutic releasing agents, and sensing paradigms (e.g., MRSw's, MR-based magnetic relaxation switches, see; Koh et al. (2008) Ang. Chem. Int'l Ed. Engl., 47(22) 4119-4121) can be used. Moreover, some embodiments mitigate biofouling and bioreactivity, convey superior permeability and less delay in response, and provide mechanical stability.

[0702] In some embodiments, enclosures can be used in non-therapeutic applications, such as in dosing probiotics in dairy products.

[0703] In some embodiments, two-dimensional materials are atomically thin, with thickness ranging from single-layer sub-nanometer thickness to a few nanometers. Two-dimensional materials include metal chalogenides (e.g., transition metal dichalogenides), transition metal oxides, hexagonal boron nitride, graphene, silicene and germanene (see: Xu et al. (2013)

"Graphene-like Two-Dimensional Materials) Chemical Reviews 113 :3766-3798).

[0704] In some embodiments, the two-dimensional material comprises a graphene-based material.

[0705] Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or a few layered sheets (e.g., about 20 or less) of fused six- membered rings forming an extended sp ² -hybridized carbon planar lattice. Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. In some embodiments, graphene-based materials also include materials which have been formed by stacking single or multilayer graphene sheets. In some embodiments, multilayer graphene includes 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In some embodiments, layers of multilayered graphene are stacked, but are less ordered in the z direction (perpendicular to the basal plane) than a thin graphite crystal.

[0706] In some embodiments, a sheet of graphene-based material may be a sheet of single or multilayer graphene or a sheet comprising a plurality of interconnected single or multilayer graphene domains, which may be observed in any known manner such as using for example small angle electron diffraction, transmission electron microscopy, etc.. In some embodiments, the multilayer graphene domains have 2 to 5 layers or 2 to 10 layers. As used herein, a domain refers to a region of a material where atoms are substantially uniformly ordered into a crystal lattice. A domain is uniform within its boundaries, but may be different from a neighboring region. For example, a single crystalline material has a single domain of ordered atoms. In some embodiments, at least some of the graphene domains are nanocrystals, having domain size from 1 to 100 nm or 10-100 nm. In some embodiments, at least some of the graphene domains have a domain size greater than from 100 nm to 1 cm, or from 100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm. In some embodiments, a domain of multilayer graphene may overlap a neighboring domain. Grain boundaries formed by crystallographic defects at edges of each domain may differentiate between neighboring crystal lattices. In some embodiments, a first crystal lattice may be rotated relative to a second crystal lattice, by rotation about an axis perpendicular to the plane of a sheet, such that the two lattices differ in crystal lattice orientation.

[0707] In some embodiments, the sheet of graphene-based material is a sheet of single or multilayer graphene or a combination thereof. In some other embodiments, the sheet of graphene-based material is a sheet comprising a plurality of interconnected single or multilayer graphene domains. In some embodiments, the interconnected domains are covalently bonded together to form the sheet. When the domains in a sheet differ in crystal lattice orientation, the sheet is polycrystalline.

[0708] In some embodiments, the thickness of the sheet of graphene-based material is from 0.3 to 10 nm, 0.34 to 10 nm, from 0.34 to 5 nm, or from 0.34 to 3 nm. In some embodiments, the thickness includes both single layer graphene and the non-graphenic carbon.

[0709] In some embodiments, a sheet of graphene-based material comprises intrinsic or native defects. Intrinsic or native defects may result from preparation of the graphene-based material in contrast to perforations which are selectively introduced into a sheet of graphene- based material or a sheet of graphene. Such intrinsic or native defects may include, but are not limited to, lattice anomalies, pores, tears, cracks or wrinkles. Lattice anomalies can include, but are not limited to, carbon rings with other than 6 members (e.g. 5, 7 or 9 membered rings), vacancies, interstitial defects (including incorporation of non-carbon atoms in the lattice), and grain boundaries. Perforations are distinct from openings in the graphene lattice due to intrinsic or native defects or grain boundaries, but testing and characterization of the final membrane such as mean pore size and the like encompasses all openings regardless of origin since they are all present. [0710] In some embodiments, graphene is the dominant material in a graphene-based material. For example, a graphene-based material may comprise at least 20% graphene, at least 30%) graphene, or at least 40% graphene, or at least 50% graphene, or at least 60%> graphene, or at least 70% graphene, or at least 80% graphene, or at least 90% graphene, or at least 95% graphene. In some embodiments, a graphene-based material comprises a range of graphene selected from 30% to 95%, or from 40% to 80% from 50% to 70%, from 60% to 95% or from 75%) to 100%). The amount of graphene in the graphene-based material is quantified as an atomic percentage utilizing known methods including scanning transmission electron microscope examination, or alternatively if STEM or TEM is ineffective another similar measurement technique.

[0711] In some embodiments, a sheet of graphene-based material further comprises non- graphenic carbon-based material located on at least one surface of the sheet of graphene-based material. In some embodiments, the sheet is exemplified by two base surfaces (e.g. top and bottom faces of the sheet, opposing faces) and side faces (e.g. the side faces of the sheet). In some further embodiments, the "bottom" face of the sheet is that face which contacted the substrate during growth of the sheet and the "free" face of the sheet opposite the "bottom" face. In some embodiments, non-graphenic carbon-based material may be located on one or both base surfaces of the sheet (e.g. the substrate side of the sheet and/or the free surface of the sheet). In some further embodiments, the sheet of graphene-based material includes a small amount of one or more other materials on the surface, such as, but not limited to, one or more dust particles or similar contaminants.

[0712] In some embodiments, the amount of non-graphenic carbon-based material is less than the amount of graphene. In some further embodiments, the amount of non-graphenic carbon material is three to five times the amount of graphene; this is measured in terms of mass. In some additional embodiments, the non-graphenic carbon material is characterized by a percentage by mass of said graphene-based material selected from the range of 0% to 80%. In some embodiments, the surface coverage of the sheet of non-graphenic carbon-based material is greater than zero and less than 80%, from 5% to 80%, from 10% to 80%, from 5% to 50% or from 10%) to 50%. This surface coverage may be measured with transmission electron microscopy, which gives a projection. In some embodiments, the amount of graphene in the graphene-based material is from 60% to 95% or from 75% to 100%. The amount of graphene in the graphene-based material is measured as an atomic percentage utilizing known methods preferentially using transmission electron microscope examination, or alternatively if STEM is ineffective using an atomic force microscope.

[0713] In some embodiments, the non-graphenic carbon-based material does not possess long range order and is classified as amorphous. In some embodiments, the non-graphenic carbon-based material further comprises elements other than carbon and/or hydrocarbons. In some embodiments, non-carbon elements which may be incorporated in the non-graphenic carbon include hydrogen, oxygen, silicon, copper, and iron. In some further embodiments, the non-graphenic carbon-based material comprises hydrocarbons. In some embodiments, carbon is the dominant material in non-graphenic carbon-based material. For example, a non-graphenic carbon-based material in some embodiments comprises at least 30% carbon, or at least 40% carbon, or at least 50% carbon, or at least 60% carbon, or at least 70% carbon, or at least 80% carbon, or at least 90% carbon, or at least 95% carbon. In some embodiments, a non-graphenic carbon-based material comprises a range of carbon selected from 30% to 95%, or from 40% to 80%), or from 50% to 70%. The amount of carbon in the non-graphenic carbon-based material is measured as an atomic percentage utilizing known methods preferentially using transmission electron microscope examination, or alternatively if STEM is ineffective using atomic force microscope.

[0714] Perforation techniques suitable for use in perforating the graphene-based materials may include described herein ion-based perforation methods and UV-oxygen based methods.

[0715] Ion-based perforation methods include methods in which the graphene-based material is irradiated with a directional source of ions. In some further embodiments, the ion source is collimated. In some embodiments, the ion source is a broad beam or flood source. A broad field or flood ion source can provide an ion flux which is significantly reduced compared to a focused ion beam. The ion source inducing perforation of the graphene or other two- dimensional material is considered to provide a broad ion field, also commonly referred to as an ion flood source. In some embodiments, the ion flood source does not include focusing lenses. In some embodiments, the ion source is operated at less than atmospheric pressure, such as at 10- ³ to 10 ^-5 torr or 10 ^-4 to 10 ^-6 torr. In some embodiments, the environment also contains background amounts (e.g. on the order of 10 ^-5 torr) of oxygen (O ₂ ), nitrogen (N ₂ ) or carbon dioxide (CO ₂ ). In some embodiments, the ion beam may be perpendicular to the surface of the layer(s) of the material (incidence angle of 0 degrees) or the incidence angle may be from 0 to 45 degrees, 0 to 20 degrees, 0 to 15 degrees or 0 to 10 degrees. In some further embodiments, exposure to ions does not include exposure to plasma.

[0716] In some embodiments, UV-oxygen based perforation methods include methods in which the graphene-based material is simultaneously exposed to ultraviolet (UV) light and an oxygen containing gas Ozone may be generated by exposure of an oxygen containing gas such as oxygen or air to the UV light. Ozone may also be supplied by an ozone generator device. In some embodiments, the UV-oxygen based perforation method further includes exposure of the graphene-based material to atomic oxygen. Suitable wavelengths of UV light include, but are not limited to wavelengths below 300 nm or from 150 nm to 300 nm. In some embodiments, the intensity from 10 to 100 mW/cm ² at 6mm distance or 100 to 1000 mW/cm ² at 6mm distance. For example, suitable light is emitted by mercury discharge lamps (e.g. about 185 nm and 254 nm). In some embodiments, UV/oxygen cleaning is performed at room temperature or at a temperature greater than room temperature. In some further embodiments, UV/oxygen cleaning is performed at atmospheric pressure (e.g. 1 atm) or under vacuum.

[0717] Perforations are sized as described herein to provide desired selective permeability of a species (atom, molecule, protein, virus, cell, etc.) for a given application. Selective

permeability relates to the propensity of a porous material or a perforated two-dimensional material to allow passage (or transport) of one or more species more readily or faster than other species. Selective permeability allows separation of species which exhibit different passage or transport rates. In two-dimensional materials selective permeability correlates to the dimension or size (e.g., diameter) of apertures and the relative effective size of the species. Selective permeability of the perforations in two-dimensional materials such as graphene-based materials can also depend on functionalization of perforations (if any) and the specific species. Separation or passage of two or more species in a mixture includes a change in the ratio(s) (weight or molar ratio) of the two or more species in the mixture during and after passage of the mixture through a perforated two-dimensional material.

[0718] In some embodiments, the characteristic size of the perforation is from 0.3 to 10 nm, from 1 to 10 nm, from 5 to 10 nm, from 5 to 20 nm, from 10 nm to 50 nm, from 50 nm to 100 nm, from 50 nm to 150 nm, from 100 nm to 200 nm, or from 100 nm to 500 nm. In some embodiments, the average pore size is within the specified range. In some embodiments, 70% to 99%, 80% to 99%, 85% to 99% or 90 to 99% of the perforations in a sheet or layer fall within a specified range, but other pores fall outside the specified range.

[0719] Nanomaterials in which pores are intentionally created may be referred to as perforated graphene, perforated graphene-based materials or perforated two-dimensional materials, and the like. Perforated graphene-based materials include materials in which non- carbon atoms have been incorporated at the edges of the pores. Pore features and other material features may be characterized in a variety of manners including in relation to size, area, domains, periodicity, coefficient of variation, etc. For instance, the size of a pore may be assessed through quantitative image analysis utilizing images preferentially obtained through transmission electron microscopy, and if TEM is ineffective, through atomic force microscopy, and if AFM is ineffective, through scanning electron microscopy, as for example presented in Figs. 60 and 61. The boundary of the presence and absence of material identifies the contour of a pore. The size of a pore may be determined by shape fitting of an expected species against the imaged pore contour where the size measurement is characterized by smallest dimension unless otherwise specified. For example, in some instances, the shape may be round or oval. The round shape exhibits a constant and smallest dimension equal to its diameter. The width of an oval is its smallest dimension. The diameter and width measurements of the shape fitting in these instances provide the size measurement, unless specified otherwise.

[0720] Each pore size of a test sample may be measured to determine a distribution of pore sizes within the test sample. Other parameters may also be measured such as area, domain, periodicity, coefficient of variation, etc. Multiple test samples may be taken of a larger membrane to determine that the consistency of the results properly characterizes the whole membrane. In such instance, the results may be confirmed by testing the performance of the membrane with test species. For example, if measurements indicate that certain sizes of species should be restrained from transport across the membrane, a performance test provides verification with test species. Alternatively, the performance test may be utilized as an indicator that the pore measurements will determine a concordant pore size, area, domains, periodicity, coefficient of variation, etc.

[0721] The size distribution of holes may be narrow, e.g., limited to 0.1-0.5 coefficient of variation. In some embodiments, the characteristic dimension of the holes is selected for the application.

[0722] In some embodiments involving circular shape fitting the equivalent diameter of each pore is calculated from the equation A= π d ² /4. Otherwise, the area is a function of the shape fitting. When the pore area is plotted as a function of equivalent pore diameter, a pore size distribution may be obtained. The coefficient of variation of the pore size may be calculated herein as the ratio of the standard deviation of the pore size to the mean of the pore size as measured across the test samples. The average area of perforations is an averaged measured area of pores as measured across the test samples.

[0723] In some embodiments, the ratio of the area of the perforations to the ratio of the area of the sheet may be used to characterize the sheet as a density of perforations. The area of a test sample may be taken as the planar area spanned by the test sample. Additional sheet surface area may be excluded due to wrinkles other non-planar features. Characterization may be based on the ratio of the area of the perforations to the test sample area as density of perforations excluding features such as surface debris. Characterization may be based on the ratio of the area of the perforations to the suspended area of the sheet. As with other testing, multiple test samples may be taken to confirm consistency across tests and verification may be obtained by performance testing. The density of perforations may be, for example, 2 per nm ² (21 nm ² ) to 1 per μηι ² (1/ μm ² ).

[0724] In some embodiments, the perforated area comprises 0.1% or greater, 1% or greater or 5%) or greater of the sheet area, less than 10% of the sheet area, less than 15% of the sheet area, from 0.1% to 15% of the sheet area, from 1% to 15% of the sheet area, from 5% to 15% of the sheet area or from 1% to 10% of the sheet area. In some further embodiments, the perforations are located over greater than 10% or greater than 15% of said area of said sheet of graphene-based material. A macroscale sheet is macroscopic and observable by the naked eye. In some embodiments, at least one lateral dimension of the sheet is greater than 3 cm, greater than 1 cm, greater than 1 mm or greater than 5 mm. In some further embodiments, the sheet is larger than a graphene flake which would be obtained by exfoliation of graphite in known processes used to make graphene flakes. For example, the sheet has a lateral dimension greater than about 1 micrometer. In an additional embodiment, the lateral dimension of the sheet is less than 10 cm. In some further embodiments, the sheet has a lateral dimension (e.g., perpendicular to the thickness of the sheet) from 10 nm to 10 cm or greater than 1 mm and less than 10 cm.

[0725] Chemical vapor deposition growth of graphene-based material typically involves use of a carbon containing precursor material, such as methane and a growth substrate. In some embodiments, the growth substrate is a metal growth substrate. In some embodiments, the metal growth substrate is a substantially continuous layer of metal rather than a grid or mesh. Metal growth substrates compatible with growth of graphene and graphene-based materials include transition metals and their alloys. In some embodiments, the metal growth substrate is copper based or nickel based. In some embodiments, the metal growth substrate is copper or nickel. In some embodiments, the graphene-based material is removed from the growth substrate by dissolution of the growth substrate.

[0726] In some embodiments, the sheet of graphene-based material is formed by chemical vapor deposition (CVD) followed by at least one additional conditioning or treatment step. In some embodiments, the conditioning step is selected from thermal treatment, UV-oxygen treatment, ion beam treatment, and combinations thereof. In some embodiments, thermal treatment may include heating to a temperature from 200 °C to 800 °C at a pressure of 10 ^-7 torr to atmospheric pressure for a time of 2 hours to 8 hours. In some embodiments, UV-oxygen treatment may involve exposure to light from 150 nm to 300 nm and an intensity from 10 to 100 mW/cm ² at 6mm distance for a time from 60 to 1200 seconds. In some embodiments, UV- oxygen treatment may be performed at room temperature or at a temperature greater than room temperature. In some further embodiments, UV-oxygen treatment may be performed at atmospheric pressure (e.g. 1 atm) or under vacuum. In some embodiments, ion beam treatment may involve exposure of the graphene-based material to ions having an ion energy from 50 eV to 1000 eV (for pretreatment) and the fluence is from 3 x 10 ¹⁰ ions/cm ² to 8 x 10 ¹¹ ions/cm ² or 3 x

10 10 ions/cm 2 to 8 x 1013 ions/cm 2 (for pretreatment). In some further embodiments, the source of ions may be collimated, such as a broad beam or flood source. In some embodiments, the ions may be noble gas ions such as Xe ⁺ . In some embodiments, one or more conditioning steps are performed while the graphene-based material is attached to a substrate, such as a growth substrate.

[0727] In some embodiments, the conditioning treatment affects the mobility and/or volatility of the non-graphitic carbon-based material. In some embodiments, the surface mobility of the non-graphenic carbon-based material is such that when irradiated with perforation parameters such as described herein, the non-graphenic carbon-based material, may have a surface mobility such that the perforation process results ultimately in perforation. Without wishing to be bound by any particular belief, hole formation is believed to be related to beam induced carbon removal from the graphene sheet and thermal replenishment of carbon in the hole region by non grapheme carbon. The replenishment process may be dependent upon energy entering the system during perforation and the resulting surface mobility of the non-graphenic carbon based material. To form holes, the rate of graphene removal may be higher than the non- graphenic carbon hole filling rate. These competing rates depend on the non-graphenic carbon flux (e.g., mobility [viscosity and temperature] and quantity) and the graphene removal rate (e.g., particle mass, energy, flux).

[0728] In some embodiments, the volatility of the non-graphenic carbon-based material may be less than that which is obtained by heating the sheet of graphene-based material to 500°C for 4 hours in vacuum or at atmospheric pressure with an inert gas.

[0729] In various embodiments, CVD graphene or graphene-based material can be liberated from its growth substrate (e.g., Cu) and transferred to a supporting grid, mesh or other supporting structure. In some embodiments, the supporting structure may be configured so that at least some portions of the sheet of graphene-based material are suspended from the supporting structure. For example, at least some portions of the sheet of graphene-based material may not be in contact with the supporting structure. [0730] In some embodiments, the sheet of graphene-based material following chemical vapor deposition comprises a single layer of graphene having at least two surfaces and non- graphenic carbon based material may be provided on said surfaces of the single layer graphene. In some embodiments, the non-graphenic carbon based material may be located on one of the two surfaces or on both. In some further embodiments, additional graphenic carbon may also present on the surface(s) of the single layer graphene.

[0731] In embodiments of the disclosure herein, the particle beam is a nanoparticle beam or cluster beam. In further embodiments, the beam is collimated or is not collimated. Furthermore, the beam need not be highly focused. In some embodiments, a plurality of the nanoparticles or clusters is singly charged. In additional embodiments, the nanoparticles comprise from 500 to 250,000 atoms or from 500 to 5,000 atoms.

[0732] A variety of metal particles are suitable for use in the methods of the present disclosure. For example, nanoparticles of Al, Ag, Au, Ti, Cu and nanoparticles comprising Al, Ag, Au, Ti, Cu are suitable. Metal Ps can be generated in a number of ways including magnetron sputtering and liquid metal ion sources (LMIS). Methods for generation of nanoparticles are further described in Cassidy, Cathal, et al. "Inoculation of silicon nanoparticles with silver atoms." Scientific reports 3 (2013), Haberland, Hellmut, et al. "Filling of micron- sized contact holes with copper by energetic cluster impact." Journal of Vacuum Science & Technology A 12.5 (1994): 2925-2930, Bromann, Karsten, et al. "Controlled deposition of size- selected silver nanoclusters." Science 274.5289 (1996): 956-958, Palmer, R. E., S. Pratontep, and H-G. Boyen. "Nanostructured surfaces from size-selected clusters." Nature Materials 2.7 (2003): 443-448, Shyjumon, L, et al. "Structural deformation, melting point and lattice parameter studies of size selected silver clusters." The European Physical Journal D-Atomic, Molecular, Optical and Plasma Physics 37.3 (2006): 409-415, Allen, L. P., et al. "Craters on silicon surfaces created by gas cluster ion impacts." Journal of applied physics 92.7 (2002): 3671-3678, Wucher, Andreas, Hua Tian, and Nicholas Winograd. "A Mixed Cluster Ion Beam to Enhance the Ionization Efficiency in Molecular Secondary Ion Mass Spectrometry." Rapid communications in mass spectrometry : RC 28.4 (2014): 396-400. PMC. Web. 6 Aug. 2015 and Pratontep, S., et al. "Size-selected cluster beam source based on radio frequency magnetron plasma sputtering and gas condensation." Review of scientific instalments 76.4 (2005): 045103, each of which is hereby incorporated by reference for its description of nanoparticle generation techniques.

[0733] Gas cluster beams can be made when high pressure gas adiabatically expands in a vacuum and cools such that it condenses into clusters. Clusters can also be made ex situ such as C60 and then accelerated towards the graphene.

[0734] In some embodiments, the nanoparticles are specially selected to introduce moieties into the graphene. In some embodiments, the nanoparticles function as catalysts. The moieties may be introduced at elevated temperatures, optionally in the presence of a gas. In other embodiments, the nanoparticles introduce" chiseling" moieties, which are moieties that help reduce the amount of energy needed to remove an atom during irradiation.

[0735] In embodiments, the size of the perforation apertures is controlled by controlling both the nanoparticle size and the nanoparticle energy. Without wishing to be bound by any particular belief, if all the nanoparticles have sufficient energy to perforate, then the resulting perforation is believed to correlated with the nanoparticle sizes selected. However, the size of the perforation is believed to be influenced by deformation of the nanoparticle during the perforation process. This deformation is believed to be influenced by both the energy and size of the nanoparticle and the stiffness of the graphene layer(s). A grazing angle of incidence of the nanoparticles can also produce deformation of the nanoparticles. In addition, if the nanoparticle energy is controlled, it is believed that nanoparticles can be deposited with a large mass and size distribution, but that a sharp cutoff can still be achieved.

[0736] Without wishing to be bound by any particular belief, the mechanism of perforation is believed to be a continuum bound by sputtering on one end (where enough energy is delivered to the graphene sheet to atomize the carbon in and around the P impact site) and ripping or fracturing (where strain induced failure opens a torn hole but leaves the graphene carbons as part of the original sheet). Part of the graphene layer may fold over at the site of the rip or fracture. In an embodiment the cluster can be reactive so as to aid in the removal of carbon (e.g. an oxygen cluster or having trace amounts of a molecule known to etch carbon in a gas cluster beam i.e. a mixed gas cluster beam). Without wishing to be bound by any particular belief, the stiffness of a graphene layer is believed to be influenced by both the elastic modulus of graphene and the tautness of the graphene. Factors influencing the elastic modulus of a graphene layer are believed to include temperature, defects (either small defects or larger defects from P irradiation), phy si sorption, chemisorption and doping. Tautness is believed to be influenced by coefficient of thermal expansion mismatches (e.g. between substrate and graphene layer) during deposition, strain in the graphene layer, wrinkling of the graphene layer. It is believed that strain in a graphene layer can be influenced by a number of factors including application of gas pressure to the backside (substrate side) of a graphene layer, straining of an elastic substrate prior to deposition of graphene, flexing of the substrate during deposition, and defecting the graphene layer in controlled regions to cause the layer to locally contract and increase the local strain.

[0737] In embodiments, nanoparticle perforation can be further controlled by straining the graphene layers during perforation to induce fracture, thereby "ripping" or "tearing" one or more graphene layers. In some embodiments, the stress is directional and used to preferentially fracture in a specific orientation. For example, ripping of one or more graphene sheets can be used to create "slit" shaped apertures; such apertures can be substantially larger than the nanoparticle used to initiate the aperture. In additional embodiments, the stress is not oriented in a particular direction.

[0738] In some embodiments, enclosures can be further modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the spirit and scope of the disclosure. Accordingly, the enclosures and methods are not limited by the foregoing description.

[0739] Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently. When a compound is described herein such that a particular isomer or enantiomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomer and enantiomer of the compound described individually or in any combination. One of ordinary skill in the art will appreciate that methods, device elements, starting materials and synthetic methods other than those specifically exemplified can be employed in the practice herein without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, starting materials and synthetic methods are intended to be included herein. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure.

[0740] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements. Embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0741] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope . Thus, it should be understood that although some embodiments have been specifically disclosed, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of embodiments as defined by the appended claims.

[0742] In general the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The preceding definitions are provided to clarify their specific use in the context herein. [0743] All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).

[0744] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which embodiments pertain. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art, in some cases as of their filing date, and it is intended that this information can be employed herein, if needed, to exclude (for example, to disclaim) specific embodiments that are in the prior art. For example, when a compound is claimed, it should be understood that compounds known in the prior art, including certain compounds disclosed in the references disclosed herein (particularly in referenced patent documents), are not intended to be included in the claims.

[0745] Working Examples

[0746] Some embodiments are further illustrated by the following examples. The examples are provided for illustrative purposes only, and are not to be construed as limiting the scope or content of embodiments any way.

[0747] Example 1: Cytotoxicity Testing: Neural Red Uptake (NRU) Cytotoxicity

[0748] The in vitro bioreactivity of L929 mouse fibroblast cell cultures were quantitatively determined in response to an extract of test material. The cells were grown to semi-confluency in 96-well tissue culture plates, so that they formed a half-confluent monolayer. An extract of the test material was prepared in Minimum Essential Medium (MEM), dipped in ethanol and allowed to dry, and then transferred onto the cell layer in the culture plate. Positive and negative controls were prepared in the same way. All extracts were dosed in 6 replicates except for the controls, which were dosed in 12 replicates. Control materials were tested at 100%

concentration (neat), and the test materials were tested at 100% (neat), 50%, 25%, and 12.5% concentrations. [0749] The plates were incubated for 24 to 26 hours at 37 + 1°C in a humified atmosphere containing 5 + 1 % CO ₂ . After examination of the plates, the culture medium was carefully removed and the cells were washed with Phosphate Buffer Saline (PBS). 100 μΙ_, of Neutral Red (NR) medium (50 μg/mL in MEM without Phenol Red, filtered through a 0.2 μπι filter and used the same day) was added to each test well, and the plates were further incubated for 3 + 0.2 hours in an incubator at 37 + 1°C. The cells were then washed with PBS and 100 μΙ_, of ethanol/acetic acid solution was added to each test well to extract the NR. The optical density (OD) of each well was measured at 540 nm.

[0750] The number of living cells was correlated to the intensity determined by photometric measurements after desorption of the NR. That is, a decrease in the number of living cells was directly correlated with the amount of NR, as monitored by absorbance at 540 nm. A reduction in viability of the test material as compared to a blank (i.e., cells exposed to extraction medium) was calculated using the following equation:

[0751] Viability % = 100 x OD _540e / OD _540b , where OD _540e is the mean value of the measured optical density of the extracts of the test material, positive control, or negative control; and OD _540b is the mean value of the measured optical density of the blanks. The test system was considered suitable if the following conditions were met: (a) the viability % for the negative control was > 70%, (b) the viability % for the positive control was < 70%, and (c) the mean of each replicate of the untreated control (i.e., each column in the tabulated results for the untreated control) was within + 15% of the untreated control mean. If the viability of test material sample was < 70%), it was considered to have cytotoxic potential.

[0752] Cytotoxicity testing was conducted with the following test materials: (i)

uncoated are substrate; (ii) unperforated graphene on substrate; and (iii) perforated graphene on substrate. Photographs of exemplary test materials are shown in Figure 72. The substrate used in the test materials was track-etched polyimide. Perforated graphene was prepared via nanoparticle perforation. Briefly, two layers of CVD graphene material were prepared. Each layer received ion beam treatment on Cu growth substrate, transfer to polymer (TEPI) for testing, UV-oxygen treatment and 300°C bakeout for 8 hours before the layers were stacked. The stacked layers were exposed to 6.5kV Ag nanoparticles (NP). The NP distribution was centered on 6nm and the fluence was approximately 5x 10 ¹⁰ Ps /cm ² . The nanoparticles were provided at an incidence angle of approximately greater than 45 degrees to the normal of the basal plane of the sheet of graphene material. The NPs were of 9-11 nm diameter at an energy of 30keV. The pores were typically 10-12 nm at their base, and varied in length from about 20nm to 70mm. Uncoated/bare substrate

[0753] Cytotoxicity of the uncoated/bare substrate was tested using the parameters in Table 1.

Table 1 : Parameters used to prepare test and control articles for uncoated/bare substrate cytotoxicity experiments

[0754] The results of the cytotoxicity testing are shown in Table 2.

Table 2: Cell viability results in experiments with uncoated/bare substrate

[0755] Based on these results, the substrate material was determined to not have a cytotoxic effect. Unperforated graphene on substrate

[0756] Cytotoxicity of uncoated graphene disposed on the substrate was tested using the parameters in Table 3.

Table 3 : Parameters used to prepare test and control articles for cytotoxicity experiments with unperforated graphene disposed on the substrate

[0757] The results of the cytotoxicity testing are shown in Table 4.

Table 4: Cell viability results in experiments with unperforated graphene disposed on the substrate

[0758] Based on these results, the unperforated graphene on substrate material was determined to not have a cytotoxic effect.

Perforated graphene on substrate

[0759] Cytotoxicity of perforated graphene disposed on the substrate was tested using the parameters in Table 5. Table 5: Parameters used to prepare test and control articles for cytotoxicity experiments with perforated graphene disposed on the substrate

[0760] The results of the cytotoxicity testing are shown in Table 6.

Table 6: Cell viability results in experiments with perforated graphene disposed on the substrate

[0761] Based on these results, the perforated graphene on substrate material was determined to not have a cytotoxic effect.

Example 2: Implantation Testing

[0762] A muscle implantation test was used to assess local effects on living tissue when test materials or devices are implanted. Test materials measured approximately 1mm in width and 10 mm in length. Test materials included (i) uncoated/bare substrate; (ii) unperforated graphene on substrate; and (iii) perforated graphene on substrate. The thickness of the materials was essentially the thickness of the polymer. Control strips also measured 1 mm in width and 10 mm in length. [0763] Prior to implantation, the materials were sterilized in 70% ethanol. Then, 6 strips were implanted into each of the paravertebral muscles of a rabbit, approximately 2.5 cm from the midline and parallel to the spinal column and approximately 2.5 cm from each other. The test material was folded in half, so that the graphene-side (if applicable) was facing out (for uncoated/bare substrate a tape layer was applied, and the test material was implanted with the taped layer facing out) and implanted on one side of the spine. In a similar fashion, negative control strips were implanted in the contralateral muscle of each animal. A total of at least 10 test material strips and 10 control strips were required for evaluation.

[0764] The animals were maintained for 2 weeks under observation to ensure proper healing of implant sites and for clinical signs of toxicity. At the end of the 2-week period, the animals were weighed and sacrificed by an injectable barbiturate. Sufficient time was allowed to lapse for the tissue to be cut without bleeding.

[0765] The paravertebral muscles in which the test or control strips were implanted were excised in toto from each animal by slicing around the implant sites with a scalpel and lifting the tissue. Excised tissue was examined grossly and placed in containers with 10% neutral buffered formalin. Axillary lymph nodes were examined and found to be free of abnormalities, and so they were not collected.

[0766] Following fixation in formalin, each of the implant sites was excised from the larger mass of tissue and examined macroscopically for signs of inflammation, encapsulation, hemorrhaging, necrosis, and discoloration using the following scale: 0 = normal; 1 = mild; 2 = moderate; and 3 = severe. In all cases, the presence, form, and location of the implanted material appeared unchanged.

[0767] After macroscopic observation, the implant material was removed and a slide of tissue containing the implant site was processed. Histologic slides of hematoxylin and eosin stained sections were prepared and evaluated by light microscopic examination. The biological reaction (inflammatory responses and healing responses) were assessed by microscopic observation and the responses graded and recorded according to Tables 7 and 8.

Table 7: Inflammatory Responses

Table 8: Healing Responses

[0768] The relative size of the involved area was scored by assessing the width of the area from the implant/tissue interface to unaffected areas which have the characteristics of normal tissue and normal vascularity. Relative size of the involved area was scored using the following scale: 0 = 0 mm, no site; 1 = up to 0.5 mm, very slight; 2 = 0.6-1.0 mm, mild; 3 = 1.1-2.0 mm, moderate; 4 = > 2.0 mm, severe.

[0769] For each implanted site, a total score was determined. The inflammatory responses were totaled for each site and weighted by a factor of 2. The healing responses were totaled separately. Inflammatory and healing responses were added together resulting in a total score for each site. The average score of the test sites was compared to the average score of the control sites for that animal. The average difference between the test and controls for all animals was calculated and a bioreactivity rating was assigned as follows: 0.0-2.9 = no reaction (negative calculations were reported as 0); 3.0-8.9 = slight reaction; 9.0-15.0 = moderate reaction; and > 15.0 = severe reaction. [0770] A pathologist reviewed the calculated level of reactivity. Based on the observation of all factors (e.g., relative size, pattern of response, inflammatory vs. resolution), the pathology observer was given leeway to revise the bioreactivity rating, if justified.

[0771] Implantation results are presented below for the following test materials: (i) uncoated are substrate; (ii) unperforated graphene on substrate; and (iii) perforated graphene on substrate.

Uncoated/bare substrate

[0772] In implantation testing with uncoated/bare substrate, all three test animals decreased a biologically insignificant amount (less than 10%) in weight, as shown in Table 9.

Table 9: Animal weights and clinical observations - 2-week implantation with uncoated/bare substrate

[0773] None of the animals exhibited signs of toxicity over the course of the study.

Macroscopic evaluation indicated no significant signs of inflammation, encapsulation, hemorrhage, necrosis, or discoloration, as shown in Table 10. Table 10: Macroscopic observations - 2-week implantation with uncoated/bare substrate

[0774] Microscopic evaluation of the implant sites indicated no significant signs of inflammation, fibrosis, neovascsularization, or fatty infiltrate as compared to control material sites, as shown in Table 1 1.

Table 1 1 : Microscopic evaluations with uncoated/bare substrate

[0775] Based on the above data, the bioreactivity rating was calculated to be 0.7.

Unperforated graphene on substrate

[0776] In implantation testing with unperforated graphene disposed on the substrate, all three test animals decreased a biologically insignificant amount (less than 6%) in weight, as shown in Table 12.

Table 12: Animal weights and clinical observations - 2-Week implantation with unperforated graphene on substrate

[0777] None of the animals exhibited signs of toxicity over the course of the study.

Macroscopic evaluation indicated no significant signs of inflammation, encapsulation, hemorrhage, necrosis, or discoloration, as shown in Table 13.

Table 13 : Macroscopic observations - 2-week implantation with unperforated graphene on substrate

[0778] Microscopic evaluation of the implant sites indicated no significant signs of inflammation, fibrosis, neovascsularization, or fatty infiltrate as compared to control material sites, as shown in Table 14. At one test site, a few MNGs were present only at the non-graphene side. Table 14: Microscopic evaluations with unperforated graphene on substrate

[0779] Based on the above data, the bioreactivity rating was calculated to be 2.2.

Perforated graphene on substrate

[0780] In implantation testing with perforated graphene disposed on the substrate, two animals lost between 3% and 10% of their body weights, and one animal maintained its weig as shown in Table 15:

Table 15: Animal weights and clinical observations - 2-week implantation with perforated graphene on substrate

[0781] None of the animals exhibited signs of toxicity over the course of the study.

Macroscopic evaluation indicated no significant signs of inflammation, encapsulation, hemorrhage, or necrosis, as shown in Table 16. Mild discoloration was noted in several control sites.

Table 16: Macroscopic observations - 2-week implantation with perforated graphene on substrate

[0782] Microscopic evaluation of the implant sites indicated no significant signs of inflammation, fibrosis, neovascsularization, or fatty infiltrate as compared to control material sites, as shown in Table 17.

Table 17: Microscopic evaluations with perforated graphene on substrate

[0783] Based on the above data, the bioreactivity rating was calculated to be 0.0.

Example 3 - Permeability Study - Allura Red AC and Silver Nanoparticles

[0784] Permeability of small (Allura Red AC) and large (silver nanoparticles) across a SiN substrate layer and a perforated graphene layer were assessed via diffusion cell experiments. Permeability was compared to a control membrane with selectivity on the order of nanometers. Results are displayed in Figure 84, where "Bare AM Chip" is the SiN substrate, "Biopore" is the control membrane, and "Nanoporous graphene" is perforated graphene. In the Figure, results were normalized to permeability of substances through the control membrane based on area of the tested substances. Also, permeability results for Allura Red AC and silver nanoparticles through the control membrane were also normalized to each other (based on the raw data, permeability of Allura Red AC through the control membrane high than permeability of silver nanoparticles through the control membrane). The data shows that less Allura Red AC permeated the perforated graphene layer than the SiN layer. Moreover, to an even larger extent, the perforated graphene layer restricted silver nanoparticles from traversing the layer, as compared to the SiN layer.

Example 4 - Diffusive Transport of Fluorescein and IgG

[0785] Diffusive transport of fluorescein conjugated to IgG was assessed via diffusion cell experiments with respect to the following materials: an SiN substrate layer (termed "Bare Chip" in Figure 85), perforated graphene, unperforated graphene, and a control membrane (termed "Biopore" in the Figure 85). The results showed that normalized flux of IgG through the SiN substrate layer was higher than through perforated graphene. The results also showed minimum flux of IgG through unperforated graphene. Without being bound by theory, it is believed that flux observed through unperforated graphene resulted from intrinsic or native defects in the graphene.

[0786] Figure 86 compares the permeability results in Figure 85 with permeability data obtained on fluorescein alone {i.e., not conjugated to IgG). The Figure demonstrates that fluorescein permeability was higher than IgG permeability.

Example 5 - Permeability Study - FluoSpheres and Fluorescein

[0787] Permeability of 100 nm diameter Red (580/605) FluoSpheres and fluorescein was assessed via diffusion cell experiments with perforated graphene. As shown in Figure 87, FluoSpheres added to the left side of diffusion cell did not traverse the perforated graphene. However, fluorescein added to the left side of the diffusion cell traversed the perforated graphene, and then was detected on the right side of the diffusion cell.

Example 6 - Permeability Study - Fluorescein Across Various Substrates

[0788] Permeability of fluorescein across various substrates was measured via Permegear cells. The experiments were conducted with 7 mm diameter test materials, and with 5 μΜ fluorescein in PBSA buffer at room temperature. As shown in Figure 88, the following test materials were used: (i) a control membrane ("Biopore"); (ii) uncoated substrate TEPI-400/7

{i.e., 400 nm pores, 7 μιη thick substrate); (ii) TEPI-400/7 coated with two layers of unperforated graphene ("SLG2 Unperf); (iii) uncoated substrate TEPI-460/25; (iv) TEPI-460/25 coated with two layers of unperforated graphene; and (v) TEPI-460/25 coated with two layers of perforated graphene, where the graphene was perforated with silver nanoparticles.

[0789] The data showed that unperforated graphene substantially reduced the amount of fluorescein that traversed the substrate layers. The data also showed the coating the substrate with perforated graphene did not substantially alter permeability of the substrate. That is, the permeability of fluorescein across uncoated TEPI-460/25 was similar to that of fluorescein across TEPI-460/25 coated with perforate graphene. This was the case even if only a small percentage of graphene suspended across the substrate was perforated. For instance, the data show similar results when 12-15%, 8-10%, 5-6%, 4-5%. 3-4% or 2-3% of the graphene suspended across the substrate was porous.

[0790] Additional data (not shown) further demonstrated that permeability of fluorescein across the substrate was enhanced by etching the substrate with NaOCl.

[0791] Figure 89A and 89B show diffusion for small fluorescent dye molecules (fluorescein, Figure 89A) and large 100nm FluoSpheres (Figure 89B) through uncoated substrate,

unperforated graphene-coated substrate, and perforated graphene-coated substrate. The plots show the relative fluorescence intensity for the low concentration solution side of the membrane over time. The uncoated substrate was highly permeable to both fluorescent analytes, whereas the unperforated graphene-coated membrane had low permeablility. Size selectivity of the nanoperforated graphene membrane was demonstrated by its relatively high permeability to the smaller analyte (fluorescein, Figure 8 A) and low permeability to the larger analyte

(FluoSpheres, Figure 89B). Both analytes were diluted in phosphate-buffered saline

solution. Donor concentrations were 5μΜ for fluorescein and 200ppm for FluoSpheres. The substrate was a track-etched polymeric membrane with pore diameters ranging between approximately 350-450 nm. Sample area available for diffusion was 49 mm ² . Testing was performed at room temperature. HEALING OF THIN GRAPHENIC -BASED MEMBRANES VIA CHARGED PARTICLE

IRRADIATION

[0792] Various aspects of the subject technology provide methods and systems for healing intrinsic or native defects, such as tears, of a graphenic-based membrane, such as a graphene- based membrane or a graphitic-based membrane.

[0793] The intrinsic or native defects in the graphenic-based membrane, in particular those of a larger size such as some tears, may adversely affect the integrity of the membrane, or may reduce its function as a filter when the graphenic-based membrane has selectively introduced perforations. In particular, when the intrinsic or native defects are of a larger size than the selectively introduced pores, the graphenic-based membrane may allow a species of a larger size to pass through the intrinsic or native defects, where the species of a larger size is intended to be restricted from passing through the membrane. On the other hand, intrinsic or native defects below a critical size required for application-specific separation may be useful from a permeability perspective, as long as such defects do not negatively impact the integrity of the membrane.

[0794] According to certain concepts disclosed herein, the region of the graphenic-based membrane where the intrinsic or native defects exist may be first identified. In other embodiments the region of the graphenic-based membrane where the intrinsic or native defects exist need not be identified prior to the healing process. If a region is to be identified, the identified region is then irradiated with charged particles of an appropriate flux, fluence and energy to heal the intrinsic or native defect in the region, thus increasing the integrity, and in certain embodiments, maintaining the filter function of the graphenic-based membrane.

[0795] Forming graphenic-based membrane

[0796] The membrane according to embodiments may be, for example, a graphenic-based membrane, such as a graphene-based membrane or a graphitic-based membrane, formed according to various methods, as described below. Once the membrane is formed as described for example in this section on forming a graphenic-based membrane, the membrane may be subject to a healing process as described later in the section on healing. [0797] In an embodiment, the membrane may be a macroscale sheet suitable for formation of perforations through exposure of the sheet to electrons or ions. In a further embodiment, the macroscale sheet may be suitable for formation of perforations through exposure of the sheet to ultraviolet light and an oxygen containing gas such as air. The perforated sheets described herein have a variety of applications including, but not limited to, filtration applications. Suspended macroscale sheets and methods for making macroscale sheets comprising single layer or multilayer graphene are also described herein.

[0798] In embodiments, the sheet of graphene-based material comprises a sheet of single layer graphene, multilayer graphene, or a combination thereof. In an embodiment, the sheet of graphene-based material may be formed by chemical vapor deposition (CVD) followed by at least one additional conditioning or treatment step. In embodiments, the conditioning step may be selected from thermal treatment, UV-oxygen treatment, ion beam treatment, or combinations thereof. The treated sheet may include reduced graphene oxide membranes and carbon nanomembranes (C M). Carbon nanomembranes are typically generated from crosslinked aromatic self-assembled monolayers and can be approximately 1 nm thick (M. Ai and A.

Golzhauser, Beilstein Bozen Symposium on Molecular Engineering and Control May 14th - 18th, 2012, Prien (Chiemsee), Germany). These Carbon nanomembranes (CNM) are appropriate for being perforated. Further embodiments include combinations of these Carbon

nanomembranes with graphene, such as graphene/CNM, CNM/graphene, and

graphene/ CNM/graphene .

[0799] In an embodiment, thermal treatment may include heating to a temperature from 200 °C to 800 °C at a pressure of 10-7 Torr to atmospheric pressure for a time of 2 hours to 8 hours. In an embodiment, UV-ozone treatment involves exposure to light from 150 nm to 300 nm and an intensity from 10 to 100 mW/cm ² or 100 to 1000 mW/cm ² at from 1 mm to 50 mm distance for a time from 60 to 600 seconds. In embodiments, UV-oxygen treatment may be performed at room temperature or at a temperature greater than room temperature. In further embodiments, UV-oxygen treatment may be performed at atmospheric pressure (e.g. 1 atm) or under vacuum. In an embodiment, ion beam treatment may involve exposure of the graphene-based material to ions having an ion energy from 50 eV to 1000 eV (for pretreatment) and the fluence is from 3 x 1010 ions/cm 2 to 8 x 1011 ions/cm 2 or 3 x 1010 ions/cm 2 to 8 x 1013 ions/cm 2 (for pretreatment). In a further embodiment, the source of ions is collimated, such as a broad beam or flood source. In an embodiment, the ions may be noble gas ions such as Xe. In an embodiment, one or more conditioning steps may be performed while the graphene-based material is attached to a substrate, such as a growth substrate.

[0800] In an embodiment, the sheet of graphene-based material following chemical vapor deposition comprises multilayer graphene having at least two surfaces and carbonaceous material provided on said surfaces of the multilayer graphene. In embodiments, the carbonaceous material may be located on one of the two surfaces or on both. In a further embodiment, additional grapheme carbon may also be present on the surface(s) of the multilayer graphene.

[0801] In an embodiment, the graphene-based material is not perforated after the

conditioning step(s). In an embodiment the conditioning/treatment process does not substantially affect the domain size or extent of defects in the material. As examples, said multilayer graphene before or after conditioning treatment may be characterized by an average size domain for long range order greater than or equal to 1 micrometer, long range lattice periodicity on the order of 1 micrometer and/or has an extent of disorder characterized by less than 1% content of lattice defects. The domains may have a size in the range from 30 micrometers to 3 millimeters, or from 100 micrometers to 1 centimeter for example. In other embodiments the

conditioning/treatment process may heal some of the defects so as to reduce the number of defects in the material.

[0802] In an embodiment, the carbonaceous material may further comprise non-carbon elements. In an embodiment, said non-carbon elements may be selected from the group consisting of hydrogen, oxygen, silicon, copper, iron, aluminum, magnesium, calcium, boron, and nitrogen and combinations thereof. In an embodiment, aluminum, magnesium, calcium, boron, and nitrogen may be present only in trace amounts. In an embodiment, the carbonaceous material may have an elemental composition comprising carbon, hydrogen and oxygen. In a further embodiment, the carbonaceous material may have a molecular composition comprising amorphous carbon, one or more hydrocarbons, oxygen containing carbon compounds, nitrogen containing carbon compounds or any combination of these. In a further embodiment, the non- carbon element, such as boron or silicon substitutes for carbon in the lattice. In an embodiment the carbonaceous material may not exhibit long range order. In an embodiment, the

carbonaceous material may be in physical contact with at least one of said surfaces of the multilayer graphene. In an embodiment, the characteristics of the carbonaceous carbon material are those as determined after at least one conditioning process.

[0803] Also provided herein are sheets of graphene-based material suspended over a supporting structure. In various embodiments, CVD graphene or graphene-based material can be liberated from its growth substrate (e.g., Cu) and transferred to a supporting grid, mesh or other porous supporting structure. In embodiments, the porous supporting structure may be polymeric, metallic or ceramic. In an embodiment, the supporting structure may be configured so that at least some portions of the sheet of graphene-based material are suspended from the supporting structure. For example, at least some portions of the sheet of graphene-based material are not in contact with the supporting structure. In an embodiment, the suspended area may be greater than 10 nm and less than 10 micrometers. In an embodiment, a sheet of graphene-based material comprising: multilayer graphene having at least two surfaces; and a carbonaceous material provided on said multilayer layer graphene; wherein exposure of said sheet of graphene-based material to ions characterized by an ion energy ranging from 10 eV to 100 keV and a fluence ranging from produces perforations in the sheet of graphene-

based material. In a further embodiment, at least a portion of the single layer graphene may be suspended. In yet a further embodiment, a mask or template is not present between the source of ions and the sheet of graphene-based material. In a further embodiment, the source of ions may be collimated, such as a broad beam or flood source. In embodiments the ions are noble gas ions, and are selected from the group consisting of Xe+, Ne+, or Ar+, or are He+ ions.

[0804] In an embodiment, the ions may be selected from the group consisting of Xe+, Ne+, or Ar+, the ion energy ranges from 5 eV to 50 eV and the ion dose ranges from 5x 10 ¹⁴ ions/cm ² to 5x 10 ¹⁵ ions/cm ² . In an embodiment, the ion energy ranges from 1 keV to 40 keV and the ion dose ranges from 1x10 19 ions/cm 2 to 1x1021 ions/cm 2. These parameters can be used for He ions. In a further embodiment, a background gas may be present during ion irradiation. For example, the sheet of graphene-based material may be exposed to the ions in an environment comprising partial pressure of 5 x 10 ^-4 Torr to 5 x 10 ^-5 Torr of oxygen, nitrogen or carbon dioxide at a total pressure of 10 ^-3 Torr to 10 ^-5 Torr. In yet a further embodiment the ion irradiation conditions when a background gas is present include an ion energy ranging from 100 eV to 1000 eV and an ion dose ranging from 1 x10 ¹³ ions/cm ² to lx10 ¹⁴ ions/cm ² . A quasi-neutral plasma may be used under these conditions.

[0805] In an aspect, the macroscale sheet of graphene-based material may be suitable for formation of perforations over greater than 10% or greater or 15% or greater of said area of said sheet of graphene-based material. In combination, at least one lateral dimension of the sheet may be from 10 nm to 10 cm, or greater than 1 mm to less than or equal to 10 cm, or lateral dimensions as described herein. In an embodiment, the mean of the pore size may be from 0.3 nm to 1 μπι. In embodiments, the coefficient of variation of the pore size may be from 0.1 to 2. In an embodiment, a perforated (hole) area correspond to 0.1% or greater of said area of said sheet of graphene-based material. In a further embodiment, the perforations may be

characterized by an average area of said perforations selected from the range of 0.2 nm ² to 0.25 μm ² .

[0806] Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or a few layered sheets (e.g., about 20 or less) of fused six- membered rings forming an extended sp2-hybridized carbon planar lattice. Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. In an embodiment, graphene-based materials also include materials which have been formed by stacking single or multilayer graphene sheets. In embodiments, multilayer graphene includes 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In an embodiment, layers of multilayered graphene may be stacked, but are less ordered in the z direction (perpendicular to the basal plane) than a thin graphite crystal.

[0807] In an embodiment, the membrane may be a sheet of graphene-based material which is a sheet of single or multilayer graphene or a sheet comprising a plurality of interconnected single or multilayer graphene domains, which may be observed in any known manner such as using for example small angle electron diffraction, transmission electron microscopy, etc. In embodiments, the multilayer graphene domains have 2 to 5 layers or 2 to 10 layers. As used herein, a "domain" refers to a region of a material where atoms are uniformly ordered into a crystal lattice. A domain is uniform within its boundaries, but different from a neighboring region. For example, a single crystalline material has a single domain of ordered atoms. In an embodiment, at least some of the graphene domains may be nanocrystals, having domain size from 1 to 100 nm or 10-100 nm. In an embodiment, at least some of the graphene domains have a domain size greater than 100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm. In an embodiment, a domain of multilayer graphene may overlap a neighboring domain. "Grain boundaries" formed by crystallographic defects at edges of each domain differentiate between neighboring crystal lattices. In some embodiments, a first crystal lattice may be rotated relative to a second crystal lattice, by rotation about an axis perpendicular to the plane of a sheet, such that the two lattices differ in "crystal lattice orientation."

[0808] In an embodiment, the membrane may be a sheet of graphene-based material comprising a sheet of single or multilayer graphene or a combination thereof. In an embodiment, the sheet of graphene-based material may be a sheet of single or multilayer graphene or a combination thereof. In another embodiment, the sheet of graphene-based material may be a sheet comprising a plurality of interconnected single or multilayer graphene domains. In an embodiment, the interconnected domains may be covalently bonded together to form the sheet. When the domains in a sheet differ in crystal lattice orientation, the sheet may be polycrystalline. In an embodiment the single layer graphene may be characterized by an average size domain for long range order greater than or equal to 1 μπι. In a further embodiment the single layer graphene has an extent of disorder characterized as an average distance between crystallographic defects of 100nm.

[0809] In embodiments, the thickness of the sheet of graphene-based material may be from 0.3 to 10 nm, 0.34 to 10 nm, from 0.34 to 5 nm, or from 0.34 to 3 nm. In an embodiment, the thickness includes both single layer graphene and the non-graphenic carbon.

[0810] In an embodiment, the membrane may be a sheet of graphene-based material comprising intrinsic or native defects. Intrinsic or native defects are those resulting from preparation of the graphene-based material in contrast to perforations which are selectively introduced into a sheet of graphene-based material or a sheet of graphene. Such intrinsic or native defects include, but are not limited to, lattice anomalies, pores, tears, cracks or wrinkles. Lattice anomalies can include, but are not limited to, carbon rings with other than 6 members (e.g. 5, 7 or 9 membered rings), vacancies, interstitial defects (including incorporation of non- carbon atoms in the lattice), and grain boundaries. As used herein, perforations do not include openings in the graphene lattice due to intrinsic or native defects or grain boundaries.

[0811] In embodiments, graphene may be the dominant material in a graphene-based material. For example, a graphene-based material comprises at least 20% graphene, 30% graphene, or at least 40% graphene, or at least 50% graphene, or at least 60% graphene, or at least 70%) graphene, or at least 80% graphene, or at least 90% graphene, or at least 95% graphene. In embodiments, a graphene-based material comprises a range of graphene selected from 30% to 95%, or from 40% to 80% from 50% to 70%, from 60% to 95% or from 75% to 100%). The amount of graphene in the graphene-based material is measured as an atomic percentage utilizing known methods including transmission electron microscope examination and the like, measuring mechanical properties such as with an atomic force microscope, etc. In an embodiment, the amount of graphene in the graphene-based material is measured as an atomic percentage.

[0812] In an embodiment, the membrane may be a sheet of graphene-based material further comprises carbonaceous material located on at least one surface of the sheet of graphene-based material. In an embodiment, the sheet may be defined by two base surfaces (e.g. top and bottom faces of the sheet) and side faces. In a further embodiment, the "bottom" face of the sheet is that face which contacted the substrate during CVD growth of the sheet and the "free" face of the sheet opposite the "bottom" face. In an embodiment, carbonaceous carbon material may be located on one or both base surfaces of the sheet (e.g. the substrate side of the sheet and/or the free surface of the sheet). In a further embodiment, the sheet of graphene-based material includes a small amount of one or more other materials on the surface, such as, but not limited to, one or more dust particles or similar contaminants.

[0813] In an embodiment, the membrane may be a layer comprising the sheet of graphene- based material further comprising carbonaceous material located on the surface of the sheet of graphene-based material. In an embodiment, the carbonaceous material does not possess long range order and may be classified as amorphous. In embodiments, the carbonaceous material further comprises elements other than carbon and/or hydrocarbons. In an embodiment, non- carbon elements which may be incorporated in the carbonaceous material include hydrogen, oxygen, silicon, copper and iron. In further embodiment, the carbonaceous material comprises hydrocarbons. In embodiments, carbon may be the dominant material in carbonaceous carbon material.

[0814] Perforation techniques suitable for use in perforating the graphene-based materials described herein include ion-based perforation methods and UV-oxygen based methods.

[0815] Ion-based perforation methods include methods in which the graphene-based material is irradiated with a directional source of ions. In a further embodiment, the ion source may be collimated. In an embodiment the ion source may be a broad beam or flood source. A broad field or flood ion source can provide an ion flux which is significantly reduced compared to a focused ion beam. The ion source inducing perforation of the graphene or other two- dimensional material in embodiments of the present disclosure is considered to provide a broad ion field, also commonly referred to as an ion flood source. In an embodiment, the ion flood source does not include focusing lenses. In embodiments, the ion source may be operated at less than atmospheric pressure, such as at 10 ^-3 to 10 ^-5 Torr or 10 ^-4 to 10 ^-6 Torr. In an embodiment, the environment also contains background amounts (e.g. on the order of 10 ^-5 Torr) of oxygen (02), nitrogen (N2) or carbon dioxide (C02). In embodiments, the ion beam may be perpendicular to the surface of the layer(s) of the material (incidence angle of 0 degrees) or the incidence angle may be from 0 to 45 degrees, 0 to 20 degrees, 0 to 15 degrees or 0 to 10 degrees. In a further embodiment, exposure to ions does not include exposure to a plasma.

[0816] UV-oxygen based perforation methods include methods in which the graphene-based material is simultaneously exposed to ultraviolet (UV) light and an oxygen containing gas.

Ozone may be generated by exposure of an oxygen containing gas such as oxygen or air to the UV light, in which case the graphene-based material is exposed to oxygen. Ozone may also be supplied by an ozone generator device. In an embodiment, the UV-ozone based perforation method further includes exposure of the graphene-based material to atomic oxygen. Suitable wavelengths of UV light include, but are not limited to wavelengths below 300 nm or from 150 nm to 300 nm. In embodiments, the intensity is from 10 to 100 mW/cm ² at 6mm distance or 100 to 1000 mW/cm ² at 6mm distance. For example, suitable light is emitted by mercury discharge lamps (e.g. about 185 nm and 254 nm). In embodiments, UV/ozone cleaning is performed at room temperature or at a temperature greater than room temperature. In further embodiments, UV/ozone cleaning is performed at atmospheric pressure (e.g. 1 atm) or under vacuum.

[0817] Perforations are sized as described herein to provide desired selective permeability of a species (atom, molecule, protein, virus, cell, etc.) for a given application. Selective

permeability relates to the propensity of a porous material or a perforated two-dimensional material to allow passage (or transport) of one or more species more readily or faster than other species. Selective permeability allows separation of species which exhibit different passage or transport rates. In two-dimensional materials selective permeability correlates to the dimension or size (e.g., diameter) of apertures and the relative effective size of the species. Selective permeability of the perforations in two-dimensional materials such as graphene-based materials can also depend on functionalization of perforations (if any) and the specific species that are to be separated. Separation of two or more species in a mixture includes a change in the ratio(s) (weight or molar ratio) of the two or more species in the mixture after passage of the mixture through a perforated two-dimensional material.

[0818] In embodiments, the characteristic size of the perforation may be from 0.3 to 10 nm, from 1 to 10 nm, from 5 to 10 nm, from 5 to 20 nm, from 10 nm to 50 nm, from 50 nm to 100 nm, from 50 nm to 150 nm, from 100 nm to 200 nm, or from 100 nm to 500 nm. In an embodiment, the average pore size is within the specified range. In embodiments, 70% to 99%, 80% to 99%, 85% to 99% or 90 to 99% of the perforations in a sheet or layer fall within a specified range, but other pores fall outside the specified range.

[0819] Nanomaterials in which pores are intentionally created may be referred to herein as "perforated graphene," "perforated graphene-based materials" or "perforated two-dimensional materials, and the like." Perforated graphene-based materials include materials in which non- carbon atoms have been incorporated at the edges of the pores. Pore features and other material features may be characterized in a variety of manners including in relation to size, area, domains, periodicity, coefficient of variation, etc. For instance, the size of a pore may be assessed through quantitative image analysis utilizing images preferentially obtained through transmission electron microscopy, and if TEM is ineffective, through atomic force microscopy, and if AFM is ineffective, through scanning electron microscopy. The boundary of the presence and absence of material identifies the contour of a pore. The size of a pore may be determined by shape fitting of an expected species against the imaged pore contour where the size measurement is characterized by smallest dimension unless otherwise specified. For example, in some instances, the shape may be round or oval. The round shape exhibits a constant and smallest dimension equal to its diameter. The width of an oval is its smallest dimension. The diameter and width measurements of the shape fitting in these instances provide the size measurement, unless specified otherwise.

[0820] Each pore size of a test sample may be measured to determine a distribution of pore sizes within the test sample. Other parameters may also be measured such as area, domain, periodicity, coefficient of variation, etc. Multiple test samples may be taken of a larger membrane to determine that the consistency of the results properly characterizes the whole membrane. In such instance, the results may be confirmed by testing the performance of the membrane with test species. For example, if measurements indicate that certain sizes of species should be restrained across the membrane, a performance test provides verification with test species. Alternatively, the performance test may be utilized as an indicator that the pore measurements will determine a concordant pore size, area, domains, periodicity, coefficient of variation, etc.

[0821] In some embodiments, the perforations are characterized by a distribution of pores with a dispersion characterized by a coefficient of variation of 0.1 to 2. The size distribution of holes may be narrow, e.g., limited to a coefficient of variation less than 2. In some embodiments involving circular shape fitting, the equivalent diameter of each pore is calculated from the equation A= π d2/4. Otherwise, the area is a function of the shape fitting. When the pore area is plotted as a function of equivalent pore diameter, a pore size distribution may be obtained. The coefficient of variation of the pore size may be calculated herein as the ratio of the standard deviation of the pore size to the mean of the pore size as measured across the test samples. The average area of perforations is an averaged measured area of the pores as measured across the test samples.

[0822] In some embodiments, the ratio of the area of the perforations to the ratio of the area of the sheet may be used to characterize the sheet as a density of perforations. The area of a test sample may be taken as the planar area spanned by the test sample. Additional sheet surface area may be excluded due to wrinkles other non-planar features. Characterization may be based on the ratio of the area of the perforations to the test sample area as density of perforations excluding features such as surface debris. Characterization may be based on the ratio of the area of the perforations to the suspended area of the sheet. As with other testing, multiple test samples may be taken to confirm consistency across tests and verification may be obtained by performance testing. The density of perforations may be, for example, 2 per nm ² (2/ nm ² ) to 1 per μm ² (1/ μm ² ).

[0823] In an embodiment, the ratio of the area of the perforations to the ratio of the area of the sheet may be used to characterize the sheet. The area of the perforations may be measured using quantitative image analysis. The area of the sheet may be taken as the planar area spanned by the sheet if it is desired to exclude the additional sheet surface area due to wrinkles or other non-planar features of the sheet. In a further embodiment, characterization may be based on the ratio of the area of the perforations to the sheet area excluding features such as surface debris. In an additional embodiment, characterization may be based on the ratio of the area of the perforations to the suspended area of the sheet.

[0824] In embodiments, the perforated area comprises 0.1% or greater, 1% or greater or 5% or greater of the sheet area, less than 10% of the sheet area, less than 15% of the sheet area, from 0.1%) to 15%) of the sheet area, from 1%> to 15%> of the sheet area, from 5% to 15%> of the sheet area or from 1%> to 10%> of the sheet area. In a further embodiment, the perforations may be located over greater than 10%> or greater than 15%> of said area of said sheet of graphene-based material. As used herein, a macroscale sheet is macroscopic and observable by the naked eye. In embodiments, at least one lateral dimension of the sheet is greater than 1 cm, greater than 1 mm or greater than 5 mm. For example, the sheet has a lateral dimension greater than about 1 micrometer. In an additional embodiment, the lateral dimension of the sheet may be less than 10 cm. In an embodiment, the sheet has a lateral dimension greater than 1 mm and less than 10 cm. As used herein, a lateral dimension is perpendicular to the thickness of the sheet.

[0825] Chemical vapor deposition growth of graphene-based material typically involves use of a carbon containing precursor material, such as methane and a growth substrate. In an embodiment, the growth substrate is a metal growth substrate. In an embodiment, the metal growth substrate is a substantially continuous layer of metal rather than a grid or mesh. Metal growth substrates compatible with growth of graphene and graphene-based materials include transition metals and their alloys. In embodiments, the metal growth substrate is copper based or nickel based. In embodiments, the metal growth substrate is copper or nickel. In embodiments, the graphene-based material is removed from the growth substrate by dissolution of the growth substrate.

[0826] Healing process

[0827] FIG. 93 illustrates a method of forming a graphenic-based membrane, including a particle irradiation healing process, according to concepts disclosed herein.

[0828] In step 9310, a graphenic-based membrane is produced, such as, for example, by the techniques described above. The graphenic-based membrane may be a graphene-based membrane. For example, the graphenic-based membrane may be produced as follows. An initial graphenic-based membrane is formed on a growth substrate, by a method such as chemical vapor deposition (CVD), for example. In the case that the graphenic-based membrane is a thin graphitic membrane, the thin graphitic membrane may be formed by, for example, electrostatic deposition, or a casting method, In the casting method, graphite is exfoliated and put into suspension, such as by using a modified hummers method. The suspension is then deposited on a substrate surface by drop casting, or if the substrate is porous, by vacuum deposited. Thin graphitic membranes may also be made by carbon nanomembranes (C M) which are formed from a deposited and crosslinked layers of polycyclic aromatic hydrocarbons (PAHs), such as phenol compounds, for example.

[0829] The initial graphenic-based membrane is removed from the growth substrate, and the removed graphenic-based membrane may be positioned on a support substrate. [0830] The initial graphenic-based membrane may be subject to a conditioning treatment, for example, while the initial graphenic-based membrane is on the growth substrate. The conditioning treatment may comprise a thermal treatment, a UV-oxygen treatment, or ion beam treatment, or combinations of these treatments. A plurality of selectively introduced perforations may also be formed in the initial graphenic-based membrane in the case the graphenic-based membrane is intended to function as filter, for example.

[0831] In general, the graphenic-based membrane produced in step 9310 has one or more layers of graphenic-based material, and further has carbonaceous material on a surface of the one or more layers of graphenic-based material. For example, if the graphenic-based membrane is a graphene-based membrane, the layers may be layers of graphene. The graphenic-based membrane further has intrinsic or native defects.

[0832] In step 9320, a region where the intrinsic or native defects exist, and are to be healed, is identified. The region may be identified according to the size of the intrinsic or native defects which exists. For example, the region may be identified only if the size of the intrinsic or native defects is above a characteristic defect size. The existence of intrinsic or native defects may be identified by methods such as measuring the electrical conductivity of the membrane, or detecting secondary electrons emitted when irradiating the membrane with primary electrons, such as during a scanning transmission electron micrograph scan. When scanning an ion beam over the membrane, defects can be detected as dark regions, which are areas where the secondary electron yield is low or non-existent, indicating that there is no material there. Methods for detecting the existence of intrinsic or native defects include Raman spectroscopy; residual gas analysis on particles being removed from the material; detecting back scattered radiation or particles; detecting Auger electrons; performing scanning probe microscopy; performing scanning tunneling microscopy; performing atomic force microscopy; performing X-ray spectroscopy; performing transmission electron microscopy; detecting nanoparticles on one or more microbalances or Faraday cups positioned behind the material.

[0833] In the case that the graphenic-based membrane comprises a plurality of selectively introduced perforations in addition to the intrinsic or native defects, the region may be identified only if the intrinsic or native defects are larger than the selectively introduced perforations. For example, the selectively introduced perforations may have a first characteristic size, and the region for healing may be identified only when intrinsic or native defects of a second characteristic size larger than the first characteristic size are present. The first characteristic size may be the equivalent diameter of the selectively introduced perforations, and the second characteristic size may be the equivalent diameter of intrinsic or native defects. Alternatively, the first characteristic size may be the minimum dimension (minor axis) of the selectively introduced perforations, and the second characteristic size may be the minimum dimension of intrinsic or native defects.

[0834] In step 9330, the identified region is subject to an intrinsic or native defect healing process. For example, the identified region may be irradiated with charged particles to heal the intrinsic or native defects. The healing process may include irradiating the identified region of the graphenic-based membrane with charged particles having an ion energy ranging from 50 eV to 1000 eV, or 50 eV to 40 keV and the fluence may be from 3 x 10 ¹⁰ ions/cm ² to 8 x 10 ¹¹ ions/cm 2 , 3 x 1010 ions/cm 2 to 8 x 1013 ions/cm 2 , or 3 x 1010 ions/cm 2 to 1 x 1019 ions/cm 2. The flux may be from 10 10 ions/cm 2 /s to 1014 ions/cm 2 /s, or from 1010 ions/cm 2 /s to 10 IT ions/cm 2 /s. The irradiation may include irradiating with a broad beam or a flood source, for example. The irradiation is done in the presence of carbonaceous material so that mobile carbonaceous surface contamination is present.

[0835] The graphenic-based membrane produced in step 9310 may have carbonaceous material on a surface of the stacked layers of the graphenic-based membrane. The carbonaceous material may be a material such as amorphous carbon, one or more hydrocarbons, oxygen containing carbon compounds, nitrogen containing carbon compounds, or combination thereof. While not being restricted to any particular theory for the mechanism for healing, the healing may be facilitated by presence of the carbonaceous material. The charged particle irradiation may provide sufficient energy for the carbonaceous material to mobilize carbon atoms which migrate and/or move to the intrinsic or native defect regions of the graphitic material in the graphenic-based membrane, where the mobilized carbon form sp2 bonds with graphitic material at the intrinsic or native defect. Thus, the intrinsic or native defect may be healed. The energy and the flux of the charged particles should be sufficient to mobilize the carbon of the carbonaceous material, but not so high as to damage the graphitic material in the graphenic- based membrane. At some energies the charged particle irradiation is milling (damaging) the graphenic-based membrane, but with sufficient carbon supply, the healing out competes the milling. The charged particle irradiation facilitates liberating the carbon from the carbonaceous material, such that it can be incorporated into the defected graphitic material.

[0836] According to an embodiment, the carbonaceous material need not initially be present in sufficient quantities on the surface of the graphenic-based membrane to allow for complete healing of the intrinsic or native defect. In this case, the carbonaceous material may be introduced to the surface of the graphenic-based membrane while the surface is irradiated with charged particles to heal the intrinsic or native defects. For example, carbon may be sputtered to the surface of the graphenic-based membrane while the surface is irradiated with charged particles. Alternatively, the graphenic-based membrane may be exposed to hydrocarbon gases while the surface is irradiated with charged particles, or the graphenic-based membrane may be exposed to carbon containing fluid at low enough temperature to be adsorbed on the membrane surface and the surface is then irradiated with charged particles. Furthermore, according to embodiments, the membrane may be heated while healing or afterwards, which further facilitates the healing.

[0837] Examples

[0838] FIG. 94 is a scanning transmission electron microscopy (STEM) micrograph of a graphene-based material before intentional charged particle irradiation.

[0839] FIG. 95 is a magnified image of the STEM micrograph of FIG. 94 with arrows pointing to some identified defects. The brighter portions of the micrograph correspond to surface contamination on the graphene-based material, such as carbonaceous material. In FIG. 95, the graphene-based material has not yet been irradiated with charged particle irradiation at a level for healing, but only for taking the STEM micrograph. The box in FIG. 95 corresponds to the portion of the graphene-based material which will be subjected to charged particle irradiation for healing.

[0840] FIG. 96 is a STEM micrograph of the region shown in FIG. 95 after some charged particle irradiation for healing. In particular the region was scanned with a highly focused electron beam at 60kV with a beam current of 40-60pA and 1024 dwells per 8nm. The defects indicated in FIG. 96 have changed relative to those in FIG. 95.

[0841] FIG. 97 is a STEM micrograph of another region of the graphene-based material shown in FIG. 94 before charged particle irradiation. Similar to FIG. 95, the box in FIG. 97 corresponds to the portion of the graphene-based material which will subjected to charged particle irradiation for healing, and the arrows point to identified defects.

[0842] FIG. 98 is a STEM micrograph of the region shown in FIG. 97 after charged particle irradiation for healing in a similar manner to that shown for FIG. 96. As can be seen, the defects indicated in FIG. 98 have changed relative to those in FIG. 97. In particular, FIG. 98 illustrates a significantly lower number of defects in the portion irradiated.

[0843] The embodiments of the concepts disclosed herein have been described in detail with particular reference to preferred embodiments thereof, but it will be understood by those skilled in the art that variations and modifications can be effected within the spirit and scope of the concepts.

PERFORATABLE SHEETS OF GRAPHENE-BASED MATERIAL

[0844] Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or a few layered sheets (e.g., about 20 or less) of fused six- membered rings forming an extended sp ² -hybridized carbon planar lattice. Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. In some embodiments, graphene-based materials also include materials which have been formed by stacking single or multilayer graphene sheets. In some embodiments, multilayer graphene includes 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In some embodiments, layers of multilayered graphene are stacked, but are less ordered in the z direction (perpendicular to the basal plane) than a thin graphite crystal.

[0845] In some embodiments, a sheet of graphene-based material is a sheet of single or multilayer graphene or a sheet comprising a plurality of interconnected single or multilayer graphene domains, which may be observed in any known manner such as using for example small angle electron diffraction, transmission electron microscopy, etc. In some embodiments, the multilayer graphene domains have 2 to 5 layers or 2 to 10 layers. As used herein, a domain refers to a region of a material where atoms are substantially uniformly ordered into a crystal lattice. A domain is uniform within its boundaries, but may be different from a neighboring region. For example, a single crystalline material has a single domain of ordered atoms. In some embodiments, at least some of the graphene domains are nanocrystals, having domain size from 1 to 100 nm or 10-100 nm. In some embodiments, at least some of the graphene domains have a domain size greater than 100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm. In some embodiments, a domain of multilayer graphene may overlap a neighboring domain. Grain boundaries formed by crystallographic defects at edges of each domain may differentiate between neighboring crystal lattices. In some embodiments, a first crystal lattice may be rotated relative to a second crystal lattice, by rotation about an axis perpendicular to the plane of a sheet, such that the two lattices differ in crystal lattice orientations.

[0846] In some embodiments, the sheet of graphene-based material comprises a sheet of single or multilayer graphene or a combination thereof. In some embodiments, the sheet of graphene-based material is a sheet of single or multilayer graphene or a combination thereof. In some embodiments, the sheet of graphene-based material is a sheet comprising a plurality of interconnected single or multilayer graphene domains. In some embodiments, the interconnected domains are covalently bonded together to form the sheet. When the domains in a sheet differ in crystal lattice orientation, the sheet is polycrystalline. In some embodiments, said single layer graphene is characterized by an average size domain for long range order greater than or equal to Ι μπι. In some embodiments, said single layer graphene has an extent of disorder characterized an average distance between crystallographic defects of 100nm.

[0847] In some embodiments, the thickness of the sheet of graphene-based material is from 0.3 to 10 nm, 0.34 to 10 nm, from 0.34 to 5 nm, or from 0.34 to 3 nm. In some embodiments, the thickness includes both single layer graphene and the non-graphenic carbon.

[0848] In some embodiments, a sheet of graphene-based material comprises intrinsic or native defects. Intrinsic or native defects may result from preparation of the graphene-based material in contrast to perforations which are selectively introduced into a sheet of graphene- based material or a sheet of graphene. Such intrinsic or native defects may include, but are not limited to, lattice anomalies, pores, tears, cracks or wrinkles. Lattice anomalies can include, but are not limited to, carbon rings with other than 6 members (e.g. 5, 7 or 9 membered rings), vacancies, interstitial defects (including incorporation of non-carbon atoms in the lattice), and grain boundaries. Perforations are distinct from openings in the graphene lattice due to intrinsic or native defects or grain boundaries, but testing and characterization of the final membrane such as mean pore size and the like encompasses all openings regardless of origin since they are all present.

[0849] In some embodiments, graphene is the dominant material in a graphene-based material. For example, a graphene-based material may comprise at least 20% graphene, 30% graphene, or at least 40% graphene, or at least 50% graphene, or at least 60% graphene, or at least 70%) graphene, or at least 80% graphene, or at least 90% graphene, or at least 95% graphene. In some embodiments, a graphene-based material comprises a range of graphene selected from 30% to 95%, or from 40% to 80% from 50% to 70%, from 60% to 95% or from 75%) to 100%). The amount of graphene in the graphene-based material is measured as an atomic percentage utilizing known methods including transmission electron microscope examination, or alternatively if TEM is ineffective another similar technique.

[0850] In some embodiments, a sheet of graphene-based material further comprises non- graphenic carbon-based material located on at least one surface of the sheet of graphene-based material. In some embodiments, the sheet is exemplified by two base surfaces (e.g. top and bottom faces of the sheet, opposing faces) and side faces. In a further embodiment, the "bottom" face of the sheet is that face which contacted the substrate during CVD growth of the sheet and the "free" face of the sheet opposite the "bottom" face. In some embodiments, non-graphenic carbon-based material may be located on one or both base surfaces of the sheet (e.g. the substrate side of the sheet and/or the free surface of the sheet). In some further embodiments, the sheet of graphene-based material includes a small amount of one or more other materials on the surface, such as, but not limited to, one or more dust particles or similar contaminants.

[0851] In some embodiments, the amount of non-graphenic carbon-based material is less than the amount of graphene. In some further embodiments, the amount of non-graphenic carbon material is three to five times the amount of graphene; this may be measured in terms of mass. In some additional embodiments, the non-graphenic carbon material is characterized by a percentage by mass of said graphene-based material selected from the range of 0% to 80%. In some embodiments, the surface coverage of the sheet by non-graphenic carbon-based material is greater than zero and less than 80%, from 5% to 80%, from 10% to 80%, from 5% to 50% or from 10%) to 50%. This surface coverage may be measured with transmission electron microscopy, which gives a projection. In some embodiments, the amount of graphene in the graphene-based material is from 60% to 95% or from 75% to 100%. The amount of graphene in the graphene-based material is measured as mass percentage utilizing known methods preferentially using transmission electron microscope examination, or alternatively if TEM is ineffective using other similar techniques.

[0852] In some embodiments, the layer comprising the sheet of graphene-based material further comprises non-graphenic carbon-based material located on the surface of the sheet of graphene-based material. In some embodiments, the non-graphenic carbon-based material does not possess long range order and may be classified as amorphous. In some embodiments, the non-graphenic carbon-based material further comprises elements other than carbon and/or hydrocarbons. In some embodiments, non-carbon elements which may be incorporated in the non-graphenic carbon include hydrogen, oxygen, silicon, copper and iron. In some further embodiments, the non-graphenic carbon-based material comprises hydrocarbons. In some embodiments, carbon is the dominant material in non-graphenic carbon-based material. For example, a non-graphenic carbon-based material in some embodiments comprises at least 30% carbon, or at least 40% carbon, or at least 50% carbon, or at least 60% carbon, or at least 70% carbon, or at least 80% carbon, or at least 90% carbon, or at least 95% carbon. In some embodiments, a non-graphenic carbon-based material comprises a range of carbon selected from 30% to 95%, or from 40% to 80%, or from 50% to 70%. The amount of carbon in the non- graphenic carbon-based material is measured as an atomic percentage utilizing known methods preferentially using transmission electron microscope examination, or alternatively if TEM is ineffective, using other similar techniques. [0853] In some embodiments, the surface mobility of the non-graphenic carbon-based material is such that, when irradiated with the perforation parameters described in this application, the non-graphenic carbon-based material has a surface mobility such that the perforation process results ultimately in perforation. Without wishing to be bound by any particular belief, hole formation is believed to related to beam induced carbon removal from the graphene sheet and thermal replenishment of carbon in the hole region by non graphenic carbon. The replenishment process is dependent upon energy entering the system during perforation and the resulting surface mobility of the non-graphenic carbon based material. To form holes, the rate of graphene removal is higher than the non-graphenic carbon hole filling rate. These competing rates depend on the non-graphenic carbon flux (mobility [viscosity and temperature] and quantity) and the graphene removal rate (particle mass, energy, flux).

[0854] In some embodiments, the volatility of the non-graphenic carbon-based material is less than that which is obtained by heating the sheet of graphene-based material to 500°C for 4 hours in vacuum or at atmospheric pressure with an inert gas.

[0855] Perforation techniques suitable for use in perforating the graphene-based may include ion-based perforation methods and UV-oxygen based methods.

[0856] Ion-based perforation methods include methods in which the graphene-based material is irradiated with a directional source of ions. In some further embodiments, the ion source is collimated. In some embodiments, the ion source is a broad beam or flood source. A broad field or flood ion source can provide an ion flux which is significantly reduced compared to a focused ion beam. The ion source inducing perforation of the graphene or other two- dimensional material is considered to provide a broad ion field, also commonly referred to as an ion flood source. In some embodiments, the ion flood source does not include focusing lenses. In some embodiments, the ion source is operated at less than atmospheric pressure, such as at 10- 3 to 10 ^-5 torr or 10 ^-4 to 10 ^-6 torr In some embodiments, the environment also contains background amounts (e.g. on the order of 10 ^-5 torr) of oxygen (O ₂ ), nitrogen (N ₂ ) or carbon dioxide (CO ₂ ). In some embodiments, the ion beam may be perpendicular to the surface of the layer(s) of the material (incidence angle of 0 degrees) or the incidence angle may be from 0 to 45 degrees, 0 to 20 degrees, 0 to 15 degrees or 0 to 10 degrees. In some further embodiments, exposure to ions does not include exposure to plasma.

[0857] In some embodiments, UV-oxygen based perforation methods include methods in which the graphene-based material is simultaneously exposed to ultraviolet (UV) light and an oxygen containing gas. Ozone may be generated by exposure of an oxygen containing gas such as oxygen or air to the UV light, in which case the graphene-based material is exposed to oxygen. Ozone may also be supplied by an ozone generator device. In some embodiments, the UV-ozone based perforation method further includes exposure of the graphene-based material to atomic oxygen. Suitable wavelengths of UV light include, but are not limited to wavelengths below 300 nm or from 150 nm to 300 nm. In some embodiments, the intensity from 10 to 100 mW/cm ² at 6mm distance or 100 to 1000 mW/cm ² at 6mm distance. For example, suitable light is emitted by mercury discharge lamps (e.g. about 185 nm and 254 nm). In some embodiments, UV/ozone cleaning is performed at room temperature or at a temperature greater than room temperature. In some further embodiments, UV/ozone cleaning is performed at atmospheric pressure (e.g. 1 atm) or under vacuum.

[0858] Perforations are sized as described herein to provide desired selective permeability of a species (atom, molecule, protein, virus, cell, etc.) for a given application. Selective

permeability relates to the propensity of a porous material or a perforated two-dimensional material to allow passage (or transport) of one or more species more readily or faster than other species. Selective permeability allows separation of species which exhibit different passage or transport rates. In two-dimensional materials selective permeability correlates to the dimension or size (e.g., diameter) of apertures and the relative effective size of the species. Selective permeability of the perforations in two-dimensional materials such as graphene-based materials can also depend on functionalization of perforations (if any) and the specific species. Separation or passage of two or more species in a mixture includes a change in the ratio(s) (weight or molar ratio) of the two or more species in the mixture during and after passage of the mixture through a perforated two-dimensional material.

[0859] In some embodiments, the characteristic size of the perforation is from 0.3 to 10 nm, from 1 to 10 nm, from 5 to 10 nm, from 5 to 20 nm, from 10 nm to 50 nm, from 50 nm to 100 nm, from 50 nm to 150 nm, from 100 nm to 200 nm, or from 100 nm to 500 nm. In some embodiments, the average pore size is within the specified range. In some embodiments, 70% to 99%, 80% to 99%, 85% to 99% or 90 to 99% of the perforations in a sheet or layer fall within a specified range, but other pores fall outside the specified range.

[0860] Nanomaterials in which pores are intentionally created may be referred to as perforated graphene, perforated graphene-based materials, perforated two-dimensional materials, and the like. Perforated graphene-based materials include materials in which non-carbon atoms have been incorporated at the edges of the pores. Pore features and other material features may be characterized in a variety of manners including in relation to size, area, domains, periodicity, coefficient of variation, etc. For instance, the size of a pore may be assessed through quantitative image analysis utilizing images preferentially obtained through transmission electron

microscopy, and if TEM is ineffective, through scanning electron microscopy and the like, as for example presented in Figs. 1 and 2. The boundary of the presence and absence of material identifies the contour of a pore. The size of a pore may be determined by shape fitting of an expected species against the imaged pore contour where the size measurement is characterized by smallest dimension unless otherwise specified. For example, in some instances, the shape may be round or oval. The round shape exhibits a constant and smallest dimension equal to its diameter. The width of an oval is its smallest dimension. The diameter and width measurements of the shape fitting in these instances provide the size measurement, unless specified otherwise.

[0861] Each pore size of a test sample may be measured to determine a distribution of pore sizes within the test sample. Other parameters may also be measured such as area, domain, periodicity, coefficient of variation, etc. Multiple test samples may be taken of a larger membrane to determine that the consistency of the results properly characterizes the whole membrane. In such instance, the results may be confirmed by testing the performance of the membrane with test species. For example, if measurements indicate that certain sizes of species should be restrained from transport across the membrane, a performance test provides verification with test species. Alternatively, the performance test may be utilized as an indicator that the pore measurements will determine a concordant pore size, area, domains, periodicity, coefficient of variation, etc. [0862] In some embodiments, the perforations are characterized by a distribution of pores with a dispersion characterized by a coefficient of variation of 0.1 to 2. The size distribution of holes may be narrow, e.g., limited to a coefficient of variation less than 2. In some

embodiments, the characteristic dimension of the holes is selected for the application. In some embodiments involving circular shape fitting, the equivalent diameter of each pore is calculated from the equation A= π d ² /4. Otherwise, the area is a function of the shape fitting. When the pore area is plotted as a function of equivalent pore diameter, a pore size distribution may be obtained. The coefficient of variation of the pore size may be calculated herein as the ratio of the standard deviation of the pore size to the mean of the pore size as measured across the test samples. The average area of perforations is an averaged measured area of the pores as measured across the test samples.

[0863] In some embodiments, the ratio of the area of the perforations to the ratio of the area of the sheet may be used to characterize the sheet as a density of perforations. The area of a test sample may be taken as the planar area spanned by the test sample. Additional sheet surface area may be excluded due to wrinkles other non-planar features. Characterization may be based on the ratio of the area of the perforations to the test sample area as density of perforations excluding features such as surface debris. Characterization may be based on the ratio of the area of the perforations to the suspended area of the sheet. As with other testing, multiple test samples may be taken to confirm consistency across tests and verification may be obtained by performance testing. The density of perforations may be, for example, 2 per nm ² (21 nm ² to 1 per μm ² (1/ μm ² ).

[0864] In some embodiments, the perforated area comprises 0.1% or greater, 1% or greater or 5% or greater of the sheet area, less than 10% of the sheet area, less than 15% of the sheet area, from 0.1% to 15% of the sheet area, from 1% to 15% of the sheet area, from 5% to 15% of the sheet area or from 1% to 10% of the sheet area. In some further embodiments, the perforations are located over greater than 10% or greater than 15% of said area of said sheet of graphene-based material. A macroscale sheet is macroscopic and observable by the naked eye. In some embodiments, at least one lateral dimension of the sheet is greater than 1 cm, greater than 1 mm or greater than 5 mm. In some further embodiments, the sheet is larger than a graphene flake which would be obtained by exfoliation of graphite in known processes used to make graphene flakes. For example, the sheet has a lateral dimension greater than about 1 micrometer. In an additional embodiment, the lateral dimension of the sheet is less than 10 cm. In some embodiments, the sheet has a lateral dimension (e.g., perpendicular to the thickness of the sheet) greater than 1 mm and less than 10 cm. Chemical vapor deposition growth of graphene-based material typically involves use of a carbon containing precursor material, such as methane and a growth substrate. In some embodiments, the growth substrate is a metal growth substrate. In some embodiments, the metal growth substrate is a substantially continuous layer of metal rather than a grid or mesh. Metal growth substrates compatible with growth of graphene and graphene-based materials include transition metals and their alloys. In some embodiments, the metal growth substrate is copper based or nickel based. In some

embodiments, the metal growth substrate is copper or nickel. In some embodiments, the graphene-based material is removed from the growth substrate by dissolution of the growth substrate.

[0865] The preferred embodiments may be further understood by the following non-limiting examples.

[0866] EXAMPLES

[0867] FIG. 99 is a transmission electron microscope image illustrating a graphene based material after conditioning treatment.

[0868] FIG. 100 is another transmission electron microscope image showing a graphene based material after conditioning treatment.

[0869] The graphene based material was synthesized using chemical vapor deposition. After synthesis, the material was exposed to an ion beam while on the copper growth substrate; the ions were Xe ions at 500V at 80°C with a fluence of 1.25 x 10 ¹³ ions/cm ² . Then the graphene based material was transferred to a TEM grid and while suspended received 120 seconds of treatment at atmospheric pressure with atmospheric gas with Ultra- Violet (UV) parameters as described herein. The graphene based material was baked at 160 °C for about 6 hours before imaging. In FIG. 99 and FIG. 100, label 9910 indicates single layer graphite regions while label 9920 indicates largely non-graphitic carbon based material. PERFORATED SHEETS OF GRAPHE E-BASED MATERIAL

[0870] Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or a few layered sheets (e.g., about 20 or less) of fused six- membered rings forming an extended sp2-hybridized carbon planar lattice. Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. In some embodiments, graphene-based materials also include materials which have been formed by stacking single or multilayer graphene sheets. In some embodiments, multilayer graphene includes 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In some embodiments, layers of multilayered graphene are stacked, but are less ordered in the z direction (perpendicular to the basal plane) than a thin graphite crystal.

[0871] In some embodiments, a sheet of graphene-based material may be a sheet of single or multilayer graphene or a sheet comprising a plurality of interconnected single or multilayer graphene domains, which may be observed in any known manner such as using for example small angle electron diffraction, transmission electron microscopy, etc.. In some embodiments, the multilayer graphene domains have 2 to 5 layers or 2 to 10 layers. As used herein, a domain refers to a region of a material where atoms are substantially uniformly ordered into a crystal lattice. A domain is uniform within its boundaries, but may be different from a neighboring region. For example, a single crystalline material has a single domain of ordered atoms. In some embodiments, at least some of the graphene domains are nanocrystals, having domain size from 1 to 100 nm or 10-100 nm. In some embodiments, at least some of the graphene domains have a domain size greater than 100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm. In some embodiments, a domain of multilayer graphene may overlap a neighboring domain. Grain boundaries formed by crystallographic defects at edges of each domain may differentiate between neighboring crystal lattices. In some embodiments, a first crystal lattice may be rotated relative to a second crystal lattice, by rotation about an axis perpendicular to the plane of a sheet, such that the two lattices differ in crystal lattice orientation.

[0872] In some embodiments, the sheet of graphene-based material is a sheet of single or multilayer graphene or a combination thereof. In some other embodiments, the sheet of graphene-based material is a sheet comprising a plurality of interconnected single or multilayer graphene domains. In some embodiments, the interconnected domains are covalently bonded together to form the sheet. When the domains in a sheet differ in crystal lattice orientation, the sheet is polycrystalline.

[0873] In some embodiments, the thickness of the sheet of graphene-based material is from 0.3 to 10 nm, 0.34 to 10 nm, from 0.34 to 5 nm, or from 0.34 to 3 nm. In some embodiments, the thickness includes both single layer graphene and the non-graphenic carbon.

[0874] In some embodiments, a sheet of graphene-based material comprises intrinsic or native defects. Intrinsic or native defects may result from preparation of the graphene-based material in contrast to perforations which are selectively introduced into a sheet of graphene- based material or a sheet of graphene. Such intrinsic or native defects may include, but are not limited to, lattice anomalies, pores, tears, cracks or wrinkles. Lattice anomalies can include, but are not limited to, carbon rings with other than 6 members (e.g. 5, 7 or 9 membered rings), vacancies, interstitial defects (including incorporation of non-carbon atoms in the lattice), and grain boundaries. Perforations are distinct from openings in the graphene lattice due to intrinsic or native defects or grain boundaries, but testing and characterization of the final membrane such as mean pore size and the like encompasses all openings regardless of origin since they are all present.

[0875] In some embodiments, graphene is the dominant material in a graphene-based material. For example, a graphene-based material may comprise at least 20% graphene, at least 30% graphene, or at least 40% graphene, or at least 50% graphene, or at least 60% graphene, or at least 70% graphene, or at least 80% graphene, or at least 90% graphene, or at least 95% graphene. In some embodiments, a graphene-based material comprises a range of graphene selected from 30% to 95%, or from 40% to 80% from 50% to 70%, from 60% to 95% or from 75%) to 100%). The amount of graphene in the graphene-based material is measured as an atomic percentage utilizing known methods including transmission electron microscope examination, or alternatively if TEM is ineffective another similar measurement technique.

[0876] In some embodiments, a sheet of graphene-based material further comprises non- graphenic carbon-based material located on at least one surface of the sheet of graphene-based material. In some embodiments, the sheet is exemplified by two base surfaces (e.g. top and bottom faces of the sheet, opposing faces) and side faces (e.g. the side faces of the sheet). In some further embodiments, the "bottom" face of the sheet is that face which contacted the substrate during growth of the sheet and the "free" face of the sheet opposite the "bottom" face. In some embodiments, non-graphenic carbon-based material may be located on one or both base surfaces of the sheet (e.g. the substrate side of the sheet and/or the free surface of the sheet). In some further embodiments, the sheet of graphene-based material includes a small amount of one or more other materials on the surface, such as, but not limited to, one or more dust particles or similar contaminants.

[0877] In some embodiments, the amount of non-graphenic carbon-based material is less than the amount of graphene. In some further embodiments, the amount of non-graphenic carbon material is three to five times the amount of graphene; this is measured in terms of mass. In some additional embodiments, the non-graphenic carbon material is characterized by a percentage by mass of said graphene-based material selected from the range of 0% to 80%. In some embodiments, the surface coverage of the sheet of non-graphenic carbon-based material is greater than zero and less than 80%, from 5% to 80%, from 10% to 80%, from 5% to 50% or from 10%) to 50%. This surface coverage may be measured with transmission electron microscopy, which gives a projection. In some embodiments, the amount of graphene in the graphene-based material is from 60%> to 95% or from 75% to 100%. The amount of graphene in the graphene-based material is measured as a mass percentage utilizing known methods preferentially using transmission electron microscope examination, or alternatively if TEM is ineffective using other similar techniques.

[0878] In some embodiments, the non-graphenic carbon-based material does not possess long range order and is classified as amorphous. In some embodiments, the non-graphenic carbon-based material further comprises elements other than carbon and/or hydrocarbons. In some embodiments, non-carbon elements which may be incorporated in the non-graphenic carbon include hydrogen, oxygen, silicon, copper, and iron. In some further embodiments, the non-graphenic carbon-based material comprises hydrocarbons. In some embodiments, carbon is the dominant material in non-graphenic carbon-based material. For example, a non-graphenic carbon-based material in some embodiments comprises at least 30% carbon, or at least 40% carbon, or at least 50% carbon, or at least 60% carbon, or at least 70% carbon, or at least 80% carbon, or at least 90% carbon, or at least 95% carbon. In some embodiments, a non-graphenic carbon-based material comprises a range of carbon selected from 30% to 95%, or from 40% to 80%), or from 50% to 70%. The amount of carbon in the non-graphenic carbon-based material is measured as an atomic percentage utilizing known methods preferentially using transmission electron microscope examination, or alternatively if TEM is ineffective, using other similar techniques.

[0879] Perforation techniques suitable for use in perforating the graphene-based materials may include described herein ion-based perforation methods and UV-oxygen based methods.

[0880] Ion-based perforation methods include methods in which the graphene-based material is irradiated with a directional source of ions. In some further embodiments, the ion source is collimated. In some embodiments, the ion source is a broad beam or flood source. A broad field or flood ion source can provide an ion flux which is significantly reduced compared to a focused ion beam. The ion source inducing perforation of the graphene or other two- dimensional material is considered to provide a broad ion field, also commonly referred to as an ion flood source. In some embodiments, the ion flood source does not include focusing lenses. In some embodiments, the ion source is operated at less than atmospheric pressure, such as at 10- 3 to 10 ^-5 torr or 10 ^-4 to 10 ^-6 torr In some embodiments, the environment also contains background amounts (e.g. on the order of 10 ^-5 torr) of oxygen (O ₂ ), nitrogen (N ₂ ) or carbon dioxide (CO ₂ ). In some embodiments, the ion beam may be perpendicular to the surface of the layer(s) of the material (incidence angle of 0 degrees) or the incidence angle may be from 0 to 45 degrees, 0 to 20 degrees, 0 to 15 degrees or 0 to 10 degrees. In some further embodiments, exposure to ions does not include exposure to plasma.

[0881] In some embodiments, UV-oxygen based perforation methods include methods in which the graphene-based material is simultaneously exposed to ultraviolet (UV) light and an oxygen containing gas Ozone may be generated by exposure of an oxygen containing gas such as oxygen or air to the UV light. Ozone may also be supplied by an ozone generator device. In some embodiments, the UV-oxygen based perforation method further includes exposure of the graphene-based material to atomic oxygen. Suitable wavelengths of UV light include, but are not limited to wavelengths below 300 nm or from 150 nm to 300 nm. In some embodiments, the intensity from 10 to 100 mW/cm ² at 6mm distance or 100 to 1000 mW/cm ² at 6mm distance. For example, suitable light is emitted by mercury discharge lamps (e.g. about 185 nm and 254 nm). In some embodiments, UV/oxygen cleaning is performed at room temperature or at a temperature greater than room temperature. In some further embodiments, UV/oxygen cleaning is performed at atmospheric pressure (e.g. 1 atm) or under vacuum.

[0882] Perforations are sized as described herein to provide desired selective permeability of a species (atom, molecule, protein, virus, cell, etc.) for a given application. Selective

permeability relates to the propensity of a porous material or a perforated two-dimensional material to allow passage (or transport) of one or more species more readily or faster than other species. Selective permeability allows separation of species which exhibit different passage or transport rates. In two-dimensional materials selective permeability correlates to the dimension or size (e.g., diameter) of apertures and the relative effective size of the species. Selective permeability of the perforations in two-dimensional materials such as graphene-based materials can also depend on functionalization of perforations (if any) and the specific species. Separation or passage of two or more species in a mixture includes a change in the ratio(s) (weight or molar ratio) of the two or more species in the mixture during and after passage of the mixture through a perforated two-dimensional material.

[0883] In some embodiments, the characteristic size of the perforation is from 0.3 to 10 nm, from 1 to 10 nm, from 5 to 10 nm, from 5 to 20 nm, from 10 nm to 50 nm, from 50 nm to 100 nm, from 50 nm to 150 nm, from 100 nm to 200 nm, or from 100 nm to 500 nm. In some embodiments, the average pore size is within the specified range. In some embodiments, 70% to 99%, 80% to 99%, 85% to 99% or 90 to 99% of the perforations in a sheet or layer fall within a specified range, but other pores fall outside the specified range.

[0884] Nanomaterials in which pores are intentionally created may be referred to as perforated graphene, perforated graphene-based materials or perforated two-dimensional materials, and the like. Perforated graphene-based materials include materials in which non- carbon atoms have been incorporated at the edges of the pores. Pore features and other material features may be characterized in a variety of manners including in relation to size, area, domains, periodicity, coefficient of variation, etc. For instance, the size of a pore may be assessed through quantitative image analysis utilizing images preferentially obtained through transmission electron microscopy, and if TEM is ineffective, through scanning electron microscopy and the like, as for example presented in Figs. 101 and 102. The boundary of the presence and absence of material identifies the contour of a pore. The size of a pore may be determined by shape fitting of an expected species against the imaged pore contour where the size measurement is characterized by smallest dimension unless otherwise specified. For example, in some instances, the shape may be round or oval. The round shape exhibits a constant and smallest dimension equal to its diameter. The width of an oval is its smallest dimension. The diameter and width measurements of the shape fitting in these instances provide the size measurement, unless specified otherwise.

[0885] Each pore size of a test sample may be measured to determine a distribution of pore sizes within the test sample. Other parameters may also be measured such as area, domain, periodicity, coefficient of variation, etc. Multiple test samples may be taken of a larger membrane to determine that the consistency of the results properly characterizes the whole membrane. In such instance, the results may be confirmed by testing the performance of the membrane with test species. For example, if measurements indicate that certain sizes of species should be restrained from transport across the membrane, a performance test provides verification with test species. Alternatively, the performance test may be utilized as an indicator that the pore measurements will determine a concordant pore size, area, domains, periodicity, coefficient of variation, etc.

[0886] The size distribution of holes may be narrow, e.g., limited to 0.1-0.5 coefficient of variation. In some embodiments, the characteristic dimension of the holes is selected for the application.

[0887] In some embodiments involving circular shape fitting the equivalent diameter of each pore is calculated from the equation A= π d ² /4. Otherwise, the area is a function of the shape fitting. When the pore area is plotted as a function of equivalent pore diameter, a pore size distribution may be obtained. The coefficient of variation of the pore size may be calculated herein as the ratio of the standard deviation of the pore size to the mean of the pore size as measured across the test samples. The average area of perforations is an averaged measured area of pores as measured across the test samples.

[0888] In some embodiments, the ratio of the area of the perforations to the ratio of the area of the sheet may be used to characterize the sheet as a density of perforations. The area of a test sample may be taken as the planar area spanned by the test sample. Additional sheet surface area may be excluded due to wrinkles other non-planar features. Characterization may be based on the ratio of the area of the perforations to the test sample area as density of perforations excluding features such as surface debris. Characterization may be based on the ratio of the area of the perforations to the suspended area of the sheet. As with other testing, multiple test samples may be taken to confirm consistency across tests and verification may be obtained by performance testing. The density of perforations may be, for example, 2 per nm ² (2/ nm ² to 1 per μm ² (1/ μm ² ).

[0889] In some embodiments, the perforated area comprises 0.1% or greater, 1% or greater or 5% or greater of the sheet area, less than 10% of the sheet area, less than 15% of the sheet area, from 0.1% to 15% of the sheet area, from 1% to 15% of the sheet area, from 5% to 15% of the sheet area or from 1% to 10% of the sheet area. In some further embodiments, the

perforations are located over greater than 10% or greater than 15% of said area of said sheet of graphene-based material. A macroscale sheet is macroscopic and observable by the naked eye. In some embodiments, at least one lateral dimension of the sheet is greater than 3 cm, greater than 1 cm, greater than 1 mm or greater than 5 mm. In some further embodiments, the sheet is larger than a graphene flake which would be obtained by exfoliation of graphite in known processes used to make graphene flakes. For example, the sheet has a lateral dimension greater than about 1 micrometer. In an additional embodiment, the lateral dimension of the sheet is less than 10 cm. In some further embodiments, the sheet has a lateral dimension (e.g., perpendicular to the thickness of the sheet) from 10 nm to 10 cm or greater than 1 mm and less than 10 cm.

[0890] Chemical vapor deposition growth of graphene-based material typically involves use of a carbon containing precursor material, such as methane and a growth substrate. In some embodiments, the growth substrate is a metal growth substrate. In some embodiments, the metal growth substrate is a substantially continuous layer of metal rather than a grid or mesh. Metal growth substrates compatible with growth of graphene and graphene-based materials include transition metals and their alloys. In some embodiments, the metal growth substrate is copper based or nickel based. In some embodiments, the metal growth substrate is copper or nickel. In some embodiments, the graphene-based material is removed from the growth substrate by dissolution of the growth substrate.

[0891] In some embodiments, the sheet of graphene-based material is formed by chemical vapor deposition (CVD) followed by at least one additional conditioning or treatment step. In some embodiments, the conditioning step is selected from thermal treatment, UV-oxygen treatment, ion beam treatment, and combinations thereof. In some embodiments, thermal treatment may include heating to a temperature from 200 °C to 800 °C at a pressure of 10-7 torr to atmospheric pressure for a time of 2 hours to 8 hours. In some embodiments, UV-oxygen treatment may involve exposure to light from 150 nm to 300 nm and an intensity from 10 to 100 mW/cm ² at 6mm distance for a time from 60 to 1200 seconds. In some embodiments, UV- oxygen treatment may be performed at room temperature or at a temperature greater than room temperature. In some further embodiments, UV-oxygen treatment may be performed at atmospheric pressure (e.g. 1 atm) or under vacuum. In some embodiments, ion beam treatment may involve exposure of the graphene-based material to ions having an ion energy from 50 eV to 1000 eV (for pretreatment) and the fluence is from 3 x 10 ¹⁰ ions/cm ² to 8 x 10 ¹¹ ions/cm ² or 3 x

10 10 ions/cm 2 to 8 x 1013 ions/cm 2 (for pretreatment). In some further embodiments, the source of ions may be collimated, such as a broad beam or flood source. In some embodiments, the ions may be noble gas ions such as Xe ⁺ . In some embodiments, one or more conditioning steps are performed while the graphene-based material is attached to a substrate, such as a growth substrate.

[0892] In some embodiments, the conditioning treatment affects the mobility and/or volatility of the non-graphitic carbon-based material. In some embodiments, the surface mobility of the non-graphenic carbon-based material is such that when irradiated with perforation parameters such as described herein, the non-graphenic carbon-based material, may have a surface mobility such that the perforation process results ultimately in perforation. Without wishing to be bound by any particular belief, hole formation is believed to related to beam induced carbon removal from the graphene sheet and thermal replenishment of carbon in the hole region by non grapheme carbon. The replenishment process may be dependent upon energy entering the system during perforation and the resulting surface mobility of the non-graphenic carbon based material. To form holes, the rate of graphene removal may be higher than the non- graphenic carbon hole filling rate. These competing rates depend on the non-graphenic carbon flux (e.g., mobility [viscosity and temperature] and quantity) and the graphene removal rate (e.g., particle mass, energy, flux).

[0893] In some embodiments, the volatility of the non-graphenic carbon-based material may be less than that which is obtained by heating the sheet of graphene-based material to 500°C for 4 hours in vacuum or at atmospheric pressure with an inert gas.

[0894] In various embodiments, CVD graphene or graphene-based material can be liberated from its growth substrate (e.g., Cu) and transferred to a supporting grid, mesh or other supporting structure. In some embodiments, the supporting structure may be configured so that at least some portions of the sheet of graphene-based material are suspended from the supporting structure. For example, at least some portions of the sheet of graphene-based material may not be in contact with the supporting structure.

[0895] In some embodiments, the sheet of graphene-based material following chemical vapor deposition comprises a single layer of graphene having at least two surfaces and non- graphenic carbon based material may be provided on said surfaces of the single layer graphene. In some embodiments, the non-graphenic carbon based material may be located on one of the two surfaces or on both. In some further embodiments, additional graphenic carbon may also present on the surface(s) of the single layer graphene.

[0896] The preferred embodiments may be further understood by the following non-limiting examples.

[0897] EXAMPLE: Perforated Graphene-Based Materials

[0898] FIGS. 101A and 101B are TEM images illustrating a portion of a sheet of graphene- based material after perforation using UV-oxygen treatment. FIG. 10 IB shows an enlarged portion of FIG. 101 A. Label 10110 indicates a region of graphene, the brighter surrounding areas include largely non-graphenic carbon and the dark regions are pores. The graphene based material was prepared by chemical vapor deposition then subjected to ion beaming while on the copper growth substrate with Xe ions at 500V at 80°C with a fluence of 1.25x 10 ¹³ ions/cm ² . Then the material was transferred to a TEM grid and then while suspended received 400 seconds of treatment at atmospheric pressure with atmospheric gas with Ultra- Violet (UV) parameters as described. The intensity was 28mW/cm ² at 6 mm.

[0899] FIGS. 102A and 102B are TEM images illustrating a portion of a sheet of graphene based material after perforation using Xe ions. FIG. 102B shows an enlarged portion of FIG. 102 A. The graphene based material was prepared by chemical vapor deposition, pretreated, then transferred to a TEM grid and irradiated with Xe ions at 20 V and 2000 nA s. 2000nA snA s = 1.25 x 10 ¹⁵ ions/cm ² . The area % of pores was 5.8%.

[0900] FIG. 103 and FIG. 104 are TEM images illustrating graphene based material after perforation using Ne ions. FIG. 104 is at higher magnification. The graphene based material was prepared by chemical vapor deposition, pretreated, then transferred to a TEM grid and irradiated with Ne ions at 23kV with a fluence of 4x 10 ¹⁷ ions/ cm.

[0901] FIG. 105 and FIG. 106 are TEM images illustrating graphene based material after perforation using He ions. FIG. 6 is at higher magnification. The graphene based material was prepared by chemical vapor deposition, pretreated, then transferred to a TEM grid and irradiated with He ions at 25kV with a fluence of lx10 ²⁰ ions/cm ² .

[0902] The perforations generally appear as darker regions in these images.

NANOPARTICLE MODIFICATION AND PERFORATION OF GRAPHENE

[0903] Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or a few layered sheets (e.g., about 20 or less) of fused six- membered rings forming an extended sp2-hybridized carbon planar lattice. Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. In

embodiments, multilayer graphene includes 2 to 25 layers, 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In an embodiment, layers of multilayered graphene are stacked, but are less ordered in the z direction (perpendicular to the basal plane) than a thin graphite crystal. [0904] In an embodiment, graphene-based materials also include materials which have been formed by stacking single or multilayer graphene sheets. Multi-layered graphene as referred to herein includes multiple sheets of graphene formed by layering or stacking independently as- synthesized sheets on a substrate. As used herein, independently as-synthesized sheets which have been layered or stacked on a substrate are termed "independently stacked." Adjacent graphene layers formed by independent stacking can be less ordered in the z direction than as- synthesized multilayer graphene. In examples, independently stacked adjacent layers do not display A-B, A-B-A or A-B-C-A stacking. In additional examples, there is no defined registry of adjacent layers of independently stacked graphene. Without wishing to be bound by any particular belief, structural differences between independently stacked multi-layer graphene and as-synthesized multi-layer graphene are believed to contribute to differences in nanoparticle perforation behavior demonstrated in Example 1. In an embodiment, layers of as-synthesized sheets of graphene which have been stacked in this fashion are less ordered in the z direction, i.e., the lattices of the sheets do not line up as well, than layers in an as-synthesized multilayer graphene sheet. Suitable as-synthesized sheets include sheets of single layer graphene (SLG), sheets of bi-layer graphene (BLG) or sheets of few layer graphene (FLG graphene, for example up to 5 layers of graphene). For example, when a "float down" transfer technique is used a sheet of single layer graphene (SLG) is layered via float-down on top of a substrate. Another sheet of the SLG is then floated down on the already prepared SLG-substrate stack. This would now be 2 layers of "as-synthesized" SLG on top of the substrate. This can be extended to few layer graphene (FLG) or a mixture of SLG and FLG; and can be achieved through transfer methods known to the art. Other transfer methods are known to the art, including stamp methods. For example, a polymer transfer method can be used to assemble the stack of polymer layers. In some instances a number of layers is intended to refer to that number of separate layers of transferred graphene. In cases where a layer of transferred graphene can have a range of graphene layers (e.g. some regions of the layer are SLG and others are BLG or FLG), the stack has a range of graphene layers. For example, if 5 layers of transferred graphene each have 1 to 5 layers, then regions where the 5 sheets line up with 5 layers, effectively have 25 layers of graphene there. Depending on the perforation conditions, the thicker regions of the stack may not perforate. In an embodiment, layering of different sheets of graphene results in a desirable membrane for filtration and separation applications.

[0905] In an embodiment, a sheet of graphene-based material is a sheet of single or multilayer graphene or a sheet comprising a plurality of interconnected single or multilayer graphene domains. In embodiments, the multilayer graphene domains have 2 to 5 layers or 2 to 10 layers. As used herein, a "domain" refers to a region of a material where atoms are uniformly ordered into a crystal lattice. A domain is uniform within its boundaries, but different from a neighboring region. For example, a single crystalline material has a single domain of ordered atoms. In an embodiment, at least some of the graphene domains are nanocrystals, having domain size from 1 to 100 nm or 10-100 nm. In an embodiment, at least some of the graphene domains have a domain size from 100 nm to 500 microns, 100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm. In an embodiment, a domain of multilayer graphene may overlap a neighboring domain. "Grain boundaries" formed by crystallographic defects at edges of each domain differentiate between neighboring crystal lattices. In some embodiments, a first crystal lattice may be rotated relative to a second crystal lattice, by rotation about an axis perpendicular to the plane of a sheet, such that the two lattices differ in "crystal lattice orientation".

[0906] In an embodiment, the sheet of graphene-based material is a sheet of multilayer graphene or a combination of single and multilayer graphene. In another embodiment, the sheet of graphene-based material is a sheet comprising a plurality of interconnected multilayer or single and multilayer graphene domains. In an embodiment, the interconnected domains are covalently bonded together to form the sheet. When the domains in a sheet differ in crystal lattice orientation, the sheet is polycrystalline.

[0907] In embodiments, the thickness of the sheet of graphene-based material is from, 0.3 to 10 nm, from 0.3 to 5 nm, or from 0.3 to 3 nm. In an embodiment, the thickness includes both single layer graphene and the non-graphenic carbon.

[0908] In an embodiment, a sheet of graphene-based material comprises intrinsic or native defects. Intrinsic or native defects are those resulting from preparation of the graphene-based material in contrast to perforations which are selectively introduced into a sheet of graphene- based material or a sheet of graphene. Such intrinsic or native defects include, but are not limited to, lattice anomalies, pores, tears, cracks or wrinkles. Lattice anomalies can include, but are not limited to, carbon rings with other than 6 members (e.g. 5, 7 or 9 membered rings), vacancies, interstitial defects (including incorporation of non-carbon atoms in the lattice), and grain boundaries. As used herein, perforations do not include openings in the graphene lattice due to intrinsic or native defects or grain boundaries.

[0909] In embodiments, graphene is the dominant material in a graphene-based material. For example, a graphene-based material comprises at least 20% graphene, 30% graphene, or at least 40%) graphene, or at least 50% graphene, or at least 60%> graphene, or at least 70% graphene, or at least 80% graphene, or at least 90% graphene, or at least 95% graphene. In embodiments, a graphene-based material comprises a range of graphene selected from 30% to 95%, or from 40% to 80% from 50% to 70%, from 60% to 95% or from 75% to 100%. In an embodiment, the amount of graphene in the graphene-based material is measured as an atomic percentage.

[0910] In an embodiment, a sheet of graphene-based material further comprises non- graphenic carbon-based material located on a surface of the sheet of graphene-based material. In an embodiment, the sheet is defined by two base surfaces (e.g. top and bottom faces of the sheet) and side faces. In a further embodiment, the "bottom" face of the sheet is that face which contacted the substrate during growth of the sheet and the "free" face of the sheet opposite the "bottom" face. In an embodiment, non-graphenic carbon-based material is located on a base surface of the sheet (e.g. the substrate side of the sheet and/or the free surface of the sheet). In a further embodiment, the sheet of graphene-based material includes a small amount of one or more other materials on the surface, such as, but not limited to, one or more dust particles or similar contaminants.

[0911] In an embodiment, the amount of non-graphenic carbon-based material is less than the amount of graphene. In embodiments, the surface coverage of the sheet of non-graphenic carbon-based material is greater than zero and less than 80%, from 5% to 80%, from 10% to 80%), from 5% to 50% or from 10% to 50%. This surface coverage may be measured with transmission electron microscopy, which gives a projection. In embodiments, the amount of graphene in the graphene-based material is from 60% to 95% or from 75% to 100%. [0912] In an embodiment, the non-graphenic carbon-based material does not possess long range order and may be classified as amorphous. In embodiments, the non-graphenic carbon- based material further comprises elements other than carbon and/or hydrocarbons. In an embodiment, non-carbon elements which may be incorporated in the non-graphenic carbon include hydrogen, oxygen, silicon, copper and iron. In further embodiment, the non-graphenic carbon-based material comprises hydrocarbons. In embodiments, carbon is the dominant material in non-graphenic carbon-based material. For example, a non-graphenic carbon-based material comprises at least 30% carbon, or at least 40% carbon, or at least 50% carbon, or at least 60%) carbon, or at least 70% carbon, or at least 80% carbon, or at least 90% carbon, or at least 95%) carbon. In embodiments, a non-graphenic carbon-based material comprises a range of carbon selected from 30% to 95%, or from 40% to 80%, or from 50% to 70%. In an

embodiment, the amount of carbon in the non-graphenic carbon-based material is measured as an atomic percentage.

[0913] In further embodiments, the sheet of graphene based material is larger than a flake which would be obtained by exfoliation. For example, the sheet has a lateral dimension greater than about 1 micrometer. As used herein, a lateral dimension is perpendicular to the thickness of the sheet.

[0914] As used herein, the term 'two-dimensional material' will refer to any extended planar structure of atomic thickness, including both single- and multi-layer variants thereof. Multi-layer two-dimensional materials can include up to about 20 stacked layers. In an embodiment, a two- dimensional material suitable for the present structures and methods can be any substance having an extended planar molecular structure and an atomic level thickness. Particular examples of two-dimensional materials include graphene films, graphene-based material, transition metal dichalcogenides, metal oxides, metal hydroxides, graphene oxide, a-boron nitride, silicone, germanene, or other materials having a like planar structure. Specific examples of transition metal dichalcogenides include molybdenum disulfide and niobium diselenide. Specific examples of metal oxides include vanadium pentoxide. Graphene or graphene-based films according to the embodiments of the present disclosure can include single-layer or multi-layer films, or any combination thereof. Choice of a suitable two-dimensional material can be determined by a number of factors, including the chemical and physical environment into which the graphene, graphene-based material or other two-dimensional material is to be terminally deployed, ease of perforating the two-dimensional material, and the like.

[0915] Nanomaterials in which pores are intentionally created will be referred to herein as "perforated graphene", "perforated graphene-based materials" or "perforated two-dimensional materials." The size distribution of holes may be narrow, e.g., limited to 0.1 to 0.5 coefficient of variation. In an embodiment, the characteristic dimension of the holes is selected for the application. For circular holes, the characteristic dimension is the diameter of the hole. In embodiments relevant to non-circular pores, the characteristic dimension can be taken as the largest distance spanning the hole, the smallest distance spanning the hole, the average of the largest and smallest distance spanning the hole, or an equivalent diameter based on the in-plane area of the pore. As used herein, perforated graphene-based materials include materials in which non-carbon atoms have been incorporated at the edges of the pores. In embodiments, the pore is asymmetric with the pore size varying along the length of the hole (e.g. pore size wider at the free surface of the graphene-based material than at the substrate surface or vice versa). In an embodiment, the pore size may be measured at one surface of the sheet of graphene based material.

[0916] Quantitative image analysis of pore features may include measurement of the number, area, size and/or perimeter of pore features. In an embodiment, the equivalent diameter of each pore is calculated from the equation A= π d ² /4. When the pore area is plotted as a function of equivalent pore diameter, a pore size distribution is obtained. The coefficient of variation of the pore size is calculated herein as the ratio of the standard deviation of the pore size to the mean of the pore size.

[0917] In an embodiment, the ratio of the area of the perforations to the ratio of the area of the sheet is used to characterize the sheet. The area of the perforations may be measured using quantitative image analysis. The area of the sheet may be taken as the planar area spanned by the sheet if it is desired to exclude the additional sheet surface area due to wrinkles or other non- planar features of the sheet. In a further embodiment, characterization may be based on the ratio of the area of the perforations to the sheet area excluding features such as surface debris. [0918] The present disclosure is directed, in part, to multi-layer graphene sheets and sheets of graphene-based material having about 2 to about 10 graphene sheets stacked upon one another and a plurality of pores penetrating through the stacked graphene sheets. The present disclosure is also directed, in part, to methods for perforating multi-layer graphene sheets and sheets of graphene-based material comprising multilayer graphene and defining pores therein that extend through the multiple graphene sheets.

[0919] Perforated graphene (i.e., graphene having a plurality of pores defined therein) has a number of possible applications including, for example, use as a molecular filter, use as a barrier material, use as a defined band gap material, and use as an electrically conductive filler material with tunable electrical properties within polymer composites. Although a number of potential uses for perforated graphene exist, there are few reliable techniques to reproducibly introduce a plurality of pores in graphene, where the pores are presented in a desired pore density and pore size. Generation of sub-nanometer pores can be particularly problematic.

[0920] In embodiments, the pretreatment step for the graphene-based material is selected from thermal treatment, UV-oxygen treatment, ion beam treatment, and combinations thereof. In an embodiment, thermal treatment includes heating to a temperature from 200 °C to 800 °C at a pressure of 10-7 torr to atmospheric pressure for a time of 2 hours to 8 hours. In an embodiment, UV-oxygen treatment involves exposure to light from 150 nm to 300 nm and an intensity from 10 to 100 mW/cm ² at 6mm distance for a time from 60 to 1200 seconds. In embodiments, UV- oxygen treatment is performed at room temperature or at a temperature greater than room temperature. In further embodiments, UV-oxygen treatment is performed at atmospheric pressure (e.g. 1 atm) or under vacuum. In an embodiment, ion beam pretreatment involves exposure one or more of the graphene layers to ions having an ion energy from 50 eV to 1000 eV (for pretreatment) and the fluence is from 3 x 10 ¹⁰ ions/cm ² to 8 x 10 ¹¹ ions/cm ² or 3 x 10 ¹⁰ ions/cm ² to 1 x 10 ¹⁴ ions/cm ² (for pretreatment). In a further embodiment, the source of ions is collimated, such as a broad beam or flood source. In an embodiment, the ions are noble gas ions such as Xe ⁺ . In modifying the sheet of perforated graphene-based material comprises creating a second set pores having a second pore size extending through the multiple graphene sheets, modifying the first pores size or combinations thereof. In an embodiment, one or more pretreatment steps are performed while the graphene-based material is attached to a substrate, such as a growth substrate. In an embodiment, the metal growth substrate is a substantially continuous layer of metal rather than a grid or mesh. Metal growth substrates compatible with growth of graphene and graphene-based materials include transition metals and their alloys. In embodiments, the metal growth substrate is copper based or nickel based. In embodiments of the present disclosure the ion source provides a broad ion field. The source of ions may be an ion flood source. In an embodiment, the ion flood source does not include focusing lenses. In embodiments, the ion source is operated at less than atmospheric pressure, such as at 10 ^-3 to 10 ^-5 torr or 10 ^-4 to 10 ^-6 torr. If perforation efficiency is lower than desired after one pretreatment step, an additional pretreatment step can be used before re-exposing the graphene layer(s) to nanoparticle or clusters.

[0921] In embodiments of the disclosure herein, the particle beam is a nanoparticle beam or cluster beam. In further embodiments, the beam is collimated or is not collimated. Furthermore, the beam need not be highly focused. In some embodiments, a plurality of the nanoparticles or clusters is singly charged. In additional embodiments, the nanoparticles comprise from 500 to 2,000,000 atoms, from 500 to 250,000 atoms or from 500 to 5,000 atoms.

[0922] A variety of metal particles are suitable for use in the methods of the present disclosure. For example, nanoparticles of Al, Ag, Au, Ti, Cu and nanoparticles comprising Al, Ag, Au, Ti, Cu are suitable. Metal Ps can be generated in a number of ways including magnetron sputtering and liquid metal ion sources (LMIS). Methods for generation of nanoparticles are further described in Cassidy, Cathal, et al. "Inoculation of silicon nanoparticles with silver atoms." Scientific reports 3 (2013), Haberland, Hellmut, et al. "Filling of micron sized contact holes with copper by energetic cluster impact." Journal of Vacuum Science & Technology A 12.5 (1994): 2925-2930, Bromann, Karsten, et al. "Controlled deposition of size- selected silver nanoclusters." Science 274.5289 (1996): 956-958, Palmer, R. E., S. Pratontep, and H-G. Boyen. "Nanostructured surfaces from size-selected clusters." Nature Materials 2.7 (2003): 443-448, Shyjumon, L, et al. "Structural deformation, melting point and lattice parameter studies of size selected silver clusters." The European Physical Journal D-Atomic, Molecular, Optical and Plasma Physics 37.3 (2006): 409-415, Allen, L. P., et al. "Craters on silicon surfaces created by gas cluster ion impacts." Journal of applied physics 92.7 (2002): 3671-3678, Wucher, Andreas, Hua Tian, and Nicholas Winograd. "A Mixed Cluster Ion Beam to Enhance the Ionization Efficiency in Molecular Secondary Ion Mass Spectrometry." Rapid communications in mass spectrometry?: RCM 28.4 (2014): 396-400. PMC. Web. 6 Aug. 2015 and Pratontep, S., et al. "Size-selected cluster beam source based on radio frequency magnetron plasma sputtering and gas condensation." Review of scientific instruments 76.4 (2005): 045103, each of which is hereby incorporated by reference for its description of nanoparticle generation techniques.

[0923] Gas cluster beams can be made when high pressure gas adiabatically expands in a vacuum and cools such that it condenses into clusters. Clusters can also be made ex situ such as C60 and then accelerated towards the graphene.

[0924] In some embodiments, the nanoparticles are specially selected to introduce moieties into the graphene. In some embodiments, the nanoparticles function as catalysts. The moieties may be introduced at elevated temperatures, optionally in the presence of a gas. In other embodiments, the nanoparticles introduce"chiseling" moieties, which are moieties that help reduce the amount of energy needed to remove an atom during irradiation.

[0925] In embodiments, the size of the perforation apertures is controlled by controlling both the nanoparticle size and the nanoparticle energy. Without wishing to be bound by any particular belief, if all the nanoparticles have sufficient energy to perforate, then the resulting perforation is believed to correlated with the nanoparticle sizes selected. However, the size of the perforation is believed to be influenced by deformation of the nanoparticle during the perforation process. This deformation is believed to be influenced by both the energy and size of the nanoparticle and the stiffness of the graphene layer(s). A grazing angle of incidence of the nanoparticles can also produce deformation of the nanoparticles. In addition, if the nanoparticle energy is controlled, it is believed that nanoparticles can be deposited with a large mass and size distribution, but that a sharp cutoff can still be achieved.

[0926] Without wishing to be bound by any particular belief, the mechanism of perforation is believed to be a continuum bound by sputtering on one end (where enough energy is delivered to the graphene sheet to atomize the carbon in and around the NP impact site) and ripping or fracturing (where strain induced failure opens a torn hole but leaves the graphene carbons as part of the original sheet). Part of the graphene layer may fold over at the site of the rip or fracture. In an embodiment the cluster can be reactive so as to aid in the removal of carbon (e.g. an oxygen cluster or having trace amounts of a molecule known to etch carbon in a gas cluster beam i.e. a mixed gas cluster beam). Without wishing to be bound by any particular belief, the stiffness of a graphene layer is believed to be influenced by both the elastic modulus of graphene and the tautness of the graphene. Factors influencing the elastic modulus of a graphene layer are believed to include temperature, defects (either small defects or larger defects from P irradiation), phy si sorption, chemisorption and doping. Tautness is believed to be influenced by coefficient of thermal expansion mismatches (e.g. between substrate and graphene layer) during deposition, strain in the graphene layer, wrinkling of the graphene layer. It is believed that strain in a graphene layer can be influenced by a number of factors including application of gas pressure to the backside (substrate side) of a graphene layer, straining of an elastic substrate prior to deposition of graphene, flexing of the substrate during deposition, and defecting the graphene layer in controlled regions to cause the layer to locally contract and increase the local strain.

[0927] In embodiments, nanoparticle perforation can be further controlled by straining the graphene layers during perforation to induce fracture, thereby "ripping" or "tearing" one or more graphene layers. In some embodiments, the stress is directional and used to preferentially fracture in a specific orientation. For example, ripping of one or more graphene sheets can be used to create "slit" shaped apertures; such apertures can be substantially larger than the nanoparticle used to initiate the aperture. In additional embodiments, the stress is not oriented in a particular direction.

[0928] In embodiments, the pores are functionalized. In some embodiments, the pores are functionalized by exposure to gas during and/or following the perforation process. The exposure to gas may occur at temperatures above room temperature. In some embodiments, the pores can have more than one effective functionalization. For example, when the top and the bottom layers of a graphene stack are exposed to different functionalizing gases, more than one effective functionalization can be produced. In further embodiments, a thin layer of a functionalizing moiety is applied to the surface before NP perforation, during NP perforation and after NP perforation. As compatible with the NP process, the thin layer may be formed by applying a fluid to the surface. In embodiments, the gas pressure is 10 ^-4 Torr to atmospheric pressure. In embodiments, functionalizing moieties include, but are not limited to water, water vapor, polyethylene glycol, oxygen, nitrogen, amines, caboxycylic acid.

[0929] The preferred gasses for before and during functionalization would depend on the reaction of graphene and the gas within the high energy environment of the particle impact. This would be within about 100 nm of the edge of the particle impact. This fits into two general classes, and the gases would be added at a partial pressure of from lx10 ^-6 Torr to lx10 ^-3 Torr. The first class would be species that reacts with radicals, carbanions (negative charge centered on a carbon) and carbocations (positive charge centered on a carbon). Representative molecules include carbon dioxide, ethylene oxide and isoprene. The second class would be species that fragment to create species that react with graphene and defective graphene. Representative molecules would be polyethylene glycol, diacytylperoxide, azobisisobutyronitrile, and phenyl diazonium iodide.

[0930] In some embodiments, a sheet of graphene-based material is perforated to create a first set of perforations, the first set of perforations are functionalized with a first moeity, the sheet is reperforated to create a second set of perforations, and the second set of perforations is functionalized with a second moiety.

[0931] In embodiments, it is desirable and advantageous to perforate multiple graphene sheets at one time rather than perforating single graphene sheets individually, since multi-layer graphene is more robust and less prone to the presence of intrinsicor native defects that align through all the layers than is single-layer graphene. In addition, the process is stepwise efficient, since perforated single-layer graphene can optionally be produced by exfoliating the multi-layer graphene after the pore definition process is completed. The pore size is also tailorable in the processes described herein. Thus, the processes described herein are desirable in terms of the number, size and size distribution of pores produced.

[0932] The multi-layer graphene contains between about 2 stacked graphene sheets and about 20 stacked graphene sheets according to the various embodiments of the present disclosure. Too few graphene sheets can lead to difficulties in handling the graphene as well as an increased incidence of intrinsic graphene defects. Having more than about 20 stacked graphene sheets, in contrast, can make it difficult to perforate all of the graphene sheets. In an embodiment, the multilayer sheets may be made by individually growing sheets and making multiple transfers to the same substrate. In various embodiments, the multi-layer graphene perforated by the techniques described herein can have 2 graphene sheets, or 3 graphene sheets, or 4 graphene sheets, or 5 graphene sheets, or 6 graphene sheets, or 7 graphene sheets, or 8 graphene sheets, or 9 graphene sheets, or 10 graphene sheets, or 11 graphene sheets, or 12 graphene sheets, or 13 graphene sheets, or 14 graphene sheets, or 15 graphene sheets, or 16 graphene sheets, or 17 graphene sheets, or 18 graphene sheets, or 19 graphene sheets, or 20 graphene sheets. Any subrange between 2 and 20 graphene sheets is also contemplated by the present disclosure.

[0933] In some embodiments, perforated graphene produced by the techniques described herein can be used in filtration processes. In addition, the perforated graphene produced by the techniques described herein can be utilized in fields such as, for example, advanced sensors, batteries and other electrical storage devices, and semiconductor devices.

[0934] In some embodiments, the perforated graphene can be placed upon a porous polymer substrate after being perforated. The combination of the porous polymer substrate and the graphene can constitute a filter in various embodiments, such as a reverse osmosis filter or a nanofiltration filter. Suitable porous polymer substrates are not believed to be particularly limited.

[0935] Although the disclosure has been described with reference to the disclosed embodiments, one having ordinary skill in the art will readily appreciate that these are only illustrative of the disclosure. It should be understood that various modifications can be made without departing from the spirit of the disclosure. The disclosure can be modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the spirit and scope of the disclosure. Additionally, while various embodiments of the disclosure have been described, it is to be understood that aspects of the disclosure may include only some of the described embodiments. Accordingly, the disclosure is not to be seen as limited by the foregoing description. [0936] Every formulation or combination of components described or exemplified can be used to practice embodiments, unless otherwise stated. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently. When a compound is described herein such that a particular isomer or enantiomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomers and enantiomer of the compound described individual or in any combination. One of ordinary skill in the art will appreciate that methods, device elements, starting materials and synthetic methods other than those specifically exemplified can be employed in the practice herein without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, starting materials and synthetic methods are intended to be included herein. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure.

[0937] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements. Embodiments illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0938] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope. Thus, it should be understood that although the embodiments have been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of embodiments as defined by the appended claims.

[0939] In general the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The preceding definitions are provided to clarify their specific use in the context herein.

[0940] All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).

[0941] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which embodiments pertain. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art, in some cases as of their filing date, and it is intended that this information can be employed herein, if needed, to exclude (for example, to disclaim) specific embodiments that are in the prior art. For example, when a compound is claimed, it should be understood that compounds known in the prior art, including certain compounds disclosed in the references disclosed herein (particularly in referenced patent documents), are not intended to be included in the claims.

[0942] EXAMPLE 1 : Comparative Nanoparticle Perforation of Bilayer Graphene and Two Stacked Layers of Graphene

[0943] FIG. 107 is a transmission electron microscopy image demonstrating perforation through two independently stacked layers of graphene by nanoparticles. Two layers of CVD graphene material were prepared. Each layer received ion beam treatment on Cu growth substrate, transfer to lacey carbon TEM grid, UV-oxygen treatment and 300°C bakeout for 8 hours before the layers were stacked. The stacked layers were exposed to 6.5kV Ag

nanoparticles (NP). The P distribution was centered on 6nm and the fluence was

approximately 5x 10 ¹⁰ NPs /cm ² .

[0944] FIG. 108 is a transmission electron microscopy image demonstrating perforation through bilayer graphene by nanoparticles. The CVD graphene was prepared then received ion beam treatment on Cu substrate, was transferred to lacey carbon TEM grid, received UV-oxygen treatment and 300°C bakeout for 8 hours. The graphene was exposed to 7.5kV Ag P with NP distribution centered on 6nm at a fluence of approximately 5x 10 ^u NPs/cm ² , followed by a 24 hour bake at 300° C in Ar with slow cool down.

[0945] EXAMPLE 2: Nanoparticle Perforation at an Angle other than Ninety Degrees with Respect to a Sheet of with Two Stacked Graphene Layers

[0946] FIG. 109 is a transmission electron microscopy image of perforations through two stacked layers of graphene made by exposure to a particle beam comprising nanoparticles. The nanoparticles were provided at an incidence angle of approximately greater than 45 degrees to the normal of the basal plane of the sheet of graphene material. The NPs were of 9-1 lnm diameter at an energy of 30keV. The pores were typically 10-12 nm at their base, and varied in length from about 20nm to 70mm. The pore size was larger than that obtained for nanoparticle perforation approximately normal to the basal sheet of graphene material under similar nanoparticle size, energy and fluence conditions. In addition, some evidence of ripping was observed on the top graphene surface.

[0947] EXAMPLE 3 : Nanoparticle Perforation Followed by Ion Irradiation

[0948] FIGS 110A and H0B illustrate a sheet of graphene based material after nanoparticle perforation (FIG. 4A) and after subsequent ion beam irradiation (FIG. H0B). The material was two layers of independently stacked graphene. The perforation conditions were 7-10nm NPs at

12 keV. The ion beam irradiation conditions were Xe+ at 300V with 2E ¹⁴ Xe+/cm ² fluence and

3E ¹⁴ Xe+/cm ² /s flux. The porosity went from 5% to 14%.

[0949] Example 4: Nanoparticle Perforation of Graphene on TEPI (460/25) [0950] FIG. 111 is a scanning electron microscopy image of two independently stacked layers of single layer graphene on a on a track etched polyimide substrate with approximately 460 nm diameter pores perforated by Ag P particles. TEPI (460/25) is track etched polyimide that has an average pore diameter (of individual, non overlapping pores) on the side that graphene is applied to it of 460nm and is approximately 25um thick. The perforation conditions were 10-15nm AgNP particles at 30keV.

METHODS FOR IN SITU MONITORING AND CONTROL OF DEFECT FORMATION OR

HEALING

[0951] Graphene has garnered widespread interest for use in a number of applications due to its favorable mechanical and electronic properties, as well as its chemical inertness.

Graphene represents an atomically thin two-dimensional layer of carbon in which the carbon atoms reside as closely spaced atoms at regular lattice positions. The regular lattice positions can have a plurality of defects present therein, which can occur natively or be intentionally introduced to the graphene basal plane. Such defects will also be equivalently referred to herein as "apertures," "perforations" or "holes." The term "perforated graphene" is used herein to denote a graphene sheet with defects in its basal plane, regardless of whether the defects are natively present or intentionally produced. Aside from such apertures, graphene and other two- dimensional materials can represent an impermeable layer to many substances. Therefore, when sized properly, the apertures in the impermeable layer of such materials can be useful for filtration and sequestration, for example.

[0952] Two-dimensional materials are, most generally, those which are atomically thin, with thickness ranging from single-layer sub-nanometer thickness to a few nanometers, and which generally have a high surface area. Two-dimensional materials include metal chalogenides (e.g., transition metal dichalogenides), transition metal oxides, hexagonal boron nitride, graphene, silicene and germanene (see: Xu et al. (2013) "Graphene-like Two-Dimensional Materials) Chemical Reviews 113 :3766-3798). Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or few layered sheets (e.g., about 20 or less) of covalently bound carbon atoms forming an extended sp ² -hybridized planar lattice. In its various forms, graphene has garnered widespread interest for use in a number of applications, primarily due to its favorable combination of high electrical and thermal conductivity values, good in- plane mechanical strength, and unique optical and electronic properties. Other two-dimensional materials having a thickness of a few nanometers or less and an extended substantially planar lattice are also of interest for various applications. In an embodiment, a two dimensional material has a thickness of 0.3 to 1.2 nm. In other embodiments, a two dimensional material has a thickness of 0.3 to 3 nm.

[0953] In various embodiments, the two-dimensional material comprises a sheet of a graphene-based material. In an embodiment, the sheet of graphene-based material is a sheet of single- or multi-layer graphene or a sheet comprising a plurality of interconnected single- or multi-layer graphene domains. In embodiments, the multilayer graphene domains have 2 to 5 layers or 2 to 10 layers. In an embodiment, the layer comprising the sheet of graphene-based material further comprises non-graphenic carbon-based material located on the surface of the sheet of graphene-based material. In an embodiment, the amount of non-graphenic carbon-based material is less than the amount of graphene. In embodiments, the amount of graphene in the graphene-based material is from 60% to 95% or from 75% to 100%. In an embodiment, the amount of graphene in the graphene-based material is measured as an atomic percentage.

[0954] In embodiments, the characteristic size of the perforations of a perforated graphene, graphene-based or two-dimensional material is from 0.3 to 10 nm, from 1 to 10 nm, from 5 to 10 nm, from 5 to 20 nm, from 10 nm to 50 nm, from 50 nm to 100 nm, from 50 nm to 150 nm, from 100 nm to 200 nm, or from 100 nm to 500 nm. In an embodiment, the average pore size of a perforated graphene, graphene-based or two-dimensional material is within the specified range. In embodiments, 70% to 99%, 80% to 99%, 85% to 99% or 90 to 99% of the perforations in a sheet or layer fall within a specified range, but other pores fall outside the specified range.

[0955] The technique used for forming the graphene or graphene-based material in the embodiments described herein is not believed to be particularly limited. For example, in some embodiments CVD graphene or graphene-based material can be used. In various embodiments, the CVD graphene or graphene-based material can be liberated from its growth substrate (e.g., Cu) and transferred to a polymer backing, or may be transferred to a porous substrate. [0956] Likewise, the techniques for introducing perforations to the graphene or graphene- based material are not believed to be particularly limited, other than being chosen to produce perforations within a desired size range. Perforations are sized as described herein to provide desired selective permeability of a species (atom, molecule, protein, virus, cell, etc.) for a given application. Selective permeability relates to the propensity of a porous material or a perforated two-dimensional material to allow passage (or transport) of one or more species more readily or faster than other species, or to block the other species from passage. Selective permeability allows separation of species which exhibit different passage or transport rates. In two- dimensional materials selective permeability correlates to the dimension or size (e.g., diameter) of apertures and the relative effective size of the species. Selective permeability of the perforations in two-dimensional materials, such as graphene-based materials, can also depend on functionalization of perforations (if any) and the specific species that are to be separated or blocked. Separation of two or more species in a mixture includes a change in the ratio(s) (weight or molar ratio) of the two or more species in the mixture after passage of the mixture through a perforated two-dimensional material.

[0957] Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. In an embodiment, graphene-based materials also include materials which have been formed by stacking single layer or multilayer graphene sheets. In embodiments, multilayer graphene includes 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In embodiments, graphene is the dominant material in a graphene-based material. For example, a graphene-based material comprises at least 30% graphene, or at least 40% graphene, or at least 50% graphene, or at least 60%) graphene, or at least 70% graphene, or at least 80% graphene, or at least 90% graphene, or at least 95% graphene. In embodiments, a graphene-based material comprises a range of graphene selected from 30% to 95%, from 40% to 80%, from 50% to 70%, from 60% to 95% or from 75%) to 100%. In an embodiment, the amount of graphene in the graphene-based material is measured as an atomic percentage.

[0958] Graphene represents a form of carbon in which the carbon atoms reside within a single atomically thin sheet or a few layered sheets (e.g., about 20 or less) of fused six- membered rings forming an extended sp ² -hybridized carbon planar lattice. Graphene-based materials include, but are not limited to, single layer graphene, multilayer graphene or interconnected single or multilayer graphene domains and combinations thereof. In

embodiments, multilayer graphene includes 2 to 25 layers, 2 to 20 layers, 2 to 10 layers or 2 to 5 layers. In an embodiment, layers of multilayered graphene are stacked, but are less ordered in the z direction (perpendicular to the basal plane) than a thin graphite crystal.

[0959] In an embodiment, graphene-based materials also include materials which have been formed by stacking single or multilayer graphene sheets. Multi-layered graphene as referred to herein includes multiple sheets of graphene formed by layering as-synthesized sheets on a substrate. In an embodiment, layers of as-synthesized sheets of graphene which have been stacked in this fashion are less ordered in the z direction than an as-synthesized multilayer graphene sheet. Suitable as-synthesized sheets include sheets of single layer graphene (SLG), sheets of bi-layer graphene (BLG) or sheets of few layer graphene (FLG graphene, for example up to 5 layers of graphene). For example, a sheet of single layer graphene (SLG) is layered via float-down on top of a substrate. Another sheet of the SLG is then floated down on the already prepared SLG-substrate stack. This would now be 2 layers of "as-synthesized" SLG on top of the substrate. This can be extended to few layer graphene (FLG) or a mixture of SLG and FLG; and can be achieved through transfer methods known to the art. For example, a polymer transfer method can be used to assemble the stack of polymer layers. In some instances a number of layers is intended to refer to that number of separate layers of transferred graphene. In cases where a layer of transferred graphene can have a range of graphene layers (e.g. some regions of the layer are SLG and others are BLG or FLG), the stack has a range of graphene layers. For example, if 5 layers of transferred graphene each have 1 to 5 layers, then regions where the 5 sheets line up with 5 layers, effectively have 25 layers of graphene at that position. Depending on the perforation conditions, the thicker regions of the stack may not perforate. In

embodiments, layering of different sheets of graphene results in a desirable membrane for filtration and separation applications.

[0960] In an embodiment, a sheet of graphene-based material is a sheet of single or multilayer graphene or a sheet comprising a plurality of interconnected single or multilayer graphene domains. In embodiments, the multilayer graphene domains have 2 to 5 layers or 2 to 10 layers. As used herein, a "domain" refers to a region of a material where atoms are uniformly ordered into a crystal lattice. A domain is uniform within its boundaries, but different from a neighboring region. For example, a single crystalline material has a single domain of ordered atoms. In an embodiment, at least some of the graphene domains are nanocrystals, having domain size from 1 to 100 nm or 10 to 100 nm. In an embodiment, at least some of the graphene domains have a domain size from 100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm. "Grain boundaries" formed by crystallographic defects at edges of each domain differentiate between neighboring crystal lattices. In some embodiments, a first crystal lattice may be rotated relative to a second crystal lattice, by rotation about an axis perpendicular to the plane of a sheet, such that the two lattices differ in "crystal lattice orientation."

[0961] In an embodiment, the sheet of graphene-based material comprises a sheet of single layer or multilayer graphene or a combination thereof. In an embodiment, the sheet of graphene- based material is a sheet of single layer or multilayer graphene or a combination thereof. In another embodiment, the sheet of graphene-based material is a sheet comprising a plurality of interconnected single or multilayer graphene domains. In an embodiment, the interconnected domains are covalently bonded together to form the sheet. When the domains in a sheet differ in crystal lattice orientation, the sheet is polycrystalline.

[0962] In embodiments, the thickness of the sheet of graphene-based material is from 0.34 to 10 nm, from 0.34 to 5 nm, or from 0.34 to 3 nm. In an embodiment, a sheet of graphene-based material comprises intrinsic or native defects. Intrinsic or native defects are those resulting from preparation of the graphene-based material in contrast to perforations which are selectively or intentionally introduced into a sheet of graphene-based material or a sheet of graphene. Such intrinsic or native defects may include, but are not limited to, lattice anomalies, pores, tears, cracks or wrinkles. Lattice anomalies can include, but are not limited to, carbon rings with other than 6 members (e.g. 5, 7 or 9 membered rings), vacancies, interstitial defects (including incorporation of non-carbon atoms in the lattice), and grain boundaries.

[0963] In an embodiment, the layer comprising the sheet of graphene-based material further comprises non-graphenic carbon-based material located on the surface of the sheet of graphene- based material. In an embodiment, the non-graphenic carbon-based material does not possess long range order and may be classified as amorphous. In embodiments, the non-graphenic carbon-based material further comprises elements other than carbon and/or hydrocarbons. Non- carbon elements which may be incorporated in the non-graphenic carbon include, but are not limited to, hydrogen, oxygen, silicon, copper and iron. In embodiments, the non-graphenic carbon-based material comprises hydrocarbons. In embodiments, carbon is the dominant material in non-graphenic carbon-based material. For example, a non-graphenic carbon-based material comprises at least 30% (weight %) carbon, or at least 40% carbon, or at least 50% carbon, or at least 60% carbon, or at least 70% carbon, or at least 80% carbon, or at least 90% carbon, or at least 95% carbon. In embodiments, a non-graphenic carbon-based material comprises a range of carbon selected from 30% to 95%, or from 40% to 80%, or from 50% to 70%). In an embodiment, the amount of carbon in the non-graphenic carbon-based material is measured as an atomic percentage.

[0964] Such nanomaterials in which pores are intentionally created will be referred to herein as "perforated graphene," "perforated graphene-based materials" or "perforated two-dimensional materials." The present disclosure is also directed, in part, to perforated graphene, perforated graphene-based materials and other perforated two-dimensional materials containing a plurality of holes of size (or size range) appropriate for a given application. The size distribution of holes may be narrow, e.g., limited to a 1-10% deviation in size or a 1-20% deviation in size. In an embodiment, the characteristic dimension of the holes is selected for the application. For circular holes, the characteristic dimension is the diameter of the hole. In embodiments relevant to non-circular pores, the characteristic dimension can be taken as the largest distance spanning the hole, the smallest distance spanning the hole, the average of the largest and smallest distance spanning the hole, or an equivalent diameter based on the in-plane area of the pore. As used herein, perforated graphene-based materials include materials in which non-carbon atoms have been incorporated at the edges of the pores.

[0965] In various embodiments, the two-dimensional material comprises graphene, molybdenum disulfide, or hexagonal boron nitride. In more particular embodiments, the two- dimensional material can be graphene. Graphene according to the embodiments of the present disclosure can include single-layer graphene, multi-layer graphene, or any combination thereof. Other nanomaterials having an extended two-dimensional molecular structure can also constitute the two-dimensional material in the various embodiments of the present disclosure. For example, molybdenum disulfide is a representative chalcogenide having a two-dimensional molecular structure, and other various chalcogenides can constitute the two-dimensional material in the embodiments of the present disclosure. Choice of a suitable two-dimensional material for a particular application can be determined by a number of factors, including the chemical and physical environment into which the graphene or other two-dimensional material is to be terminally deployed.

[0966] The process of forming holes in graphene and other two-dimensional materials will be referred to herein as "perforation," and such nanomaterials will be referred to herein as being "perforated." In a graphene sheet an interstitial aperture is formed by each six-carbon atom ring structure in the sheet and this interstitial aperture is less than one nanometer across. In particular, this interstitial aperture is believed to be about 0.3 nanometers across its longest dimension (the center to center distance between carbon atoms being about 0.28 nm and the aperture being somewhat smaller than this distance). Perforation of sheets comprising two-dimensional network structures typically refers to formation of holes larger than the interstitial apertures in the network structure.

[0967] Due to the atomic-level thinness of graphene and other two-dimensional materials, it may be possible to achieve high fluid throughput fluxes during separation or filtration processes, even with holes that are in the ranges of 1-200 nm, 1-100 nm, 1-50 nm, or 1-20 nm.

[0968] Chemical techniques can be used to create holes in graphene and other two- dimensional materials. Exposure of graphene or another two-dimensional material to ozone or atmospheric pressure plasma (e.g., an oxygen/argon or nitrogen/argon plasma) can effect perforation. Other techniques, such as ion bombardment, can also be used to remove matter from the planar structure of two-dimensional materials in order to create holes. All such methods can be applied for preparation of perforated two-dimensional materials for use herein dependent upon the hole sizes or range of hole sizes desired for a given application. [0969] In various embodiments of the present disclosure, the holes produced in the graphene or other two-dimensional material can range from about 0.3 nm to about 50 nm in size. In a more specific embodiment, hole sizes can range from 1 nm to 50 nm. In a more specific embodiment, hole sizes can range from 1 nm to 10 nm. In a more specific embodiment, hole sizes can range from 5 nm to 10 nm. In a more specific embodiment, hole sizes can range from 1 nm to 5 nm. In a more specific embodiment, the holes can range from about 0.5 nm to about 2.5 nm in size. In an additional embodiment, the hole size is from 0.3 to 0.5 nm. In a further embodiment, the hole size is from 0.5 to 10 nm. In an additional embodiment, the hole size is from 5 nm to 20 nm. In a further embodiment, the hole size is from 0.7 nm to 1.2 nm. In an additional embodiment, the hole size is from 10 nm to 50 nm. In embodiments where larger hole sizes are preferred, the hole size is from 50 nm to 100 nm, from 50 nm to 150 nm, or from 100 nm to 200 nm.

[0970] In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The following definitions are provided to clarify their specific use in the context herein.

[0971] As used herein, the term "two-dimensional material" will refer to any extended planar structure of atomic thickness, including both single- and multi-layer variants thereof. Multi-layer two-dimensional materials can include up to about 20 stacked layers.

[0972] A "defect" refers to an opening in a plane of a two-dimensional material. In an embodiment, the defect may be an intrinsic or native defect. Intrinsic or native defects are those resulting from preparation of the two-dimensional material in contrast to perforations which are selectively introduced into a sheet of two-dimensional material. Such intrinsic or native defects include, but are not limited to, lattice anomalies, pores, tears, cracks or wrinkles. Lattice anomalies can include, but are not limited to, carbon rings with other than 6 members (e.g. 5, 7 or 9 membered rings) in graphene or graphene-based materials, vacancies, interstitial defects (including incorporation of non-carbon atoms in the lattice), and grain boundaries. In an embodiment, the defect may be a non-intrinsic defect. Non-intrinsic defects are nanoscale apertures (e.g., pores, holes) formed by a defect formation process, wherein energy (e.g., heat, pressure, electromagnetic radiation and combinations and variations thereof) sufficient to break the chemical bonds of the two-dimensional material is applied to at least one target location of the material. A plurality of non-intrinsic defects may be provided in a uniform or non -uniform (i.e., random) distribution or pattern. Typically, non-intrinsic defects are produced in at a target location of a two-dimensional material with precision of ± 50 nm, ± 10 nm or ± 5 nm. In some embodiments, nanoscale apertures in a two-dimensional material are separated by an average closest edge-to-edge distance less than or equal to 20 nm or less than or equal to 15 nm or less than or equal to 10 nm.

[0973] A defect healing process, as used herein, refers to a process for partially or completely closing one or more openings (defects) in a two-dimensional material. A defect healing process may transform a perforated two-dimensional material into a less perforated or unperf orated two-dimensional material using chemical techniques (e.g., bonding), physical techniques (e.g., blocking) or a combination of chemical and physical techniques. Exemplary healing techniques include, but are not limited to, reforming the crystallographic lattice of the two-dimensional material within the defect area, filling the defect with a material other than the two-dimensional material (e.g., epoxy), and covering the defect with a section of the same or different material, which at least partially overlaps the two-dimensional material. In an embodiment, the process of reforming the crystallographic lattice of the two-dimensional material within the defect area utilizes hydrocarbon-based surface contamination that is mobilized by the addition of energy. In an embodiment, the healing may be performed via a reorganization of existing defects without adding any new material.

[0974] "In situ" methods are performed on a sample that remains in position throughout the method. For example, a sample that remains "in position" is stationary or does not leave a sample chamber during the in situ method. In situ methods according to the concepts disclosed herein are useful for providing data indicative of a spatial and/or temporal change of a sample that remains in its original position. In some embodiments, an in situ method processes (e.g., perforates or heals) the sample and simultaneously interrogates the sample to provide substantially instantaneous, real-time data. In some embodiments, an in situ method processes (e.g., perforates or heals) the sample and performs nearly simultaneously interrogation of the sample to provide substantially near real-time data.

[0975] As described above, the incident radiation, and the scattered, emitted or transmitted radiation may be any one or more of electromagnetic radiation, electrons, ions, nanoparticles, or plasma. In an embodiment, incident radiation is processing radiation, such as perforating or healing radiation or interrogating radiation. The incident radiation may also be both processing radiation and interrogating radiation. As described herein, processing radiation performs a process on the material when incident thereon.

[0976] In some embodiments, the incident perforating or healing radiation may also be interrogating radiation, which interacts with the two-dimensional material to produce scattered, emitted or transmitted radiation that is detected by a detector to provide data indicative of defect formation or healing. In an embodiment, the incident perforating or healing radiation is separate from interrogating radiation, which is produced by an alternate source and which interacts with the two-dimensional material to produce scattered, emitted or transmitted radiation that is detected by a detector to provide data indicative of defect formation or healing.

[0977] In an embodiment, the incident radiation does not perforate the two-dimensional material. The incident non-perforating radiation may interrogate the two-dimensional material by interacting with the two-dimensional material to produce scattered, emitted or transmitted radiation that is detected by a detector to provide data indicative of defect formation or healing.

[0978] In some embodiments, especially for methods configured for local sampling, micromechanical shutters disposed between the two-dimensional material and the detector could be opened and closed to allow for targeted sampling. Activation of the micromechanical shutters could be implemented by each shutter being biased to collect current from a respective local portion of the material. In an embodiment, the shutters may be arranged in an array over the two dimensional material, each shutter corresponding to a respective local portion. Thus, the respective local portion of the material may be electronically monitored by actuating its respective shutter to open to allow for appropriate radiation to be incident on the respective portion, and the electrical conductivity data of the local portion may be acquired. The shutters may be electrostatically actuated. When the emitted current divided by the bias current is not equal to 1, the actuator may move because the charge on the shutter moves the shutter by coulombic force. Other ways of moving the actuator once the appropriate signal is received are contemplated. In an embodiment, the shutters may be arranged in a two-dimensional array. In another embodiment, the shutters may be arranged in a one-dimensional array.

[0979] In some embodiments, probes, such as wires, could be contacted with respective local portions of the two-dimensional material to acquire electrical conductivity data. Thus, the electrical conductivity of the respective local portions may be monitored.

[0980] In embodiments, the two-dimensional material is a graphene-based material. In embodiments, the two-dimensional material is graphene.

[0981] In embodiments, at least a portion of the holes in the two-dimensional material are functionalized.

[0982] Additionally, the conductive properties of graphene-based or other two-dimensional membranes can allow for electrification to take place from an external source. In exemplary embodiments, an AC or DC voltage can be applied to conductive two-dimensional materials.

[0983] In some embodiments, the two-dimensional material, such as graphene, can be affixed to a suitable porous substrate. Suitable porous substrates can include, for example, thin film polymers and ceramics. Useful exemplary ceramics include nanoporous silica or SiN. Useful porous polymer substrates include track-etched polymers, expanded polymers or non- woven polymers. The substrate material can be porous or permeable.

[0984] FIG. 58 shows a graphene sheet 5810 of carbon atoms defining a repeating pattern of hexagonal ring structures that collectively form a two-dimensional honeycomb lattice. An interstitial aperture 5812 of less than 1 nm in diameter is formed by each hexagonal ring structure in the sheet. More particularly, the interstitial aperture in a perfect crystalline graphene lattice is estimated to be about 0.28 nanometers across its longest dimension. Accordingly, graphene materials preclude transport of any molecule across the graphene sheet's thickness unless there are pores, perforation-induced or intrinsic. The thickness of a theoretically perfect single graphene sheet is approximately 0.3 nm. Further, graphene has a breaking strength about 200 times that of steel, a spring constant in the range 1 N/m to 5 N/m and a Young's modulus of about 1 TPa. [0985] FIG. 112 shows a flowchart 11200 for a method for monitoring defect formation or healing via detection of scattered, emitted or transmitted radiation, according to an embodiment of the concepts disclosed herein. Step 11202 involves providing a material having a surface. In step 11204, the surface of the material is exposed to incident radiation. In step 11206, scattered, emitted or transmitted radiation are detected from at least a portion of the material exposed to the incident radiation, and in step 11208 data indicative of defect formation or healing is generated. Typically, the method is performed in situ and the data indicative of defect formation or healing provide a rate of defect formation or healing, a temporal change in the rate of defect formation or healing, a temporal change in the size of the defects, a spatial change in the rate of defect formation or healing, a spatial change in the size of the defects, or combinations thereof.

[0986] FIG. 113 shows a flowchart 11300 for a method for monitoring defect formation or healing via detection of movement of an analyte, according to an embodiment according to concepts disclosed herein. Step 11302 involves providing a material having a surface. In step 11304, the surface of the material is exposed to incident radiation. In step 11306, movement of an analyte through defects in the material is detected, and in step 11308 data indicative of defect formation or healing is generated. Typically, the method is performed in situ and the data indicative of defect formation or healing provide a rate of defect formation or healing, a temporal change in the rate of defect formation or healing, a temporal change in the size of the defects, a spatial change in the rate of defect formation or healing, a spatial change in the size of the defects, a maximum size of the defects, or combinations thereof.

[0987] FIG. 114 shows a flowchart 11400 for a method for monitoring defect formation or healing via measurement of electrical conductivity, according to an embodiment according to concepts disclosed herein. In step 11402, a material having a surface is provided and in step 11404 the surface of the material is exposed to incident radiation. In step 11406, an electrical bias is applied to the material. In step 11408, electrical conductivity is measured with a conductive probe in electrical contact with the material. In step 11410, data indicative of defect formation or healing is generated. Typically, the method is performed in situ and the data indicative of defect formation or healing provide a rate of defect formation or healing, a temporal change in the rate of defect formation or healing, a temporal change in the size of the defects, a spatial change in the rate of defect formation or healing, a spatial change in the size of the defects, or combinations thereof. In an embodiment, a conductive probe is a conductive grid or a local probe. In an embodiment, defect density and electrical conductivity are inversely related such that an increase in defect density is observed as a decrease in electrical conductivity.

[0988] FIG. 115 shows a flowchart 11500 for a method for monitoring defect formation or healing via Joule heating and temperature measurement, according to an embodiment according to concepts disclosed herein. In step 11502, a material having a surface is provided and the surface of the material is exposed to incident radiation, in step 11504. In step 11506, the material is heated. Subsequently, temperature of the surface of the material is measured in step 11508. In step 11510, data indicative of defect formation or healing is generated. Typically, the method is performed in situ and the data indicative of defect formation or healing provides a rate of defect formation or healing, a temporal change in the rate of defect formation or healing, a temporal change in the size of the defects, a spatial change in the rate of defect formation or healing, a spatial change in the size of the defects, or combinations thereof. In an embodiment, the step of heating the material comprises applying a potential to the two-dimensional material to induce joule heating. In an embodiment, defect density and thermal conductivity are inversely related such that an increase in defect density is observed as a decrease in thermal conductivity.

[0989] In embodiments, examples of which are schematically illustrated in FIGS. 116A and 116B, a system 11600 for monitoring a two-dimensional material comprises a source 11602 for delivering incident radiation 11604 to a two-dimensional material 11606, a detector 11608 for receiving scattered, emitted or transmitted radiation or particles 11610 from the two-dimensional material 11606, and a processor 11612 for receiving at least one signal 11614 from the detector 11608 and transforming the signal 11614 into data indicative of defect formation or healing 11622. The data 11622 may be stored in a register or memory of the processor. In the embodiment shown in FIG. 116A, the detector 11608 is positioned on the same side of the two- dimensional material 11606 as the source 11602 to receive scattered or emitted radiation or particles 11610. In the embodiment shown in FIG. 116B, the detector 11608 is positioned on the opposite side of the two-dimensional material 11606 relative to the source 11602 so that the detector 11608 can receive transmitted radiation or particles 11610. In an embodiment (not shown), a system 11600 may include two or more detectors 11608 positioned on one or both sides of the two-dimensional material 11606. Optionally, the system 11600 may also include a controller 11616 that receives input 11617 from the processor and provides control signals 11618 to adjust the incident radiation 11604 or a rate of sample translation in response to the data indicative of defect formation or healing 11622 and/or a display 11620 for visualizing the data. The rate of sample translation may be controlled by translation means, such as rollers 11624 and/or a translation stage. Those of skill in the art will appreciate that for some methods disclosed herein the detector may be located on the same side of the two-dimensional material as the incident radiation source. Such configurations typically utilize detectors that are off-axis between about 15° and 75° relative to the trajectory of the incident radiation beam in order to protect the detector from damage. However, other methods disclosed herein may include a detector located on the opposite side of the two-dimensional material from the incident radiation source. Detectors that may be used in the methods disclosed herein include, but are not limited to, electron detectors, mass spectrometers, electromagnetic spectrometers, microbalances, Faraday cups, charge-coupled devices, ion detectors, resistors, capacitors, thermocouples, microchannel plates, phosphor screens, photodiodes and thermistors.

[0990] Concepts disclosed herein will now be described with reference to the following non- limiting example.

EXAMPLE

IN SITU MONITORING OF GRAPHENE DEFECT FORMATION OR HEALING

[0991] Suspended graphene on a substrate is loaded into an ion chamber on a platen and pumped down to 10 ^-6 - 10 ^-7 Torr while being heated to 50° C. Once the pressure is achieved the ion source (Kaufman source), which is a Xe ⁺ beam of approximately 1mm diameter at 300V and a beam current of 100nA/mm ² (6.24x 10 ¹³ Xe ⁺ /cm ² s), is rastered across the sample. The beam dwells such that the FWHM of the beam profile touches the previous dwell location. The dwell time for each spot is determined by monitoring the secondary electron (SE) emission from the incoming Xe ⁺ using an Everhart-Thornley detector and is compared to known yields (defined as SE emitted for given incident ions in this case) for a given desired pore size, on a particular substrate, with these conditions (i.e. ion voltage, flux, etc.) which were previously acquired empirically. Once the proper SE electron yield is achieved (e.g., actual values match target values on a look-up table), a processor of the control system sends instructions to the instrument to move the beam to the next defect or healing location. The system also accounts for changes in expected yield over time as irradiation progresses.

METHOD FOR TREATING GRAPHENE SHEETS FOR LARGE-SCALE TRANSFER

USING FREE-FLOAT METHOD

[0992] Some embodiments provide a system and method for treating graphene sheet that has been grown on a growth substrate before the growth substrate is removed and the graphene sheet transferred to a functional substrate using the free-float transfer method. The treatment provides a pristine (e.g., substantially residual/contaminant-free) graphene sheet having little to no unintended defects, which is capable of being transferred from the growth substrate with reduced risk of failure (e.g., little risk of tearing, cracking, or forming other undesirable defects) in transferring the sheet to a functional substrate during the free-float transfer method. In some embodiments, the graphene sheet is modified, and thus prepared for transfer, through an application of energy to the graphene sheet while it is disposed on the growth substrate. The energetic application may be in the form of a broad beam ion source configured to irradiate the graphene sheet with ions (e.g., group 18 element ions) such that the graphene sheet is prepared for reliable, large-scale transfer while disposed on the growth substrate. Thus, some of the systems and methods described herein eliminate the need of secondary coating materials (e.g., polymers) to aid in the transfer of the graphene sheet to the functional substrate, thus eliminating the risk of lowering the quality of the graphene sheet through contaminants introduced by the use of secondary coating materials. Accordingly, the transfer preparation method of some of the embodiments allows for the reliable transfer of high quality graphene sheets on a large-scale (i.e., 1 cm ² or larger) using the free-float transfer method.

[0993] FIGS. 117A-117B illustrate a method for growing a large-scale graphene or graphene-based sheet onto a growth substrate according to some embodiments. FIG. 117A shows a first step of preparing a growth substrate 11710 for use in the production of a graphene sheet. The growth substrate 11710 may be any growth substrate appropriate for the production of graphene. For example, in some embodiments, the growth substrate 11710 is a metal catalyst, such as copper or nickel. As shown in FIG. 117A, the growth substrate 11710 is a copper substrate, which is prepared by cleaning the surface with a solvent and annealing the substrate 11710 at a high temperature.

[0994] After preparation of the growth substrate 11710, graphene is grown on both the upper and bottom surface of the growth substrate 11710, which may be accomplished through chemical vapor deposition (CVD) by exposing the growth substrate 11710 to gaseous reactants until graphene is formed. The CVD process results in graphene sheets being synthesized on both a bottom surface of the growth substrate 11710 and an upper surface of the growth substrate 11710. As shown in FIG. 117B, the graphene sheet synthesized on the bottom surface is removed, while the graphene sheet 11710 synthesized on the upper surface is utilized for transfer to a functional substrate. After growth, the graphene sheet 11710 may have carbonaceous material on its surface which, in some cases, may be the result of the growth of the graphene sheet 11710 on the copper substrate. The carbonaceous material may be a material such as amorphous carbon, one or more hydrocarbons, oxygen-containing carbon compounds, nitrogen- containing carbon compounds, or combinations thereof. In the embodiment shown in FIG.

117B, the graphene sheet 11710 is a large-scale sheet having a cross-sectional area in the planar direction of at least 1 cm ² or greater.

[0995] Once the graphene sheet 11720 has been deposited onto the upper surface of the growth substrate 11710, the graphene sheet 11720 may then be transferred to a substrate for a desired application. As shown in FIG. 118, before the graphene sheet 11720 is removed from the growth substrate 11710, the graphene sheet 11720 is prepared for transfer using a transfer preparation apparatus 117100. The transfer preparation apparatus 117100 is configured to impart energy to the graphene sheet 11720 and growth substrate 11710 structure. For example, the transfer preparation apparatus 117100 may be configured to impart ion irradiation to the graphene sheet 11720 and growth substrate 11710. As shown in FIG. 118, the transfer preparation apparatus 117100 may be an ion source configured to supply a plurality of ions 11750 to the graphene sheet 11720. [0996] In certain embodiments, the transfer preparation apparatus 117100 may be configured to provide broad beam ion irradiation to the graphene sheet 11720 and the growth substrate 11710. The broad beam ion source may be collimated or substantially collimated (e.g., five degrees from normal). The plurality of ions 11750 may comprise of ions that are singly charged or multiply charged. In some embodiments, the plurality of ions 11750 may be noble gas ions, such as ions of an element from Group 18 of the periodic table. In some embodiments, the plurality of ions 11750 may be organic ions or organometallic ions. The organic or

organometallic ions may have an aromatic component. In addition, the molecular mass of the organic or organometallic ions may range from 75 to 200 or 90 to 200. In some embodiments, the plurality of ions 11750 may comprise Ne+ ions, Ar+ ions, tropylium ions, and/or ferrocenium ions. In certain embodiments, the plurality of ions 1 1750 comprises Xe+ ions.

[0997] The ion source may be configured to supply the plurality of ions 11750 at a voltage in a range of about 100 V to about 1500 V. In some embodiments, the plurality of ions 11750 may be applied at a voltage in a range of about 250 V to about 750 V. In certain embodiments, the plurality of ions 11750 (e.g., Xe+ ions) may be applied at a voltage of about 500 V.

[0998] During the transfer preparation process, the graphene sheet 11720 and the growth substrate 11730 may be heated to a temperature ranging from about 50°C to about 100°C. In some embodiments, the graphene sheet 11720 and the growth substrate 11730 may be heated to a temperature of about 80°C. In other embodiments, the graphene sheet 11720 and the growth substrate 11730 may be kept at room temperature. In addition, the graphene sheet 11720 and the growth substrate 11730 may be exposed to a pressure of less than 5x 10 ^-7 Torn In some embodiments, the graphene sheet 11720 and the growth substrate 11730 may be exposed to a pressure ranging from 1 x 10 ^-7 Torr to 5 x 10 ^-6 Torn In some embodiments, this process may be set to occur over several hours or overnight.

[0999] The ion source may be configured to provide the plurality of ions 11750 at a flux of about 1 nA/ram ² (6.24 x 10 ¹¹ ions/cm ² /s) to about 1000 nA/ram ² (6.24 x 10 ¹⁴ ions/cm ² /s).. In some embodiments, the plurality of ions 11750 is provided at a flux of about 10 nA/mm ² (6.24 x

10 12 ions/cm 2 /s) to about 100 nA/mm 2 (6.24 x 1013 ions/cm 2 /s). In certain embodiments, the plurality of ions 11750 is provided at a flux of about 40 nA/mm ² (2.5 x 10 ¹³ ions/cm ² /s) to about 80 nA/mm ² (5.0 x 10 ¹³ ions/cm ² /s). In certain embodiments, the plurality of ions 11750 is provided at a flux of about 60 nA/mm ² (3.75 x 10 ¹³ ions/cm ² /s). In embodiments where the plurality of ions 11750 comprises Xe ⁺ ions, the plurality of ions 11750 may be provided at a flux of about 6.24 x 10 ¹¹ Xe ⁺ /cm ² /s to about 6.24 x 10 ¹⁴ Xe ⁺ /cm ² /s. In other embodiments, the plurality of ions 11750 comprises Xe ⁺ ions provided at a flux of about 6.24 x 10 ¹² Xe ⁺ /cm ² /s to about 6.24 x 10 ¹³ Xe ⁺ /cm ² /s. In other embodiments, the plurality of ions 11750 comprises Xe ⁺ ions provided at a flux of about 3.75 x 10 ¹³ Xe ⁺ /cm ² /s.

[1000] The graphene sheet 11720 and the growth substrate 11730 may be exposed to the ion source for a contact time resulting in a total fluence of about 10 nA snA s/mm ² (6.24 x 10 ¹² ions/cm ² ) to about 40 nA snA s/mm ² (2.5 x 10 ¹³ ions/cm ² ). In certain embodiments, the graphene sheet 11720 and the growth substrate 11730 are exposed for under a second such that the total fluence is 20 nA snA s/mm ² (1.25 x 10 ¹³ ions/cm ² ). In embodiments where the plurality of ions comprises Xe ⁺ ions, the graphene sheet 11720 and the growth substrate 11730 may be exposed for a contact time that results in a total fluence of about 10 nA s/mm ² to about 40 nA s/mm ² (or about 6.24 x 10 ¹² XeVcm ² to about 2.5 x 10 ¹³ XeVcm ² ). In certain

embodiments where the plurality of ions 11750 comprises Xe ⁺ ions, the total exposure time results in a total fluence of about 1.25 x 10 ¹³ XeVcm ² . The upper limit of total fluence for the transfer preparation process may increase as the atomic number of the plurality of ions 11750 decreases. In some embodiments, the upper limit of the total fluence may be about 120 nA s/mm ² . In other embodiments, the upper limit of the total fluence may be about 500 nA s/mm ² . In some embodiments, the upper limit of the total fluence may be about 1000 nA s/mm ² . For example, in embodiments where the plurality of ions comprises Ne ⁺ ions, the graphene sheet 11720 and the growth substrate 11730 may be exposed for a contact time that results in a total fluence of about 10 nA s/mm ² (6.24 x 10 ¹² ions/cm ² ) to about 120 nA s/mm ² (7.5 x 10 ¹³ ions/cm ² /s). In some embodiments, the graphene sheet 20 and the growth substrate 30 may be exposed to a plurality of neon ions for a contact time that results in a total fluence of about 10 nA s/mm ² to about 500 nA s/mm2. In other embodiments, the graphene sheet 20 and the growth substrate 30 may be exposed to a plurality of neon ions for a contact time that results in a total fluence of about 10 nA s/mm ² to about 1000 nA s/mm ² . In yet other embodiments, the graphene sheet 20 and the growth substrate 30 may be exposed to a plurality of neon ions for a contact time that results in a total fluence of up to 2 x 10 ¹⁴ ions/cm ² .

[1001] After the above treatment, the graphene sheet 11720 and the growth substrate 11730 may be exposed to about 1 atm of N ₂ as a final step in the process before transferring of the graphene sheet 11720 to the functional substrate. The result of the preparation process is, in effect, a "toughened" graphene sheet 11720 that may be reliably transferred to a functional substrate using the unsupported free-float transfer method while being resistant to forming or inducing unintentional defects (tears, cracks, wrinkles, unintentionally-created pores) in the graphene sheet 11720 during the free-float transfer process. The treatment thus provides a toughened graphene sheet 11720 that is capable of providing a high coverage area (e.g., 99% or more of the functional substrate is covered by the graphene sheet) over the functional substrate and a clean surface for effective use of other treatment processes (e.g., perforating processes). While not being restricted to any particular theory for the mechanism that prepares or toughens the graphene sheet 11720 for transfer, the toughening may be facilitated by the presence of the carbonaceous material and the interaction between the graphene sheet 11720 and the copper growth substrate 11710 interface. The ion beam irradiation may provide sufficient energy to the carbonaceous material to reform the graphene sheet 11720 while on the copper substrate 1 1710 to a pristine layer due to the sputtering of the carbon atoms present in and/or on the surface of the graphene sheet 11720.

[1002] Once the graphene sheet 11720 has been prepared using the transfer preparation apparatus 117100, the graphene sheet 11720 and the growth substrate 11710 composite is placed in an etchant bath 11730, as shown in FIG. 119 A. The etchant bath 11730 allows the growth substrate 10 to be etched away such that a clean graphene sheet 11720 remains. The etchant bath 11730 may be any appropriate etchant capable of etching the growth substrate 11710 from the graphene sheet 11720. For example, for copper-based growth substrates, the etchant bath 11730 may include iron chloride, iron nitrate, and/or ammonium persulfate. In some embodiments, the graphene sheet 11720 and the growth substrate 11710 composite may be placed in a second etchant bath 11730, which may include the same or a different etchant, to further aid in the complete etching of the growth substrate 11710 from the graphene sheet 11720. [1003] As shown in FIG. 119B, the etchant bath 11730 is then gradually removed and replaced with a floating bath 11735 that may serve as a floating mechanism to transfer the graphene sheet 11720 to a functional substrate 11740. The floating bath 11735 may be a water- based solution, such as water (e.g., deionized water) or a mixture of water and a solvent (e.g., isopropyl alcohol). For example, in some embodiments, the etchant bath 11730 may be removed by the gradual introduction of deionized water, which may then be additionally introduced as a mixture of deionized water and isopropyl alcohol. As the graphene sheet 11720 floats in the floating bath 11735, the functional substrate 11740 may be introduced below a bottom surface of the graphene sheet 11720, as shown in FIG. 119B. In some embodiments, a floating frame (not shown) may be disposed around the graphene sheet 11720 during this process to provide stability to the graphene sheet 11720 as it floats in the solution and then applied to the functional substrate 11740. The floating bath 11735 is then gradually removed such that the fluid level decreases to lower the graphene sheet 1 1720 onto the substrate 11740. One or more additional graphene sheets 11720 that have been prepared for transfer using the transfer preparation apparatus 117100 may be stacked onto the functional substrate 11740 as needed using the free- float transfer method.

[1004] FIGS. 120 and 121 show images of a graphene sheet that was prepared for transfer by an embodiment of a transfer preparation apparatus configured to supply collimated broad beam ion irradiation using Xe+ ions. FIG. 120 shows a prepared graphene sheet after removal of the copper growth substrate by chemical etching. The prepared graphene sheet shown in FIG. 120 is large-scale sheet having dimensions approximately 9 cm by 14 cm (or about 126 cm ² extended planar area). The black circular markings shown in FIG. 120 delineate the boundaries of the graphene sheet.

[1005] FIG. 121 shows a prepared graphene sheet like that shown in FIG. 120 after it has been transferred to a functional substrate (a polymer membrane substrate in the embodiment shown in FIG. 121). Like FIG. 120, the prepared graphene sheet is a large-scale sheet having dimensions approximately 9 cm by 14 cm. As shown in FIG. 121, the graphene sheet and functional substrate composite shows a graphene sheet that is free of visible, unintentional defects. While some defects may occur along the edges due to collisions with the walls of the etchant bath tank while the sheet was free-floating, the prepared graphene sheet does not show any visible defects (e.g., visible tears, crack, or wrinkles) within the main body of the sheet even after the free-float and lowering of the graphene sheet onto the functional substrate without the use of secondary polymer support materials. This indicates that the preparation process of the graphene sheet using the transfer preparation apparatus results in a graphene sheet that is toughened to be resistant to unintentional defects that may arise during the free-float transfer process.

[1006] FIGS. 122 and 123 show SEM images of a prepared graphene sheet that was prepared for transfer by an embodiment of a transfer preparation apparatus configured to supply collimated broad beam ion irradiation using Xe ⁺ ions. After preparation, the prepared graphene sheet was transferred to a functional substrate in the form of a track-etched polymer substrate having a plurality of pores using the free-float transfer method as described above. In the embodiment shown in the figures, the plurality of pores has a nominal pore size ranging from 350 nm to 450 nm. The total field of view shown in FIG. 122 is approximately 0.036 mm ² (about 225 μπι x 160 μπι), while FIG. 123 shows a detailed area of the top-left quadrant of the graphene sheet shown in FIG. 122.

[1007] The pores present in the polymer substrate that are covered by the prepared graphene sheet are shown as medium gray in FIGS. 122 and 123. Pores that are uncovered due to unintentional defects present in the prepared graphene sheet due to the transfer process are shown in black. As shown in FIGS. 122 and 123, greater than 99% of the substrate pores are covered by the prepared graphene sheet indicating high coverage area of the prepared graphene sheet over the polymer substrate.

[1008] Although embodiments have been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that these only illustrative herein. It should be understood that various modifications can be made without departing from the spirit herein. Embodiments can be modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the spirit and scope herein. Additionally, while various embodiments herein have been described, it is to be understood that aspects herein may include only some of the described embodiments. Accordingly, embodiments are not to be seen as limited by the foregoing description.

[1009] Every formulation or combination of components described or exemplified can be used to practice embodiments, unless otherwise stated. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently. When a compound is described herein such that a particular isomer or enantiomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomers and enantiomer of the compound described individual or in any combination. One of ordinary skill in the art will appreciate that methods, device elements, starting materials and synthetic methods other than those specifically exemplified can be employed in the practice herein without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, starting materials and synthetic methods are intended to be included herein. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure.

[1010] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements. Embodiments illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. [1011] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope. Thus, it should be understood that although embodiments have been specifically disclosed by preferred

embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of embodiments as defined by the appended claims.

[1012] In general the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The preceding definitions are provided to clarify their specific use in the context herein.

[1013] All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).

[1014] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which embodiments pertain. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art, in some cases as of their filing date, and it is intended that this information can be employed herein, if needed, to exclude (for example, to disclaim) specific embodiments that are in the prior art. For example, when a compound is claimed, it should be understood that compounds known in the prior art, including certain compounds disclosed in the references disclosed herein (particularly in referenced patent documents), are not intended to be included in the claim.