BACKGROUND

Nanoconfinement-based manipulation is a powerful approach for controlling the conformation of single DNA molecules. When single polymer chains are squeezed into environments confined at length scales below their diameter of gyration in free solution, the polymer equilibrium conformation will be molded by the surrounding nanoscale geometry.

Nanoconfinement-based manipulation, compared with competing techniques for single-molecule manipulation such as tweezer technology and surface/hydrodynamic-based stretching, has several advantages. It is highly parallel, providing the high throughput essential for mapping large-scale genomes. It can be efficiently integrated with microfluidics to rapidly cycle molecules through the channel arrays for upstream/downstream pre- and postprocessing of DNA. It does not require applied flow or electric force to maintain the DNA extension.

SUMMARY

Nanoconfinement-based approaches have a key difficulty inherent to the use of nanoscale dimensions: the need to bridge length scales differing by up to 5 orders of magnitude (submillimeter scale of a pipette tip to channels in the 10-100 nm range) in the same fluidic device. This introduces challenges in device design and manufacture. There is a need to drive single-molecule analytes across a very high free-energy barrier at the edge of nanoconfined regions. There is inefficient fluid transport due to the high hydraulic resistance of channels with nanoscale dimensions. These challenges have limited the practical range of nanochannel dimensions to 40-50 nm. Moreover, the high hydraulic resistance of nanoscale features (for a slit of depth h, the hydraulic resistance scales as 1/h ³ , compared with 1/h for electrical resistance) can require that electrophoretic actuation or some other substantial force, be used to drive DNA into sub-100-nm nanochannels. This in turn necessitates the use of special high-salt electrophoresis buffers [e.g. 2-5xTris/borate/EDTA (TBE)], which reduces DNA extension and constrains the imaging buffer used. Other methods also introduce constraints and limits.

One answer to these challenges is to develop specialized grayscale lithography approaches that can create gently funneling channel dimensions, reducing the free-energy barrier. Gray- scale approaches, although they are feasible technologically, are still highly challenging to implement and remain limited in the range of confinement that can be varied continuously in both lateral and vertical dimensions.

To overcome the challenges faced by classical nanofluidic technology, described herein is a new approach for introducing tunable nanoscale confinement, in combination with electrophoresis, to trap and align DNA molecules for further analysis including optical analysis, electron-optical analysis, high resolution sequencing and functional genomic analysis.

The devices described herein provide the ability to isolate and purify single, long (>10kb) DNA molecules. In addition to DNA sequencing, the device allows researchers and clinical investigators to perform single molecule analyses on multiple analytes as well as perform additional investigations beyond DNA sequencing.

Presented herein are methods for analyzing biological analytes, including single biological molecules, combinations of different biological molecules of the same type, and combinations of biological molecules of different types using the device platform. In addition to the biological molecules, cells or exosomes can also be analyzed using the devices, as described further herein.

According to one aspect, nanofluidic devices are provided including a deformable flow-cell formed between flow-cell surfaces, wherein at least one of the flow-cell surfaces comprises an array of posts, and an electric field generator that provides an electric field capable of pulling or driving biological molecules into and/or about the flow-cell. In some embodiments,

In some embodiments, the post array ensures an even confinement around the region of contact of the post array and the flow-cell surfaces. In some embodiments, one or more of the flow-cell surfaces comprise embedded micro- and/or nano-topographies. In some embodiments, one or more of the flow-cell surfaces are formed by a coverslip. In some embodiments, one or more of the flow-cell surfaces comprise glass, quartz, silicon, or silicon wafer derived material surfaces or are formed of glass, quartz, silicon, or silicon wafer derived materials.

In some embodiments, one or more of the flow-cell surfaces comprises a hexagonal array of post extrusions. In some embodiments, the posts forming the array are 30-μιη- spaced posts. In some embodiments, the posts forming the array are 5-100 nm tall. In some embodiments, the post array forms a nanoslit.

In some embodiments, one of the flow-cell surfaces comprises linear embedded nanogrooves. In some embodiments, the nanogrooves are about 20-30 nm, 30-40 nm, or 40- 50 nm deep; about 50-nm wide; and about 500-μιη long.

In some embodiments, walls of the flow-cell are coated with a surface-passivation agent, optionally polyvinyl pyrrolidone (PVP).

In some embodiments, the flow-cell comprises a floor, and the floor of the flow-cell comprises a microchannel. In some embodiments, the microchannel is about 30^m-deep and about 200^m-wide. In some embodiments, the microchannel encircles the nanogroove array and imaging region.

In some embodiments, the electric field generator comprises internal electrodes. In some embodiments, at least one of the internal electrodes is centrally located relative to the flow-cell. In other embodiments, the internal electrodes are non-centrally located relative to the flow-cell. In some embodiments, the internal electrodes are located outside of the flow- cell or peripherally to the flow-cell.

In some embodiments, the electric field generator comprises external electrodes.

In some embodiments, the electric field generator comprises a combination of external and internal electrodes.

According to another aspect, nanofluidic devices are provided including a deformable flow-cell formed between flow-cell surfaces, and an electric field generator that provides an electric field capable of pulling or driving biological molecules into and/or about the flow- cell, wherein the electric field generator comprises (1) external electrodes; (2) internal electrodes that are non-centrally located relative to the flow-cell; or (3) a combination of external and internal electrodes.

In some embodiments, at least one of the flow-cell surfaces comprises an array of posts that ensures an even confinement around the region of contact of the post array and the flow-cell surfaces.

In some embodiments, one or more of the flow-cell surfaces comprise embedded micro- and/or nano-topographies. In some embodiments, one or more of the flow-cell surfaces are formed by a coverslip. In some embodiments, one or more of the flow-cell surfaces comprise glass, quartz, silicon, or silicon wafer derived material surfaces or are formed of glass, quartz, silicon, or silicon wafer derived materials.

In some embodiments, one or more of the flow-cell surfaces comprises a hexagonal array of post extrusions. In some embodiments, the posts forming the array are 30-μιη- spaced posts. In some embodiments, the posts forming the array are 5-100 nm tall. In some embodiments, the post array forms a nanoslit.

In some embodiments, one of the flow-cell surfaces comprises linear embedded nanogrooves. In some embodiments, the nanogrooves are about 20-30 nm, 30-40 nm, or 40- 50 nm deep; about 50-nm wide; and about 500-μιη long.

In some embodiments, walls of the flow-cell are coated with a surface-passivation agent, optionally polyvinyl pyrrolidone (PVP).

In some embodiments, the flow-cell comprises a floor, and the floor of the flow-cell comprises a microchannel. In some embodiments, the microchannel is about 30^m-deep and about 200^m-wide. In some embodiments, the microchannel encircles the nanogroove array and imaging region.

In some embodiments, the electrodes are located outside of the flow-cell or peripherally to the flow-cell.

According to another aspect, methods for analyzing the interaction of biomolecules are provided. The methods include loading one or more biomolecules and optionally one or more reagents into the deformable flow-cell of the nanofluidic device described above having an array of posts, and an electric field generator comprising electrodes, applying pressure on a deformable flow-cell surface, such that the flow-cell surfaces contact the post array, and applying voltage to the electric field generator to pull or drive the biological molecules into the flow-cell.

In some embodiments, the pressure is applied by contacting the upper flow-cell surface with a Convex Lens-Induced Confinement (CLIC)-lens or Convex Lens-Induced Nanoscale Templating (CLINT) device.

In some embodiments, the methods also include obtaining images of the one or more biomolecules.

In some embodiments, the methods also include altering the buffer used in loading the one or more biomolecules into the flow-cell prior to analysis of the biomolecules or during analysis of the biomolecules. In some embodiments, the step of altering the buffer includes removing the buffer used in loading the one or more biomolecules from the flow-cell and replacing the buffer with a different buffer.

According to another aspect, methods for analyzing the interaction of biomolecules are provided. The methods include loading one or more biomolecules and optionally one or more reagents into the deformable flow-cell of the nanofluidic device described above having an electric field generator that includes (1) external electrodes; (2) internal electrodes that are non-centrally located relative to the flow-cell; or (3) a combination of external and internal electrodes, applying pressure on a deformable flow-cell surface, and applying voltage to the one or more electrodes to pull or drive the biological molecules into the flow-cell.

In some embodiments, the pressure is applied by contacting the upper flow-cell surface with a Convex Lens-Induced Confinement (CLIC)-lens or Convex Lens-Induced Nanoscale Templating (CLINT) device.

In some embodiments, the methods also include obtaining images of the one or more biomolecules.

In some embodiments, the methods also include altering the buffer used in loading the one or more biomolecules into the flow-cell prior to analysis of the biomolecules or during analysis of the biomolecules. In some embodiments, the step of altering the buffer includes removing the buffer used in loading the one or more biomolecules from the flow-cell and replacing the buffer with a different buffer.

According to another aspect, methods for analyzing biological analytes are provided.

The methods include using the devices described herein that include an analysis chamber that permits analysis of one or more single biological molecules, multiple biological molecules, combinations of biological molecules, cells or exosomes. In some embodiments, the one or more single biological molecules, multiple biological molecules, or combinations of biological molecules is/are DNA, RNA, and/or protein. In some embodiments, the device provides for repetitive analysis of the one or more single biological molecule, set of multiple biological molecules, combinations of biological molecules, cells or exosomes.

In some embodiments, the method includes introducing one or more single biological molecules, multiple biological molecules, combinations of biological molecules, cells or exosomes into the analysis chamber of the device, subjecting the one or more single biological molecules, multiple biological molecules, combinations of biological molecules, cells or exosomes to analysis in the analysis chamber using a first set of reagents, removing the first set of reagents from the analysis chamber, and subjecting the one or more single biological molecules, multiple biological molecules, combinations of biological molecules, cells or exosomes to analysis in the analysis chamber using a second set of reagents. In some embodiments, first and second sets of reagents are used to query the properties or identity of a subset of multiple biological molecules, combination of biological molecules, cells or exosomes. In some embodiments, wherein the first and second sets of reagents are used to query different properties of the biological molecule, multiple biological molecules or a subset thereof, combination of biological molecules or a subset thereof, cells or exosomes.

In some embodiments, the biological molecule, a set of multiple biological molecules, a combination of biological molecules, cells or exosomes is not destroyed during the analysis, thereby permitting repetitive analysis of the biological molecule, the set of multiple biological molecules, the combination of biological molecules, cells or exosomes.

In some embodiments, the device traps the biological molecule, set of multiple biological molecules, combination of biological molecules, cells or exosomes in the analysis chamber.

In some embodiments, the device includes a nanofluidic flow cell. In some embodiments, the analysis chamber of the device includes a nanofluidic flow cell.

In some embodiments, the biological molecule, set of multiple biological molecules, combination of biological molecules, cells or exosomes are trapped by deposition in the analysis chamber. In some embodiments, the deposition includes tethering of the biological molecule, set of multiple biological molecules, combination of biological molecules, cells or exosomes in the analysis chamber.

In some embodiments, the sample volume used is less than 1 milliliter. In some embodiments, the sample volume is 1 μΐ, 2 μΐ, 3 μΐ, 4 μΐ, 5 μΐ, 6 μΐ, 7 μΐ, 8 μΐ, 9 μΐ, 0.01 ml, 0.02 ml, 0.03 ml, 0.04 ml, 0.05 ml, 0.06 ml, 0.07 ml, 0.08 ml, 0.09 ml, 0.10 ml, 0.20 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, or 0.9 ml.

In some embodiments, the biological molecule(s) is one or more DNA molecules, and wherein analysis of the DNA molecules includes analysis of DNA sequence, optionally including heavy atom labeling.

In some embodiments, the biological molecule(s) is one or more DNA molecules, and wherein analysis of the DNA molecules includes analysis of ultra-long DNA molecules (>500kb), optionally wherein the analysis is without functionalization. In some embodiments, the biological molecule(s) is one or more DNA molecules, and wherein analysis of the DNA molecules includes fluorescent in situ hybridization (FISH) analysis; karyotyping; comparative genomic hybridization (CGH); non-invasive prenatal testing

(NIPT); aneuploidy analysis; mutation detection; analysis of inherited disorders; analysis of hyper/hypo methylation; genome and nucleic acid mapping analysis; analysis of phasing mutations; analysis of paternal or maternal origin; analysis of microbiome(s); or analysis of

DNA modifications, optionally epigenetic modifications.

In some embodiments, the biological molecule(s) is one or more RNA molecules, and wherein analysis of the RNA molecules includes analysis of RNA sequence; analysis of microRNA, such as identification and quantification; RNA identification and quantification; and analysis of RNA modifications.

In some embodiments, the biological molecule(s) is one or more protein molecules, and wherein analysis of the protein molecules includes identification; quantification; analysis of protein/protein interactions; and analysis of protein modifications, such as acetylation, phosphorylation, ubiquitination and sumoylation.

In some embodiments, the biological molecules are a combination of biological molecules, and wherein analysis of the combination of biological molecules includes analysis of DNA and RNA, analysis of DNA and protein, analysis of RNA and protein, or analysis of

DNA and RNA and protein.

In some embodiments, the biological analyte is one or more cells and/or one or more exosomes.

In some embodiments, a plurality of analyses are performed sequentially. In some embodiments, the sequential analysis includes observing the same information multiple times for confirmation or statistical analysis; observing similar information over time (e.g., sequential DNA probes of different sequence); and/or observing different information types over time (DNA probes, binding proteins, melting point).

In some embodiments, the surfaces of the analysis chamber are locally planar.

In some embodiments, one or more of the surfaces of the analysis chamber include cavities for capturing the biological molecule(s), cells or exosomes.

In some embodiments, the surfaces of the analysis chamber are movable relative to one another, or wherein the surfaces optionally have a fixed proximity to one another. In some embodiments, the surfaces of the analysis chamber are substantially parallel to one another, or locally parallel to one another. In some embodiments, the analysis chamber is a nanofluidic flow cell, and wherein the top and bottom surfaces of the nanofluidic flow cell are parallel.

In some embodiments, the surfaces of the analysis chamber include variable geometry, such that there are changes in distance between surfaces.

In some embodiments, the includes a controller for controlling the distance between surfaces.

In some embodiments, the analysis chamber of the device includes fixed posts on at least one of the surfaces to regulate the minimum distance between the surfaces of the analysis chamber.

In some embodiments, the device includes a dynamically adjustable nanofluidic platform as described in Henkin et al., Anal Chem. 2016 (PMID: 27767294; DOI:

10.1021/acs.analchem.6b03149).

In some embodiments, the device captured materials by moving surfaces relative to one another.

In some embodiments, the surfaces can come into contact, preventing flow of materials in the regions of contact.

In some embodiments, the biological molecule(s) are selected and/or trapped in a cavity in a surface of the analysis chamber, such as a nanochannel or other nano-cavity, according to one or more properties of the biological molecule(s), cells or exosomes. In some embodiments, the one or more properties of the biological molecule(s), cells or exosomes is size, shape, duration in the cavity, charge, mass, density, dipole moment, surface attraction, biological interaction, chemical interaction, and/or environmental impacts.

In some embodiments, the device includes a detector that detects the biological molecule(s), cells or exosomes by electron microscopy, light microscopy, fluorescent microscopy, atomic force microscopy, pH, heat discharge, and/or mass.

Other aspects, embodiments and features of the invention will become apparent from the following detailed description when considered in conjunction with the accompanying drawing(s). The accompanying figures are schematic and are not intended to be drawn to scale. In the figures, each identical, or substantially similar component that is illustrated in various figures may be represented by a single numeral or notation (though not always). For purposes of clarity, not every component is labeled in every figure. Nor is every component of each embodiment shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. All patent applications and patents incorporated herein by reference are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

DETAILED DESCRIPTION

Nanochannel arrays can be used for massively parallel extension of DNA across an optical field, serving as the basis for a high throughput optical or electron-optical mapping of genomes, high resolution sequencing and functional genomic analysis. More varied manipulations can be performed based on the design of the surrounding nanotopology, such as using nanocavities embedded in a nanoslit to trap single DNA molecules.

Confinement-based imaging technology combines nanotemplated substrates with a single-molecule imaging technique called "convex lens-induced confinement" (CLIC). Fig. 1 of Berard et al., ("Convex lens-induced nanoscale templating" Proc Natl Acad Sci U S A. 2014 Sep 16; 111(37): 13295-300) illustrates a flow cell implementation, in which molecules are initially loaded into a planar micron-scale chamber. To form the imaging chamber, the upper chamber surface is subsequently pressed into contact with the lower surface. The final vertical confinement profile varies gradually away from the contact point, typically increasing by tens of nanometers over a field of view of a hundred microns.

When performed over a surface containing nano-templated structures such as nanocavities and nanogrooves, the vertical confinement imposed in CLIC drives the single- molecule analytes into the embedded topology. In the CLIC approach, molecules are loaded gently by imposing confinement from above, eliminating the need for high pressures or electric fields to introduce single-molecule analytes into the confined region of the device.

The ease of loading in CLIC ensures that, when used to confine macromolecules in structures with dimensions smaller than their persistence length, the process is compatible with a much wider range of ionic conditions than classic direct-bonded devices that rely on electrophoresis for loading. This "physical" loading of the chambers allows for multiple different solvents or buffers to be used. Thus loading of the DNA is not solvent dependent as is electrophoresis based loading. In addition, the procedure of sample loading in CLIC allows the user to alter the solvents to achieve specific applications or ask specific biological questions requiring different biological conditions that can be controlled by utilizing different solvents. Complex reaction combinations can be performed with sequential buffer/solvent/reagent combinations that would not be possible when constrained by the need to maintain compatibility with electrophoretic conditions.

Loading molecules from above can also reduce the sensitivity of the technique to fabrication defects that can lead to clogging of direct-bonded channels loaded from the side. As shown in Berard et al. (Proc Natl Acad Sci U S A. 2014 Sep 16; 111(37): 13295-300), convex lens-induced nanoscale templating (CLINT) can efficiently load DNA into nanochannels less than 30 nm in size, imposing sub-persistence length confinement at which stretching is high, polymer back- folding is energetically unfeasible, and thermal fluctuations are suppressed (i.e., the Odijk regime).

The ability to operate devices in the Odijk regime is critical as the suppression of back-bending and thermal fluctuations leads to more efficient alignment of optically mapped DNA fragments to a reference genome. Finally, unlike enclosed classical nanofluidic devices, a CLINT device is an open system, whereby the interior can be easily exposed, leading to greater reusability and ease of access. This feature can be used, for example, to load single cells at precise locations on the chip adjacent to nanofluidic features and to apply surface coatings to suppress nonspecific interactions of fluorescent probes and proteins with exposed device surfaces.

DNA molecules are loaded into a chamber formed by two transparent surfaces, such as a fused silica coverslip and substrate, separated by a spacer (typically 5- to 30^m-thick adhesive tape). Alternatively, other materials can be used which provide the mechanical, chemical and optical properties appropriate to the application. Flowcells can be made from the 3-component (two transparent plus one spacer) design or the spacer can be integrated into one of the transparent layers.

Molecules loaded into the chamber are initially unconfined (Berard et al., Fig. 1A, Proc Natl Acad Sci U S A. 2014 Sep 16; 111(37): 13295-300). A convex lens mounted on a nanopositioner presses down on the upper coverslip, deforming it (Berard et al., Figs. IB and 2B, Proc Natl Acad Sci U S A. 2014 Sep 16; 111(37): 13295-300). The upper surface bows downward until it comes into contact with the lower planar coverslip at a single point, forming the chamber geometry (Berard et al., Fig. 2C, Proc Natl Acad Sci U S A. 2014 Sep 16; 111(37): 13295-300). The applied vertical confinement causes DNA molecules to favor extended conformations due to self-exclusion interactions in the region surrounding the contact point, where the chamber height is less than the diameter of gyration D _g of DNA in free solution. The lower surface of the imaging chamber contains embedded nano- lithography (e.g., nanochannels) subjecting the DNA to additional transverse confinement.

When the chamber height is small enough, the molecules' free energy is minimized when they are maximally extended in the channels (Berard et al., Fig. IB, Proc Natl Acad Sci U S A. 2014 Sep 16; 111(37): 13295-300), which were observed to occur spontaneously. The CLINT technique can be applied to other nanotopologies (e.g., nanopits) and other nanomaterials (e.g., actin, microtubules, DNA nanotubes, nanowires).

When DNA is confined to a nanochannel of dimensions less than D _g , it will stretch out along the channel axis due to excluded volume interactions. If the width D of a cross- section nanochannel is larger than the 50 nm persistence length P of the DNA, the polymer is described by the de Gennes confinement regime and will coil up into multiple blobs along the nanochannel axis. When D < P, coiling within the channel is suppressed and the molecule undergoes periodic deflections along the walls with no back-bending (i.e., the Odijk regime). When the chamber height has microscale vertical dimensions, DNA molecules are unconfined and take on coiled conformations. When the chamber height is reduced, the imposed vertical nanoscale confinement causes DNA molecules to align in the nanochannels, their energetically preferred state.

This can be implemented using a microscopy device shown in Fig. 2A of Berard et al. (Proc Natl Acad Sci U S A. 2014 Sep 16; 111(37): 13295-300), mounted on an inverted fluorescence microscope. The imaging chamber is formed between two confining surfaces. In this instance, the lower surface is a fused silica substrate containing embedded

nano topography. In this instance, the upper surface is a coverslip with two small holes sandblasted into the corners for fluid insertion and recovery. Double sided adhesive tape (30- or 10-μιη thick; Nitto Denko) separates the upper and lower chamber surfaces, and is laser-cut to create channels for liquid to flow into a main central chamber.

Computer-controlled syringe pumps insert and retrieve the sample from the imaging chamber, facilitating serial measurements and sample recovery. Chemically inert fluorinated ethylene propylene tubing connects the pump outlets to the holes in the upper coverslip and a seal is formed between them by a thick silicone gasket. To form the imaging chamber, a lens mounted on a nanopositioner presses down on the upper coverslip, causing it to deform around its curved surface.

The lower surface of the imaging chamber contains nano- and microscale features, including nanochannels. These channels can be of various lengths and distances from one another. The depths and widths are generally below the persistence length of the DNA under the confinement conditions (temperature, ionic strength of solution, etc.). These features can be patterned in fused silica using electron-beam lithography (e.g., VB6 UHR EWF; Vistec Lithography) and etched using reactive ion etching (RIE). One-micron-deep microchannels, can be defined using contact UV photolithography (EVG620; EVG) and etched using RIE. The microchannels enable buffer and reagent exchanges.

Flowcells

As described further below (see "Entropic Confinement Chamber" section) and elsewhere herein, a flowcell can be deformable to provide tunable nanoscale confinement. The flowcells can be of any convenient size. The 25mm x 25mm format, referred to commonly as "coverslip size," is compatible with many general purpose lab tools, especially microscopic tools. The 3mm format is compatible with industry- standard electron microscope sample holders. Other formats and sizes are compatible with different production methods. For example, for lithographically defined components, smaller sizes results in a larger number of devices or components per wafer processed, creating scale economies.

Flowcells can be of differing materials. Optically transparent flowcells have advantages when used in applications taking advantage of optical properties of analytes, reagents and samples. Other materials are advantageous for electron optical applications, especially taking advantage of thin support materials for transmission electron microscopy and electrically conductive materials for managing static charge buildup. Materials can include a combination of soft (PDMS, thermoplastic, etc.) lithographic materials and hard (glass, quartz, silicon, silicon wafer derived materials, etc.) lithographic materials.

Flowcells can be designed for reprocessing and reuse. Such reuasable flowcells can be designed to be disassembled, cleaned and reassembled. Assembly can include sacrificial layers destroyed in reprocessing or reversible bonding of components. Alternatively, flowcells can be partially disposable, with some components reprocessed and reused while others are used once and disposed of.

Flowcells can be sealed, or allow additions, removals, and exchange of materials. A sealed flowcell would have analytes sealed within chambers or channels. Open cells allow for materials to be added to the flowcell after sealing the components together. Mixed flowcells can have a blend of open features and sealed features.

Flowcell chambers and separations can be conditional, depending on bringing the "ceiling" and "floor" into proximity. These regions can be isolated from one another, or connected by distinct fluidic elements (flow channels, valves, etc.) which are partially defined by the "roof and partially defined by the "floor" of the flowcell.

Flowcells are not, of necessity, assembled from the three components in the example (floor, ceiling and sticky tape "walls"). They can be assembled from more, or fewer components, based on manufacturing priorities. Entropic Confinement Chamber

The "chamber" is the region of a deformable flowcell that is compressed, evacuated, otherwise deformed or with intrinsically variable dimensions in order to create a region of confinement. The surfaces brought together are referred to, alternately as the "ceiling" and "floor". These terms are meant only to differentiate one from the other. Different features and functionality can be embedded in either the ceiling or floor or in both, in whatever combination is appropriate for the application at hand.

Alternatively, the chamber can be made with fixed dimensions that allow for entropic confinement of molecules that are brought into the chamber. Flow Features

Flow features include systems that allow for reagents to be directed to and from different parts of the flowcells, especially flow channels, valves and junctions. Flow features can include valves that seal portions of the flowcells from one another. Flow features can include compression zones of the flowcell that allow for the chamber height to vary, without changing the volume of the flowcell outside of the confinement chamber/region. Flow features can include pressure management inlets/outlets that allow for the internal pressure to be changed either as part of the chamber compression (e.g. internal vacuum) or independently of chamber compression.

Flow features can include posts or other obstructions that prevent the chamber from reaching a zero height, i.e., different that used in CLIC or CLINT devices. Such features can be used to control the degree of chamber compression. This degree of control can vary from one portion of the flowcell to another. For example, maintaining a minimal chamber height of tens of nanometers in the vicinity of one or more nanochannels, but allowing a lesser or greater dimension in other areas.

Microfluidic flow features or elements can be directly connected to nanofluidic components. This will allow additional flow into the compression chamber and along the nano-channels independent of chamber compression.

Inlets/Outlets

Inlets and outlets can be provided in several geometries. They can be a part of any component, from the top, bottom or side of the chamber.

Surface Chemistry/Modifications

Different surfaces of the flowcells can be modified chemically prior to flowcell assembly. These modifications can include, but are not limited to: changes of

hydrophilicity/hydrophobicity, linkers with specific chemical or biological reactivities, changes to electrical conductance, changes to optical properties, modifications of surface roughness, application of adhesives.

Surface modification can be different for different elements or regions of surfaces. For example, the nanochannels in the chamber can be functionalized differently from the surfaces of the chamber. Or the area opposite the nanofeatures, which are brought into close proximity to the nanofeatures when the chamber is compressed, can be differentially functionalized.

Morphological Features

Morphological features of the chamber include features to control the chamber dimensions (posts of varying size, shape, pitch, pattern) and recessed for trapping reagents or analytes (linear or curved nanochannels, pits and depressions matched to particular analytes or classes of analytes (polymerases, restriction enzymes, probes). Morphological features can also be functionalized, either uniformly or differentially. Recessed morphological features (including nanochannels) can be connected to one another or separated. Connections can be extensions of nanochannels; for example, channels at right angles to parallel channels or hemi-circular channels connecting adjacent parallel channels to one another.

In some embodiments, at least one of the flow-cell surfaces (e.g., the top and/or bottom surfaces) includes an array of posts. In some embodiments the top flow-cell surface will include the post array. The post array ensures an even confinement around the region of contact of the post array and the flow-cell surfaces, in contrast to prior devices in which deformation of the top surface of a flow cell resulted in a convex area of confinement.

In some embodiments, post array formed on one or more of the flow-cell surfaces is a hexagonal array of post extrusions. Other geometries of posts can also be used. In instances where posts are formed on both of the top and bottom flow-cell surfaces, the respective arrays each form complementary parts of the whole post array. The spacing of the posts in the array can be determined according to the use of the device, the dimensions of the flow-cell, the height of the posts, the materials used, etc. For example, the posts forming the array can be spaced about 30-μιη apart, but other spacings of posts can be used also, such as 10-μιη apart, 20-μιη apart, 30-μιη apart, 40-μιη apart, 50-μιη apart, 60-μιη apart, 70-μιη apart, and so on.

The height of the posts in the post array can be selected depending on the particular use of the device, the dimensions of the flow-cell, the spacing of the posts in the array, the materials used, etc. In some embodiments, the posts forming the array are 5-100 nm tall, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm tall. The posts in the array are preferably substantially the same height to provide a uniform distance between the top and bottom surfaces of the flow-cell, e.g., such that the top and bottom surfaces of the flow-cell are substantially parallel. In some embodiments, the post array forms a nanoslit that can be used for introduction and exchange of reagents.

One or more of the flow-cell surfaces includes embedded micro- and/or nano- topographies. Typically the bottom surface (also referred to as the floor herein) contains the embedded micro- and/or nano-topographies. In some embodiments, one of the flow-cell surfaces includes linear embedded nano grooves. The nanogrooves may be between 10-100 nm deep, 10-100 nm wide, and 100-1000μιη long, though the depth, width and length are only limited by the materials used and the size of the device. In some embodiments, the nanogrooves are about 40-50 nm deep, about 50-nm wide and about 500- μιη long. Other micro- and/or nano-topographies can be included instead of nanogrooves, and mixtures of different micro- and/or nano-topographies can be included.

The flow-cell can include a microchannel in the floor of the flow-cell, which can be used for delivery of reagents to the flow-cell. The shape and dimensions of the microchannel can be determined by the size of the device and the particular use of the microchannel. In some embodiments, the microchannel is about 30^m-deep and about 200^m-wide. In some embodiments, the microchannel can encircle the embedded nano-topographies, such as a nanogroove array, and the imaging region.

Multiplicity of Chambers

There can be more than one chamber, with analytes flowing from one to another after being processed.

Compressor/Jig

The flowcells are integrated into a system via a jig. This jig can be a single, integrated device or a set of devices that comprise the functions of a single, integrated device. The primary functions of the jig are to:

1) Mechanically connect and align a flowcell or flowcells with an optical microscope.

This includes providing optical paths for effective operation of the optical

microscope;

2) Provide fluidic connections between the flowcell and external fluidic components, especially including reservoirs, tubing, pumps and sensors; and

3) Provide a force that compresses, or otherwise reduces the chamber height. This can be a mechanical, pneumatic, hydrostatic, electrical, magnetic or other force.

Electrodes and Electrokinetic Methods

Electrokinetic methods of DNA injection into micron- and nanoscale fluidic devices offer numerous advantages over hydrodynamic flows for lab-on-a-chip applications.

Provided an electrical field between two electrodes, DNA will migrate along the field lines with a speed of V _D NA = μΕ, where μ is the electrophoretic mobility and E is the magnitude of electrical field. Electrokinetic DNA injection can be used to (1) work around hydrodynamic resistance in very confined environments, (2) purify the material entering the device, (3) avoid possible device clogging due to debris in the original sample, (4) concentrate DNA, and (5) localize DNA to certain device sub-domains.

Described herein are devices that provide varied uses of electrokinetic DNA manipulation. In one embodiment, DNA is delivered steadily into the interrogation region of the device (e.g., imaging or analysis region, such as a flow cell) under the influence of the electrical field. In this embodiment the DNA concentration does not change appreciably from the original sample concentration. In another embodiment the DNA is concentrated within the device against a nanoporous matrix like a hydrogel. In this embodiment, the smaller ions that carry the electrical current flow freely through the matrix but the DNA compacts against the gel boundary. In yet another non-limiting embodiment the DNA is driven from one region of the device to another using multiple electrode / obstacle pairings.

In the first embodiment, electrokinetic DNA injection provides the first three advantages described above: lower resistance, purification, and boosted device reliability. A minimum of two electrodes with an imposed voltage are required to complete the electrical circuit, plus a biological pH buffer with suitable ionic strength to carry the current such as TBE (Tris-borate-EDTA: 45 mM Tris, 45 mM boric acid, and 1 mM EDTA, pH 8.3). This embodiment can be considered a replacement of (or supplement to) the hydrodynamic flow, and the device otherwise functions similarly.

In the next embodiment, DNA is concentrated on-device in addition to the other advantages of the first embodiment. There are numerous advantages to on-device concentrating, including maximizing device throughput and increasing the efficiency of sample usage. In the invention, a nanoporous obstacle blocks the migration of DNA within the device. In the preferred embodiment, the obstacle is a polyacrylamide microgel (for example, see Meltzer et al, 2010, DOI: 10.1039/c01c00477d) with buffer back sweeping to avoid ion depletion. However, the devices and methods disclosed herein can use any selective boundary that maintains electrical current but inhibits DNA migration.

In yet further embodiments, concentrated DNA is shuttled about the device as a localized group (a "band") impinging on different obstacles— driven by different

electrodes— as the needs of the assay require. Entanglement of polymers, like DNA, becomes an issue at very high DNA concentration. However, the disclosed devices and methods avoid this by impacting and immobilizing the DNA against the obstacles. When the electrode polarities are reversed, the concentrated DNA emerges as a tight band yet does not have time for entanglement to occur. Full morphological equilibration of a polymer solution can take days. So long as the device operation completes substantially faster than the equilibration time, then entanglement is not an issue.

The devices and methods disclosed herein contemplate all applications of the combination of electrical DNA manipulation with nanoconfinement. One significant but non-limiting application is the delivery and localization of a highly concentrated band of DNA adjacent or overlapping with the nanochannel array. In this method, the obstacle is patterned within the device and some advantage is taken that the obstacle is compliant, such as with hydrogels, to permit CLIC lens actuation. DNA is concentrated against the obstacle, then the current is reversed to position the band of highly concentrated DNA over the nanochannel region, and lastly the CLIC lens is lowered. In other embodiments, the use of posts facilitates movement of DNA molecules without alterations of the dimensions of a flow cell or nanoconfinement area.

Each of the embodiments describe DNA manipulation with regard to an applied electrical field provided by an electric field generator. The electric field generator can include electrode locations in varied configurations. In some embodiments, the electrodes are all external to the device. In other embodiments the device contains internal electrodes (such as electrodes that are "on-chip") that are used in conjunction with external electrodes. And in other embodiments the device only uses internal electrodes.

The electrode(s) used in the device can be located in any of several places. The placement of one or more electrodes includes: a central positive electrode, peripherally located negative electrode(s) and positive electrode(s), negative electrode(s) and positive electrode(s), negative electrode(s) and positive electrode(s) located at inlet and outlet positions, etc. Moreover, the attracting electrode can be an array of electrodes rather than a single electrode. There can be electrodes that operate in series, at different steps in the process.

The electrode voltage applied can be varied for different steps in the process, though it need not necessarily be varied. For example, a certain first voltage can be used to drive DNA molecules into the chamber, and a second, different voltage can be used to retain the DNA molecules while the chamber height is changed. Other analytes can use different voltages or combinations of voltages, as appropriate.

The voltage can be varied, or turned off after the DNA is trapped, or if deposition is used, after the DNA is deposited. The voltage can be varied, or turned off before or after buffer is exchanged.

Electrokinetic methods comprise electrophoretic and electroosmotic components. In the preferred embodiment, electroosmosis is suppressed for fast and reliable DNA transport. Many methods of electroosmotic flow suppression are known to those skilled in the art, and the invention considers all such methods. Dynamic coatings such as UltraTrol™ are a preferred balance of simplicity, effectiveness, and reusability.

Operation of the System

The system can be operated in many ways, depending on the objective at hand. In one example:

1) An assembled flowcell is mounted in the Compressor/Jig.

2) DNA is introduced into the flowcell via external fluidic connections and pumps mated to the flowcell via the compressor/jig. DNA is subsequently presented to the

Chamber via internal fluidic elements. Alternatively, DNA is delivered to the flowcell and chamber by a combination of fluidic forces and

electrokinetic/electrophoretic forces .

3) The Chamber is compressed by forces applied by the compressor/jig, decreasing the Chamber geometry. DNA entropic confinement ensues.

Electrophoretic/electrokinetic forces optionally are used to retain DNA in chamber while the volume is reduced and subsequent fluid outflow occurs.

4) Reagents are introduced into the flowcell via external fluidic connections and pumps mated to the flowcell via the compressor/jig. Reagents are subsequently presented to the Chamber via internal fluidic elements.

5) Reagents interact with the DNA, inducing deposition of the DNA onto one or more surfaces of the flowcell. Optionally, reagents are used to interrogate the DNA. These steps are optionally repeated with either the same reagents or different reagents.

6) The flowcell is disassembled. 7) The component of the flowcell with the deposited DNA is used for subsequent analysis.

Alternatively:

1) An assembled flowcell is mounted in the Compressor/Jig

2) DNA is introduced into the flowcell via external fluidic connections and pumps mated to the flowcell via the compressor/jig. DNA is subsequently presented to the

Chamber via internal fluidic elements. Alternatively, DNA is delivered to the flowcell and chamber by a combination of fluidic forces and

electrokinetic/electrophoretic forces .

3) The Chamber is compressed by forces applied by the compressor/jig, decreasing the Chamber geometry. DNA entropic confinement ensues.

Electrophoretic/electrokinetic forces optionally are used to retain DNA in chamber while the volume is reduced and subsequent fluid outflow occurs.

4) Reagents are introduced into the flowcell via external fluidic connections and pumps mated to the flowcell via the compressor/jig. Reagents are subsequently presented to the Chamber via internal fluidic elements.

5) Reagents interact with the DNA, binding to and labeling the DNA in a sequence dependent manner. Optionally, reagents are used to interrogate the DNA. These steps are optionally repeated with either the same reagents or different reagents.

6) The labeled DNA is interrogated with the optical microscope that is optically

connected to the flowcell via the Compressor/Jig.

Certain steps are optional. Such optional steps can include:

1) Presenting reagents, then then flushing reagents out of the flowcell or chamber.

2) Repeating the presentation of reagents, either the same as before, or a different

reagent or mix of reagents.

3) Between cycles of reagent presentation, interrogating the flowcell via optical

microscopy or other analytical techniques. 4) The order of operations can vary. For example the chamber can be compressed before it is loaded with DNA, then loaded using electrokinetic and/or fluidic forces. This takes advantage of the posts which prevent the chamber from reaching complete closure. Similarly, DNA can be deposited while the chamber is compressed, then the chamber can be expanded for subsequent cycling of reagents, including reagents that can reverse the deposition.

5) Deposition of DNA or other analyte onto one or more internal flowcell surfaces. This allows for immobilization of the analyte for further processing, with advantages similar to other immobilization techniques such as bead-binding or precipitation. 6) Temperature control and variation. Temperature can be changed to aid or suppress certain reactions at different steps in the overall process.

7) Sequential processing of flowcell elements. A flowcell can be partially disassembled, then reassembled with other flowcell components for subsequent processing. For example, DNA could be deposited onto one surface of a flowcell. The component including that surface could be removed from the flowcell and assembled with similar or different components into a second flowcell for further processing steps.

Another unique benefit of the device is the reversibility of the nanoconfinement. Because the confining roof is held in place by a lowered push-lens, the lens can be lifted in order to retrieve molecules that have been confined in reactions. In particular, a bifunctional linker, e.g., APTES, can be introduced that binds DNA to exposed hydroxyl groups on the clean silica surface. Other bifunctional linkers are appropriate for other analytes and other surface reactivities.

Modification of selected surfaces with a fluorinated silane (or other passivating treatment) renders those confining surfaces inert - thusly, the user can select the surface that DNA interacts with. Relaxing the compressor, removing the flow cell from the Jig, and disassembling the flowcell allows high-yield retrieval of templated, extended DNA. This technique can be extended to SEM and TEM-compatible membranes with silica or similarly treatable surfaces, such as silicon nitride.

A new method of linearizing long DNA molecules and depositing them on selected surfaces is described herein, which allows high-yield retrieval of extended and deposited molecules. Modifications allow chemical treatment of DNA molecules while they are linearized: an array of posts that suppresses the confinement gradient and stabilizes the chamber, and a microchannel that delivers reagents to the nanochannel region. The chamber can be disassembled for further processing.

Additional features of the devices described herein and useful in the methods described herein are known in the art, such as in: Henkin et al. (Anal. Chem. 2016, 88:

11100-11107); Berard et al. (Proc Natl Acad Sci U S A. 2014 Sep 16; 111(37): 13295-300); Berard et al. (Appl. Phys. Lett. 2016, 109: 033702); Berard et al. (Advanced Photonics OSA Technical Digest (online) (Optical Society of America, 2014), paper SeM4C.3,

https://doi.org/10.1364/SENSORS.2014.SeM4C3); Berard et al. (Rev Sci Instrum. 2013 Oct;84(10): 103704); and Ahamed et al. (Macromolecules. 2016, 49(7): 2853-2859). Each of these articles is incorporated by reference for the teaching contained therein of CLIC, CLINT, and other nanofluidic devices.

Provided herein are methods for analyzing biological molecules, cells or exosomes using the device platform described herein that permits analysis of single biological molecules (DNA, RNA, or protein), multiple biological molecules (multiple DNAs, multiple RNAs, or multiple proteins), combinations of biological molecules (DNA, RNA, and/or protein), cells or exosomes. For example, the platform provides for analysis of long DNA molecules, including single molecule DNA molecules, which in some embodiments can be amplified (such as by PCR amplification), or in other embodiments are not amplified.

The device platform provides for repetitive analysis of a biological molecule, a set of multiple biological molecules, a combination of biological molecules, cells or exosomes. For example, the biological molecule, multiple biological molecules, combination of biological molecules, cells or exosomes can be introduced into an analysis chamber, subjected to analysis using a first set of reagents, subjected to removal of the first set of reagents, and subjected to analysis using a second set of reagents. In some embodiments, the first and second sets of reagents are used to query the properties or identity of a subset of multiple biological molecules, combination of biological molecules, cells or exosomes. In other embodiments, the first and second sets of reagents are used to query the different properties (including identity) of the biological molecule, multiple biological molecules (or a subset thereof), combination of biological molecules (or a subset thereof), cells or exosomes.

Further analyses can use additional sets of reagents, such as a third set of reagents, fourth set of reagents, fifth set of reagents, and so on. The repetitive analysis is possible because the device platform permits analysis of the biological molecule, set of multiple biological molecules, combination of biological molecules, cells or exosomes without sample destruction. In some embodiments, the device platform traps the biological molecule, set of multiple biological molecules, combination of biological molecules, cells or exosomes in an analysis chamber, such as a nanofluidic flow cell. For example, the device platform can trap the biological molecule, set of multiple biological molecules, combination of biological molecules, cells or exosomes by deposition in the analysis chamber. In some instances, the deposition can include tethering of the biological molecule, set of multiple biological molecules, combination of biological molecules in the analysis chamber, cells or exosomes. "Tethering" as used herein includes, for example, connecting a surface of the analysis chamber to the biological molecule, set of multiple biological molecules, combination of biological molecules, cells or exosomes. In other instances, the biological molecule, set of multiple biological molecules, combination of biological molecules, cells or exosomes is connected by a linker, a covalent bond, a non- covalent bond, a hydrophobic interaction, a protein-protein bond, a protein-nucleic acid bond, or a nucleic acid-nucleic acid bond. In other instances, the biological molecule, set of multiple biological molecules, combination of biological molecules, cells or exosomes is retained in the analysis chamber without tethering. The sample can be deposited on one or more surfaces of the analysis chamber, such as on a bottom surface or top surface (e.g., a lid) of the analysis chamber.

The ability to use the device platform to repetitively analyze the biological molecule, set of multiple biological molecules, combination of biological molecules, cells or exosomes means that only one sample having a small sample volume is needed (e.g., of blood or tissue) to do multiple analyses on same biological molecule(s) , cells or exosomes. This provides for analysis of rare samples or small samples, and also provides for greater compliance and comfort for patients because larger quantities of biological samples (e.g., blood or tissue biopsies) are not required. The sample volume in some embodiments is less than 1 milliliter, such as 0.01 ml, 0.02 ml, 0.03 ml, 0.04 ml, 0.05 ml, 0.06 ml, 0.07 ml, 0.08 ml, 0.09 ml, 0.10 ml, 0.20 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, or 0.9 ml.

Analysis of DNA molecules using the device platform includes: analysis of DNA sequence (e.g., by heavy atom labeling, e.g., as described in US patents 7288379, 7291468, 7291467, 7604942, 7604943, 7910311, 8697432, or PCT application PCT/US2016/059058); fluorescent in situ hybridization (FISH); karyotyping; comparative genomic hybridization (CGH); non-invasive prenatal testing (NIPT); aneuploidy analysis; mutation detection;

analysis of inherited disorders, such as CFTR, DMD, etc.; analysis of hyper/hypo

methylation; genome and nucleic acid mapping analysis; analysis of phasing mutations; analysis of paternal or maternal origin; analysis of microbiome(s); and analysis of DNA modifications such as epigenetic modifications, including methylation. One particular advantage of the device platform is the ability to analyze ultra-long DNA molecules

(>500kb), including analysis without functionalization.

Analysis of RNA molecules using the device platform includes: analysis of RNA sequence; analysis of microRNA, such as identification and quantification; RNA

identification and quantification; and analysis of RNA modifications.

Analysis of protein molecules using the device platform includes: identification; quantification; analysis of protein/protein interactions; and analysis of protein modifications, such as acetylation, phosphorylation, ubiquitination and sumoylation).

Analysis of a combination of biological molecules using the device platform includes: analysis of DNA and RNA, analysis of DNA and protein, analysis of RNA and protein, or analysis of DNA and RNA and protein. Such analysis of a combination of biological molecules using the device platform includes: multi-omic analysis, e.g., analysis of genome, epigenome, transcriptome, proteome, etc. of a single sample; analysis of nucleic acid-protein binding and enzymatic modification of nucleic acids; and analysis of the effects of real-time change(s) in reaction conditions.

Advantages of the device platform include that: the biological molecule(s), cells or exosomes in a sample stay wet and localized in an analysis chamber; the biological molecule(s), cells or exosomes in a sample need not be tethered, but can be tethered for certain applications at certain points in the process of analysis; the ability to add, remove, and/or flush reagents. Other analytical platforms flush materials of interest out of analysis chambers along with reagents.

Advantages of the device platform also include that there is no pressure applied, or substantially no pressure applied. Other analytical platforms can require large hydrostatic pressures.

Advantages of the device platform also include that the analysis is non-destructive, with no sample damage, or minimal sample damage. Other analytical platforms/methods consume or alter the materials of interest in the sample such that they cannot be further analyzed.

Another advantage flows from the ability to run multiple analyses on a single sample.

For example, a first analytical reagent set may be applied to analyze a subset of biological materials, a subset of a combination of biological materials, cells or exosomes. The first

analysis may result in selective sample damage of the analyzed subset of biological materials, subset of a combination of biological materials, cells or exosomes, but not other subsets of biological materials, subsets of a combination of biological materials, cells or exosomes.

This permits further analysis of the other subsets of biological materials, subsets of a

combination of biological materials, cells or exosomes, because those remain undamaged by the first analysis.

Advantages of the device platform also include the ability to perform sequential

analysis. This includes: observing the same information multiple times for confirmation or statistical analysis; observing similar information over time (e.g., sequential DNA probes of different sequence); and observing different information types over time (DNA probes,

binding proteins, melting point).

Advantages of the device platform also include the aforementioned ability to perform multi-omic analysis using a single device platform. This provides advantages in speed of

analysis, consistency of analysis, relevance of analysis (e.g., the same sample is used for

multiple analyses), cost of analysis, and capital cost.

The current procedure for fluorescence in situ hybridization (FISH) performs analysis on intact cells, but the chromosomal location during analysis requires the identification of other cellular characteristics to confirm that the analytical signals generated are representative of the molecular state and not due to random probe association. The technology described herein has the advantage of creating addressable positions on a chip that can be queried for heavy atoms to determine sequence in electron microscopy nucleic acid sequencing. The "addressable" property of the technology coupled with the ability to analyze single molecule and very long molecules is also an advantage in FISH analysis. Additionally, by assuring a single molecule analytical scenario, with large enough chip capacity that could reach statistical significance, by counting single molecules and identifying the percentages of observed chromosome abnormalities to normal sequences present, one can determine the heterogeneity of a tumor or cancer sample. This is a very important question associated within oncology research, and is currently being developing utilizing single cells. The single "long" molecules on the chip essentially represent a single cell. Also, the device platform described herein results in much higher throughput, and lower cost due to easier analysis and its ability to be automated.

Comparative genomic hybridization (CGH) analysis uses FISH analysis in a parallel manner with the comparison of hybridization strength to detect major disruptions in the duplication process of the DNA sequences in the genome. The technology described herein creates single, long and addressable molecules. CGH analysis results in differences in hybridization intensities. With the device platform described herein, one can count a statistically significant number of molecules and determine with much greater precision the differences in the numbers of deletions and amplifications present in the sample being analyzed. Also, the device platform described herein provides in much higher throughput, and lower cost due to easier analysis and its ability to be automated.

The device platform described herein also has advantages in targeted single gene disorder mutation detection. The growing standard for "multi-plex" mutation detection for prenatal diagnosis and carrier status is to analyze a patients genomic DNA with sequencing technology. Although this approach has the capabilities of analyzing for the presence of a large number of mutations, there is important clinical information lost by not being able to "phase" mutations, and to know if the mutations are located on the same chromosome or different chromosomes. Also, being able to phase other DNA abnormalities (e.g. structural variants, "SVs") with the presence of Single Nucleotide Variants ("SNVs") has important clinical value. With the ability to analyze long, single molecule addressable DNA fragments, the device platform described herein provides the capability of accurately quantifying the number of abnormal molecules as well as understanding the association between the abnormalities relative to their presence on the same chromosome or different chromosomes.

The device platform described herein also has advantages in analysis of tumor

heterogeneity. Although there is growing literature supporting the fact that tumor

heterogeneity and mutation burden within a tumor adds important clinical information

beyond known pathway associated abnormalities directly contributing to malignancy,

currently there is no standard methodology to determine tumor heterogeneity. Pathology

approaches and more recently digital pathology and single cell analyses are being developed.

The device platform described herein provides for generation of long, single molecule

addressable DNA fragments. Given the length of the molecules, each molecule theoretically represents a single genome equivalent. By accurately quantifying abnormalities (SNV, SVs, etc.) associated with the DNA one could more readily and inexpensively determine tumor heterogeneity.

The device platform described herein also has advantages for whole genome hyper and hypo methylation analysis. Presently, there are a number of approaches taken to analyze for sequence specific hypermethylation and hypomethylation. Most of the approaches are

PCR based, and therefore association between varying regions within the genome that may be hypermethylated and hypomethylated are very targeted and not quantitative. The device

platform described herein provides isolated single long addressable DNA molecules; having done so, one can query the genomic DNA simultaneously for regions of hypermethylation and hypomethylation while quantifying and understanding the special association of the

hyper, and hypomethylated regions.

The device platform described herein also has advantages in microbiome analysis. Currently scientists in the field determine species and categorize microbial species by the 16S RNA sequence. Although somewhat effective, there is not enough diversity within the 16S RNA sequence to get maximum species resolution. As we move into the clinical microbiome world, and start to ask how the microbiome influences human disease, there is a clear need for methodologies that can resolve large number of species quantitatively and qualitatively. The device platform described herein provides a platform with single molecule long DNA molecules that can be utilized to obtain more precise mapping and resolution between bacterial species. Such analysis would also allow the technology to be introduced a Point of Care basis. This will be important due to the very large impact microbiome analysis will have on a population basis.

The device platform described herein also has advantages for multi-omic analysis. There is no single platform available today that can analyze DNA, RNA, protein etc. due to the fact that each analyte requires different analytical and biochemical conditions for analysis. The device platform described herein provides the ability to secure analytes at addressable position within an analysis chamber. As such, different zones can be set up in an analysis chamber, such as a DNA zone, RNA zone, and protein zone, etc. With the capabilities of changing the reaction conditions by flushing and changing components within the analysis chamber, one can analyze the DNA, change the reaction solution, flush, add RNA specific analysis components, flush, add protein analysis components etc.

The device platform includes the following features which provide the

aforementioned advantages over other analytical devices. The surfaces of the analysis

chamber of the device, such as a nanofluidic flow cell, are locally planar. One or more of the surfaces of the analysis chamber of the device include cavities for capturing materials, e.g., the biological molecule(s). In addition, the surfaces can be moved relative to one another, or the surfaces can have a fixed proximity to one another. The surfaces in some embodiments are substantially parallel to one another, or at least locally parallel to one another. For example, the top and bottom surfaces of a nanofluidic flow cell can be parallel. In addition, the device can provide for the surfaces having variable geometry, such that there are changes distance between surfaces. Also includes are features for controlling the distance between surfaces, such as controlling the maximum and/or minimum distances between the surfaces (such as top and bottom surfaces of the analysis chamber). In one embodiment, fixed posts are provided on at least one of the surfaces to regulate the minimum distance between the surfaces.

The device platform can be used to capture materials by moving surfaces relative to one another. For example, the top and bottom surfaces of the analysis chamber can be moved closer to one another to force the biological molecule(s), cells or exosomes in a sample into a cavity in a surface, e.g., which may impose a particular configuration or conformation on the biological molecule(s), cells or exosomes. After material is captured, the surfaces can be moved and remain out of contact to allow for flow of subsequent materials, such as analytical reagents, wash reagents, etc. Optionally, in some regions, the surfaces can come into contact, preventing flow of materials in the regions of contact. Steps of analysis can be repeated, with variation in order if so desired.

The device platform is fabricated from materials such as silicon, glass, soft lithography materials (PDMS and similar), or hard lithography materials (Si, SiOx, SixNy, GaAs, etc.). The different parts of the device platform can include different materials. For example, the analysis chamber may have surfaces made of glass fully or partially (e.g., the layer forming the inside of the chamber), while other parts of the device can be made of different materials for purposes of strength, cost, ability, and so on.

The biological molecule(s) (e.g., DNA, RNA and/or protein, etc.), cells or exosomes can be selected and/or trapped in a cavity, such as a nanochannel or other nano-cavity, according to one or more properties of the biological molecule(s), cells or exosomes.

In some embodiments, the property of the biological molecule(s), cells or exosomes is size. For example, the cavity can have dimensions that allow for selection of certain biological molecule(s), cells or exosomes, and prohibit other biological molecule(s), cells or exosomes from entry into the cavity.

In some embodiments, the property of the biological molecule(s), cells or exosomes is shape. For example, the cavity can have dimensions that allow for trapping certain biological molecule(s), cells or exosomes based on shape (e.g., long and narrow for DNA; a unique shape for a protein of a particular shape, such as a figure 8 shape, or shape of a polymerase).

In some embodiments, the property of the biological molecule(s), cells or exosomes is duration. For example, the biological molecule(s), cells or exosomes are allowed to be in the presence of cavities for a limited time. This can increase selectivity by ensuring that only the biological molecule(s), cells or exosomes having the property or properties most appropriate to the cavities will be selected.

In some embodiments, the property of the biological molecule(s), cells or exosomes is charge. For example, the cavity or nearby region of the analysis chamber is functionalized with charge-bearing or charge- supporting materials for attracting or repelling the biological molecule(s), cells or exosomes. In another example, an electric field is applied in vicinity of a cavity to attract or repel the biological molecule(s), cells or exosomes.

In some embodiments, the property of the biological molecule(s), cells or exosomes is mass or density.

In some embodiments, the property of the biological molecule(s), cells or exosomes is dipole moment. For example, a cavity or the analysis chamber exposed to a magnetic field to attract or repel biological molecule(s), cells or exosomes having a selected dipole moment.

In some embodiments, the property of the biological molecule(s), cells or exosomes is surface attraction. For example, the cavity is functionalized or passivated to attract or repel materials biological molecule(s), cells or exosomes having selected surface attraction properties.

Biological materials that can be selected and/or trapped in one or more nanochannels or other nano-cavity for subsequent analysis include: DNA, RNA, cells, such as circulating tumor cells, exosomes, and specific proteins. The biological materials in some instance are biological molecule(s), while in other instances the biological materials include or encompass biological molecule(s), such as cells or exosomes. For example, cells or exosomes including DNA, RNA, and/or proteins can be analyzed; alternatively, after selecting and/or trapping the cells or exosomes, the biological molecule(s) of interest can be selected or extracted from the cells or exosomes for further analysis in the device platform.

The properties of biological molecule(s), cells or exosomes that can be observed using the device platform, individually or in combination, include: size, shape, charge, biological or chemical interaction, and environmental impacts. Thus, observation of the presence of one or more biological molecule(s), cells or exosomes in a size- selective cavity will confer information about the size of the biological molecule(s), cells or exosomes. Observation of the presence of one or more biological molecule(s), cells or exosomes in a shape-selective cavity will confer information about the shape of the biological molecule(s), cells or exosomes. Observation of the presence of one or more biological molecule(s), cells or exosomes in a charge- selective cavity will confer information about the charge of the biological molecule(s), cells or exosomes. Observation of the interaction of one or more biological molecule(s), cells or exosomes with biological or chemical materials present in the vicinity of a cavity (or in the cavity) will confer information about the biological or chemical interaction of the biological molecule(s), cells or exosomes. Observation of the effect of environmental properties on one or more biological molecule(s), cells or exosomes in a cavity will confer information about the environmental impacts of materials on the biological molecule(s), cells or exosomes. The absence of observation of one or more of these properties also confers information about the biological molecule(s), cells or exosomes.

Different portions of the device can be arranged for observation of different properties. For example, some cavities in the analysis chamber may be configured to analyze size of one or more biological molecule(s), cells or exosomes, while other cavities in the analysis chamber may be configured to analyze shape of the one or more biological molecule(s), cells or exosomes, while still other cavities in the analysis chamber may be configured to analyze charge, biological or chemical interaction, etc. of the one or more biological molecule(s), cells or exosomes.

The same biological molecule(s) can be observed for different properties sequentially, such as by presenting different materials to the region of the cavity at different times.

In addition, the foregoing properties of one or more biological molecule(s), cells or exosomes can be observed as a function of temperature, pressure, pH, salt concentrations, salt species, etc., alone or in combination. The device platform also detects the properties of the one or more biological molecule(s), cells or exosomes. Depending on the properties being analyzed by the device platform, one or more of the following modes of detection can be included in or applied to the device platform.

Electron microscopy can be used, such as TEM, SEM, STEM or related charged particle microscopy.

Optical microscopy can be used, such as light microscopy, e.g., for unlabeled samples; or fluorescent microscopy, e.g., for biological molecule(s) that have natural fluorescence, are fluorescently labeled directly or by binding of a fluorescently-labeled reagent to the biological molecule(s), or by use of reagents that fluoresce when in proximity to the biological molecule(s).

Other modes of detection or properties detected include: atomic force microscopy (AFM), pH (observed via proximity to pH meter); heat discharge (observed via proximity to calorimeter), and mass (observed by impact of mass on nearby materials, such as by vibration mode of oscillator, etc.)

Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description is by way of example only.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. In addition, any combination of two or more of such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or," as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.