MAKING SAME

TECHNICAL FIELD

[001] The present disclosure relates to diagnostic devices and more particularly to encapsulated porous substrate-based (e.g., paper-based) medical and food safety diagnostic devices and methods of making the same.

BACKGROUND

[002] Simple, low-cost diagnostic technologies are an important component of strategies for improving healthcare and access to healthcare in developing nations and resource-limited settings. According to the World Health Organization, diagnostic devices for use in developing countries should be ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end users).

[003] Inexpensive, portable, and easy-to-use diagnostic devices have used a porous substrate including a reagent selected to rapidly perform quantitative or qualitative analysis of a fluid sample such as, for example, a bodily fluid, an industrial fluid, food samples or water, in the field when laboratory facilities are not available or easily accessed for sample analysis. In one example, a paper- based diagnostic device includes a colorimetric immunoassay reagent with a color change as a readout, and the color change readout can be detected visually or with a machine to provide a rapid, low-cost diagnosis of the presence of an infectious disease.

[004] In various examples, analytes in a sample can be rapidly detected using the diagnostic devices include viral antigens, bacterial antigens, fungal antigens, parasitic antigens, cancer antigens, metabolic markers, and combinations thereof. In one example, in an immune -chromatographic diagnostic assay, antibodies acting as binding proteins can be used to capture disease-relevant biomarkers from the patient sample, and then produce a visible diagnostic signal resulting from the binding event.

[005] In some examples, the diagnostic devices include multiple layers of a porous material disposed in planes parallel to one another and in face-to-face contact. The various layers of the diagnostic device include fluid impermeable hydrophobic regions and hydrophilic water absorbent regions arranged to provide a sample flow path configured such that a fluid sample can wick or flow from one layer to another. At least some of the layers may include reagents, buffer salts, analytes (for example antigens) and binders (for example, antibodies) selected to perform a multiplexed assay.

[006] U.S, 8,628,729 describes a method for making paper-based devices that includes building fluid- impermeable areas in paper. U.S. 9,669,638 describe ink-jet printing methods for making hydrophobic areas in otherwise hydrophilic media. U.S. 9,597,684 describes other methods for forming liquid- impermeable barriers in hydrophilic media. SUMMARY

[007] In general, the present disclosure is directed to inexpensive, easy to use diagnostic devices for quantitative or qualitative analysis of a sample fluid including an analyte. Suitable sample fluids include, but are not limited to, body fluids (e.g., blood, sputum, saliva, or urine), industrial fluids, water samples, and the like. The diagnostic device includes at least two substantially planar portions, each planar portion made from a hydrophilic material such as paper. The planar portions are stacked on each other and each occupy a different and substantially parallel plane to form a three- dimensional porous structure.

[008] To manufacture a diagnostic device including multiple planar regions of a porous substrate, optionally having different reagents or different patterns of hydrophobic and hydrophilic regions, multiple layers must be individually produced, accurately stacked and aligned to provide the sample flow path, and adhered together in order to maintain the continuity of the sample flow path and form an operable stack. In practice it can be difficult to produce a low-cost diagnostic device using such a complex series of steps, and to date manufacturing costs have limited deployment of these types of diagnostic devices, particularly to resource-limited settings such as developing nations. To provide enhanced diagnostic devices and improve health care, particularly in these developing countries, there remains a need for multiplexed assay devices that are inexpensive, portable, and easy to construct and use.

[009] When a porous substrate-based (e.g. , paper-based) diagnostic device is made from multiple layers of the porous substrate (e.g. , paper), intimate contact between the porous substrate (e.g., paper) layers is critical for device performance. If a gap is formed between layers of paper, testing fluid will flow laterally in that gap, in addition to normal flow across the layers of paper. When the testing liquid flows laterally in the gaps instead of normal to the layers of paper, it will not react with reagents deposited within the pores of the paper layers. In addition, the time that it takes for liquid to flow a given distance through the layers becomes unpredictable.

[010] In particular, when a porous substrate-based diagnostic device is formed by folding the porous substrate (e.g., paper), the folds function like springs that push the porous substrate (e.g., paper) layers apart. Methods need to be developed to manage that spring action and ensure that the multilayer porous substrate (e.g., paper) stack is compressed to provide intimate contact between the various layers in the multilayer porous substrate (e.g., paper) stack.

[Oil] This disclosure describes multiple methods for maintaining a porous substrate stack (e.g., a paper substrate stack) in a multi-layer porous substrate-based (e.g., paper-based) diagnostic device without significant gaps between the layers of the porous substrate (e.g., paper). The gaps between the layers of a multilayer porous substrate stack is considered significant when the thickness of the fully compressed multilayer porous substrate stack is less than 95% of the thickness of the uncompressed multilayer porous substrate stack. For example, an uncompressed multilayer porous substrate stack with ten layers of the porous substrate, where each layer of porous substrate has a porosity of 30% and a thickness of 180 micrometers would theoretically have an uncompressed thickness of 1800 micrometers and a fully- compressed thickness of 1,260 micrometers, corresponding to a 70% air gap or approximately a 60 micrometers-thick air layer for each porous substrate layer in the multilayer porous substrate stack.

[012] Briefly, in one aspect, the present disclosure describes diagnostic devices including a first metallic or (co)polymeric substrate, a second metallic or (co)polymeric substrate opposite the first (co)polymeric substrate and an elongate substantially planar porous substrate positioned between the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate such that the first and the second metallic or (co)polymeric substrates together substantially surround and encapsulate the porous substrate.

[013] An optional compressible layer is preferably positioned between at least one of the first metallic or (co)polymeric substrate and the porous substrate and/or the second metallic or (co)polymeric substrate and the porous substrate.

[014] In further optional exemplary embodiments, a first portion of the porous substrate lies in a first plane with respect to the folded region, wherein the first portion of the porous substrate comprises a first hydrophobic region and a first hydrophilic region, wherein the first hydrophobic region comprises a first low surface energy (co)polymeric material extending from a first major surface of the first portion of the porous substrate to a second major surface of the first portion of the porous substrate. The first hydrophobic region includes an arrangement of interconnected open pores providing at least one uninterrupted path extending from the first major surface of the first portion of the porous substrate to the second major surface of the first portion of the porous substrate.

[015] Preferably, a second portion of the porous substrate lies in a second plane with respect to the folded region, wherein the second plane is substantially parallel to the first plane. The second portion of the porous substrate includes a second hydrophilic region and a second hydrophobic region comprising a second low surface energy (co)polymeric material, which may be the same or different from the first low surface energy (co)polymeric material, extending from a first major surface of the second portion of the porous substrate to second major surface of the second portion of the porous substrate. The second hydrophobic region includes an arrangement of interconnected open pores providing at least one uninterrupted path extending from the first major surface of the second portion of the porous substrate to the second major surface of the second portion of the porous substrate.

[016] Preferably, at least one connective region is positioned between the first portion of the porous substrate and the second portion of the porous substrate. At least one connective region is configured to maintain alignment of the first hydrophilic region and the second hydrophilic region sufficient to provide a sample flow path between the first portion of the porous substrate and the second portion of the porous substrate along a direction normal to the first plane and the second plane.

[017] Preferably, a reagent is positioned along the sample flow path. The reagent is selected to detect at least one of a presence, an absence or a concentration of an analyte present in a sample applied to the diagnostic device. [018] In another aspect, the present disclosure describes a diagnostic device including a first (co)polymeric substrate, a second (co)polymeric substrate opposite the first (co)polymeric substrate, and an elongate substantially planar porous substrate comprising a multiplicity of fibers. The porous substrate is positioned between the first (co)polymeric substrate and the second (co)polymeric substrate such that the first and the second (co)polymeric substrates together substantially surround and encapsulate the porous substrate.

[019] An optional compressible layer is preferably positioned between at least one of the first metallic or (co)polymeric substrate and the porous substrate and/or the second metallic or (co)polymeric substrate and the porous substrate.

[020] In further optional exemplary embodiments, the porous substrate includes a multiplicity of folded regions between a first end and a second end of the porous substrate. The multiplicity of folded regions divide the planar porous fibrous substrate into a multilayer stack of overlying substantially planar panels, wherein each of the panels in the multilayer stack occupies a different substantially parallel plane. A connective region is configured to attach adjacent panels to each other.

[021] Preferably, the planar panels include at least one hydrophobic region including fibers coated with a hydrophobic low surface energy (co)polymeric ink such that open areas remain between the fibers, the open areas between the fibers providing at least one uninterrupted open flow path between a first major surface of the panel and a second major surface of the panel, and at least one hydrophilic region. The hydrophobic regions and hydrophilic regions in adjacent panels of the stack are aligned with each other to provide the sample flow path between the hydrophilic regions thereof along a direction normal to the first plane and the second plane such that the sample contacts the reagent disposed in the flow path to provide an indication of at least one of the presence, absence or concentration of the analyte in the sample.

[022] Preferably, a reagent is positioned along the sample flow path. The reagent is selected to detect at least one of a presence, an absence or a concentration of an analyte present in a sample applied to the diagnostic device.

[023] In certain exemplary embodiments of each of the foregoing aspects of the disclosure, at least one of the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate is a heat-sealable (co)polymeric film. In some such embodiments, both of first the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrates are heat-sealable (co)polymeric films. In further such embodiments, the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate further comprise at least one access port created by removing a portion of at least one of the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate, optionally in a region which overlaps a first hydrophilic region of the porous substrate. [024] In additional such exemplary embodiments, the elongate porous substrate is chosen from paper, nonwoven materials, (co)polymeric fdms, and combinations thereof. It is presently preferred that the elongate porous substrate includes paper.

[025] The disclosed diagnostic devices are particularly well adapted to conduct immunoassays, such as sandwich or competitive immunoassays, although they may be readily adapted to execute assay formats including steps such as, for example, fdtration, multiple incubations with different reagents or combinations of reagents, serial or timed addition of reagents, various incubation times, washing, and the like. The diagnostic devices are particularly effective for executing colorimetric assays, e.g., immunoassays with a color change as a readout, and are easily adapted to execute multiple assays simultaneously. They are extremely sensitive, simple to manufacture, inexpensive, and versatile.

[026] In a further aspect, the present disclosure is directed to a method of making a diagnostic device, the method including:

[027] applying a hydrophobic hardenable (co)polymeric ink composition to an elongate web of a fibrous material, wherein the hydrophobic hardenable (co)polymeric ink composition is applied in a plurality of adjacent web regions extending from a first edge of the web to a second edge of the web, wherein each web region is separated from adjacent web regions by a border region; each and wherein each web region includes a hydrophobic area including the hydrophobic (co)polymeric ink composition, a hydrophilic area substantially free of the hydrophobic (co)polymeric ink composition;

[028] positioning a second metallic or (co)polymeric substrate opposite the (co)polymeric film such that the folded web of fibrous material is positioned between the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate; and

[029] bonding the first metallic or (co)polymeric substrate to the second metallic or (co)polymeric substrate to encapsulate the porous substrate.

[030] In some exemplary optional embodiments, the method further includes providing an optional compressible layer preferably positioned between at least one of the first metallic or (co)polymeric substrate and the porous substrate and/or the second metallic or (co)polymeric substrate and the porous substrate.

[031] In some exemplary embodiments of the foregoing method, at least one of the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate comprises a heat-sealable (co)polymeric film and wherein the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate are bonded to each other at a plurality of edges which do not overlap the porous substrate.

[032] In certain such embodiments, the first and second (co)polymeric substrate heat-sealable (co)polymeric films are independently selected from the group consisting of polyethylene films, polypropylene films, polybutylene films, polycarbonate films and polyester films.

[033] In some presently preferred embodiments, the method further includes creating at least one access port in the diagnostic device by removing a portion of at least one of the first metallic or (co)polymeric film or the second metallic or (co)polymeric film in a region which overlaps the first hydrophilic region of the porous substrate. In certain presently preferred embodiments, the elongate porous substrate is chosen from paper, nonwoven materials, (co)polymeric films, and combinations thereof. In additional presently preferred embodiments, the ink composition is applied to the elongate web of porous substrate by at least one of printing, coating, physical vapor deposition, and combinations thereof. In other presently preferred embodiments, the method further includes disposing a reagent in at least one of the hydrophilic areas along the flow path, wherein the reagent is selected to provide an indication of at least one of a presence, an absence, and a concentration of an analyte in the sample.

[034] Various unexpected results and advantages are obtained in exemplary embodiments of the disclosure. One such advantage of exemplary embodiments of the present disclosure is that the reliability and reproducibility of the diagnostic device is improved by providing a controlled, repeatable compressive force to the multilayer paper stack to eliminate air gaps which can adversely affect both the time required to obtain a test result and the dependability and repeatability of the test result. An additional advantage of exemplary embodiments is to provide an adhesive-free assembly method for the diagnostic device, as some adhesives can penetrate into the pores of the multilayer paper stack and inhibit or prevent the wicking of the test sample through the multilayer paper stack in either a vertical or lateral direction.

[035] Another advantage of exemplary embodiments is to enable a high speed, roll-to-roll continuous production process for the paper substrate and, in certain embodiments, even the paper-based diagnostic device. A further advantage of exemplary embodiments is the ability to achieve a fully automated assembly process for the paper-based diagnostic device. These and other unexpected results and advantages are within the scope of the following exemplary embodiments.

Listing of Exemnlarv Embodiments Embodiment A: A diagnostic device, comprising: a first metallic or (co)polymeric substrate; a second metallic or (co)polymeric substrate opposite the first (co)polymeric substrate; and an elongate substantially planar porous substrate positioned between the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate such that the first and the second metallic or (co)polymeric substrates together substantially surround and encapsulate the porous substrate, wherein the porous substrate comprises at least one folded region between the first end and the second end, optionally wherein a compressible layer is positioned between at least one of the first metallic or (co)polymeric substrate and the porous substrate and/or the second metallic or (co)polymeric substrate and the porous substrate, and optionally further wherein: a first portion of the porous substrate lies in a first plane with respect to the folded region, wherein the first portion of the porous substrate comprises a first hydrophobic region and a first hydrophilic region, wherein the first hydrophobic region comprises a first low surface energy (co)polymeric material extending from a first major surface of the first portion of the porous substrate to a second major surface of the first portion of the porous substrate, and wherein the first hydrophobic region comprises an arrangement of interconnected open pores providing at least one uninterrupted path extending from the first major surface of the first portion of the porous substrate to the second major surface of the first portion of the porous substrate; and a second portion of the porous substrate that lies in a second plane with respect to the folded region, wherein the second plane is substantially parallel to the first plane, the second portion of the porous substrate comprising a second hydrophilic region and a second hydrophobic region comprising a second low surface energy (co)polymeric material, which may be the same or different from the first low surface energy (co)polymeric material, extending from a first major surface of the second portion of the porous substrate to second major surface of the second portion of the porous substrate, and wherein the second hydrophobic region comprises an arrangement of interconnected open pores providing at least one uninterrupted path extending from the first major surface of the second portion of the porous substrate to the second major surface of the second portion of the porous substrate; at least one connective region between the first portion of the porous substrate and the second portion of the porous substrate, wherein the at least one connective region is configured to maintain alignment of the first hydrophilic region and the second hydrophilic region sufficient to provide a sample flow path between the first portion of the porous substrate and the second portion of the porous substrate along a direction normal to the first plane and the second plane; and a reagent along the sample flow path, wherein the reagent is selected to detect at least one of a presence, an absence or a concentration of an analyte present in a sample applied to the diagnostic device.

Embodiment B: A diagnostic device, comprising: a first (co)polymeric substrate; a second (co)polymeric substrate opposite the first (co)polymeric substrate; and an elongate substantially planar porous substrate comprising a plurality of fibers, wherein a first end and a second end of the porous substrate is positioned between the first (co)polymeric substrate and the second (co)polymeric substrate such that the first and the second (co)polymeric substrates together substantially surround and encapsulate the porous substrate, wherein the porous substrate comprises; a plurality of folded regions between the first end and the second end, the plurality of folded regions dividing the planar porous fibrous substrate into a stack of overlying substantially planar panels, wherein each of the panels in the stack occupies a different substantially parallel plane, optionally wherein a compressible layer is positioned between at least one of the first(co)polymeric substrate and the porous substrate or the second (co)polymeric substrate and the porous substrate and optionally further wherein each of the panels comprises: at least one hydrophobic region comprising the fibers coated with a hydrophobic low surface energy (co)polymeric ink such that open areas remain between the fibers, the open areas between the fibers providing at least one uninterrupted open path between a first major surface of the panel and a second major surface of the panel, and at least one hydrophilic region; and wherein at least some of the panels comprise: a reagent selected to detect an analyte present in a sample, and a connective region configured to attach adjacent panels to each other; and wherein the hydrophobic regions and hydrophilic regions in adjacent panels of the stack are aligned with each other to provide a sample flow path between the hydrophilic regions thereof along a direction normal to the first plane and the second plane such that the sample contacts the reagent disposed in the flow path to provide an indication of at least one of the presence, absence or concentration of the analyte in the sample.

Embodiment C: The diagnostic device of Embodiment A or B, wherein at least one of the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate comprises a heat- sealable (co)polymeric film.

Embodiment D: The diagnostic device of Embodiment C, wherein both of first the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrates comprise heat-sealable (co)polymeric films.

Embodiment E: The diagnostic device of any preceding Embodiment, wherein the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate further comprise at least one access port created by removing a portion of at least one of the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate, optionally in a region which overlaps the first hydrophilic region of the porous substrate.

Embodiment F : The diagnostic device of any preceding Embodiment, wherein the elongate porous substrate is chosen from paper, nonwoven materials, (co)polymeric films, and combinations thereof.

Embodiment G: The diagnostic device of Embodiment F, wherein the elongate porous substrate comprises paper.

Embodiment H: The diagnostic device of any preceding Embodiment, wherein the optional hydrophobic regions substantially surround the hydrophilic regions.

Embodiment I: The diagnostic device of Embodiments A, C, D, E, G, or H, wherein the optional first and second low surface energy (co)polymeric materials each comprise a radiation curable (co)polymeric ink.

Embodiment J: The diagnostic device of Embodiments A, C, D, E, G, H or I, wherein the optional first and second low surface energy (co)polymeric materials comprise monomers, oligomers, or (co)polymers chosen from fluorocarbons, silicones, or hydrocarbons. Embodiment K: The diagnostic device of Embodiments A, C, D, E, G, H, I or J, wherein the optional first and second low surface energy (co)polymeric materials comprise a hydrophobic ink with a surface energy lower than 35 dynes/cm.

Embodiment L: The diagnostic device of Embodiments B, C, D, E, G or H, wherein the optional at least one connective region occupies a periphery of the panels and substantially surrounds the optional hydrophobic regions.

Embodiment M: A method of making a diagnostic device, the method comprising: applying a hydrophobic hardenable (co)polymeric ink composition to an elongate web of a fibrous material, wherein the hydrophobic hardenable (co)polymeric ink composition is applied in a plurality of adjacent web regions extending from a first edge of the web to a second edge of the web, wherein each web region is separated from adjacent web regions by a border region; each and wherein each web region comprises: a hydrophobic area comprising the hydrophobic (co)polymeric ink composition, a hydrophilic area substantially free of the hydrophobic (co)polymeric ink composition, and at least partially hardening the hardenable (co)polymeric ink composition in the hydrophobic areas of each web region to provide a hydrophobic ink on fibers of the fibrous material and open areas between the fibers, the open areas between the fibers providing at least one uninterrupted open ink-free flow path between a first major surface of the web and a second major surface of the web; and folding the web of fibrous material along the border regions to form a stack of overlying substantially planar panels, wherein each of the overlying planar panels in the stack occupies a different substantially parallel plane, and wherein each of the overlying planar panels comprises registered hydrophilic areas forming a sample flow path therebetween; positioning a first metallic or (co)polymeric substrate adjacent to the folded web of fibrous material; positioning a second metallic or (co)polymeric substrate opposite the (co)polymeric film such that the folded web of fibrous material is positioned between the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate; and bonding the first metallic or (co)polymeric substrate to the second metallic or (co)polymeric substrate to encapsulate the porous substrate, optionally wherein a compressible layer is positioned between the first metallic or (co)polymeric substrate and the folded web of fibrous material and/or the second metallic or (co)polymeric substrate and the folded web of fibrous material.

Embodiment N: The method of Embodiment M, wherein at least one of the first metallic or

(co)polymeric substrate and the second metallic or (co)polymeric substrate comprises a heat-sealable (co)polymeric film and wherein the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate are bonded to each other at a plurality of edges which do not overlap the porous substrate. Embodiment O: The method of Embodiment N, wherein each of the first (co)polymeric substrate and the second (co)polymeric substrate comprise a heat-sealable (co)polymeric film, and wherein the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate are bonded to each other at a plurality of edges which do not overlap the porous substrate.

Embodiment P: The method of Embodiment N or O, wherein the first and second (co)polymeric substrate heat-sealable (co)polymeric films are independently selected from the group consisting of polyethylene films, polypropylene films, polybutylene films, polycarbonate films and polyester films. Embodiment Q: The method of Embodiment M, N, O or P, further comprising creating at least one access port in the diagnostic device by removing a portion of at least one of the first metallic or (co)polymeric film or the second metallic or (co)polymeric film in a region which overlaps the first hydrophilic region of the porous substrate.

Embodiment R: The method of Embodiment M, N, O, P or Q, wherein the elongate porous substrate is chosen from paper, nonwoven materials, (co)polymeric films, and combinations thereof. Embodiment S: The method of Embodiment M, N, O, P, Q or R, wherein the ink composition is applied to the elongate web of porous substrate by at least one of printing, coating, physical vapor deposition, and combinations thereof.

Embodiment T: The method of Embodiment M, N, O, P, Q, R or S, further comprising disposing a reagent in at least one of the hydrophilic areas along the flow path, wherein the reagent is selected to provide an indication of at least one of a presence, an absence, and a concentration of an analyte in the sample.

[036] Various aspects and advantages of exemplary embodiments of the disclosure have been summarized. The above Summary is not intended to describe each illustrated embodiment or every implementation of the present certain exemplary embodiments of the present disclosure. The Drawings and the Detailed Description that follow more particularly exemplify certain preferred embodiments using the principles disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[037] The disclosure may be more completely understood in consideration of the following detailed description of various embodiments of the disclosure in connection with the accompanying figures, in which:

[038] FIG. 1 is a schematic side view of a porous substrate-based (e.g., paper-based) diagnostic device prior to encapsulation according to an embodiment of the present disclosure.

[039] FIG. 2A is a schematic side view of an encapsulated porous substrate-based (e.g., paper-based) diagnostic device after encapsulation according to an embodiment of the present disclosure.

[040] FIG. 2B is a schematic side view of an encapsulated porous substrate-based (e.g., paper-based) diagnostic device after encapsulation according to an alternative embodiment of the present disclosure. [041] FIG. 3 is a schematic side view of an encapsulated porous substrate -based (e.g., paper-based) diagnostic device after encapsulation according to yet another alternative embodiment of the present disclosure

[042] FIG. 4 is a schematic side view of an encapsulated porous substrate -based (e.g., paper-based) diagnostic device after encapsulation according to still another alternative embodiment of the present disclosure.

[043] In the drawings, like reference numerals indicate like elements. While the above-identified drawings, which may not be drawn to scale, sets forth various embodiments of the present disclosure, other embodiments are also contemplated, as noted in the Detailed Description. In all cases, this disclosure describes the presently disclosed disclosure by way of representation of exemplary embodiments and not by express limitations. It should be understood that numerous other modifications and embodiments can be devised by those skilled in the art, which fall within the scope and spirit of this disclosure.

DETAILED DESCRIPTION

[044] Certain terms are used throughout the description and the claims that, while for the most part are well known, may require some explanation. For the following Glossary of defined terms, these definitions shall be applied for the entire application, unless a different definition is expressly provided in the claims or elsewhere in the specification.

Glossary

[045] The terms “(co)polymer” and “(co)polymer” include homopolymers and copolymers, such as homopolymers or copolymers that may be formed in a miscible blend, e.g., by coextrusion or by reaction, including, e.g., transesterification. The term “copolymer” includes both random and block copolymers. [046] The term “coupling agent” means a compound which provides a chemical bond between two dissimilar materials, usually an inorganic and an organic material. Coupling agents are typically multi functional molecules or oligomers which can act to effect crosslinking during chemical reactions, for example, a chemical reaction such as free radical polymerization to form a (co)polymer.

[047] The term “layer” refers to a single stratum within a multi-strata (multilayer) assembly.

[048] The term “encapsulate” or “encapsulated” means enclosed or encased within.

[049] The term "(meth)acryl" or “(meth)acrylate” with respect to a monomer, oligomer, (co)polymer or compound means a vinyl-functional alkyl ester formed as the reaction product of an alcohol with an acrylic or a methacrylic acid.

[050] The term "crosslinked" (co)polymer refers to a (co)polymer whose (co)polymer chains are joined together by covalent chemical bonds, usually via crosslinking molecules or groups, to form a network (co)polymer. A crosslinked (co)polymer is generally characterized by insolubility but may be swellable in the presence of an appropriate solvent. [051] The term “cure” refers to a process that causes a chemical change, e.g., a reaction that creates a covalent bond to solidify a multilayer film layer or increase its viscosity.

[052] The term "cured (co)polymer" includes both crosslinked and uncrosslinked polymers.

[053] By using the term "T _g ", we refer to the glass transition temperature of a (co)polymer when evaluated in bulk rather than in a thin film form. In instances where a (co)polymer can only be examined in thin film form, the bulk form T _g can usually be estimated with reasonable accuracy. Bulk form T _g values usually are determined by evaluating the rate of heat flow vs. temperature using differential scanning calorimetry (DSC) to determine the onset of segmental mobility for the (co)polymer and the inflection point (usually a second-order transition) at which the (co)polymer can be said to change from a glassy to a rubbery state. Bulk form T _g values can also be estimated using a dynamic mechanical thermal analysis (DMTA) technique, which measures the change in modulus of the (co)polymer as a function of temperature and frequency of vibration of a test sample.

[054] The term “adjoining” with reference to a particular layer means joined with or attached to another layer, in a position wherein the two layers are either next to (i.e.. adjacent to) and directly contacting each other, or contiguous with each other but not in direct contact (i.e.. there are one or more additional layers intervening between the layers).

[055] By using terms of orientation such as “atop”, “on”, “over,” “covering”, “uppermost”, “underlying” and the like for the location of various elements in the disclosed coated articles, we refer to the relative position of an element with respect to a horizontally-disposed, upwardly-facing substrate. However, unless otherwise indicated, it is not intended that the substrate or articles should have any particular orientation in space during or after manufacture.

[056] By using the term “overcoated” to describe the position of a layer with respect to a substrate or other element of an article of the present disclosure, we refer to the layer as being atop the substrate or other element, but not necessarily contiguous to either the substrate or the other element.

[057] By using the term “separated by” to describe the position of a layer with respect to other layers, we refer to the layer as being positioned between two other layers but not necessarily contiguous to or adjacent to either layer.

[058] The terms “about” or “approximately” with reference to a numerical value or a shape means +/- five percent of the numerical value or property or characteristic, but expressly includes the exact numerical value. For example, a viscosity of “about” 1 Pa-sec refers to a viscosity from 0.95 to 1.05 Pa- sec, but also expressly includes a viscosity of exactly 1 Pa-sec. Similarly, a perimeter that is “approximately square” is intended to describe a geometric shape having four lateral edges in which each lateral edge has a length which is from 99% to 101% of the length of any other lateral edge, but which also includes a geometric shape in which each lateral edge has exactly the same length.

[059] Unless otherwise indicated, all numbers expressing quantities or ingredients, measurement of properties and so forth used in the specification and embodiments are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the foregoing specification and attached listing of embodiments can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claimed embodiments, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[060] The term “substantially” with reference to a property or characteristic means that the property or characteristic is exhibited to a greater extent than the opposite of that property or characteristic is exhibited. For example, a substrate that is “substantially” transparent refers to a substrate that transmits more radiation (e.g. visible light) than it fails to transmit (e.g. absorbs and reflects). Thus, a substrate that transmits more than 50% of the visible light incident upon its surface is substantially transparent, but a substrate that transmits 50% or less of the visible light incident upon its surface is not substantially transparent.

[061] As used in this specification and the appended embodiments, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to fine fibers containing “a compound” includes a mixture of two or more compounds. As used in this specification and the appended embodiments, the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.

[062] As used in this specification, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.8, 4, and 5).

[063] By definition the total weight percentages of all ingredients in a composition equals 100 weight percent.

[064] Various exemplary embodiments of the disclosure will now be described. Exemplary embodiments of the present disclosure may take on various modifications and alterations without departing from the spirit and scope of the present disclosure. Accordingly, it is to be understood that the embodiments of the present disclosure are not to be limited to the following described exemplary embodiments but is to be controlled by the limitations set forth in the claims and any equivalents thereof.

Encapsulated Paper-based Diagnostic Devices

[065] In one exemplary embodiment, the present disclosure describes diagnostic devices including a first metallic or (co)polymeric substrate, a second metallic or (co)polymeric substrate opposite the first (co)polymeric substrate and an elongate substantially planar porous substrate positioned between the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate such that the first and the second metallic or (co)polymeric substrates together substantially surround and encapsulate the porous substrate. The first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate are preferably bonded together at a plurality of edge regions proximate at least a portion of the perimeter of the porous substrate. [066] In other exemplary embodiments, the present disclosure describes a diagnostic device including a first (co)polymeric substrate, a second (co)polymeric substrate opposite the first (co)polymeric substrate, and an elongate substantially planar porous substrate comprising a multiplicity of fibers. The porous substrate is positioned between the first (co)polymeric substrate and the second (co)polymeric substrate such that the first and the second (co)polymeric substrates together substantially surround and encapsulate the porous substrate. The first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate are preferably bonded together at a plurality of edge regions proximate at least a portion of the perimeter of the porous substrate.

[067] In any of the foregoing embodiments, an optional compressible layer is preferably positioned between at least one of the first metallic or (co)polymeric substrate and the porous substrate and/or the second metallic or (co)polymeric substrate and the porous substrate.

[068] In practice, when a diagnostics device is made from multiple layers (e.g., paper), intimate contact between layers is important for reproducible device performance. When an air gap is formed between adjacent or adjoining layers of the porous substrate such as paper, the testing fluid may flow in that gap, in addition to flowing within the porous structure of the porous substrate. When liquid flows in the gaps instead of within a porous layer of the porous substrate, it may not react with reagents deposited in that layer. In addition, liquid flow in the device may become unpredictable or non-reproducible.

[069] Thus, in some embodiments, the optional first compressible layer may be advantageously applied to compress the stacked layers tightly against each other to eliminate or mitigate a significant gap between the adjacent porous substrate layers or portions thereof. In some cases, a gap is considered significant when the gap is greater than 75 micrometers, greater than 50 micrometers, greater than 25 micrometers, greater than 10 micrometers, greater than 5 micrometers, greater than 2 micrometers, greater than one micrometer and the like. In some cases, a gap is considered significant when the fraction of fluid sample flowing into the gap is more than 20 weight %, more than 10 weight %, 5 weight %, 2 weight %, 1 weight % and the like.

[070] Suitable metallic or (co)polymeric substrates, porous substrates, optional compressible layers and diagnostic devices are described in co-pending U.S. Prov. Patent Application 63/003,169, filed March 31, 2020 and titled “DIAGNOSTIC DEVICE”; co-pending U.S. Prov. Patent Application 63/164,144, filed March 22, 2021 and titled “DIAGNOSTIC DEVICE”; co-pending U.S. Prov. Patent Application 63/200,871, filed March 31, 2021 and titled “ULTRASONICALLY-BONDED POROUS SUBSTRATE DIAGNOSTIC DEVICES AND METHODS OF MAKING SAME”; co-pending U.S. Prov. Patent Application 63/200,844, filed March 31, 2021 and titled “ENCAPSULATED POROUS SUBSTRATE DIAGNOSTIC DEVICES AND METHODS OF MAKING SAME”; co-pending U.S. Prov. Patent Application 63/200,849, filed March 31, 2021 and titled “EDGE-SEALED POROUS SUBSTRATE DIAGNOSTIC DEVICES AND METHODS OF MAKING SAME”; co-pending U.S. Prov. Patent Application 63/200,869, filed March 31, 2021 and titled “CURVED DIAGNOSTIC DEVICE”; as well as any patent applications claiming priority to these provisional patent applications, the entire disclosures of which are incorporated herein by reference in their entireties.

[071] In certain exemplary embodiments of each of the foregoing diagnostic devices of the present disclosure, at least one of the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate is a heat-sealable (co)polymeric film. In some such embodiments, both of first the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrates are heat- sealable (co)polymeric films. In further such embodiments, the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate further comprise at least one access port created by removing a portion of at least one of the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate, optionally in a region which overlaps a first hydrophilic region of the porous substrate.

[072] In additional such exemplary embodiments, the elongate porous substrate is chosen from paper, nonwoven materials, (co)polymeric films, and combinations thereof. It is presently preferred that the elongate porous substrate includes paper.

[073] The disclosed diagnostic devices are particularly well adapted to conduct immunoassays, such as sandwich or competitive immunoassays, although they may be readily adapted to execute assay formats including steps such as, for example, filtration, multiple incubations with different reagents or combinations of reagents, serial or timed addition of reagents, various incubation times, washing, and the like. The diagnostic devices are particularly effective for executing colorimetric assays, e.g., immunoassays with a color change as a readout, and are easily adapted to execute multiple assays simultaneously. They are extremely sensitive, simple to manufacture, inexpensive, and versatile.

[074] Turning now to FIG. 1, a paper-based diagnostic device 100 according to an embodiment of the present disclosure is shown prior to compression and encapsulation. The device 100 comprises a porous substrate 102, which may be a paper web, having a plurality of substantially planar opposed faces 102 (a- e) and a plurality of edges 104 for folding the porous substrate 102 upon itself to form a multilayer porous substrate (e.g., paper) stack ( see Fig. 2, reference numeral 202).

[075] FIG. 2A is a schematic side view of an encapsulated porous substrate (e.g. paper-based) diagnostic device 200 after encapsulation according to a first embodiment of the present disclosure. As shown in Fig. 2A, the folded multilayer porous substrate (e.g., paper) stack 202 includes a plurality of folded edges 204 and is encapsulated by a first (co)polymeric substrate 206 and a second (co)polymeric substrate 208 opposite the first (co)polymeric substrate such that the first and the second (co)polymeric substrates 206 and 208 substantially surround and encapsulate the porous substrate ((e.g., paper) stack 202

[076] An optional first compressible layer 210b is shown positioned between the first (co)polymeric substrate 208 and the porous substrate (e.g., paper) stack 202. An optional second compressible layer 210a is shown positioned between the second (co)polymeric substrate 206 and the porous substrate (e.g., paper) stack 202. [077] FIG. 2B is a schematic side view of an encapsulated paper-based diagnostic device 200 after encapsulation according to an alternative embodiment of the present disclosure. As shown in Fig. 2B, the folded multilayer porous substrate (e.g., paper) stack 202 includes a plurality of folded edges 204 and is encapsulated by a first (co)polymeric substrate 208 and a second (co)polymeric substrate 206 opposite the first (co)polymeric substrate such that the first and the second (co)polymeric substrates 208 and 206 substantially surround and encapsulate the porous substrate ((e.g., paper) stack 202. An optional first compressible layer 210b is shown positioned between the first (co)polymeric substrate 208 and the porous substrate (e.g., paper) stack 202.

[078] FIG. 3 is a schematic side view of an encapsulated paper-based diagnostic device 300 after encapsulation according to yet another alternative embodiment of the present disclosure.

As shown in Fig. 3, the folded multilayer porous substrate (e.g., paper) stack 302 includes a plurality of folded edges 304 and is encapsulated by a first (co)polymeric substrate 306 and a second (co)polymeric substrate 312a opposite the first (co)polymeric substrate such that the first and the second (co)polymeric substrates 306 and 312a substantially surround and encapsulate the porous substrate ((e.g., paper) stack 302.

[079] The first (co)polymeric substrate 306 may be an enclosure such as a pouch or bag formed of the first (co)polymeric substrate 306, or may be an adhesive-coated film comprised of the first (co)polymeric substrate 306. The first (co)polymeric substrate 306 preferably comprises a heat-sealable or heat- shrinkable (co)polymeric film.

[080] An optional first compressible layer 310b is shown positioned between the first (co)polymeric substrate 308 and the porous substrate (e.g., paper) stack 302. An optional second compressible layer 310a is shown positioned between the second (co)polymeric substrate 306 and the porous substrate (e.g., paper) stack 302. An optional adhesive layer 312b may be positioned between the first (co)polymeric substrate 306 and one or both of the optional first and/or second compressible layers 310a and 310b.

[081] FIG. 4 is a schematic side view of an encapsulated paper-based diagnostic device 400 after encapsulation according to still another alternative embodiment of the present disclosure. As shown in Fig. 4, the folded multilayer porous substrate (e.g., paper) stack 402 includes a plurality of folded edges 404 and is encapsulated by an enclosure 406 comprised of the first and second (co)polymeric substrates such that the first and the second (co)polymeric substrates substantially surround and encapsulate the porous substrate ((e.g., paper) stack 402.

[082] The enclosure 406 may be a pouch or bag formed of the first (co)polymeric substrate, or may be an adhesive-coated film. The enclosure 406 preferably comprises a heat-sealable or heat-shrinkable (co)polymeric film.

[083] An optional first compressible layer 410b is shown positioned between the enclosure 406 comprised of the first (co)polymeric substrate and the porous substrate (e.g., paper) stack 402. An optional second compressible layer 410a is shown positioned between the enclosure 406 comprised of the second (co)polymeric substrate and the porous substrate (e.g., paper) stack 402. [084] In some exemplary embodiments, the diagnostic device includes an elongate substantially planar hydrophilic porous substrate with a first (terminal) end, a second, opposed (terminal) end and at least one folded region between the first and the second ends. The folded region separates the hydrophilic substrate into a first sheet-like portion and a second sheet-like portion, each occupying a substantially parallel plane with respect to the folded region.

[085] The first porous substrate portion includes a first major surface and a second opposed major surface, while the second substrate portion includes a first major surface and a second opposed major surface. In the embodiment of FIG. 2A, the first portion of the substrate and the second portion of the substrate overlie one another such that the respective major surfaces are adjacent to each other.

[086] The first substrate portion includes a first hydrophobic region and a first hydrophilic region, while the second substrate portion includes a second hydrophobic region and a second hydrophilic region. The fibers of the substrate in the hydrophobic regions have applied thereto a low surface energy polymeric material, and as such resist unassisted capillary fluid flow or wicking of a selected fluid, such as, for example, a sample fluid including, for example, an analyte, or a buffer or a wash solution, therethrough. As a result of this resistance, the selected fluid is passively transported (requiring no external pressure gradients, gravitational or electrostatic forces) between the hydrophilic regions.

[087] The hydrophobic regions substantially confine the flow of the fluid to a direction normal to the planar surface of the folded paper stack and which is aligned along thickness of the substrate portions or along the z-axis of the three-dimensional diagnostic device normal to the major surfaces of the porous substrate. The hydrophilic regions are sufficiently aligned with each other such that a fluid sample placed on the first hydrophilic region (not shown in FIGS.2A-2B) can be passively transported using, for example, wicking or capillary action, along a sample flow path to provide fluid communication between the first substrate portion and the second substrate portion such that the fluid sample wicks into the second hydrophilic region.

[088] The shapes and sizes of the hydrophobic regions and the hydrophilic regions may vary widely depending on the intended use of the device. Any number of hydrophobic regions and hydrophilic regions may be used. However, since mass transfer of a fluid is proportional to the area of the wicking, varying the size of the hydrophilic regions or a coating pattern of the hydrophobic regions can be used to control mass transfer by wicking or capillary action across layers. For example, smaller hydrophilic regions will reduce mass transfer in direct relation to the reduced area of the substrate occupied by the hydrophilic regions. In some embodiments, which are provided by way of example and are not intended to be limiting, to provide good wicking between overlying layers the hydrophilic regions should occupy about 10% to about 90%, or about 25% to about 50%, of the total cross-sectional area of the substrate. [089] In some embodiments, the separation regions are free of the hydrophobic regions, but such an arrangement is not required. The hydrophobic regions and the hydrophilic regions may have the same shape, but in some embodiments the hydrophobic regions and hydrophilic regions can have different shapes, depending on the requirements of a specific diagnostic assay. [090] In the embodiment of FIG. 2A, the web regions further include connective regions that surround the hydrophilic regions. In addition, in the embodiment of FIG. 2A, the web region includes a patterned connective region of, for example, a pressure sensitive adhesive (PSA).

[091] As shown in FIG. 2B, the web may be folded along the separation regions to form a folded paper diagnostic device including overlying and substantially parallel panels. When so folded, the connection regions come together to adhere and maintain registration of the panels, and the patterned connective region maintains the registration of the panels. The registration of the panels maintains alignment of the hydrophilic regions, which allows flow of sample fluid along a sample flow path through the hydrophilic regions.

[092] While not shown in FIGS.2A-2B, additional connective regions of any suitable shape or configuration may be used to temporarily maintain alignment of the hydrophilic regions in the panels. In some embodiments, mechanical fasteners (not shown in FIGS. 2A-2B) may also be used, alone or in combination with adhesive connective regions, to temporarily maintain alignment of any or all of the panels.

[093] In another embodiment a diagnostic device includes a sensor stack formed from a plurality of overlying substantially planar panels of a porous material. Each of the panels of the sensor stack include a hydrophobic region and a hydrophilic region. The hydrophilic regions are substantially aligned with each other to provide a sample flow path along the direction substantially normal to a first major surface to a second major surface thereof.

[094] The hydrophilic region provides an exposed sample port upon which a sample fluid is placed for analysis in the sensor stack, and as discussed in detail above the sample fluid moves through the panels by wicking or capillary action. An optional wicking layer includes a first major surface on the second major surface of the sensor stack, as well as a second major surface.

[095] In some embodiments, the sample port formed by the hydrophilic region may optionally be colored to enhance contrast with the surrounding hydrophobic region and provide a guide for sample placement on the sensor stack. In some embodiments, the hydrophilic region may also be colored to provide a color zone for reading the results of an assay performed with the sensor stack.

[096] The sensor stack is preferably overlain by a packaging layer. The packaging layer may vary widely, and can include, for example, a (co)polymeric film, a plastic molded cover, and the like. In some embodiments, the packaging layer is an elastic polymeric film that contacts the first major surface of the sensor stack and compresses the sensor stack against the first major surface of the wicking layer. In some examples, such compression can improve wicking through the panels in the sensor stack.

[097] In other embodiments, the packaging layer comprises at least on thermoplastic (co)polymeric film that contacts the first major surface of the sensor stack and compresses the sensor stack against the first major surface of the wicking layer. Preferably, the thermoplastic (co)polymeric film is a heat- sealable film. [098] In some examples, the packaging layer may optionally include an access port aligned with the hydrophilic region of the sensor stack. The packaging layer may be attached to the first major surface of the wicking layer by a hinge.

[099] In another exemplary embodiment, a diagnostic device includes a sensor stack formed from a plurality of overlying substantially planar panels of a porous material such as, for example, paper. As discussed above, the panels may be discrete pieces of the porous material or may be a continuous web of porous material that is folded to form the individual panels. Each of the panels of the sensor stack may preferably include a hydrophobic region and a hydrophilic region. The hydrophilic regions are substantially aligned with each other to provide a sample flow path through the thickness of the sensor stack from a first major surface to a second major surface thereof.

[0100] The hydrophilic region preferably provides an exposed sample port upon which a sample fluid is placed for analysis in the sensor stack, and as discussed in detail above the sample fluid moves through the panels in direction substantially normal to the first and second major surfaces by wicking or capillary action.

[0101] An optional wicking layer includes a first major surface on the second major surface of the sensor stack, as well as an exposed second major surface. The optional wicking layer, which in various embodiments is made of paper, woven materials, non-woven materials, glass fibers, and the like, helps to draw the sample fluid through the hydrophilic regions in the respective panels to increase the speed and efficiency of the capillary action process.

[0102] In some embodiments, the sample port formed by the hydrophilic region may optionally be colored to enhance contrast with the surrounding hydrophobic region and provide a guide for sample placement on the sensor stack. In some embodiments, the hydrophilic region may also be colored to provide a color zone for reading for reading the results of an assay performed with the sensor stack. [0103] Another exemplary embodiment of an assay device includes a sensor stack formed from multiple overlying planar panels of a porous material. In some embodiments, the sensor stack is formed from a single piece of porous material that is folded into multiple overlying planar panels. The sensor stack includes a first major surface and a second major surface. The panels of the sensor stack preferably include hydrophobic regions and hydrophilic regions, wherein the hydrophilic regions in each panel overlie and are aligned with each other to provide a sample flow path for migration of a fluid sample through the sensor stack from one panel to another along a direction substantially normal to the first an second major surfaces.

[0104] As discussed in the embodiments described above, the assay device may further include an optional wicking layer with a first major surface adjacent to the second major surface of the sensor stack, as well as an opposed second major surface. As noted above, the wicking layer may be a single layer, or may include multiple sublayers.

[0105] In some embodiments, the sublayers of the wicking layer may optionally additionally be bonded together with adhesives, and in some examples the adhesives may be positioned to further enhance flow between the hydrophilic regions of the sensor stack, or to enhance the performance of lateral flow control features in the sublayers.

[0106] In some embodiments, the assay device further includes an outer packaging layer that overlies the cover layer, if present, or the first major surface of the sensor stack. In some embodiments, the outer packaging layer is a polymeric tape construction with an adhesive layer that adheres to a surface of the backing layer.

[0107] In some presently preferred embodiments, the outer packaging layer is an adhesive-free (co)polymeric film that is heat-sealed or ultrasonically welded to the surface of the backing layer. In some example, the outer packaging layer includes an “easy open” feature such as a tab or a strippable portion that can provide access to the hydrophilic regions of the sensor stack.

[0108] In some embodiments, the polymeric film of the outer packaging layer has elastic properties and exerts a compressive force on the sensor stack in a direction substantially normal to the first and second major surfaces of the porous stack to facilitate the optimal fluid transport rate and avoid lateral capillary flow between the stacked or folded panels thereof. In some embodiments, the outer packaging layer includes apertures (not shown in FIG. 2A-2B) that overlie the apertures in the cover layer and the hydrophilic regions of the sensor stack. The overlying apertures in the cover layer and the outer packaging layer allow application of a sample fluid through the packaging layer and the cover layer onto the hydrophilic regions of the sensor stack.

[0109] In some embodiments, one or more optional adhesive layers, which may be single-sided or double-sided, may optionally be used to bond the wicking layer and the compression layer, or to bond the compression layer to the backing layer, or both. In some examples, an optional arrangement of adhesive tabs on the second major surface of the sensor stack may be used to securely bond the sensor stack to the outer packaging layer. In some examples, some or all of the adhesive layers or the adhesive tabs may be replaced with ultrasonic welds.

[0110] In various embodiments, the elongate hydrophilic substrate may be made from any porous, hydrophilic, adsorbent material capable of wicking a sample fluid by capillary action. In one or more embodiments, the substrate is a paper product such as, for example, chromatographic paper, filter paper, and the like, but may also be chosen from woven or nonwoven fabrics, or from (co)polymer films such as, for example, nitrocellulose, cellulose acetate, polyesters, and polyurethane, and the like.

[0111] The first and second hydrophobic regions may be formed by applying a desired pattern of a low surface energy polymeric material such as, for example, a polymeric ink composition that forms a low surface energy polymeric ink when cured, to the substrate.

[0112] The hydrophobic regions thus resist absorption of a liquid applied to, for example, the hydrophilic region of the first substrate portion, and the liquid is passively transported via capillary action or wicking between the hydrophilic regions. While not wishing to be bound by any theory, currently available evidence indicates that the relative difference in absorption between the hydrophobic regions and the hydrophilic regions is a function of difference between the surface energy of the fibers in the hydrophilic regions for a selected liquid such as, for example, a sample fluid, a buffer, and the like, which are intended to flow between the substrate portions, and the surface energy of the fibers coated with an ink composition deposited or patterned onto the substrate and subsequently cured to form the low surface energy ink in the hydrophobic regions. The larger this difference, the larger the resistivity to absorption of the selected fluid in the hydrophobic regions. The difference may also depend on, for example, the uniformity of ink coverage, the structure of the fibers, and the like.

[0113] In one example, if the sample fluid selected to flow by wicking or capillary action between the substrate portions is a bodily fluid, the surface energy of the fibers with the low surface energy hydrophobic ink applied thereto in the hydrophobic regions should be lower than the lowest value of the surface tension of the bodily fluid. Because bodily fluids have a range of surface tensions, the surface energy of the fibers in the hydrophobic regions should be at least 10 dynes/cm lower than the lowest surface tension of the bodily fluid, or at least 15 dynes/cm lower, or at least 20 dynes/cm lower, or even at least 30 dynes/cm lower.

[0114] For example, it is reported that human urine has a minimum surface tension of about 55 dynes/cm, and human saliva has a surface tension of about 40 dynes/cm, so to resist absorption of these bodily fluids by wicking or capillary action the hydrophobic regions 24, 28 surface energy of ink should have a surface tension of less than about 45 dynes/cm, or less than about 40 dynes/cm, or less than about 35 dynes/cm, or less than about 30 dynes/cm, or less than about 25 dynes/cm, or less than about 20 dynes/cm.

[0115] In another example, to resist capillary flow or wicking of a selected fluid, presently available evidence indicates that the hydrophobic ink compositions in the regions, when hardened, provide a contact angle for the selected fluid of greater than about 90°, or greater than about 95°, or greater than about 100°, or greater than about 105°, or greater than about 110°, or greater than about 115°, or greater than about 120°, or greater than about 125°, or greater than about 130°, or greater than about 135°, or even greater than about 140°.

[0116] Contact angles and wettability may be measured using the techniques described in, for example, CAPILLARITY AND WETTING PHENOMENA DROPS, BUBBLES, PEARLS, WAVES by Francoise Brochard-Wyart; David Quere, Hardcover; New York: Springer, September 12, 2003; WETTABILITY (SURFACTANT SCIENCE) by John Berg, ed., CRC Press; 1 edition, April 20, 1993, each of which are incorporated herein by reference in their entirety.

[0117] In various embodiments, the hydrophobic ink composition includes at least one polymerizable low surface energy monomer, oligomer, or (co)polymer that can provide a desired resistance to absorption of a selected liquid or sample fluid. This low surface energy monomer, oligomer, or (co)polymer can be a fluorocarbon, silicone, or hydrocarbon. The low surface energy monomer, oligomer, or (co)polymer is added to the formulation to reduce the surface energy of the cured hydrophobic coating to a wetting tension of from about 30 to less than about 38 mJ/m ² as measured by ASTM D 2578-08. Examples of suitable polymerizable low surface energy monomers, oligomers and polymers are described in WO2011/094342, which is incorporated by reference herein in its entirety. [0118] In general, a wide variety of reagents may be disposed in, or in fluid communication with, the test area in hydrophilic regions of the diagnostic device to detect one or more analytes in a sample fluid. These reagents include, but are not limited to, antibodies, nucleic acids, aptamers, molecularly-imprinted polymers, chemical receptors, proteins, peptides, inorganic compounds, and organic small molecules. In a given device, one or more reagents may be adsorbed to one or more hydrophilic regions (non- covalently through non-specific interactions), or covalently (as esters, amides, imines, ethers, or through carbon-carbon, carbon-nitrogen, carbon-oxygen, or oxygen-nitrogen bonds).

[0119] Any reagent needed in the assay may be provided within, or in a separate adsorbent layer in fluid communication with the test area within the hydrophilic regions and the sample flow path. Exemplary assay reagents include protein assay reagents, immunoassay reagents (e.g., ELISA reagents), glucose assay reagents, sodium acetoacetate assay reagents, sodium nitrite assay reagents, or a combination thereof. In various embodiments, which are not intended to be limiting, the diagnostic device may include, a blocking agent, enzyme substrate, specific binding reagent such as an antibody or sFv reagent, labeled binding agent, e.g., labeled antibody, may be disposed in the device within or in flow communication with one or more of the hydrophilic regions, or in a specific area thereof configured as a test area.

[0120] In some embodiments, a binder, e.g., an antibody, may be labeled with an enzyme or a colored particle to permit colorimetric assessment of analyte presence or concentration in a sample fluid. For example, the binder may be labeled with gold colloidal particles or the like as the color forming labeling substance. Where an enzyme is involved as a label, e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or b-galactosidase, an enzyme substrate may be disposed in the device within or in flow communication with one of the hydrophilic regions.

[0121] Exemplary substrates for these enzymes include BCIP/NBT, 3,3',5,5'-Tetramethylbenzidine (TMB), 3,3'-Diaminobenzidine (DAB), and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 4-methylumbelliferphosphoric acid, 3-(4-hydroxyphenyl)-propionic acid, or 4- m ethyl um be 11 i fc r-b- D-gal actos i dc. or the like.

[0122] In various embodiments, the reagent(s) develop color in one or more test areas 42 along the sample path (including gradations from white to black) as an indication of the presence, absence or concentration of an analyte in a sample.

[0123] In some embodiments, a device may include many reagents disposed along the sample flow path, each of which can react with a different analyte to produce a detectable effect. Alternatively, the reagents may be sensitive to a predetermined concentration of a single analyte.

[0124] In some embodiments, the reagent may include a washing reagent, or plural wash reagents such as buffers or surfactant solutions, within or in fluid communication with a hydrophilic region or the sample flow path. Washing reagent(s) function to wash an analyte by removing unbound species within the hydrophilic regions. For example, a suitable washing buffer may comprise phosphate buffered saline (PBS), detergent, surfactants, water, and salt. The composition of the washing reagent will vary in accordance with the requirements of the specific assay such as, for example, the particular capture reagent and indicator reagent employed to determine the presence of a target analyte in a test sample, as well as the nature of the analyte itself.

[0125] Alternatively, steps of a reaction using the devices disclosed herein may be washed as follows. In certain embodiments, defined hydrophilic regions do not contain a reagent In such case, water or buffer is then added to the hydrophilic regions of the device and the fluid passes through the device along the sample flow path to provide a washing step for the analytes in the fluid sample. Such washing steps can be used to remove unbound analyte or other components added for the detection of the presence of an analyte.

[0126] The hydrophilic regions can include one or more test areas that can be used to perform one or more assays for the detection of multiple analytes in the sample fluid. One or more of the hydrophilic regions can be treated with reagents that respond to the presence of analytes in a sample fluid and provide an indicator of the presence of an analyte in the sample fluid. In some embodiments, the detection of an analyte in the sample fluid is visible to the naked eye and can provide a color indicator of the presence of the analyte. In various embodiments, indicators may include molecules that become colored in the presence of the analyte, change color in the presence of the analyte, or emit fluorescence, phosphorescence, or luminescence in the presence of the analyte. In other embodiments, radiological, magnetic, optical, and/or electrical measurements can be used to determine the presence of proteins, antibodies, or other analytes in the sample flow path.

Methods of Making Encapsulated Paper-based Diagnostic Devices

[0127] In further exemplary embodiments, the present disclosure describes methods of making diagnostic devices, the methods including:

[0128] applying a hydrophobic hardenable (co)polymeric ink composition to an elongate web of a fibrous material, wherein the hydrophobic hardenable (co)polymeric ink composition is applied in a plurality of adjacent web regions extending from a first edge of the web to a second edge of the web, wherein each web region is separated from adjacent web regions by a border region; each and wherein each web region includes a hydrophobic area including the hydrophobic (co)polymeric ink composition, a hydrophilic area substantially free of the hydrophobic (co)polymeric ink composition;

[0129] positioning a second metallic or (co)polymeric substrate opposite the (co)polymeric film such that the folded web of fibrous material is positioned between the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate; and

[0130] bonding the first metallic or (co)polymeric substrate to the second metallic or (co)polymeric substrate to encapsulate the porous substrate, optionally wherein a compressible layer is positioned between the first metallic or (co)polymeric substrate and the folded web of fibrous material and/or the second metallic or (co)polymeric substrate and the folded web of fibrous material.

[0131] In some exemplary optional embodiments, the method further includes providing an optional compressible layer preferably positioned between at least one of the first metallic or (co)polymeric substrate and the porous substrate and/or the second metallic or (co)polymeric substrate and the porous substrate.

[0132] In some exemplary embodiments of the foregoing method, at least one of the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate comprises a heat-sealable (co)polymeric film and wherein the first metallic or (co)polymeric substrate and the second metallic or (co)polymeric substrate are bonded to each other at a plurality of edges which do not overlap the porous substrate.

[0133] In certain such embodiments, the first and second (co)polymeric substrate heat-sealable (co)polymeric films are independently selected from the group consisting of polyethylene films, polypropylene films, polybutylene films, polycarbonate films and polyester films.

[0134] In some presently preferred embodiments, the method further includes creating at least one access port in the diagnostic device by removing a portion of at least one of the first metallic or (co)polymeric film or the second metallic or (co)polymeric film in a region which overlaps the first hydrophilic region of the porous substrate. In certain presently preferred embodiments, the elongate porous substrate is chosen from paper, nonwoven materials, (co)polymeric films, and combinations thereof. In additional presently preferred embodiments, the ink composition is applied to the elongate web of porous substrate by at least one of printing, coating, physical vapor deposition, and combinations thereof. In other presently preferred embodiments, the method further includes disposing a reagent in at least one of the hydrophilic areas along the flow path, wherein the reagent is selected to provide an indication of at least one of a presence, an absence, and a concentration of an analyte in the sample. [0135] Preferably, a roll-to-roll process is used to produce the diagnostic devices. In a roll-to-roll process, printing on the porous substrate is performed on a web line where the hydrophobic ink composition is printed on the substrate and then transported over an array of web idlers surfaces until it reaches a UV lamp, which quickly solidifies the ink with UV radiation. To create a barrier print in the paper, it is a requirement that the hydrophobic ink reach the opposing surface of the paper before it is solidified. Between the time when the liquid ink reaches the opposing surface and when it is solidified, there is opportunity for traces of the liquid hydrophobic ink to contact and transfer to web line idlers. This residual un-solidified liquid ink can then transfer to unpattemed hydrophilic regions of the substrate that passes over the idler at a subsequent time, which can potentially damage later-manufactured devices. [0136] In addition, for UV-cured inks and thick substrates, in some cases the total volume of printed ink can be a challenge to cure completely. In this case, it may be necessary to run the porous substrate through multiple UV radiation stations, and it may be difficult to achieve sufficient or complete polymerization of the radiation curable ink. If some of the ink remains unsolidified, the unhardened or partially-solidified ink can contaminate the processing equipment and the hydrophilic regions of the paper - making the diagnostic device ineffective. In some cases, splitting the UV-curing between two layers can more fully polymerize the hydrophobic ink composition.

[0137] In some embodiments, mechanical fasteners may be used to temporarily maintain the alignment of one or more of the hydrophilic regions in overlying layers or panels of the diagnostic device, either alone or in combination with any of the adhesive layers described above. Suitable mechanical fasteners include, but are not limited to, plastic or metal clips, staples, elastic bands such as plastic or rubber bands, plastic zip ties and combinations thereof.

[0138] The mechanical fasteners may also be used to compress the stack of porous substrate layers and maintain intimate contact between layers, which in some examples can reduce wicking times between the layers. The hydrophilic regions in the layers should be in intimate contact with each other so that a test liquid or analyte flows inside paper pores and not in gaps between the layers of the stack. In some examples, intimate contact between the layers of the stack can be enhanced by placing the arms of a clamping mechanical fastener along the edges or folds of the porous substrate.

[0139] Compression can be applied across the whole device or at the folded edges. Compressing the device can provide intimate contact between the layers of the diagnostic device, which can improve the flow of fluid through the layers by avoiding air gaps. In an embodiment, it may be advantageous to only compress the device at the edges. It was found that over-compression of the device decreases the porosity of the hydrophilic regions and inhibits the flow of fluid through the layers.

[0140] Compression of the device can be achieved when the edges of the device are dry or wet. Dry compression can be advantageous when wetting the device is undesirable. Wet compression can be advantageous by decreasing the resistance to bending the fibers at the edge of the device. Wetting of the device can be accomplished by applying a small amount of water along the edges of the device. Compression decreases the springy-ness of the folded device by bending or breaking the fibers at the edge of the device through high pressure. Additional methods of compressing the device include an arbor press, nipped-roller, other methods known in the art.

[0141] The operation of the present disclosure will be further described with regard to the following detailed examples. These examples are offered to further illustrate the various specific and preferred embodiments and techniques. It should be understood, however, that many variations and modifications may be made while remaining within the scope of the present disclosure.

Methods of Using Encapsulated Paper-based Diagnostic Devices [0142] In one embodiment, which is not intended to be limiting and provided as an example, a sample solution (not shown in FIGS. 2A-2B) including a sample fluid, solvents, reagents, and the like, is placed on the sample port. A wash solution is then applied to the sample port, and the sample solution moves downward via capillary action through the hydrophilic regions of the respective panels of the sensor stack. [0143] In certain embodiments, analytes may be detected by direct or indirect detection methods that apply the principles of immunoassays (e.g., a sandwich or competitive immunoassay or ELISA).

[0144] In some embodiments, to detect a specific protein, one or more areas of the hydrophilic regions can be derivatized with reagents, such as antibodies, ligands, receptors, or small molecules that selectively bind to or interact with a protein in the sample fluid. For example, to detect a specific antigen in a sample, a test area of the hydrophilic regions can be derivatized with reagents such as antibodies that selectively bind to or interact with that antigen. Alternatively, to detect the presence of a specific antibody in the sample fluid, a test area of the hydrophilic regions may be derivatized with antigens that bind or interact with that antibody.

[0145] For example, reagents such as small molecules and/or proteins can be covalently linked to the hydrophilic regions using similar chemistry to that used to immobilize molecules on beads or glass slides, or using chemistry used for linking molecules to carbohydrates. In alternative embodiments, the reagents may be applied and/or immobilized in the hydrophilic regions by applying a solution containing the reagent and allowing the solvent to evaporate (e.g., depositing reagent into the hydrophilic region). The reagents can be immobilized by physical absorption onto the porous substrate by other non-covalent interactions.

[0146] The interaction of certain analytes with some reagents may not result in a visible color change unless the analyte was previously labeled. The devices disclosed herein may be additionally treated to add a stain or a labeled protein, antibody, nucleic acid, or other reagent that binds to the target analyte after it binds to the reagent disposed in the sample flow path 32, which produces a visible color change. For example, the device may include a separate area that already contains the stain, or labeled reagent, and includes a mechanism by which the stain or labeled reagent can be easily introduced into the sample flow path to bond to the target analyte after it binds to the reagent.

[0147] Or, for example, the device can be provided with a separate channel that can be used to flow the stain or labeled reagent from a different area of the hydrophilic regions into test area along the sample flow path to the target analyte after it binds to the reagent in the sample flow path. In one embodiment, this flow is initiated with a drop of water, or some other fluid. In another embodiment, the reagent and labeled reagent are applied at the same location in the device, for example, in a test area of one of the hydrophilic regions along the sample flow path.

[0148] In one exemplary embodiment, ELISA may be used to detect and analyze a wide range of analytes and disease markers with the high specificity, and the result of ELISA can be quantified colorimetrically with the proper selection of enzyme and substrate.

[0149] Detection of an analyte in a sample fluid may include an additional step of creating digital data indicative of an image of a developed test area and the assay result, and transmitting the data remotely for further analysis to obtain diagnostic information, or to store assay results in an appropriate database. [0150] Some embodiments further include equipment that can be used to image the device after deposition of the liquid to obtain information about the quantity of analyte(s) based on the intensity of a colorimetric response of the device. In some embodiments, the equipment establishes a communication link with off-site personnel, e.g., via cellular phone communication channels, who perform the analysis based on images obtained by the equipment.

[0151] In some example embodiments, which are not intended to be limiting, the entire assay can be completed in less than 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes. In some example embodiments, the device can have a detection limit of about 500 pM, 250 pm, 100 pM, 1 pM, 500 fM, 250 fM, or 100 fM.

[0152] The diagnostic device of the present disclosure can be used for assaying small volumes of fluid samples. In various embodiments, the fluid samples that can be assayed include, but are not limited to, biological samples such as urine, whole blood, blood plasma, blood serum, sputum, cerebrospinal fluid, ascites, tears, sweat, saliva, excrement, gingival cervicular fluid, or tissue extract. In some embodiments, the volume of fluid sample to be assayed may be a drop of blood, e.g., from a finger prick, or a small sample of urine, e.g., from a newborn or a small animal. In some embodiments, the sample fluid is an environmental sample such as a water sample obtained from a river, lake, ocean or the like, or a sample of an industrial fluid. The device may also be adapted for assaying non-aqueous fluid samples for detecting environmental contamination.

[0153] In some embodiments, a single drop of liquid, e.g., a drop of blood from a pinpricked finger, is sufficient to perform assays providing a simple yes/no answer to determine the presence of an analyte in a sample fluid, or a semi-quantitative measurement of the amount of analyte that is present in the sample, e.g., by performing a visual or digital comparison of the intensity of the assay to a calibrated color chart. However, to obtain a quantitative measurement of an analyte in the liquid, a defined volume of fluid is typically deposited in the device.

[0154] Thus, in some embodiments, a defined volume of fluid (or a volume that is sufficiently close to the defined volume to provide a reasonably accurate readout) can be obtained by patterning the hydrophilic substrate to include a sample well that accepts a defined volume of fluid. For example, in the case of a whole blood sample, the subject's finger could be pinpricked, and then pressed against the sample well until the well was full, thus providing a satisfactory approximation of the defined volume. [0155] The assay reagents included in the device are selected to provide a visible indication of the presence of one or more analytes in the sample fluid. The source or nature of the analytes that may be detected using the disclosed devices are not intended to be limiting. Exemplary analytes include, but are not limited to, toxins, organic compounds, proteins, peptides, microorganisms, bacteria, viruses, amino acids, nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs, pollutants, pesticides, and metabolites of or, antibodies to, any of the above substances. Analytes may also include any antigenic substances, haptens, antibodies, macromolecules, and combinations thereof. For example, immunoassays using the disclosed devices could be adopted for antigens having known antibodies that specifically bind the antigen. [0156] In exemplary embodiments, the disclosed devices may be used to detect the presence or absence of one or more viral antigens, bacterial antigens, fungal antigens, or parasite antigens, cancer antigens. [0157] Exemplary viral antigens may include those derived from, for example, the hepatitis A, B, C, or E virus, human immunodeficiency virus (HIV), herpes simplex virus, Ebola virus, varicella zoster virus (virus leading to chicken pox and shingles), avian influenza virus, SARS virus, MERS virus, Epstein Barr virus, rhinoviruses, coronaviruses (such as, for example, the COVID19 coronavirus), and coxsackieviruses.

[0158] Exemplary bacterial antigens may include those derived from, for example, Staphylococcus aureus, Staphylococcus epidermis, Helicobacter pylori, Streptococcus bovis, Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium leprae, Corynebacterium diphtheriae, Borrelia burgdorferi, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Salmonella typhi, Vibrio chloerae, Haemophilus influenzae, Bordetella pertussis, Yersinia pestis, Neisseria gonorrhoeae, Treponema pallidum, Mycoplasm sp., Legionella pneumophila, Rickettsia typhi, Chlamydia trachomatis, Shigella dysenteriae, and Vibrio cholera.

[0159] Exemplary fungal antigens may include those derived from, for example, Tinea pedis, Tinea corporus, Tinea cruris, Tinea unguium, Cladosporium carionii, Coccidioides immitis, Candida sp., Aspergillus fumigatus, and Pneumocystis carinii.

[0160] Exemplary parasite antigens include those derived from, for example, Giardia lamblia, Leishmania sp., Trypanosoma sp., Trichomonas sp., and Plasmodium sp.

[0161] Exemplary cancer antigens may include, for example, antigens expressed, for example, in colon cancer, stomach cancer, pancreatic cancer, lung cancer, ovarian cancer, prostate cancer, breast cancer, liver cancer, brain cancer, skin cancer (e.g., melanoma), leukemia, lymphoma, or myeloma.

[0162] In other embodiments, the assay reagents may react with one or more metabolic compounds. Exemplary metabolic compounds include, for example, proteins, nucleic acids, polysaccharides, lipids, fatty acids, amino acids, nucleotides, nucleosides, monosaccharides and disaccharides. For example, the assay reagent is selected to react to the presence of at least one of glucose, protein, fat, vascular endothelial growth factor, insulin-like growth factor 1, antibodies, and cytokines.

[0163] As noted above, one or more reagents (not shown in FIGs. 2A-2B) may be included in any or all of the hydrophilic regions, and one or more of the panels may include a test area to indicate at least one of the presence, the absence, or the concentration of an analyte in a sample fluid.

[0164] In some embodiments (not shown in FIGS. 2A-2B), each web region may be printed on a separate web or area of a web. After the web is further processed, the individual web regions may then be aligned, placed over each other in a desired order, and stacked to form a suitable diagnostic device. However, in some cases the alignment and stacking steps in such a process may increase the overall manufacturing cost of the diagnostic device compared to the folding process described above. [0165] In yet another aspect, the present disclosure is directed to assay methods including any of the embodiments of the diagnostic devices shown above. With reference to the diagnostic device 10 shown in FIGS. 2A-2B, example assay methods include adding a fluid sample including an analyte to the hydrophilic regions such that the sample fluid enters and wicks along the sample flow path by capillary action. In some embodiments, water or a buffer may also be added to the hydrophilic regions to assist in the movement of the sample fluid along the sample flow path.

[0166] Visual or machine examination of the test area within the hydrophilic regions, or over the entire hydrophilic regions, permits determination of at least one of a presence, absence, or concentration of the analyte in the fluid sample. For example, in some embodiments, the assay protocol produces a color reaction, which includes the development of a grey scale from black to white, and the examination of the development of or, intensity of, the color in the test area within the hydrophilic regions, or within the entire hydrophilic regions, to determine the presence, absence, or concentration of the analyte.

[0167] In one embodiment, an ELISA test may be conducted using the disclosed device. The method may include the steps of: (1) addition of a sample to the device, wherein the sample is wicked directly through the hydrophilic regions along the sample flow path (2) binding an analyte with a labeled antibody along the flow path and into the test area; and binding the analyte binds to an antigen in the test area; and optionally washing the hydrophilic regions with a buffer such as, for example, PBS, to observe the results in the test area.

[0168] In one embodiment, which is not intended to be limiting and provided as an example, a sample solution (not shown in FIGS. 2A-2B) including a sample fluid, solvents, reagents, and the like, is placed on the sample port. The sample solution and fluids may optionally be inserted through the access port in the packaging layer. A wash solution is then applied to the sample port, and the sample solution moves downward via capillary action through the hydrophilic regions of the respective panels of the sensor stack.

EXAMPLES

[0169] These Examples are merely for illustrative purposes and are not meant to be overly limiting on the scope of the appended claims. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Summary of Materials

[0170] Unless otherwise noted, all parts, percentages, ratios, etc. in the Examples and the rest of the specification are by weight. Solvents and other reagents used may generally be obtained from Sigma- Aldrich Chemical Company (Milwaukee, WI) unless otherwise noted.

[0171] The first and second (co)polymeric substrates used to encapsulate the porous substrates (e.g., paper) was a 5 mil thick PET film manufactured by 3M Company (Greenville, NC). Other materials are described in co-pending U.S. Prov. Patent Application 63/003,169, filed March 31, 2020 and titled “DIAGNOSTIC DEVICE” and co-pending U.S. Prov. Patent Applications 63/164,144, filed March 22, 2021 and titled “DIAGNOSTIC DEVICE.”

Test Methods

[0172] The importance of a predictable flow rate (equated to the time to flow through a diagnostic device to achieve a diagnostic result) was recognized as a key parameter for success of the final device. [0173] Lateral Wicking Rate was determined for lateral flow diagnostic devices by visual inspection of wicking of a test solution across a pre-determined measured lateral distance of the porous substrate. A stop-watch with 0.1 second resolution was used to measure the time required for the test solution to traverse the pre-determined measured lateral distance.

[0174] Vertical Wicking Rate was determined for vertical flow diagnostic devices using an electronic timing device that measured the time required for wicking of a test solution across a pre-determined measured vertical distance of the porous substrate.

[0175] Permeability of the porous substrate was measured to show that hydrophobic areas of selected porous substrates retained sufficient fluid permeability to be useful in a diagnostic device according to the present disclosure. A 5.08 cm (2 inch) diameter round disc was cut out of the porous substrate. The porous substrate samples were then inserted into a standard filter housing. A water line with one meter of static head pressure was then connected to the filter housing, and the outflow flow rate was measured. Lateral Flow Device Examples

[0176] Lateral flow devices with a H/H (hydrophobic/hydrophilic) pattern with at least one “oval” (as opposed to an H/H pattern that contained only circles) were tested. The lateral flow devices were wetted at an inlet port on the front face of the device and observed for wetting at the first circle and the end of the channel on the back (viewing) side of the device. Binder clipped (CE-1), plastic clipped (CE-2), and a device according to the present disclosure as shown in Fig. 3 (EX-1) were tested, the results of which are shown in Table 1.

Table 1: Results for Lateral Flow Devices Vertical Flow Devices Examples

[0177] The vertical flow devices were wetted at an inlet port on the front face of the device and observed for wetting on the back (viewing) side of the device. Observation was difficult by visual inspection, therefore an electronic measurement device described in the test methods was used to determine the time for flow through the device. A binder clipped (CE-3) device similar to Fig. 1 without encapsulation and a device encapsulated according to FIG. 4 of the present disclosure (EX-2) were tested, the results of which are shown in Table 2.

Table 2: Results for Vertical Flow Devices

Example 3:

[0178] A diagnostic device or assay device substantially similar to that described schematically in FIG. 2B was prepared that included a thick (about 60 mils or 1.5 mm) cardboard backer card and a clear pressure sensitive adhesive (PSA) tape outer packaging layer fixing the sensor stack to the baker card. The outer packaging layer included 2 apertures to provide access to the sensor stack, as well as a pull tab configured to strip away a predetermined portion of the outer packaging layer.

[0179] A compression layer of 90 mil (2.3 mm) closed cell polyethylene (PE) foam was attached to the backer card with an adhesive transfer tape. Above the foam layer was a wicking element including 2 sublayers of 0.9 mm wicking pads. The bottom sublayer of the wicking layer was attached to the top of the foam layer with an adhesive transfer tape. The top sublayer wicking pad may have optional laser-cut lateral flow control features.

[0180] The wicking pads were attached to each other with disks of an adhesive transfer tape which also blocked the wicking flow and facilitated the lateral flow control features. Above the top wicking pad the sensor stack was attached to the cover PSA tape with adhesive tape tabs.

[0181] A cover layer above the sensor stack was a 14 mil (0.36 mm) polyethylene terephthalate (PET) sheet. The cover layer included 2 apertures underlying the apertures in the outer packaging layer, which provided access to the sensor stack from above.

[0182] In another embodiment, the backing layer was a PET film that was ultrasonically welded about

[0183] In another embodiment, the backing layer was 14 mil (0.36 mm) PET, and the outer packaging layer was an injection molded plastic shell. The plastic shell included apertures on an upper surface to provide access to the sensor stack. [0184] The follow Preparative Examples illustrate methods of making various porous substrates suitable for encapsulation within a diagnostic device according to embodiments of the present disclosure.

Preparative Example 1 :

[0185] Flexographic ink (9418 obtained from NAZDAR Ink Technologies of Shawnee, KANS) was printed on a WHATMAN Grade 1 filter (obtained from GE Healthcare Life Sciences of Piscataway, NJ) paper substrate busing a FLEXIPROOF 100 printing system (obtained from RK Industries, Herts, United Kingdom). Printing was accomplished by using a 38.75 micrometer (25 billions of cubic microns (BCM)), 35.4 lines per centimeter (90 lines per inch) anilox roll to form a 5.08 cm (2 inch) diameter circle. After printing, the printed paper sample was heated for eight minutes at 177°C (350°F) and the ink was cured by exposure to UV radiation by a FUSION High Intensity UV curing system (obtained from FUSION UV Systems Inc of Hampshire, United Kingdom) outfitted with an H-bulb and conveyed at 1.5 meters (5 feet) per minute to form a hydrophobic region on the paper sample. After curing, the printed paper sample was tested for performance by depositing dyed deionized water into non-printed areas and visually inspecting the spread of the dyed water. The dye was added to water to help with observations. [0186] A test was performed to show that volumetrically hydrophobic areas of the paper retained sufficient fluid permeability. A 5.08 cm (2 inch) diameter round disk was cut out of the printed paper. The paper samples were inserted into a standard filter housing and a water line with one meter of head pressure was connected to the filter housing and outflow was measured. Table 3 summarizes these test results. Table 3: Permeability Testing of Porous Substrates

Preparative Example 2:

[0187] A sample was created as described in Preparative Example 1 using NAZDAR OP 1028 ink (obtained from NAZDAR Ink Technologies of Shawnee, KANS) instead of the 9418 ink. A test was performed to show that volumetrically hydrophobic areas of the printed paper retained sufficient fluid permeability. A 5.08 cm (2 inch) diameter round disk was cut out of the printed paper. The paper samples were inserted into a standard fdter housing and a water line with one meter of head pressure was connected to the fdter housing and outflow was measured. Refer to Table 3 for test results.

Preparative Examples 3 - 5:

[0188] Three samples were created as described in Preparative Example 1 using a release ink UVF03408 (UV Easy Release) (obtained from Flint Group, Rogers, MN) instead of the 9418 ink. The first sample was undiluted. Before applying the ink, the second sample diluted the release ink by adding 20 percent isopropyl alcohol (IP A) solvent Before applying the ink, the third sample diluted the release ink by adding 40 percent IPA solvent. Samples with the solvent-containing ink were dried at room temperature for 15 minutes. A test was performed to show that volumetrically hydrophobic areas of the printed paper retained sufficient fluid permeability. A 5.08 cm (2 inch) diameter round disk was cut out of the printed paper. The paper samples were inserted into a standard filter housing and a water line with one meter of head pressure was connected to the filter housing and outflow was measured. Refer to Table 3 for test results.

Preparative Example 6:

[0189] A CH 265 self-adhering adhesive (obtained from Valpac Industries, Federalsburg, MD) was manually applied by a cotton swab onto the printed regions on both sides of the sample created in Preparatory Example 1. After drying at room temperature for one hour, the sample was folded and lightly pressed together. Dyed water was placed on one side of the sample and wicking to the other side was observed after 25 seconds indicating that fluid transport across layers was successful.

Comparative Example 4:

[0190] A wax-saturated paper was made by melting Batik Wax (available from Jacquard Products, Healdsburg, CA) at 65.6°C (150°F) and dripping it on WHATMAN Grade 1 paper pre-heated to the same temperature until saturation of less than about 5 minutes. A test was performed to show that volumetrically hydrophobic areas of the printed paper retained sufficient fluid permeability. A 5.08 cm (2 inch) diameter round disk was cut out of the printed paper. The paper samples were inserted into a standard filter housing and a water line with one meter of head pressure was connected to the filter housing and outflow was measured. Refer to Table 3 for test results.

Preparative Example 7:

[0191] An adhesive was printed in an open mesh pattern onto the hydrophobic printed regions of a specific region of the configuration as described in Preparative Example 1.

[0192] The measured flow was highest for unprinted paper, followed by the printed paper. This shows that while printed paper remained permeable to water, flow through the wax-saturated disk was very low, which likely was due to de lamination of wax under one meter of water pressure.

Preparative Example 8:

[0193] Flint Group Easy Release Coating (available from Flint Group, Rogers, MN) was flexographically printed on a 12-wide roll of Great Lakes filter paper (equivalent to #1 Whatman, Grade 601, available from Great Lakes Filters, Bloomfield Hills, MI) on custom-made flexographic printing line using 24 bcm (billion cubic microns), 100 lines per inch (about 40 lines per cm) anilox roll at 10 feet per minute (fpm; 5 cm/sec) line speed in a pattern representing an array of 5 folds of bio-diagnostics devices.

[0194] The ink was in-line UV cured on both sides in two passes. Single 5-fold devices were cut out of the roll of paper and folded along unprinted spaces between the prints. 3M spray adhesive (3M Spray Adhesive Super 77®, available from 3M Company, St. Paul, MN) was lightly sprayed by hand on both side of the device over both hydrophobic and hydrophilic areas.

[0195] After drying for 2 minutes, device was folded. Dyed water was placed on the top hydrophilic circle (covered with sprayed adhesive) and left to wick. Wicking to the other side was observed in about 50 seconds indicating that fluid transport across layers was successful.

Preparative Example 9:

[0196] In this example, we demonstrated dual-side registered patterning of a hydrophobic ink via the following steps:

[0197] In step (1), a hydrophobic ink available from Flint Group under the trade designation Easy Release (UVF03408) UV curable ink was flexographically printed using a 12 BCM/in ² anilox roll and a patterned LUX ITP60 flexographic printing plate (MacDermid Graphics Solutions, Atlanta, GA) at 20 ft/min (10 cm/sec) onto filter paper onto Great Lakes Filter Paper (Grade: CP51232 - Grade 601) obtained from Ahlstrom-Munksjo Filtration LLC), and the ink was transported to the UV curing station and solidified. The time between printing and curing was such that when the paper was transported at 20 ft/min (10 cm/sec), it provided enough time for the ink to wick approximately half the thickness of the filter paper.

[0198] In step (2), the printed paper from step (1) was then re-inserted through the printing line at 20 ft/min (10 cm/sec), and a second matching reverse-image pattern of the Flint Group Easy Release ink was printed in registration to the backside (i.e., opposing paper surface to the first pass of printing) of the filter paper. The printing was performed with a 12 BCM/in ² anilox roll and patterned LUX ITP60 flexographic printing plate. The printed paper was transported to the UV curing station and solidified at 20 ft/min (10 cm/sec), which was sufficient time for the hydrophobic ink to penetrate the paper and reach the other half-printed hydrophobic barrier layer, completing a barrier layer for a diagnostic device.

[0199] The sample from step (2) of this example was tested with red-dyed water to ensure a complete seal was created throughout the thickness of the paper. The red-dyed water was deposited into the channels formed by the hydrophobic barriers, dried, and then photo-graphed.

Prophetic Example 1 :

[0200] Any of the porous substrates of Preparatory Examples 1-9 may be incorporated into an encapsulated diagnostic device according to the present disclosure. The porous substrate may be positioned between first and second (co)polymeric substrates (e.g., 5 mil thick PET film manufactured by 3M Company, Greenville, NC) and then passed through a heated laminator to encapsulate the porous substrate. Alternatively, an ultrasonic bonding device may be used to bond together the first and second (co)polymeric substrates, thereby fully encapsulating the porous substrate.

[0201] Reference throughout this specification to "one embodiment," "certain embodiments," "one or more embodiments" or "an embodiment," whether or not including the term "exemplary" preceding the term "embodiment," means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the certain exemplary embodiments of the present disclosure. Thus, the appearances of the phrases such as "in one or more embodiments," "in certain embodiments," "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the certain exemplary embodiments of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0202] While the specification has described in detail certain exemplary embodiments, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing, may readily conceive of alterations to, variations of, and equivalents to these embodiments. Accordingly, it should be understood that this disclosure is not to be unduly limited to the illustrative embodiments set forth hereinabove. Furthermore, all publications and patents referenced herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0203] Various exemplary embodiments have been described. These and other embodiments are within the scope of the following claims.