Diagnostic device

Diagnostic devices for quantitative or qualitative analysis of a sample fluid including an analyte include at least two portions made from a hydrophilic material. The planar portions are stacked on each other and each occupy a different and substantially parallel plane to form a three-dimensional structure. At least one of the planar portions includes a hydrophobic region formed by applying a low surface energy material that extends through a thickness of the substrate portion from a first major surface to a second major surface thereof. The hydrophilic regions in the overlying substantially parallel substrate portions can be aligned with each other such that a fluid is passively transported between adjacent hydrophilic regions to provide a sample flow path between adjacent substrate portions.

BACKGROUND

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).

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, 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. 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 immunochromatographic 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.

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 include reagents, buffer salts, analytes (for example antigens) and binders (for example, antibodies) selected to perform a multiplexed assay.

To manufacture a diagnostic device including multiple planar regions 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 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 to resource-limited settings such as developing nations. To provide enhanced diagnostic resources and improve health care these areas, there remains a need for multiplexed assay devices that are inexpensive, portable, and easy to construct and use.

SUMMARY

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 portions, each 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 structure. At least one of the planar portions includes a hydrophobic region and a hydrophilic region. The hydrophobic region in each substrate portion is formed by applying a low surface energy material, such as a hydrophobic ink, which extends through a thickness of the substrate portion from a first major surface to a second major surface thereof. The hydrophobic region in each substrate portion includes an arrangement of interconnected pores having at least one uninterrupted path that extends between the first major surface and the second major surface. The hydrophilic regions in the overlying substantially parallel substrate portions can be aligned with each other such that a fluid is passively transported between adjacent hydrophilic regions to provide a sample flow path between adjacent substrate portions that is substantially normal to the overlying planes of the substrate portions.

In some embodiments, some surfaces of the overlying substrate portions may optionally include connective regions that maintain the alignment of the hydrophobic and hydrophilic regions and the sample flow path. In some embodiments, the diagnostic device can include a mechanical fastener to maintain the alignment of the hydrophobic and hydrophilic regions.

In various embodiments, a reagent is within the sample flow path, in fluid communication with the sample flow path, or may be applied to the sample flow path, to provide an indication of at least one of a presence, absence, or concentration of an analyte in the sample. For example, in some embodiments, the indication includes an easily readable color change.

In one embodiment, the diagnostic device includes an elongate hydrophilic substrate with folded regions dividing the substrate into at least two portions, each portion occupying a different and substantially parallel plane. When the substrate is folded, the planar portions are stacked adjacent to each other to provide a diagnostic device with a three-dimensional structure.

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.

In one aspect, the present disclosure is directed to a diagnostic device including an elongate substantially planar porous substrate with a first end and a second end, wherein the substrate has at least one folded region between the first end and the second end. A first portion of the substrate lies in a first plane with respect to the folded region, wherein the first portion of the substrate includes a first hydrophobic region and a first hydrophilic region, wherein the first hydrophobic region includes a first low surface energy polymeric material extending from a first major surface of the first portion of the substrate to a second major surface of the first portion of the substrate, and wherein the first hydrophobic region has an arrangement of interconnected open pores providing at least one uninterrupted path extending from the first major surface of the first portion of the substrate to the second major surface of the first portion of the substrate. A second portion of the 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 substrate including a second hydrophilic region and a second hydrophobic region having a second low surface energy polymeric material, which may be the same or different from the first low surface energy polymeric material, extending from a first major surface of the second portion of the substrate to second major surface of the second portion of the substrate, and wherein the second hydrophobic region has an arrangement of interconnected open pores providing at least one uninterrupted path extending from the first major surface of the second portion of the substrate to the second major surface of the second portion of the substrate. At least one connective region is between the first portion of the substrate and the second portion of the 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 substrate and the second portion of the substrate along a direction normal to the first plane and the second plane. A reagent is 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.

In another aspect, the present disclosure is directed to a diagnostic device that includes an elongate substantially planar porous fibrous substrate with a first end and a second end. The substrate includes a plurality of folded regions between the first end and the second end, the plurality of folded regions dividing the planar porous substrate into a stack of overlying substantially planar panels, wherein each of the panels in the stack occupies a different substantially parallel plane, and wherein each of the panels includes: a hydrophobic area with fibers coated with a hydrophobic low surface energy 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 a hydrophilic area. At least some of the panels includes 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 areas and hydrophilic areas in adjacent panels of the stack are aligned with each other to provide a sample flow path between the hydrophilic areas 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.

In another aspect, the present disclosure is directed to a diagnostic method, the method including: providing a diagnostic device including an elongate substantially planar porous fibrous substrate with a first end and a second end, wherein the substrate has a plurality of folded regions between the first end and the second end, the plurality of folded regions dividing the planar porous substrate into a stack of overlying planar panels each occupying a different substantially parallel plane, and wherein each of the panels includes: a hydrophobic area and a hydrophilic area arranged such that the hydrophilic areas in the panels are registered with each other to provide a sample flow path therebetween, the hydrophobic areas including fibers coated with a low surface energy polymeric material 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; a reagent disposed in the sample flow path, and a connector between at least some of the panels that maintains the alignment of the hydrophilic regions along the sample flow path; applying a sample to the sample flow path; and flowing the sample by capillary action along the sample flow path such that the reagent provides an indication of at least one of a presence, absence, or a concentration of the analyte in the sample.

In another aspect, the present disclosure is directed to a method of making a diagnostic device, the method including: applying a hydrophobic hardenable polymeric ink composition to an elongate web of a fibrous material, wherein the hydrophobic hardenable 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 polymeric ink composition, a hydrophilic area substantially free of the hydrophobic polymeric ink composition, and at least partially hardening the hardenable 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 path between a first major surface of the web and a second major surface of the web; and folding the web of porous 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 includes registered hydrophilic areas forming a sample flow path therebetween.

In another aspect, the present disclosure is directed to a system, including a diagnostic device with an elongate substantially planar porous substrate with a first end and a second end, wherein the substrate has at least one folded region between the first end and the second end, and wherein: a first portion of the substrate lies in a first plane with respect to the folded region, wherein the first portion of the substrate has a first hydrophobic region and a first hydrophilic region, wherein the first hydrophobic region includes a hydrophobic polymeric low surface energy material extending from a first major surface of the first portion of the substrate to a second major surface of the first portion of the substrate, and wherein 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 substrate to the second major surface of the first portion of the substrate; and a second portion of the substrate, different from the first portion of the substrate, wherein the second portion of the 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 substrate including a second hydrophilic region and a second hydrophobic region including the hydrophobic polymeric low surface energy material and extending from a first major surface of the second portion of the substrate to second major surface of the second portion of the substrate, and wherein the second hydrophobic region has an arrangement of interconnected open pores providing at least one uninterrupted path extending from the first major surface of the second portion of the substrate to the second major surface of the second portion of the substrate; at least one connective region between the first portion of the substrate and the second portion of the 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 passive sample flow path between the first portion of the substrate and the second portion of the substrate along a direction normal to the first plane and the second plane; and a reagent selected to detect at least one of a presence, an absence or a concentration of an analyte present in a sample fluid applied to flow path of the diagnostic device.

Like symbols in the drawings indicate like elements.

DETAILED DESCRIPTION

Referring now toFIGS.1A-1B, an embodiment of a diagnostic device10includes an elongate substantially planar hydrophilic substrate12with a first end13, a second end15, and at least one folded region14between the first and the second ends13,15. The folded region14separates the hydrophilic substrate12into a first sheet-like portion16and a second sheet-like portion18, each occupying a substantially parallel plane with respect to the folded region14. The first substrate portion16includes a first major surface17and a second major surface19, while the second substrate portion18includes a first major surface21and a second major surface23. In the embodiment ofFIG.1A, the first portion of the substrate16and the second portion of the substrate18overlie one another such that the respective major surfaces19and21are adjacent to each other.

The first substrate portion16includes a first hydrophobic region24and a first hydrophilic region26, while the second substrate portion18includes a second hydrophobic region28and a second hydrophilic region30. The fibers of the substrate12in the hydrophobic regions24,28have 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 regions26,30. The hydrophobic regions24,28substantially confine the flow of the fluid along the direction of the arrow A, which is aligned along thickness of the substrate portions16,18, or along the z-axis of the three-dimensional diagnostic device10. The hydrophilic regions26,30are sufficiently aligned with each other such that a fluid sample placed on the first hydrophilic region26(not shown inFIGS.1A-1B, seeFIGS.1C-1D) can be passively transported using, for example, wicking or capillary action, along a sample flow path32to provide fluid communication between the first substrate portion16and the second substrate portion18such that the fluid sample wicks into the second hydrophilic region30.

Referring now to the magnified schematic cross-sectional diagrams inFIGS.1C-1D, the diagnostic device10ofFIGS.1A-1Bincludes a hydrophilic substrate12with a first substrate portion16. The substrate portion16of the hydrophilic substrate12includes a hydrophobic portion24and a hydrophilic portion26. The hydrophilic portion26includes an arrangement of entangled fibers80. In some example embodiments, which are not intended to be limiting and are provided only as an illustrative example, the fibers80in the hydrophilic region26have a surface energy σ at a selected temperature for a selected liquid84of about 40 to about 65 dynes/cm. In the hydrophobic portion24, at least a portion of the fibers80are coated with a low-surface energy polymeric material82, which limits capillary flow (or wicking) of a fluid into the hydrophobic portion24. In some embodiments, if the low surface energy polymeric material82is deposited on the fibers80such that it only coats the surface of the fibers, at least some interconnected interstitial passages83remain between the fibers. The passages83remain open such that a gas (which is a fluid) may freely move through the porous substrate12in the hydrophobic regions24. After coating with the low surface energy polymeric material82, the fibers in the hydrophobic regions24have a surface energy at least 10 dyne/cm less than the surface tension of the liquid84.

If the liquid84is placed on the surface17of the hydrophilic region26at a time t=0, after a saturation time tsatgreater than t=0 has elapsed, the fluid84will wick and be passively transported along the fibers80and occupy interstitial regions85in the hydrophilic region26. The low surface energy polymeric material in the hydrophobic regions24tends to repel or resist intrusion of the fluid84into the interstitial regions83therein, thereby forming a flow path88through the hydrophilic region26for the fluid84.

Referring again toFIGS.1A-1B, all or a portion of one or both of the hydrophilic regions26,30can include a test area42where an analytical result or output of the device10can be displayed for a user, as well as one or more reagents40in the test area42or in fluid communication with the test area42. The reagents40are selected to provide an indication of at least one of a presence, absence or concentration of an analyte in the fluid sample are disposed in the sample flow path32. In various embodiments, the reagent40is applied to all or a portion of one or both of the hydrophilic regions26,30, can be in another portion of the device10and in fluid communication with the flow path32, or can be applied to the sample flow path32before or after the application of the fluid sample to the sample flow path32.

In some embodiments, the diagnostic device10includes an optional first connection region34on the second major surface19of the first substrate portion16. In some embodiments, the diagnostic device10further includes an optional second connection region36on the first major surface21of the second substrate portion18. Either or both of the adjacent major surfaces19,21of the overlying substrate portions16,18can include connection regions, which adhere the first substrate portion16to the second substrate portion18and maintain the registration of the hydrophilic regions26,30to preserve the sample flow path32(FIG.1B).

In various embodiments, the elongate hydrophilic substrate12may be made from any porous, hydrophilic, adsorbent material capable of wicking a sample fluid by capillary action. In one or more embodiments, the substrate12is 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 polymer films such as, for example, nitrocellulose, cellulose acetate, polyesters, and polyurethane, and the like.

The first and second hydrophobic regions24,28may be formed by applying a desired pattern of a low surface energy polymeric material such as, for example, a polymeric ink composition, to the substrate12. As shown schematically inFIGS.1C-1D, the hydrophobic ink composition wicks along the fibers of the hydrophilic substrate12and coats the fibers thereof, leaving open at least some interstitial regions between the fibers. When subsequently cured or hardened, the polymeric ink composition provides open interstitial regions that form at least one uninterrupted open path between the respective major surfaces17,19of the first substrate portion16and major surfaces21,23of the second substrate portion18. The hydrophobic regions24,28thus resist absorption of a liquid applied to, for example, the hydrophilic region26of the first substrate portion16, and the liquid is passively transported via capillary action or wicking between the hydrophilic regions26,30.

While not wishing to be bound by any theory, currently available evidence indicates that the relative difference in absorption between the hydrophobic regions24,28and the hydrophilic regions26,30is 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 portions16,18, and the surface energy of the fibers coated with the low surface energy ink in the hydrophobic regions24,28. The larger this difference, the larger the resistivity to absorption of the selected fluid in the hydrophobic regions24,28. The difference may also depend on, for example, the uniformity of ink coverage, the structure of the fibers, and the like.

In one example, if the sample fluid selected to flow by wicking or capillary action between the substrate portions16,18is a bodily fluid, the surface energy of the fibers with the low surface energy hydrophobic ink applied thereto in the hydrophobic regions24,28should 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 regions24,28should be at least 10 dyne/cm lower than the lowest surface tension of the bodily fluid, or at least 15 dyne/cm lower, or at least 20 dyne/cm lower, or even at least 30 dyne/cm lower. For example, it is reported that human urine has a minimum surface tension of about 55 dyne/cm, and human saliva has a surface tension of about40dyne/cm, so to resist absorption of these bodily fluids by wicking or capillary action the hydrophobic regions24,28surface energy of ink should have a surface tension of less than about 45 dyne/cm, or less than about 40 dyne/cm, or less than about 35 dyne/cm, or less than about 30 dyne/cm, or less than about 25 dyne/cm, or less than about 20 dyne/cm.

In another example, to resist capillary flow or wicking of a selected fluid, presently available evidence indicates that the hydrophobic ink compositions in the regions24,28, 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°.

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, Sep. 12, 2003; WETTABILITY (SURFACTANT SCIENCE) by John Berg, ed., CRC Press; 1 edition, Apr. 20, 1993, each of which are incorporated herein by reference in their entirety.

In various embodiments, the hydrophobic ink composition includes at least one polymerizable low surface energy monomer, oligomer, or polymer that can provide a desired resistance to absorption of a selected liquid or sample fluid. This low surface energy monomer, oligomer, or polymer can be a fluorocarbon, silicone, or hydrocarbon. The low surface energy monomer, oligomer, or 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/m2 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.

In some embodiments, the hydrophobic region24,28includes a non-tacky crosslinked polymeric layer. This polymeric layer is made from a radiation curable coating formulation containing at least one low surface energy monomer, oligomer, or polymer chosen from the group of polymerizable fluorocarbon, silicone, or hydrocarbon monomers.

The non-tacky crosslinked polymeric layer may be formed by polymerizing a precursor composition, although other methods (e.g., crosslinking of a polymer or blend thereof using chemical means or ionizing radiation) may also be used. Useful precursor compositions typically include one or more polymerizable materials (e.g., monomers and/or oligomers, which may be monofunctional and/or polyfunctional), a curative, and optionally inorganic particles. Polymerizable materials may be, for example, free- radically polymerizable, cationically polymerizable, and/or condensation polymerizable.

Useful polymerizable materials include, for example, acrylates and methacrylates, epoxies, polyisocyanates, and trialkoxysilane terminated oligomers and polymers. Preferably, the polymerizable material includes a free-radically polymerizable material.

Exemplary free-radically polymerizable oligomers include those marketed by UCB Chemicals, Smyrna, Georgia (e.g., under the trade designation “EBECRYL”), and those marketed by Sartomer Company, Exton, PA (e.g., under the trade designations “KAYARAD” or “CN”).

Depending on the choice of polymerizable material, the precursor composition may, optionally, contain one or more curatives that assist in polymerizing the polymerizable material. The choice of curative for specific polymerizable materials depends on the chemical nature of the copolymerizable material. For example, in the case of epoxy resins, one would typically select a curative known for use with epoxy resins (e.g., dicyandiamide, onium salt, or polymercaptan). In the case of free-radically polymerizable resins, free radical thermal initiators and/or photoinitiators are useful curatives.

Typically, the optional curative(s) is used in an amount effective to facilitate polymerization of the monomers and the amount will vary depending upon, for example, the type of curative, the molecular weight of the curative, and the polymerization process. The optional curative(s) is typically included in the precursor composition in an amount in a range of from about 0.01 percent by weight to about 10 percent by weight, based on the total weight of the precursor composition, although higher and lower amounts may also be used. The precursor composition may be cured, for example, by exposure to a thermal source (e.g., heat, infrared radiation), electromagnetic radiation (e.g., ultraviolet and/or visible radiation), and/or particulate radiation (e.g., electron beam of gamma radiation).

A variety of curing strategies can be readily selected, determined in part upon the characteristics of the curable coating composition, other components of the article, as well as manufacturing facilities. Illustrative techniques for maximizing the cure of a UV cured coating composition include curing under nitrogen, using new UV bulbs, cleaning the UV bulbs before use, matching the output spectrum of the UV bulb to the absorption of the initiator, and treatment at a slow speed and/or for a longer time. In some embodiments, a certain amount of post-exposure cure may take place over time as the dry erase article ages at room temperature.

A second cure treatment may be required in addition to the first cure described above. The second cure may use the same radiation source as the first cure, or it may use a different radiation source. Preferred second cure methods include heat, electron beam, and gamma ray treatment.

If the optional curative is a free-radical initiator, the amount of curative is preferably in a range of from about 1 percent by weight to about 5 percent by weight, based on the total weight of the precursor composition, although higher and lower amounts may also be used. Useful free-radical photoinitiators include, for example, benzoin ethers such as benzoin methyl ether and benzoin isopropyl ether, substituted benzoin ethers (e.g., anisoin methyl ether), substituted acetophenones (e.g., 2,2- dimethoxy-2-phenylacetophenone), substituted alpha-ketols (e.g., 2-methyl-2-hydroxypropiophenone), benzophenone derivatives (e.g., benzophenone), and acylphosphine oxides. Exemplary commercially available photoinitiators include photoinitiators under the trade designation “IRGACURE” (e.g., IRGACURE 651, IRGACURE 184, and IRGACURE 819) or “DAROCUR” (e.g., DAROCUR 1173, DAROCUR 4265) from Ciba Specialty Chemicals, Tarrytown, New York, and under the trade designation “LUCIRIN” (e.g., “LUCIRIN TPO”) from BASF, Parsippany, New Jersey.

The low surface energy monomers, oligomers, or polymers may be chosen from the group of fluorocarbon, silicone, or hydrocarbon monomers. Fluorocarbon monomers suitable for the hydrophobic ink composition include but are not limited to perfluoro acrylates or methacrylates, e.g., C4F9 based sulfonamide acrylates and C3F7 based sulfonamide acrylates.

Hydrocarbon monomers can be used to reduce the surface energy of a coating. Those hydrocarbon monomers are characterized by a long side chain that can form a crystalline structure on a surface. Suitable hydrocarbon monomers include but are not limited to octadecyl acrylate.

In one embodiment, the low surface energy monomers, oligomers, or polymers are added to a coating formulation in a concentration sufficient to produce a cured coating with a wetting tension of from about 20 to about 40 mJ/m2. In some embodiments, the wetting tension of the cured coating is from about 30 to about 36 mJ/m2.

In some embodiments, the radiation curable material includes the foregoing oligomer(s), monomer(s) and/or polymer(s) in one or more solvents along with a volume of optional particles or nanoparticles, eg., to impart increased hardness and durability to the writing member. In some cases, dilution of the hydrophobic ink in solvent can promote faster wicking into the porous or fibrous substrate (by lowering the viscosity of the ink) and can leave more interconnected space between the fibers.

Nanoparticles can be surface modified which refers to the fact that the nanoparticles have a modified surface so that the nanoparticles provide a stable dispersion. “Stable dispersion” refers to a dispersion in which the colloidal nanoparticles do not agglomerate after standing for a period of time, such as about 24 hours, under ambient conditions, e.g., room temperature (i.e., about 20 to about 22° C.), and atmospheric pressure, without extreme electromagnetic forces.

Surface-modified colloidal nanoparticles can optionally be present in a polymer coating used as a coatable composition herein with nanoparticles present in an amount effective to enhance the durability of the finished or optical element. The surface- modified colloidal nanoparticles described herein can have a variety of desirable attributes, including, for example, nanoparticle compatibility with a coatable composition such that the nanoparticles form stable dispersions within the coatable composition, reactivity of the nanoparticle with the coatable composition making the composite more durable, and a low impact or uncured composition viscosity. A combination of surface modifications can be used to manipulate the uncured and cured properties of the composition. Surface-modified nanoparticles can improve optical and physical properties of the coatable composition such as, for example, improved resin mechanical strength, minimized viscosity changes while increasing solids volume loading in the coatable composition and maintain optical clarity while increasing solid volume loading in the coatable composition.

In some embodiments, the nanoparticles are surface-modified nanoparticles. Suitable surface-modified colloidal nanoparticles can comprise oxide particles. Nanoparticles may comprise a range of particle sizes over a known particle size distribution for a given material In some embodiments, the average particle size may be within a range from about 1 nm to about 100 nm. Particle sizes and particle size distributions may be determined in a known manner including, for example, by transmission electron microscopy (“TEM”). Suitable nanoparticles can comprise any of a variety of materials such as metal oxides selected from alumina, tin oxide, antimony oxide, silica, zirconia, titania and combinations of two or more of the foregoing. Surface-modified colloidal nanoparticles can be substantially fully condensed.

In some embodiments, silica nanoparticles can have a particle size ranging from about 5 to about 100 nm. In some embodiments, silica nanoparticles can have a particle size ranging from about 10 to about 30 nm. Silica nanoparticles can be present in the coatable composition in an amount from about 10 to about 100 phr. In some embodiments, silica nanoparticles can be present in the coatable composition in an amount from about 30 to about 90 phr. Silica nanoparticles suitable for use in the coatable compositions of the present disclosure are commercially available from Nalco Chemical Co. (Naperville, IL) under the product designation NALCO COLLOIDAL SILICAS. Suitable silica products include NALCO products 1040, 1042. 1050, 1060, 2327 and 2329. Suitable turned silica products include, for example, products sold under the AEROSIL series OX-50, -130, -150, and -200 available from DeGussa AG. (Hanau, Germany), and CAB-O-SPERSE 2095, CAB-O-SPERSE A 105, and CAB-O-SIL MS available from Cabot Corp. (Tuscola, IL). Surface-treating the nanosized particles can provide a stable dispersion in the coatable composition (e.g.. a polymeric resin). Preferably, the surface- treatment stabilizes the nanoparticles so that the particles will be well dispersed in the coatable composition and results in a substantially homogeneous composition.

In some embodiments, the average particle sizes (e.g., particle diameter) may be within the range from about 1 nm to about 1000 nm. In addition to the foregoing particle sizes, use of smaller and larger average particle sizes are also contemplated. In embodiments of the disclosure, at least a portion of the foregoing particles may be surface modified in the manner described above. In outer embodiments, all the particles are surface modified. In still other embodiments, none of the particles are surface modified.

As will be understood, coating compositions used to make the hydrophobic regions of the present disclosure may include optional additives to enhance or control characteristics as desired, e.g., rheology modifiers such as JAYLINK Rheology Modifiers, colorants (e.g.. dyes and/or pigments). fire retardants, antioxidants, stabilizers, antiozonants, plasticizers, UV absorbers, hindered amine light stabilizers (HALS), etc

The hydrophobic ink compositions suitable to form the hydrophobic regions24,28may include any commercially available ink that creates a desired resistance to capillary flow or wicking of a selected liquid such as, for example, a sample fluid. Suitable examples include, but are not limited to, NAZDAR 9400 Series UV Flexo Inks or OP Series Inks (available from NAZDAR Ink Technologies of Shawnee, KS, United States) such as 9418 or OP 1028. In some embodiments, the ink composition is hardenable or curable with radiation, such as, for example ultraviolet (UV) light.

In some embodiments, the hydrophobic ink composition may include a solvent selected to provide, for example, optimal wicking properties along the fibers of the substrate12. Suitable solvents include, but are not limited to, water, alcohols, ethers, ketones, esters, and mixtures and combinations thereof.

The hydrophobic regions24,28may be patterned with the hardenable hydrophobic ink by any suitable technique including, but not limited to coating, screening, stamping, printing, photolithography, and combinations thereof. In some embodiments, the patterning technique may include heating the ink composition to a suitable temperature such that the ink wicks and flows along the fibers of the substrate, but does not occupy interstitial regions between the fibers. The interstitial regions in the hydrophobic regions24,28are sufficiently open and interconnected to allow some fluid flow between the major surfaces17,19and21,23of the substrate12, but a fluid flow rate between the major surfaces of the substrate12in the hydrophobic regions24,28is significantly lower that that of the hydrophilic regions26,30, so that a fluid placed in the hydrophilic regions26,30avoids the hydrophobic regions24,28and remains in the hydrophilic regions26,30to proceed along the sample flow path32.

In various embodiments, the optional connection regions34,36may vary widely, and can include any type of adhesive such as, for example, pressure sensitive adhesives, hot-melt adhesives, cohesive adhesives, and mixtures and combinations thereof. In the present application, the term cohesive adhesive refers to adhesive materials that adhere to each other, but have low adhesion, or no adhesion, to other non-adhesive surfaces.

Suitable pressure-sensitive adhesives (“PSAs”) are defined herein as adhesives which exhibit permanent tack at room temperature. This property allows pressure-sensitive adhesives to adhere tenaciously upon application with only light finger pressure. PSAs have a balance of properties: adhesion, cohesion, stretchiness, and elasticity. Adhesion refers both to immediate adhesion to a surface and to the bond strength which develops upon application of pressure (often measured as “peel strength”). Cohesion refers to the “shear strength” or resistance of the applied PSA to failure when subjected to shearing forces. Stretchiness refers to the ability to elongate under low stresses. Elasticity refers to a property wherein the material exhibits a retractive force when stretched and retracts when the force is released. A general description of pressure-sensitive adhesives may be found in the Encyclopedia of Polymer Sciences and Engineering, Vol. 13, Wiley-Interscience Publishers (New York, 1988).

In one example embodiment, a suitable cohesive adhesive as utilized herein includes quick-drying adhesives that, once dried, will create a surface with essentially no tack and will only adhere to other surfaces coated with the same adhesive when placed under pressure. Cohesive adhesives bond to themselves at ambient temperature with pressure, yet are essentially tack free to the touch, allowing coated substrates to be folded or wound upon themselves and stored without adhering to the opposing face of the substrate backing.

In various embodiments, suitable cohesive adhesives include latex or water-based adhesive compositions that, after drying, are substantially tack free to the touch, yet will adhere to themselves at ambient temperature with a pressure of 100 psi, and preferably at a pressure of about 60 psi or less. The bond strength of the self-seal may vary depending on the coat weight, pressure, and dwell time used. However, at minimum the removal force is at least about 10 g/linear inch, typically at least about 20 g/linear inch, preferably at least 50 g/linear inch, and most preferably at least about 100 g/linear inch. Substantially tack free to the touch means that the dried composition is nonblocking.

The cohesive adhesive is further capable of being applied to a hydrophilic substrate material at a relatively high rate of production and of being dried relatively quickly. As a result, the cohesive adhesive enables the manufacture of relatively low-cost diagnostic devices at production rates much faster than conventional adhesive materials used in the art.

Adhesives of this type have been employed in a variety of packaging applications including food (i.e. flexible packaging for candy wrappers, chips etc.); medical packaging; self-seal and tamper evident envelopes; banding for paper money, napkins, and clothing; and protective packaging such as fold over “blister” packages for hardware and small parts.

In some embodiments, for example, the cohesive adhesive may be applied using a highspeed printing process to reduce film thickness, further enabling the manufacture of a diagnostic device at production rates much faster than conventional adhesive materials used in the art. In some embodiments, which are provided as examples and not intended to be limiting, suitable cohesive adhesives include emulsions of natural and/or synthetic latex rubber in aqueous solution of ammoniated water with a solids content between 15 and 65 percent by weight.

In some example embodiments, which are not intended to be limiting, the viscosity of a suitable cohesive adhesive may be between 10 and 450 centipoise (cP) at 20 revolutions per minute and 23° C. per ASTM D1084 Test Method B. In some embodiments, the density of cohesive adhesive may be between 8.0 and 9.0 pounds per gallon (lb/gal) at 25° C., and the basicity or pH may be between 9.5 and 12.

In various embodiments, the cohesive adhesive may optionally contain dispersants, surfactants, tackifiers, isocyanates, antioxidants, and antifoaming agents, as is well known in the art, without deviating from the scope of the disclosure.

In at least one embodiment of the present disclosure, which is not intended to be limiting, the cohesive adhesive has the following properties: the solids content is 57.5 percent by weight, the viscosity is 75 cP at 25° C., the density is 8.3 lb/gal, and the pH is 10.0. In at least one embodiment of the present disclosure, the adhesive has a solids content between 45 and 58 percent by weight, a viscosity between 75 and 200 cP at 23° C., a density between 8.3 and 8.7 lb/gal at °C, and a pH of 10 to 11.

In some embodiments, mechanical fasteners may be utilized to 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.

In general, a wide variety of reagents40may be disposed in, or in fluid communication with, the test area42in hydrophilic regions26,30of the diagnostic device10to 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 regions26,30(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).

Any reagent40needed in the assay may be provided within, or in a separate adsorbent layer in fluid communication with the test area42within the hydrophilic regions26,30and the sample flow path32. 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 device10may 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 regions26,30, or in a specific area thereof configured as a test area42.

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 β-galactosidase, an enzyme substrate may be disposed in the device within or in flow communication with one of the hydrophilic regions26,30. 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-methylumbellifer-β-D-galactoside, or the like. In various embodiments, the reagent(s)40develop color in one or more test areas42along the sample path32(including gradations from white to black) as an indication of the presence, absence or concentration of an analyte in a sample.

In some embodiments, a device may include many reagents40disposed along the sample flow path, each of which can react with a different analyte to produce a detectable effect. Alternatively, the reagents40may be sensitive to a predetermined concentration of a single analyte.

In some embodiments, the reagent40may include a washing reagent, or plural wash reagents such as buffers or surfactant solutions, within or in fluid communication with a hydrophilic region26,30or the sample flow path32. Washing reagent(s) function to wash an analyte by removing unbound species within the hydrophilic regions26,30. For example, a suitable washing buffer may comprise 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.

Alternatively, steps of a reaction using the devices disclosed herein may be washed as follows. In certain embodiments, defined hydrophilic regions26,30do not contain a reagent40. In such case, water or buffer is then added to the hydrophilic regions26,30of the device10and the fluid passes through the device along the sample flow path32to 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.

The hydrophilic regions26,30can include one or more test areas42that 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 regions26,30can be treated with reagents40that 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 path32.

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).

In some embodiments, to detect a specific protein, one or more areas of the hydrophilic regions26,30can be derivatized with reagents40, 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 area42of the hydrophilic regions26,30can 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 area42of the hydrophilic regions26,30may be derivatized with antigens that bind or interact with that antibody. For example, reagents40such as small molecules and/or proteins can be covalently linked to the hydrophilic regions26,30using 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 reagents40may be applied and/or immobilized in the hydrophilic regions26,30by 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.

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 reagent40disposed in the sample flow path32, which produces a visible color change. For example, the device10may 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 reagent40. Or, for example, the device10can be provided with a separate channel that can be used to flow the stain or labeled reagent from a different area of the hydrophilic regions26,30into test area42along the sample flow path32to 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 area42of one of the hydrophilic regions26,30along the sample flow path32.

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.

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 area42and the assay result, and transmitting the data remotely for further analysis to obtain diagnostic information, or to store assay results in an appropriate database. 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 cell phone communication channels, who perform the analysis based on images obtained by the equipment.

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 device10can have a detection limit of about 500 pM, 250 pm, 100 pM, 1 pM, 500 fM, 250 fM, or 100 fM.

The diagnostic device10of 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 device10may also be adapted for assaying non-aqueous fluid samples for detecting environmental contamination.

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. 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 substrate12to 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.

The assay reagents included in the device10are 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.

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.

Exemplary parasite antigens include those derived from, for example,Giardia lamblia,Leishmaniasp.,Trypanosomasp.,Trichomonassp., andPlasmodiumsp.

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.

Referring now toFIGS.2A-2B, another embodiment of a diagnostic device110includes an elongate substantially planar hydrophilic substrate112with a first end113, a second end115, and at least one folded region114between the first and the second ends113,115. The folded region114separates the hydrophilic substrate112into a first sheet-like portion116and a second sheet-like portion118, each occupying a substantially parallel plane with respect to the folded region114. The first substrate portion116includes a first major surface117and a second major surface119, while the second substrate portion118includes a first major surface121and a second major surface123. In the embodiment ofFIG.2A, the first portion of the substrate116and the second portion of the substrate118overlie one another such that the respective major surfaces119and121are adjacent to each other.

The first substrate portion116includes a first hydrophobic region124and a first hydrophilic region126, while the second substrate portion118includes a second hydrophobic region128and a second hydrophilic region130. The hydrophobic regions124,128each resist fluid flow along the direction of the arrow A, which is aligned along thickness of the substrate portions116,118, or along the z-axis of the three-dimensional diagnostic device110. The hydrophilic regions126,130are aligned in registration with each other such that a fluid sample placed on the first hydrophilic region126(not shown inFIG.2A) can flow using, for example, wicking or capillary action, along a sample flow path132to provide fluid communication between the first substrate portion116and the second substrate portion118such that the fluid sample wicks into the second hydrophilic region130.

All or a portion of one or both of the hydrophilic regions126,130can include a test area142where an analytical result or output of the diagnostic device110can be displayed for a user, as well as one or more reagents140in the test area142or in fluid communication with the test area142. The one or more reagents140disposed in the sample flow path132are selected to provide an indication of at least one of a presence, absence or concentration of an analyte in the sample fluid. In various embodiments, the reagent140is applied to all or a portion of one or both of the hydrophilic regions126,130, can be in another portion of the device110and in fluid communication with the flow path132, or can be applied to the sample flow path132before or after the application of the fluid sample to the sample flow path132.

The diagnostic device110includes regular or irregular grid or mesh-like first connection region154on the second major surface119of the first substrate portion116. As shown schematically in the example embodiment ofFIG.2C, the first connection region154includes grid lines153,155aligned substantially normal to each other.

In some embodiments, the diagnostic device110further includes an optional grid or mesh-like second connection region156on the first major surface121of the second substrate portion118.

The grid-like connection regions154,156are configured to include sufficient open areas160between the grid lines153,155to allow a sample fluid to wick and flow from the first hydrophilic region126to the second hydrophilic region130along the sample flow path132, while adhering the first substrate portion116to the second substrate portion118and maintaining the registration of the hydrophilic regions126,130to preserve the alignment of the sample flow path132(FIG.2B). The grid lines153,155in the connection regions154,156, along with the hydrophobic regions124,128, prevent flow of the sample fluid along the direction B normal to the direction A of the sample flow path132.

In various embodiments, the connection regions154,156can include any type of adhesive described above such as, for example, pressure sensitive adhesives, hot-melt adhesives, cohesive adhesives, and the like. In some embodiments, the adhesive can be applied by spraying, printing, or use of a transfer adhesive, which provide a sufficiently open structure to allow wicking of the sample fluid between layers or panels of the device.

Referring now toFIG.3A, a portion of an elongate web200includes a hydrophilic substrate212including a first end213and a second end215. The web200includes a plurality of web regions270A-270E, which are separated by separation regions272A-272D. In the embodiment ofFIG.3A, each web region270A-270E includes a hydrophobic region224A-224E and a hydrophilic region226A-226E. In some embodiments, the separation regions272are free of the hydrophobic regions, but such an arrangement is not required. In the embodiment ofFIG.3A, the hydrophobic regions224A-224E and the hydrophilic regions226A-226E 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.

In the embodiment ofFIG.3A, the web regions270A and270B further include connective regions234A,234B that surround the hydrophilic regions226A,226B. In addition, in the embodiment ofFIG.3A, the web region270D includes a patterned connective region254of, for example, a pressure sensitive adhesive (PSA).

As shown inFIG.3B, the web200ofFIG.3Amay be folded along the separation regions272A-D in the direction of the arrows C to form a diagnostic device300including overlying and substantially parallel panels270A-270E. When so folded, the connection regions234A and234B come together to adhere and maintain registration of the panels270A-270B, and the patterned connective region254maintains the registration of the panels270C-270D. The registration of the panels270A-270E maintains alignment of the hydrophilic regions226A-226E, which allows flow of sample fluid along a sample flow path232through the hydrophilic regions226A-226E. While not shown inFIGS.3A-3B, additional connective regions of any suitable shape or configuration may be used to maintain alignment of the hydrophilic regions in the panels270B-C and270D-E. In some embodiments, mechanical fasteners (not shown inFIGS.3A-3B) may also be used, alone or in combination with adhesive connective regions, to maintain alignment of any or all of the panels270A-270E.

As noted above, one or more reagents (not shown inFIG.3B) may be included in any or all of the hydrophilic regions226A-226E (FIG.3A), and one or more of the panels270A-270E 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.

In some embodiments (not shown inFIGS.3A-3B), each web region270A-270E may be printed on a separate web or area of a web. After the web is further processed, the individual web regions270-270E 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 inFIGS.3A-3B.

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 device10shown inFIGS.1A-1B, example assay methods include adding a fluid sample including an analyte to the hydrophilic regions26,30such that the sample fluid enters and wicks along the sample flow path32by capillary action. In some embodiments, water or a buffer may also be added to the hydrophilic regions26,32to assist in the movement of the sample fluid along the sample flow path32.

Visual or machine examination of the test area42within the hydrophilic regions26,30, or over the entire hydrophilic regions26,30, 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 area42within the hydrophilic regions26,30, or within the entire hydrophilic regions26,30, to determine the presence, absence, or concentration of the analyte.

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 regions26,30along the sample flow path32; (2) binding an analyte with a labeled antibody along the flow path32and into the test area42; and binding the analyte binds to an antigen in the test area42; and optionally washing the hydrophilic regions26,30with a buffer such as, for example, PBS, to observe the results in the test area42.

In another aspect, the present disclosure is directed to a kit including the diagnostic device10and other equipment useful in performing an assay for a selected analyte. For example, the kit may optionally include one or more vials of purified water and/or buffer, e.g., PBS, one or vials of a suitable reagent, a device to obtaining a blood sample (e.g., a device of making a needle stick), a device for collecting a urine sample or saliva sample or other body fluid, or a pipette for transferring water and/or buffer to the device. Further, the kit may include instructions or color charts for quantitating a color reaction.

The devices and methods of the present disclosure will now be further described in the following non-limiting examples.

EXAMPLES

A 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.

FIGS.4A-4Bshow images of the printed paper after testing with dyed water. The dyed water saturated most of the unprinted area but did not wick into the printed areas as well as into the areas close to the border of the printed areas. This ink has pigment particulates in it (they show as blue), that did not wick along the fibers, while polymer component of the ink did, creating hydrophobic areas around printed pattern. Grey-colored water stopped at the created hydrophobic border.

A test was performed to show that volumetrically hydrophobic areas of the paper retained sufficient fluid permeability. A 5.08 cm (2 inch) dimeter round disc 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 1 below andFIG.5for test results.

A sample was created as described in Example 1 using 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) dimeter round disc 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 1 andFIG.5for test results.

Three samples were created as described in Example 1 using a release ink UVF03408 (UV Easy Release) (obtained from FlintGroup, 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 (IPA) 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) dimeter round disc 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 1 andFIG.5for test results.

Comparative Example 1

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 preheated 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) dimeter round disc 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 1 andFIG.5for test results.

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 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.

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

Table 1 andFIG.5show in pertinent part that flow was highest for unprinted paper, followed by the printed paper. This shows that while printed paper remained permeable to water. Flow through wax-saturated disc was very low and was due to delamination of wax under one meter of water pressure.

Flint Group Easy Release Coating (available from FlintGroup, 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 anilox roll at 10 fpm line speed in a pattern representing an array of 5 folds of bio-diagnostics devices.

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 77, available from 3M Company) was lightly sprayed by hand on both side of the device over both hydrophobic and hydrophilic areas.

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.