Microfluidic assay device and method

Abstract

Disclosed herein are microfluidic assay devices that have improved versatility and reliability. An example microfluidic assay device includes a cassette having a body formed by a plurality of layers. The body includes a reaction chamber having an inlet, an outlet downstream from the inlet, and a non-fouling polymer brush with a plurality of assay reagents. The reaction chamber is configured to house a fluid sample. The body also includes a seal disposed adjacent the outlet of the reaction chamber for preventing fluid flow through the outlet, and an actuator configured to break the seal upon actuation to create a fluid flow path for the fluid sample to exit from the outlet of the reaction chamber.

Description

Docket No.028193-0056-WO01 MICROFLUIDIC ASSAY DEVICE AND METHOD CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 678,589 filed on August 2, 2024, which is incorporated fully herein by reference. FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Federal Grant no. MCC- 1909-01 awarded by the ATI / Medical CBRN Defense Consortium, Federal Grant no. CB11194 awarded by the Naval Medical Research Center, and Federal Grant no. AI177098 awarded by the National Institutes of Health. The government has certain rights to this invention. TECHNICAL FIELD

[0003] This disclosure relates to an assay device, and, more particularly, to an assay device including a multi-layered cassette having an actuator. INTRODUCTION

[0004] Point of care assay devices are conventionally utilized to detect, identify, and / or quantify the presence of analytes in a fluid sample. Conventional assay devices typically utilize timing channels (e.g., small fluid channels) built into the assay device for dictating the residence time of the fluid sample. However, depending on the type of fluid sample, the timing channels have to be redesigned for each unique fluid sample to account for unique residence times. Therefore, it would be useful to develop a single assay device capable of receiving different types of fluid samples that can account for each of the fluid sample’s unique residence times. SUMMARY

[0005] In one aspect, disclosed is a microfluidic assay device including a cassette having a body formed by a plurality of layers. The body includes a reaction chamber having an inlet, an outlet downstream from the inlet, and a non-fouling polymer brush with a plurality of assay reagents. The reaction chamber is configured to house a fluid sample. The body also includes a seal disposed adjacent the outlet of the reaction chamber for preventing fluid flow through the outlet, and an actuator configured to break the seal upon actuation to create a fluid flow path for the fluid sample to exit from the outlet of the reaction chamber.Docket No.028193-0056-WO01

[0006] In another aspect, disclosed is a method for analyzing a fluid sample, the method includes orienting the microfluidic assay device in a substantially vertical upright position, loading the fluid sample into the inlet of the reaction chamber, loading a wash buffer into the inlet of the reaction chamber, and waiting for an incubation period. The method also includes breaking the seal by actuating the actuator to allow the fluid sample to exit the reaction chamber; and analyzing the device to measure a signal for a target analyte and a signal for a control to determine a concentration of the analyte.

[0007] In another aspect, disclosed is a kit including the microfluidic assay device, a wash buffer, a conditioning agent, and a sample applicator. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG.1 is a perspective view of an example microfluidic assay device.

[0009] FIG.2 is a front view of the microfluidic assay device of FIG.1 with portions made transparent for clarity.

[0010] FIG.3 is a partial cross-sectional view of the microfluidic assay device of FIG.1 taken along section line 3—3.

[0011] FIG.4 is a schematic view of a reaction chamber of an example microfluidic assay device.

[0012] FIG.5 is an exploded view of the microfluidic assay device of FIG.1.

[0013] FIG.6A illustrates a schematic view of an actuator of an example microfluidic assay device showing during incubation the reaction chamber is isolated by a breakable seal from the outlet.

[0014] FIG.6B illustrates a schematic view of an actuator of an example microfluidic assay device showing a user pressing the flexible button, which pushes a solid pin down, breaking the acrylic seal.

[0015] FIG.6C illustrates a schematic view of an actuator of an example microfluidic assay device showing a user releasing the button, creating a fluid path for the sample and upstream wash buffer to exit the reaction chamber.

[0016] FIG.7A shows design details of an example of a first layer used to fabricate the microfluidic assay device.

[0017] FIG.7B shows design details of an example of a second layer used to fabricate the microfluidic assay device.Docket No.028193-0056-WO01

[0018] FIG.7C shows design details of an example of a third layer used to fabricate the microfluidic assay device.

[0019] FIG.7D shows design details of an example of a fourth layer used to fabricate the microfluidic assay device.

[0020] FIG.7E shows design details of an example of a fifth layer used to fabricate the microfluidic assay device.

[0021] FIG.7F shows design details of an example of an outline for the microfluidic assay device to be cut by a cutter.

[0022] FIG.8A shows design details of an example flow resistor (top) and an example button seal (bottom).

[0023] FIG.8B shows design details of an example resistor cover (top) and an example inlet backing (bottom).

[0024] FIG.8C shows design details of an example pad spacer (top) and an example pad cover (bottom).

[0025] FIG.9 is a plot showing multiplexed dose response curves on an example microfluidic assay device. Whole blood with 10% (v:v) added lysis agent with spiked with serial dilution of three antigens (IL-6, Zika NS1, and inactivated native antigen of Salmonella typhi) was added to each cassette (n = 3). Incubation time = 30 minutes. Imaged on the (A) Genepix, and (B) D4Scope.

[0026] FIG.10A is a plot showing storage and operation analysis of example devices. Device storage was at 4°C (cold) and run at 4°C (cold). Target analyte was IL-6 and Zika NS1 antigen.

[0027] FIG.10B is a plot showing storage and operation analysis of example devices. Device storage was at 40°C (hot) and assay was run at 4°C (cold). Target analyte was IL-6 and Zika NS1 antigen.

[0028] FIG.10C is a plot showing superimposed curve fits for all four experimental conditions for IL-6 shown in FIG.10A, FIG.10B, FIG.10D, and FIG.10E.

[0029] FIG.10D is a plot showing storage and operation analysis of example devices. Device storage was at 4°C (cold) and assay was run at 40°C (hot). Target analyte was IL-6 and Zika NS1 antigen.

[0030] FIG.10E is a plot showing storage and operation analysis of example devices. Device storage was at 40°C (hot) and assay was run at 40°C (hot). Target analyte was IL-6 and Zika NS1 antigen.Docket No.028193-0056-WO01

[0031] FIG.10F is a plot showing superimposed curve fits for all four experimental conditions for NS1 shown in FIG.10A, FIG.10B, FIG.10D, and FIG.10E.

[0032] FIG.11 is a plot showing 6-plex detection of cytokines in fetal bovine serum (n = 3, blanks are n = 4) on an example microfluidic assay device.

[0033] FIG.12 is a plot showing detection of fungal biomarker Mp1p in human urine (n = 3) on an example microfluidic assay device.

[0034] FIG.13 is a plot showing detection of a toxin in human whole blood (n = 3, blanks are n = 4) on an example microfluidic assay device.

[0035] FIG.14 is a plot showing detection of canine biomarkers for traumatic brain injury in canine serum (n=2) on an example microfluidic assay device. DETAILED DESCRIPTION 1. Definitions

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0037] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0038] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%”Docket No.028193-0056-WO01 may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0039] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are contemplated, and for the range 1.5-2, the numbers 1.5, 1.6, 1.7, 1.8, 1.9, and 2 are contemplated.

[0040] The terms “fluid sample,” “biological sample,” or “sample,” as used herein, refer to any material that is taken from its native or natural state, so as to facilitate any desirable manipulation or further processing and / or modification. A fluid sample or a biological sample can include a cell, a tissue, a fluid (e.g., a biological fluid), a protein (e.g., antibody, enzyme, soluble protein, insoluble protein), a polynucleotide (e.g., RNA, DNA), a membrane preparation, and the like, that can optionally be further isolated and / or purified from its native or natural state. Example fluid samples include, but are not limited to, blood, serum, plasma, lymph fluid, bile fluid, urine, saliva, mucus, sputum, tears, cerebrospinal fluid (CSF), bronchioalveolar lavage, nasopharyngeal lavage, rectal lavage, vaginal lavage, colonic lavage, nasal lavage, throat lavage, synovial fluid, semen, ascites fluid, pus, maternal milk, ear fluid, sweat, and amniotic fluid. A fluid sample may be in its natural state or in a modified state by the addition of components such as reagents, or removal of one or more natural constituents (e.g., blood plasma).

[0041] The term “detection moiety,” as used herein, refers to a moiety or compound that is detectable by methods including, but not limited to, spectroscopic, photochemical, biochemical, immunochemical, chemical, electrochemical, radioactivity, and other physical means. A detection moiety may be detectable directly or indirectly. A non-limiting example of an indirectly detectable detection moiety is biotin, which may bind to avidin or streptavidin comprising a detection moiety such as a fluorophore. Example detection moieties include, but are not limited to, fluorophores, chromophores, radiolabels, polynucleotides, small molecules, enzymes, nanoparticles, and upconverters.

[0042] A “protein” or “polypeptide” is a linked sequence of 50 or more amino acids linked by peptide bonds. A peptide is a linked sequence of 2 to 50 amino acids linked by peptide bonds. The polypeptide and peptide can be natural, synthetic, or a modification or combination of natural and synthetic. Proteins and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide,” and “protein” are used interchangeablyDocket No.028193-0056-WO01 herein. “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tall domains, “Domains” are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Example domains include domains with enzymatic activity or ligand binding activity. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units. A “motif” is a portion of a polypeptide sequence and includes at least two amino acids. A motif may be 2 to 20, 2 to 15, or 2 to 10 amino acids in length, in some embodiments, a motif includes 3, 4, 5, 6, or 7 sequential amino acids. A domain may be comprised of a series of motifs, which may be similar or different.

[0043] The term “specifically binds,” as used herein, is generally meant that a molecule binds to a target molecule when it binds to that target molecule more readily than it would bind to a random, unrelated target. 2. Example Devices

[0044] Example devices herein relate to a point of care immunoassay device, e.g., for target analyte detection, fabricated using a non-fouling polymer brush, such as poly(oligo ethylene glycol methyl ether methacrylate) (POEGMA). The disclosed devices take advantage of a microfluidic architecture that allows automation of most if not all user intervention steps in detection of a target analyte. Furthermore, the disclosed devices include a mechanism to control the interaction time of the target analyte with the device assay reagents. This mechanism, e.g., an actuator, significantly simplifies analyte detection and improves versatility and reliability of the disclosed device. As a result, the disclosed devices can be used for a variety of different sample without having to significantly alter the structure of the device. Microfluidic Assay Device

[0045] FIG.1, FIG.2, and FIG.3 illustrate a microfluidic assay device 100 according to an embodiment of the present disclosure. The device 100 includes a cassette having a body 104 formed by a plurality of layers stacked on top of each other that is described in more detail below. The body 104 includes a reaction chamber 108 having an inlet 112 and an outlet 116Docket No.028193-0056-WO01 located downstream from the inlet 112, an actuator 120 supported on the body 104, a flow resistor 124 located downstream from the outlet 116 of the reaction chamber 108, and a first waste pad 131 located downstream from the flow resistor 124 that is protected by a cover 139 coupled to the body 104. The device 100 also includes a reservoir 126 coupled to the body 104 and located upstream from the inlet 112 of the reaction chamber 108. The reservoir 126 is in fluid communication with the inlet 112 of the reaction chamber 108 and includes a plurality of inlets. In the illustrated embodiment, the reservoir 126 includes a first inlet 127 for loading a wash buffer and a second inlet 128 for loading a fluid sample. In other embodiments, the reservoir 126 may include fewer or more inlets for receiving both the fluid sample and the wash buffer. The reservoir 126 also defines an angled interior surface 133 that angles the inlets 127, 128 with respect to the inlet 112 of the reaction chamber 108 to permit the fluid sample and the wash buffer to laterally enter the reaction chamber 108. Lateral, or side entry of the fluid sample and wash buffer into the reaction chamber 108 can prevent the clogging, bridging, and / or trapping of air bubbles in the fluid sample and wash buffer mixture before entering the reaction chamber 108. By reducing the number of bubbles in the fluid sample and wash buffer mixture, the device 100 is able to produce more accurate results.

[0046] With reference to FIG.2, FIG.3, and FIG.4, the reaction chamber 108 includes a non-fouling polymer brush 132 (FIG.5) that stores and stabilizes bioreagents (e.g., assay reagents) for detecting a target analyte. In the illustrated embodiment, the polymer brush 132 is an inkjet-printed poly(oligo ethylene glycol methyl ether methacrylate) (POEGMA) brush having a plurality of assay reagent layers inkjet printed on a glass slide. In other embodiments, the polymer brush 132 may be another type of polymer brush known in the art and as disclosed herein, such as POEGMA-based copolymers (e.g., poly(oligo ethylene glycol methyl ether methacrylate-co-glycidyl methacrylate) (POEGMA-co-GMA)). In the illustrated embodiment, the POEGMA brush 132 includes a plurality of detection antigens (e.g., detection reagent) printed onto a plurality of corresponding excipient pads / layers, such as trehalose pads 136, and a capture array region 140 having a plurality of capture antigens (e.g., capture reagents) printed directly on the POEGMA brush 132. The POEGMA brush 132 also includes a plurality of control spot regions 148 adjacent the capture array region 140.

[0047] In other embodiments, the polymer brush 132 includes a plurality of capture reagents printed directly onto the brush 132, and the fluid sample includes a plurality of detection reagents incorporated within the fluid sample.

[0048] The non-fouling polymer brush can decrease non-specific binding and / or adsorption of non-target analytes. Non-fouling, as used herein, relates to the inhibition (e.g., reduction orDocket No.028193-0056-WO01 prevention) of growth of an organism as well as to non-specific or adventitious binding interactions between the non-fouling polymer brush and an organism or biomolecule (e.g., cell, protein, nucleotide, etc.). The non-fouling property of the polymer brush can be instilled through the inclusion of a brush-like polymer. Generally, brush-like polymers are formed by the polymerization of monomeric core groups having one or more groups that function to inhibit binding of a biomolecule (e.g., cell, protein, nucleotide, carbohydrate / lipid) coupled thereto. The monomeric core group can be coupled to a protein-resistant head group. In some embodiments, the brush-like polymer includes a monomeric core group, and a protein-resistant head group coupled to the monomeric core group.

[0049] Brush-like polymers can be synthesized using radical polymerization techniques, such as catalytic chain transfer polymerization, iniferter mediated polymerization (e.g., photoiniferter mediated polymerization), free radical polymerization, stable free radical mediated polymerization (SFRP), atom transfer radical polymerization (ATRP), and reversible addition- fragmentation chain transfer (RAFT) polymerization. For example, free radical polymerization of monomers to form brush-like polymers can be carried out in accordance with known techniques, such as described in U.S. Pat. Nos.6,423,465; 6,413,587; and 6,649,138; U.S. Patent Application Publication No. US 2003 / 0108879 – all of which are incorporated herein by reference in their entirety, and variations thereof which will be apparent to those skilled in the art. Atom transfer radical polymerization of monomers to form brush-like polymers can also be carried out in accordance with known techniques, such as described in U.S. Pat. Nos. 6,541,580 and 6,512,060; U.S. Patent Application Publication No. US 2003 / 0185741 - all of which are incorporated herein by reference in their entirety, and variations thereof which will be apparent to those skilled in the art.

[0050] Any suitable core vinyl monomer polymerizable by the processes discussed above can be used, including but not limited to styrenes, acrylonitriles, acetates, acrylates, methacrylates, acrylamides, methacrylamides, vinyl alcohols, vinyl acids, and combinations thereof.

[0051] Protein resistant groups can be hydrophilic head groups or kosmotropes. Examples include, but are not limited to, oligosaccharides, tri(propyl sulfoxide), hydroxyl, glycerol, phosphorylcholine, tri(sarcosine) (Sarc), N-acetylpiperazine, betaine, carboxybetaine, sulfobetaine, permethylated sorbitol, hexamethylphosphoramide, an intramolecular zwitterion (for example, –CH2N+(CH3)2CH2CH2CH2S03) (ZW), and mannitol.

[0052] Additional examples of kosmotrope protein resistant head groups can include: –(OCH2CH2)6OH;Docket No.028193-0056-WO01 –O(Mannitol);–N(CH3)2+CH2CH2SO3; –C(O)Pip(NAc) (Pip=piperazinyl); –N(CH3)2+CH2CO2; –O([Glc- (1,4)-Glc- (1) ]);–C(O)(N(CH3)CH2C(O))3N(CH3)2; –N(CH3)2+CH2CH2CH2SO3; –C(O)N(CH3)CH2CH2N(CH3)P(O)(N(CH3)2)2; and –S(O)CH2CH2CH2)3S(O)CH3.

[0053] In some embodiments, a protein resistant head group includes poly(ethylene glycol) (PEG), for example PEG of from 1 to 30 monomeric units, such as 2 to 25 monomeric units, 3 to 20 monomeric units, 4 to 18 monomeric units, or 2 to 15 monomeric units.

[0054] In some embodiments, the non-fouling polymer brush is formed by surface-initiated ATRP (SI-ATRP) of oligo(ethylene glycol)methyl methacrylate (OEGMA) to form a poly(OEGMA) (POEGMA). In some embodiments, the non-fouling polymer brush includes a functionalized POEGMA prepared by copolymerization of a methacrylate and methoxy terminated OEGMA. The POEGMA polymer can be formed in a single step. In some embodiments, the non-fouling polymer brush includes POEGMA.

[0055] Prior to deposition of further components, such as the assay reagents, onto the polymer brush, the polymer brush can be dry or at least macroscopically dry (that is, dry to the touch or dry to visual inspection, but retaining bound water or water of hydration in the polymer layer). For example, to enhance immobilization of a capture reagent, the polymer brush can suitably retain bound water or water of hydration, but not bulk surface water. If the polymer brush has been stored in desiccated form, bound water or water of hydration can be reintroduced by quickly exposing the polymer brush to water (e.g., by dipping in to water) and subsequently blow-drying the surface (e.g., with a nitrogen or argon jet). Alternatively, bound water or water of hydration can be reintroduced by exposing the polymer brush to ambient air for a time sufficient for atmospheric water to bind to the polymer brush.

[0056] As described herein, “reagents” refer to “capture arrays,” “capture reagents,” “capture antigens,” “detection reagents,” “detection antigens,” and “controls” that are deposited on a non- fouling polymer brush spatially separated. The detection reagent and the capture reagent can each, individually, be a peptide, a protein, a carbohydrate, a lipid, a small molecule ligand, or aDocket No.028193-0056-WO01 combination thereof. In some embodiments, the detection reagent and the capture reagent each, individually, include a peptide, a protein, or a combination thereof. In some embodiments, the detection reagent and the capture reagent each, individually, include a peptide or a protein. In some embodiments, the detection reagent and the capture reagent each, individually, include an antibody or fragment thereof.

[0057] In some embodiments, the capture arrays comprise capture antigens and the detection reagents comprise fluorescently labeled detection antigens (dAgs) disposed on trehalose “pads.” Both the capture arrays and detection reagents can be printed onto the non- fouling polymer brush (e.g., (POEGMA) disposed on a substrate. The detection reagent can further include a detectable moiety that, directly or indirectly, provides a detectable signal. Example detection moieties include, but are not limited to, fluorophores, chromophores, radiolabels, polynucleotides, small molecules, enzymes, nanoparticles, and upconverters. In some embodiments, the detection moiety may be a fluorophore such as a cyanine (e.g., CyDyes such as Cy3 or Cy5), a fluorescein, a rhodamine, a coumarin, a fluorescent protein or functional fragment thereof, or it may include a small molecule such as biotin, or it may include gold, silver, or latex particles. In some embodiments, the detection reagent includes a detection moiety selected from the group consisting of a chromophore, a fluorophore, a radiolabel, a polynucleotide, a small molecule, an enzyme, a nanoparticle, a microparticle, a quantum dot, and an upconverter.

[0058] The excipient, e.g., of the excipient pad / layer, is a molecule or a combination of molecules that is selected as to allow for a stable, but non-permanent, association between the detection reagent and the non-fouling polymer brush. In some embodiments, the excipient can be partially soluble, substantially soluble or soluble in an aqueous solution (e.g., buffer, water, sample, biological fluid, etc.). In such embodiments, the excipient can be selected from the non- limiting examples of salts, carbohydrates (e.g., sugars, such as glucose, fucose, fructose, maltose and trehalose), polyols (e.g., mannitol, glycerol, ethylene glycol), emulsifiers, water- soluble polymers, and any combination thereof. Such excipients are well known in the art and can be selected based on the interaction between the excipient and detection reagent, the excipient and the brush-like polymer, the solubility of the excipient in a particular medium, and any combination of such factors.

[0059] In some embodiments, the excipient includes a salt, a carbohydrate, a polyol, an emulsifier, a water soluble polymer, or a combination thereof. In some embodiments, the excipient includes a salt, a carbohydrate, a water soluble polymer, or a combination thereof. In some embodiments, the excipient includes a salt, a carbohydrate, or a combination thereof. InDocket No.028193-0056-WO01 some embodiments the excipient includes PEG. In some embodiments, the excipient includes trehalose.

[0060] FIG.5 illustrates the layers that form the body 104 of the cassette. In the illustrated embodiment, the layers include a first substrate layer 144 having cut-outs 149, 152 for receiving the POEGMA brush 132 and a second waste pad 156, respectively, a first adhesive layer 160 that covers the first substate layer 144 and has a cut-out 164 to form part of the reaction chamber 108, and first and second reaction layers 168, 172 stacked on top of each other and fastened to the first adhesive layer 160. The first and the second reaction layers 168, 172 each include cut-outs 176, 180, respectively, for forming part of the reaction chamber 108. In the illustrated embodiment, the cut-out 180 in the second reaction layer 172 defines first and second inlets regions 135, 137 that are in fluid communication with and correspond to the first and the second inlets 127, 128 of the reservoir 126. Each of the first and second inlet regions 135, 137 fluidly communicates with the inlet 112 of the reaction chamber 108. The layers also include a second substrate layer 184 affixed to the second reaction layer 172. The second substrate layer 184 is configured to seal the reaction chamber 108 to the first substrate layer 144 to both enclose and form the reaction chamber 108 between the first and the second substrate layers 144, 184. The second substrate layer 184 defines a plane and includes a cut- out 188 that further defines the first and second inlet regions 135, 137. In some embodiments, the reservoir 126 is coupled to the second substrate layer 184. The second substrate layer 184 also includes another cut-out 192 for receiving the first waste pad 131 and a seal 196. The seal 196 is a breakable seal that is broken by the actuator 120 upon actuation. In some embodiments, the seal is a breakable substrate formed from acrylic. The seal 196 lies in the same plane as the second substrate layer 184 and is positioned adjacent the outlet 116 of the reaction chamber 108 to seal the reaction chamber 108 and prevent fluid flow out of the outlet 116 of the reaction chamber 108. In some embodiments, the seal 196 is affixed to or integrated within the second substrate layer 184.

[0061] The layers also include a channel layer 200 affixed to the second substrate layer 184, and a cover layer 204 affixed to the channel layer 200. The channel layer 200 includes a channel 208 forming the flow resistor 124. The flow resistor is not limited to using capillary action via, e.g., a channel 208. Alternatively, the flow resistor 124 can be an absorbable membrane or pad, such a nitrocellulose, an absorbent cotton pad, a glass fiber pad, a porous polymer, or hydrogel. The channel 208 includes an inlet 210 that is in fluid communication with the outlet 116 of the reaction chamber 108 when the seal 196 is broken. Otherwise, the seal 196 seals the fluid sample and the wash buffer within the reaction chamber 108 until that sealDocket No.028193-0056-WO01 196 is broken by the actuator 120 to release the fluid sample and the wash buffer into the channel 208. In the illustrated embodiment, the channel 208 is a microfluidic channel that forms a serpentine pattern for routing a processed fluid sample and the wash buffer toward the first waste pad 131.

[0062] The cover layer 204 includes a cut-out 212 for receiving the first waste pad 131 and shielding the first waste pad 131 from outside interference to reduce exposure of the waste to the end user. The cover layer 204 also includes a through hole 216 extending through the cover layer 204 and aligned with the inlet 210 of the channel 208 on the channel layer 200 and the seal 196 on the second substrate layer 184. In the illustrated embodiment, the actuator 120 is coupled to the cover layer 204; however, in other embodiments, the actuator 120 can be coupled to other areas or parts of the body 104 of the cassette. The actuator 120 includes an actuator cover 122, and a body 123 received within the cover 122 having a pin 125 extending from the body 123 and into the through hole 216 on the cover layer 204. The actuator cover 122 includes a resilient pressable portion 129 configured to be pressed by a user. In some embodiments, the actuator is a push-button and the cover 122 is formed from an elastomeric material. In other embodiments, the actuator 120 can be triggered by a user manually, or by an external actuator (not shown) that can be programmed to actuate the actuator 120 according to a predetermined time corresponding to an incubation time of the fluid sample. In other embodiments, the actuator 120 can act as a diaphragm valve for the reaction chamber 108 to modulate pressure and introduce active mixing of the fluid sample. In other embodiments, the actuator 120 can include an agitator configured to mix the fluid sample and the wash buffer within the reaction chamber 108. In yet other embodiments, the actuator may be a screw valve, a magnetic flap, or a peelable adhesive.

[0063] In some embodiments, each of the substrates 144, 184 and / or layers 168, 172, 200, 204 can be comprised of a thermoplastic polymer, such as acrylic. In such embodiments, the substrates 144, 184 and / or layers 168, 172, 200, 204 can each have a thickness between 0.2 millimeters to 2.0 millimeters. However, in other embodiments, other thicknesses may be used. Example Method of Using the Microfluidic Assay Device

[0064] With reference to FIG.2, FIG.3, FIG.4, FIG.5, FIG.6A, FIG.6B, and FIG.6C, to use the microfluidic assay device 100, a fluid sample from a sample source is first gathered. The fluid sample may comprise whole blood, serum, plasma, urine, tears, sweat, saliva, lymph, cerebrospinal fluid, fecal extract, cellular or tissue extracts, or any other aqueous sample. In some embodiments, the fluid sample is non-diluted. A conditioning, or lysis agent is then addedDocket No.028193-0056-WO01 to the fluid sample. In some embodiments, detection reagents may also be added to the fluid sample at this time in addition to the conditioning agent. The conditioning agent can include a surfactant. In some embodiments, the surfactant includes a non-ionic detergent, such as a Triton X-100 surfactant, for increasing the fluid sample’s ability to spread easily over surfaces and homogenize by breaking up cellular debris and clots. In other embodiments, the surfactant can include other known non-ionic detergents in the art, such as NP-40, Tween-20, poloxamer 188, etc., or a combination of known detergents, salts, or additives. In some embodiments, the non-ionic detergent comprises Triton-X, NP-40, Tween-20, poloxamer, or a combination thereof. In other embodiments, the fluid sample does not need to be mixed with a conditioning agent; instead, the inner surfaces of the various components can be coated with a material (e.g., hydrophilic polymer, surfactant, etc.) that lowers the surface energy and / or lowers the contact angle of the inner surfaces of the device 100 (e.g., the reaction chamber 108, inlets 127, 128 on the reservoir 126, the angled interior surface 133 of the reservoir 126, etc.). Next, the device 100 is oriented in a substantially vertical orientation for the duration of use. In other embodiments, the device 100 is configured to operate in an orientation other than the substantially vertical orientation. In such embodiments, the device 100 includes a hydrophobic vent (e.g., a PTFE vent) located downstream of the reaction chamber 108 that is configured to displace air bubbles if air bubbles form within the reaction chamber 108. The fluid sample and conditioning agent mixture is then loaded into the second inlet 128 on the reservoir 126 and a wash buffer is also loaded into the first inlet 127 on the reservoir 126. Together, the wash buffer and the fluid sample are housed in the reaction chamber 108 for a predetermined residence time according to the type of fluid sample. While in the reaction chamber 108, the detection antigens disposed on the trehalose pads 136 are released from the pads 136 and antibodies targeting each antigen then bridge the capture antigens from the capture array region 140 to the detection antigens, resulting in a fluorescence signal that scales with antibody concentration.

[0065] Analyte detection can be performed by known specific / selective binding techniques known within the art. Some non-limiting examples include antigen detection via sandwich fluorescent immunoassay and antibody detection via double antigen, and in-direct assay formats. In, e.g., a sandwich assay, detection reagents can specifically bind to the target analyte and form a first complex. The detection reagent can be added directly to the fluid sample or can be disposed on the non-fouling polymer brush. The capture reagent can then specifically bind to the first complex to form a second complex. Within the second complex the detection reagent may have a detectable moiety, or a second detection reagent having a detection moiety can be present that binds to the second complex. Further description of assays that can be used on theDocket No.028193-0056-WO01 disclosed device and POEGMA related layers and reagents thereon can be found in U.S. Pat. No.11,169,150, which is incorporated by reference herein in its entirety.

[0066] Once the predetermined residence time has concluded, or the user has decided to dispose of the fluid sample, the user actuates the actuator 120, which breaks the seal 196 and opens up a fluid flow path 220 between the outlet 116 of the reaction chamber 108 and the inlet 210 of the flow resistor 124. In other embodiments that do not include the flow resistor 124 and / or the waste pads 131, 156, the fluid flow path 220 extends between the outlet 116 of the reaction chamber 108 and an outlet of the device 100. Opening up the fluid flow path 220 allows the fluid sample and wash buffer mixture to exit the reaction chamber 108 and move into the flow resistor 124. In some embodiments, opening up the fluid flow path 220 with the actuator 120 allows the fluid sample and wash buffer mixture to exit the device 100 without being routed to the flow resistor 124. In the illustrated embodiment, the actuator 120 is a push- button, and the user depresses the pressable portion 129 (FIG.6B) of the actuator 120, which pushes the pin 125 on the actuator 120 through the through hole 216 in the cover layer 204 and through the second substrate layer 184 into the reaction chamber 108 to puncture the seal 196 and create the fluid flow path 220 (FIG.6C). Once the fluid sample and wash buffer mixture enters the flow resistor 124, the flow resistor 124 controls the rate in which the processed fluid sample and the wash buffer empty the outlet 116 of the reaction chamber 108. Controlling the fluid flow rate can increase the likelihood that the reaction chamber 108 is adequately washed and ready to be analyzed. In some embodiments, the fluid flow rate can be altered by changing the geometry or length of the channel 208 of the flow resistor 124. After the processed fluid sample and the wash buffer mixture pass through the flow resistor 124, the sample and buffer mixture is absorbed by the first and / or second waste pads 131, 156. In some embodiments, the waste pads 131, 156 are cotton absorbent pads; however, in other embodiments, the waste pads 131, 156 may be other absorbent materials commonly known and used in the art.

[0067] After the fluid sample and wash buffer mixture has exited the reaction chamber 108, been absorbed by the waste pad 131, and the reaction chamber 108 is sufficiently dried, the reaction chamber 108 can be imaged and scanned by an external device, and the results may be interpreted. In some embodiments, the external device is a D4Scope, such as the D4Scope described in U.S. Patent Application No.18 / 042,032, which is incorporated herein by reference.

[0068] In other embodiments, before the fluid sample and wash buffer are added to the reservoir 126, a pre-evacuated blister (i.e., a negative pressure space) can be inserted downstream of the reaction chamber 108 to functionally draw the fluid sample into the reaction chamber 108.Docket No.028193-0056-WO01

[0069] By utilizing the device 100, the user is able to trigger the wash and dry steps of the device 100 by a user-determined action (i.e., depressing the actuator 120 to break the seal 196). This allows the same device 100 to be used for all sample types and desired incubation times without test-specific design optimization in contrast to conventional assay devices. Example Methods of Fabricating the Microfluidic Assay Device

[0070] In one example, the device 100 is fabricated as a multilayer laminate, where laser-cut Clarex® acrylic sheets, or panels of varying thicknesses (e.g., 0.2, 0.5, 1.0, and 2.0 millimeters) are alternated with precision-cut doubled-sided adhesives (e.g., 3M® 9474LE and 468MP) to form both the microfluidic features of the device 100 and bond the stack of laminate. Panel production for the laminate stack utilizes alignment fiducials to line up individual layers of the stack for layer-by-layer assembly. Assembly steps can include placing each layer in a jig, peeling protective liners in sequence, and pressing the stack of layers together to seal. The panels are then perimeter-cut to isolate individual cassettes. The flow resistor 124 and the waste pad assembly are then added to each of the cassettes. Accessories are then added, such as guillotine-cut Cytiva® CF7 absorbent pads, a resin-printed sample / wash reservoir, and a 3-D printed punch pin topped with an elastic button cover. A POEGMA-coated glass slide bearing the inkjet-printed reagents is bonded into the back of the reaction chamber 108 at the final step. The finished cassette is sealed in a foil pouch with molecular sieve desiccant to maintain stability long term at room temperature.

[0071] With reference to FIG.7A, FIG.7B, FIG.7C, FIG.7D, FIG.7E, FIG.7F, FIG.8A, FIG. 8B, and FIG.8C, another example method of fabricating and assembling the microfluidic assay device 100 is described. In one example, the device 100 is fabricated as a multilayer laminate, where laser-cut Clarex® acrylic sheets, or panels of varying thicknesses (e.g., 0.2, 0.5, 1.0, and 2.0 millimeters) are alternated with precision-cut doubled-sided adhesives (e.g., 3M® 9474LE and 468MP) to form both the microfluidic features of the device 100 and bond the stack of laminate. In some examples, a LS900 Gravograph® laser cutter is used, but any laser cutter with 0.1 millimeter laser diameter and autofocus features can be used. For a process of 12 cassettes, six layers of panels are used. The first five layers includes various internal features of the cassette built across multiple layers in panel form. The sixth layer is an outline for the cassettes that is used to separate the individual backbones from the panel after full assembly. In some examples, the first layer corresponds to the first substrate layer 144, the second layer corresponds to the first adhesive layer 160, the third layer corresponds to the first reaction layer 168, the fourth layer corresponds to the second reaction layer 172, and the fifth layerDocket No.028193-0056-WO01 corresponds to the second substrate layer 184, The holes on the outside of each of the layers are alignment fiducials that interface with an alignment device including vertical pillars in the same pattern that can be used to precisely hold each layer in the XY dimension. The layers can be placed into the alignment rig and fabricated into individual cassettes in the following steps: 1. Place layer 2 into the alignment rig. 2. Remove top 200 mp liner from layer 2. 3. Remove bottom 0.5 mm acrylic liner from layer 3. 4. Place layer 3 onto layer 2. 5. Remove top 0.5 mm acrylic liner from layer 3. 6. Remove bottom 200 mp liner from layer 4. 7. Place layer 4 onto layer 3. 8. Remove top 200 mp liner from layer 4. 9. Remove layer 5 from laser cut sheet, this will likely remove the bottom 0.2 mm acrylic liner. If it doesn't, remove bottom 0.2 mm acrylic liner. Be careful not to bend and crack this layer. 10. Remove entire assembly from rig. 11. Place layer 1 into rig. 12. Remove top 1mm acrylic liner from Layer 1. 13. Carefully remove the sacrificial layer of layer 2 from the previously made assembly by peeling off layers (bottom 0.5 mm acrylic liner through bottom 200 mp liner). 14. Place the assembly onto layer 1 that is in the rig, such that the 1 mm acrylic of layer 1 is bonded to the 200 mp layer of layer 2. 15. Press layers to make sure they are bound. 16. Remove assembly from rig. 17. Use a fresh sheet of 300 LSE adhesive to completely cover both sides to passivate all inlets and outlets. 18. Make sure the honeycomb bed of the laser is top left justified. Place the assembly into the top left corner of the honeycomb cut bed. 19. Cut using file "final cut" to separate the 12 individual cassettes.

[0072] Once the individual cassettes are formed, the accessories can be manufactured. The punch pins can be formed by 3-D printing or injection molding. The reservoir and punch cover can be formed by 3-D printing. After the accessories are formed, the acrylic and POEGMA slides are assembled onto the adhesive cut channels in the following steps:Docket No.028193-0056-WO01 1. Peal the top 300LSE liner from the Flow Resistor. 2. Remove the bottom 2 mm acrylic liner from the Resistor Cover. 3. Stick the Resistor Cover to the Flow Resistor. 4. Remove assembly making sure both layers of adhesive come up with the piece. 5. Carefully place the subassembly onto the main assembly. Line up the holes for the punch on both designs. The reservoir can be assembled onto respective cassettes using the following steps: 1. Peel the front passivation layer of the inlet backing adhesive and attach to the sample and wash reservoir. 2. Remove the sacrificial acrylic attached to the sample / wash adhesive to expose the adhesive surface. 3. Affix the sample and wash assembly to the rest of the cassette on the top front edge. The punch pin and the punch cover can be assembled and affixed onto respective cassettes using the following steps: 1. Peel the top passivation layer of the Resistor cover. 2. Peel the top passivation layer of the button seal adhesive and attach it exposed button hole on top of the resistor cover, exposed adhesive side down. 3. Remove the sacrificial acrylic attached to the adhesive to expose the adhesive surface. 4. Insert the punch pin into hole with the slots of the pin aligned vertically with the cassette. 5. Affix the button cover on top of the pin attaching it to the exposed adhesive. Following the assembly of the previous components, wicking pads that were cut by a cutter, such as a guillotine cutter, are assembled to respective cassettes in the following steps: 1. Following the assemble of the previous components, the wicking pads that had been cut are assembled onto the chips. 2. Peel the bottom 200 mp liner off of the Pad Spacer (see SOP-006). 3. Stick it onto the main assembly, justifying with the bottom of the cassette. 4. Place one 22 mm x 9.5 mm piece of pad in the back opening. 5. Place one 11 mm x 9.5 mm piece of pad in the small front opening. 6. Place a third 22 mm x 9.5 mm piece on top of the front piece. 7. Peel the bottom acrylic liner from the Pad cover. 8. Peel the top 200 mp liner off of the Pad Spacer. 9. Stick the Pad cover onto the Pad Spacer, sealing in the pad.Docket No.028193-0056-WO01 10. Use tape to seal off the back of the cassette to lock in the pad on that side (temporary measure for prototype devices). Once the wicking pads have been added, each of the cassettes are assembled. Kits

[0073] Also disclosed are kits including the microfluidic assay device, a wash buffer, a conditioning agent, and a sample applicator as disclosed herein. The kit may also include other reagents to facilitate using the microfluidic assay device and methods thereof. In addition, the kit may include a packaging configured to contain the microfluidic assay device, the wash buffer, the conditioning agent, and the sample applicator. The packaging may be a sealed packaging, such as a sterile sealed packaging. By “sterile” it is meant that there are substantially no microbes (such as fungi, bacteria, viruses, spore forms, etc.). In some embodiments, the packaging may be configured to be sealed, e.g., a water vapor-resistant packaging, optionally under an air-tight and / or vacuum seal.

[0074] The kits may further include instructions for using the microfluidic assay device. These instructions may be present in the kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Another form for the instructions could be a computer readable medium, e.g., computer-readable memory (e.g., flash memory), etc., on which the information has been recorded or stored. Yet another form for the instructions that may be present is a website address which may be used via the Internet to access the information at a removed site. Any convenient means may be present in the kits.

[0075] The present disclosure has multiple aspects, illustrated by the following non-limiting examples. 3. Examples Example 1 Multiplexed Dose Response Curves on the Cassette

[0076] Whole blood with 10% (v:v) added lysis agent spiked with serial dilution of three antigens (IL-6, Zika NS1, and inactivated native antigen of salmonella typhi) was added to each cassette (n=3). Incubation time was 30 minutes. Imaged on the (A) Genepix, and (B) D4Scope. See FIG.9.Docket No.028193-0056-WO01

[0077] These results illustrate functionality in whole blood samples at longer incubation times, and the ability to effectively detect multiple targets on the same cassette. The resulting sensitivity of the cassette is comparable on both the commercial benchtop Genepix microarray scanner, and a custom-built handheld D4Scope. Example 2 Storage and Operational Temperature Study of the Cassette

[0078] Cassettes were assembled and stored for 8 days at either 4 °C or 40 °C. After storage cassettes were tested using human whole blood with 10% (v:v) added lysis buffer and spiked with various concentrations of IL-6 and Zika NS1 antigen. All conditions were incubated for 30 minutes (n=2). (FIG.10A) Storage 4°C (cold) and run at 4°C (cold). (FIG.10B) Storage 40°C (hot) and run at 4°C (cold). (FIG.10D) Storage 4°C (cold) and run at 40°C (hot). (FIG.10E) Storage 40°C (hot) and run at 40°C (hot). (FIG.10C) Superimposed curve fits for all four experimental conditions for IL-6 only. (FIG.10F) Superimposed curve fits for all four experimental conditions for NS1 only. In the legend “S : (H or C)” and “R : (H or C)” refers to S = storage condition or R = Run condition, where H = 40 °C and C = 4 °C.

[0079] These results illustrate that the device remains functional after storage for many days and subsequent operation at temperature extremes. The resulting does response curves do not appreciably differentiate highlighting the resilience of the platform. Example 3

[0080] This example illustrates 6-plex detection of cytokines in fetal bovine serum (n=3,blanks are n=4). The incubation time was 30 minutes. Concentrations of IL-1 , IL-2, IL-6, IL-10, IFN- , and TNF- were spiked into fetal bovine serum. A triton X-100 based sampleconditioner was added to each sample at a 1:10 ratio (1 part conditioner, 9 parts sample). Approximately 70 μL of sample was loaded into each cassette, followed immediately by 700 μL of wash buffer. The sample incubated for 30 minutes at room temperature, after which the user punched the cassette to finish running the test. Each cassette was fluorescently scanned using the companion reader (The D4Scope fluorescence scanner). Output is the result of the automated analysis by the D4Scope including spot finding, and outlier removal. Curve fit is a 5- parameter asymmetric logarithmic regression. The LOD is calculated as: LOD = mean of the blank + 3*standard deviation of the blank.Docket No.028193-0056-WO01

[0081] These results (FIG.11) illustrate the capability to multiplex large numbers of targets simultaneously and maintain quantitative capabilities. These results also illustrate optimal sensitivities in the single to double digit pg / mL. Example 4

[0082] This example illustrates detection of fungal biomarker Mp1p in human urine (n=3). The incubation time was 30 minutes. Concentrations of rMp1p antigen were spiked into commercially purchased single donor human urine. A triton X-100 based sample conditioner was added to each sample at a 1:10 ratio (1 part conditioner, 9 parts sample). Approximately 70 μL of sample was loaded into each cassette, followed immediately by 700 μL of wash buffer. The sample incubated for 30 minutes at room temperature, after which the user punched the cassette to finish running the test. Each cassette was fluorescently scanned using the companion reader (The D4Scope fluorescence scanner). Output is the result of the automated analysis by the D4Scope including spot finding, and outlier removal. Curve fit is a 5-parameter asymmetric logarithmic regression. The LOD is calculated as: LOD = mean of the blank + 3*standard deviation of the blank.

[0083] These results (FIG.12) illustrate that even with complex sample types like human urine, the device is able to exhibit both strong sensitivities and quantitative capabilities with low error. Furthermore, these results highlight the ability of the device to accurately analyze an undiluted sample with the addition of a sample conditioner without significantly diluting the sample. Many conventional antibody immunoassay devices are unable to produce as accurate results as the disclosed device. Example 5

[0084] This example illustrates detection of a toxin in human whole blood (n=4, blanks are n=4). Concentrations of the toxin were spiked into commercially purchased single donor human whole blood. The incubation time was 15 minutes. A triton X-100 based sample conditioner was added to each sample at a 1:10 ratio (1 part conditioner, 9 parts sample). Approximately 70 μL of sample was loaded into each cassette, followed immediately by 700 μL of wash buffer. The sample was incubated for 30 minutes at room temperature, after which the user punched the cassette to finish running the test. Each cassette was fluorescently scanned using the companion reader (The D4Scope fluorescence scanner). Output is the result of the automated analysis by the D4Scope including spot finding, and outlier removal. Curve fit is a 5-parameter asymmetric logarithmic regression. The LOD is calculated as: LOD = mean of the blank + 3*standard deviation of the blank.Docket No.028193-0056-WO01

[0085] These results (FIG.13) illustrate that even with complex sample types like human whole blood (without processing to serum), the device can exhibit both strong sensitivities and quantitative capabilities with low error. Furthermore, these results highlight the ability of the device to accurately and quickly (e.g., in about 15 minutes) analyze an undiluted sample with the addition of a sample conditioner without significantly diluting the sample. Example 6

[0086] This example illustrates detection of canine biomarkers for traumatic brain injury in canine serum (n=2). This sample was run as described in Examples 3-5. FIG.14 shows that the disclosed device is also capable of detecting analytes in a complex sample, such as canine serum.

[0087] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure.

[0088] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosed technology, may be made without departing from the spirit and scope thereof.

[0089] For reasons of completeness, the following Embodiments are provided.

[0090] Clause 1. A microfluidic assay device comprising: a cassette including a body formed by a plurality of layers, the body having a reaction chamber with an inlet, an outlet downstream from the inlet, and a non-fouling polymer brush including a plurality of assay reagents, the reaction chamber configured to house a fluid sample, and a seal disposed adjacent the outlet of the reaction chamber for preventing fluid flow through the outlet, and an actuator configured to break the seal upon actuation to create a fluid flow path for the fluid sample to exit from the outlet of the reaction chamber.

[0091] Clause 2. The microfluidic assay device of clause 1, further comprising: a flow resistor fluidly coupled to the outlet of the reaction chamber and configured to control a fluid flow rate of the fluid sample exiting from the outlet of the reaction chamber; and a waste pad downstream from the flow resistor and configured to collect the fluid sample.

[0092] Clause 3. The microfluidic assay device of clause 1 or 2, wherein the plurality of layers include: a first substrate layer having the non-fouling polymer brush; a reaction layer having a cut-out that forms the reaction chamber; a channel layer having a serpentine channel in fluid communication with the reaction chamber, the serpentine channel forming the flowDocket No.028193-0056-WO01 resistor; and a second substate layer configured to seal the reaction chamber to the first substrate layer.

[0093] Clause 4. The microfluidic assay device of any one of the clauses 1-3, wherein the actuator is supported on the body, and wherein the actuator is a push-button, a screw valve, a magnetic flap, or a peelable adhesive.

[0094] Clause 5. The microfluidic assay device of clause 4, wherein the push-button includes a punch.

[0095] Clause 6. The microfluidic assay device of clause 5, wherein the punch extends through the second substrate and into the reaction chamber to break the seal upon actuation of the push-button.

[0096] Clause 7. The microfluidic assay device of any of the clauses 1-6, wherein the assay reagents comprise one or more capture reagents disposed on the non-fouling polymer brush.

[0097] Clause 8. The microfluidic assay device of any of the clauses 1-7, wherein the assay reagents comprise one or more detection reagents and one or more capture reagents that are spatially separated and disposed on the non-fouling polymer brush.

[0098] Clause 9. The microfluidic assay device of clause 8, wherein the one of more detection reagents are disposed on an excipient layer, wherein the one or more detection reagents solubilize upon contact with the fluid sample.

[0099] Clause 10. The microfluidic assay device of clause 9, wherein the excipient layer comprises a salt, a carbohydrate, a polyol, an emulsifier, a water soluble polymer, or a combination thereof.

[0100] Clause 11. The microfluidic assay device of any of the clauses 1-10, wherein the reaction layer is disposed between the first substrate layer and the channel layer, and wherein the seal lies in a same plane as the second substrate layer.

[0101] Clause 12. The microfluidic assay device of any of the clauses 1-11, wherein the waste pad is a first waste pad disposed in a cut-out on the second substrate layer, and wherein the first substrate includes a cut-out for receiving a second waste pad.

[0102] Clause 13. The microfluidic assay device of any of the clauses 1-12, wherein the non- fouling polymer brush comprises poly(oligo ethylene glycol methyl ether methacrylate) (POEGMA).

[0103] Clause 14. The microfluidic assay device of any of the clauses 1-12, wherein the non- fouling polymer brush comprises poly(oligo ethylene glycol methyl ether methacrylate-co- glycidyl methacrylate) (POEGMA-co-GMA).Docket No.028193-0056-WO01

[0104] Clause 15. The microfluidic assay device of any one of the clauses 1-14, further comprising a reservoir mounted to the second substrate, the reservoir being fluidly coupled to the reaction chamber and configured to receive the fluid sample.

[0105] Clause 16. The microfluidic assay device of any of the clauses 1-15, wherein the seal is a breakable substrate.

[0106] Clause 17. Use of the microfluidic assay device of any of the clauses 1-16 for analyzing the fluid sample by measuring a concentration level of an analyte in the fluid sample.

[0107] Clause 18. A method for analyzing a fluid sample, the method comprising: orienting the device of clause 1 in a substantially vertical upright position; loading the fluid sample into the inlet of the reaction chamber; loading a wash buffer into the inlet of the reaction chamber; waiting for an incubation period; breaking the seal by actuating the actuator to allow the fluid sample to exit the reaction chamber; and analyzing the device to measure a signal for a target analyte and a signal for a control to determine a concentration of the analyte.

[0108] Clause 19. The method of clause 18, further comprising adding a conditioning agent, detection reagent, or a combination thereof to the fluid sample before loading the sample into the inlet.

[0109] Clause 20. The method of clause 19, wherein the conditioning agent comprises a surfactant.

[0110] Clause 21. The method of clause 20, wherein the surfactant is a non-ionic detergent.

[0111] Clause 22. The method of any of the clauses 18-21, wherein the device further comprises: a reservoir mounted to the body and configured to be in fluid communication with the inlet of the reaction chamber, the reservoir including an inlet for receiving the fluid sample and the wash buffer.

[0112] Clause 23. The method of any of the clauses of 18-22, wherein the device further comprises: a flow resistor fluidly coupled to the outlet of the reaction chamber and configured to control a fluid flow rate of the fluid sample exiting from the outlet of the reaction chamber; and a waste pad downstream from the flow resistor and configured to collect the fluid sample.

[0113] Clause 24. The method of clause 23, further comprising: washing the fluid sample from the reaction chamber according to the fluid flow rate of the flow resistor; and collecting a processed fluid sample and the wash buffer in the waste pad.

[0114] Clause 25. The method of clause 18-24, wherein the actuator is supported on the body, and wherein the actuator is a push-button including a punch.Docket No.028193-0056-WO01

[0115] Clause 26. The method of any of the clauses of 18-25, wherein the seal is a breakable substrate.

[0116] Clause 27. The method of any of the clauses of 18-26, wherein the fluid sample comprises whole blood, serum, plasma, urine, tears, sweat, saliva, lymph, cerebrospinal fluid, fecal extract, cellular or tissue extracts, or any other aqueous sample.

[0117] Clause 28. A kit comprising: the microfluidic assay device of clause 1; a wash buffer; a conditioning agent; and a sample applicator.

[0118] Clause 29. The kit of clause 28, wherein the conditioning agent comprises a surfactant.

Claims

Docket No.028193-0056-WO01 CLAIMS What is claimed is:

1. A microfluidic assay device comprising: a cassette including a body formed by a plurality of layers, the body having a reaction chamber with an inlet, an outlet downstream from the inlet, and a non-fouling polymer brush including a plurality of assay reagents, the reaction chamber configured to house a fluid sample, and a seal disposed adjacent the outlet of the reaction chamber for preventing fluid flow through the outlet, and an actuator configured to break the seal upon actuation to create a fluid flow path for the fluid sample to exit from the outlet of the reaction chamber.

2. The microfluidic assay device of claim 1, further comprising: a flow resistor fluidly coupled to the outlet of the reaction chamber and configured to control a fluid flow rate of the fluid sample exiting from the outlet of the reaction chamber; and a waste pad downstream from the flow resistor and configured to collect the fluid sample.

3. The microfluidic assay device of claim 2, wherein the plurality of layers include: a first substrate layer having the non-fouling polymer brush; a reaction layer having a cut-out that forms the reaction chamber; a channel layer having a serpentine channel in fluid communication with the reaction chamber, the serpentine channel forming the flow resistor; and a second substate layer configured to seal the reaction chamber to the first substrate layer.

4. The microfluidic assay device of claim 3, wherein the actuator is supported on the body, and wherein the actuator is a push-button, a screw valve, a magnetic flap, or a peelable adhesive.

5. The microfluidic assay device of claim 4, wherein the push-button includes a punch.Docket No.028193-0056-WO01 6. The microfluidic assay device of claim 5, wherein the punch extends through the second substrate and into the reaction chamber to break the seal upon actuation of the push-button.

7. The microfluidic assay device of claim 1, wherein the assay reagents comprise one or more capture reagents disposed on the non-fouling polymer brush.

8. The microfluidic assay device of claim 1, wherein the assay reagents comprise one or more detection reagents and one or more capture reagents that are spatially separated and disposed on the non-fouling polymer brush.

9. The microfluidic assay device of claim 8, wherein the one of more detection reagents are disposed on an excipient layer, wherein the one or more detection reagents solubilize upon contact with the fluid sample.

10. The microfluidic assay device of claim 9, wherein the excipient layer comprises a salt, a carbohydrate, a polyol, an emulsifier, a water soluble polymer, or a combination thereof.

11. The microfluidic assay device of claim 3, wherein the reaction layer is disposed between the first substrate layer and the channel layer, and wherein the seal lies in a same plane as the second substrate layer.

12. The microfluidic assay device of claim 3, wherein the waste pad is a first waste pad disposed in a cut-out on the second substrate layer, and wherein the first substrate includes a cut-out for receiving a second waste pad.

13. The microfluidic assay device of claim 3, wherein the non-fouling polymer brush comprises poly(oligo ethylene glycol methyl ether methacrylate) (POEGMA).

14. The microfluidic assay device of claim 3, wherein the non-fouling polymer brush comprises poly(oligo ethylene glycol methyl ether methacrylate-co-glycidyl methacrylate) (POEGMA-co-GMA).Docket No.028193-0056-WO01 15. The microfluidic assay device of claim 3, further comprising a reservoir mounted to the second substrate, the reservoir being fluidly coupled to the reaction chamber and configured to receive the fluid sample.

16. The microfluidic assay device of claim 1, wherein the seal is a breakable substrate.

17. Use of the microfluidic assay device of claim 1 for analyzing the fluid sample by measuring a concentration level of an analyte in the fluid sample.

18. A method for analyzing a fluid sample, the method comprising: orienting the device of claim 1 in a substantially vertical upright position; loading the fluid sample into the inlet of the reaction chamber; loading a wash buffer into the inlet of the reaction chamber; waiting for an incubation period; breaking the seal by actuating the actuator to allow the fluid sample to exit the reaction chamber; and analyzing the device to measure a signal for a target analyte and a signal for a control to determine a concentration of the analyte.

19. The method of claim 18, further comprising adding a conditioning agent, detection reagent, or a combination thereof to the fluid sample before loading the sample into the inlet.

20. The method of claim 19, wherein the conditioning agent comprises a surfactant.

21. The method of claim 20, wherein the surfactant is a non-ionic detergent.

22. The method of claim 18, wherein the device further comprises: a reservoir mounted to the body and configured to be in fluid communication with the inlet of the reaction chamber, the reservoir including an inlet for receiving the fluid sample and the wash buffer.

23. The method of claim 18, wherein the device further comprises: a flow resistor fluidly coupled to the outlet of the reaction chamber and configured to control a fluid flow rate of the fluid sample exiting from the outlet of the reaction chamber; andDocket No.028193-0056-WO01 a waste pad downstream from the flow resistor and configured to collect the fluid sample.

24. The method of claim 23, further comprising: washing the fluid sample from the reaction chamber according to the fluid flow rate of the flow resistor; and collecting a processed fluid sample and the wash buffer in the waste pad.

25. The method of claim 18, wherein the actuator is supported on the body, and wherein the actuator is a push-button including a punch.

26. The method of claim 18, wherein the seal is a breakable substrate.

27. The method of claim 18, wherein the fluid sample comprises whole blood, serum, plasma, urine, tears, sweat, saliva, lymph, cerebrospinal fluid, fecal extract, cellular or tissue extracts, or any other aqueous sample.

28. A kit comprising: the microfluidic assay device of claim 1; a wash buffer; a conditioning agent; and a sample applicator.

29. The kit of claim 28, wherein the conditioning agent comprises a surfactant.

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