Sample analysis device and methods

Abstract

The present disclosure provides devices and methods for separation of capture particles into wells of an array of wells and sealing the wells with a hydrophobic liquid. In certain embodiments, the methods of the present disclosure involve detecting presence of a signal from the array of wells, wherein the presence of the signal is indicative of the presence of an analyte immobilized on capture particles. In certain embodiments, the device for separation of capture particles into wells of an array of wells and sealing the wells with a hydrophobic liquid includes protrusions at an opening configured to prevent the hydrophobic liquid from reaching the opening before the hydrophilic liquid is removed.

Description

[0001] SAMPLE ANALYSIS DEVICE AND METHODS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 601,654, filed November 21, 2023, which application is incorporated herein by reference in its entirety.

[0004] INTRODUCTION

[0005] Assaying an analyte in the sample can involve capturing the analyte on a solid support, such as, magnetic or non-magnetic particles. These particles often need to be transported into a sample analysis instrument for determining the presence of the captured analyte. The instrument may perform digital detection and / or analog detection and report on the presence or absence of the analyte and / or the amount of the analyte.

[0006] Regardless of digital or analog methods for detection of analyte, it is desirable to spatially separate the particles. Spatially separated particles can require sealing with a hydrophobic liquid to reduce evaporation and isolate reactions during analysis of the particles which can make these detection methods cumbersome.

[0007] As such, there is interest in methods and devices that can be used for sample analysis involving spatial separation of particles.

[0008] SUMMARY

[0009] The present disclosure provides devices, systems, kits, and methods for separation of capture particles into wells of an array of wells and sealing the wells with a hydrophobic liquid.

[0010] A device for sealing capture particles in an array of wells with a hydrophobic liquid is disclosed. The device may include a first plate comprising an upper surface and a lower surface and a second plate comprising an upper surface and a lower surface, the first plate positioned in a spaced-apart manner from the upper surface of the second plate, an array of wells disposed on the upper surface of the second plate, the lower surface of the first plate and the upper surface of the second plate defining a wall-less chamber comprising the array of wells; a first opening extending between the upper and lower surfaces in the first plate and positioned adjacent to a first side of the array of wells, wherein the first opening is configured for introducing an aqueous liquid over the array of wells and removing the aqueous liquid; and a second opening extending between the upper and lower surfaces in the first plate and positioned adjacent to a second side of the array of wells, e.g., opposite to the first side, wherein the second opening is configured for introducing the hydrophobic liquid over the array of wells. The first and second openings may be positioned directly opposite to each other on opposite sides of the array of wells. The wall-less chamber may also be referred to as a liquid holding region and all references to the wall-less chamber may be considered as references to the liquid holding region; in this manner, the terminology “wall-less chamber” is intended to be interchangeable with the terminology “liquid holding region”.

[0011] A sealing pad may be delineated in the lower surface of the first plate, wherein the pad region is closer to the upper surface of the second plate than the regions surrounding it. The sealing pad may define a liquid holding region (wall-less chamber) in conjunction with the upper surface of the second plate. The sealing pad holds a liquid present in the chamber by surface tension.

[0012] The protrusions extending from the lower surface around the first opening may extend partially towards the second plate. The protrusions may partially occlude the first opening. The protrusions extending from the lower surface around the first opening may extend completely to the second plate such that they contact the second plate.

[0013] The scaling pad may comprise a pinning wall, for controlling movement of aqueous and / or hydrophobic liquid in the chamber, extending towards the second plate, wherein the pinning wall does not extend to the second plate, wherein the pinning wall is positioned in a space apart manner from the first opening and surrounds the first opening and extends towards the second opening.

[0014] A method for using this device for seeding capture particles and substrate solution in the wells of the array and sealing the wells with a hydrophobic liquid that minimizes evaporation of the substrate solution and limits egress of molecules from the wells is also provided.

[0015] The methods of the present disclosure involve depositing capture particles over the array of wells; flowing a substrate solution over the array of wells; removing the substrate solution and flowing hydrophobic liquid over the array of wells to cover the array of wells with the hydrophobic liquid; and detecting presence of a signal from the array of wells, wherein the presence of the signal is indicative of the presence of an analyte immobilized on capture particles. Removing the substrate solution and flowing hydrophobic liquid over the array of wells may be performed simultaneously.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS. 1A-1C illustrates partial views of a device comprising an array of wells and use of the device for seeding capture particles into the wells, contacting the capture particles with an aqueous liquid (e.g., a substrate solution) and sealing the wells with a hydrophobic liquid (e.g., an oil).

[0018] FIGS. 1D-1E provide detailed views of the sealing pad present in the device for seeding and sealing capture particles into an array of wells.

[0019] FIGS. 1F-1G provide examples of scaling pads present in the device for seeding and sealing capture particles into an array of wells (FIG. IF) and exemplary dimensions of the openings in the sealing pad (FIG. 1G).

[0020] FIGS. 1H-1I provide examples of shapes of the sealing pads. In FIG. 1H, the sealing pad (3) is formed by an abrupt indentation in the top plate (1). In FIG. 1G, the sealing pad (3) is formed by a gradually sloping indentation in the top plate (1). An image taken from below the bottom plate (2) is shown in FIG. 1H and II.

[0021] FIGS. 1J-1M. FIGS. 1J, IK, and IM show details of the pinning wall included in the sealing pad. FIG. IL shows a schematic in which a pinning wall is not present.

[0022] FIG. 2A shows a schematic illustration of filling of the chamber defined by the sealing pad and actual images during phases of filling of the device illustrated in Figs. 1A-1M. The seeding process is not depicted. The sealing process where an aqueous liquid is introduced from opening

[0023] (10) and covers that array of wells (4), followed by simultaneous aspiration of the aqueous liquid via opening (10) and introduction of hydrophobic liquid via opening (11) is illustrated.

[0024] FIG. 2B-2D. FIGS. 2B and 2C show the effect of presence of an aqueous liquid stopping feature on the scaling pad which helps stop the spread of the aqueous liquid to the second opening

[0025] (11) through which the hydrophobic liquid is to be introduced. FIG. 2D shows that in this scenario, in the absence of the aqueous liquid stopping feature, the aqueous liquid can spread to the second opening (11).

[0026] FIG. 3 shows examples of shapes of the wells in an array for depositing capture particles. Exemplary directions used for seeding, substrate filling, and sealing are depicted for one of the array of wells.

[0027] FIG. 4 shows examples of shapes of the pad region, formed on the first plate, for optimizing simultaneous aspiration of the aqueous liquid and introduction of hydrophobic liquid.

[0028] FIG. 5 depicts exemplary substrate retention features (6) that aid in aspiration of the aqueous liquid.

[0029] FIG. 6 depicts exemplary retention features and shapes of pad region for optimal aspiration of hydrophilic liquid and / or limit aspiration of the hydrophobic liquid and / or egress of the hydrophobic liquid from the chamber.

[0030] FIG. 7 depicts exemplary pinning wall (15) that aids in aspiration of the hydrophilic liquid and / or limit aspiration of the hydrophobic liquid.

[0031] FIGS. 8A-8D show different paths for dragging magnetic capture particles over the array of wells for reducing debris present on the array .

[0032] FIG. 9A provides a schematic of flow rate of an aqueous liquid (e.g., a substrate solution) during aspiration and flow rate of hydrophobic liquid (e.g., oil) during introduction of the hydrophobic liquid into the chamber.

[0033] FIG. 9B shows position of the probe for injecting the hydrophobic liquid (e.g., oil) and position of the probe for aspirating the hydrophilic liquid (e.g., a substrate solution) relative to the distance from the upper surface of the second plate. This schematic also shows that the diameter of the probe for dispensing the hydrophobic liquid (e.g., oil) is smaller than the diameter of the probe for dispensing the hydrophilic liquid (e.g., a substrate solution) while the diameter of the opening for the probe for dispensing the hydrophobic liquid is larger than the diameter of the opening for the probe for dispensing and aspirating the hydrophilic liquid. FIG. 10A is a schematic of contact angle of the hydrophobic liquid relative to the surface of the second plate. A hydrophobic liquid with an obtuse contact angle (due to low affinity for the surface) tends to seal the wells without displacing the hydrophilic liquid (e.g., a substrate solution) present in the wells. A hydrophobic liquid with an acute contact angle (due to higher affinity for the surface) tends to displace the hydrophilic liquid (e.g., a substrate solution) present in the wells.

[0034] FIG. 10B shows volume of hydrophilic liquid (e.g., a substrate solution) remaining in the wells over time. Sealing of wells with a hydrophobic liquid having a low affinity for the surface of the lower plate and thus having an obtuse contact angle relative to the surface results in more retention of the of hydrophilic liquid in the wells as compared to the retention of the hydrophilic liquid in the wells upon sealing with a hydrophobic liquid having a high affinity for the surface and thus having an acute contact angle relative to the surface.

[0035] FIG. IOC shows the relationship between contact angle and fluorescence detected from the wells for fluorinated hydrophobic liquids (Novec 7500, FC-40, and Galden HT200) and nonfluorinated hydrophobic liquids (soybean oil, Silicone 317667, Silicone 378356), and Galden HT200. Fluorinated hydrophobic liquids do not displace the substrate solution from the wells.

[0036] FIGS. 11A-11C. FIGS. 11A-11B provide images of a droplet of an oil with a greater submerged contact angle (SC A) (FIG. 11 A) and of a droplet of an oil with a lower SCA. FIG. 11C is a schematic showing relationship between SCA scaling of wells.

[0037] FIG. 12A shows the variation in SCA between different types of hydrophobic liquids.

[0038] FIG. 12B shows effect of formulation of the aqueous solution on SCA of silicone oil.

[0039] FIG. 12C Comparison of effectiveness of sealing array of wells with FC-40 or with silicone oil when using the ABJ assay buffer.

[0040] FIG. 13. illustrate a problem that has been solved by the pinning wall.

[0041] FIG. 14. Addition of proclin 950 and pyranine phosphate increase the SCA.

[0042] FIG. 15. Addition of Korasilon®® elevates the SCA. FIG. 16. Schematic for a method of seeding and sealing wells of an array of wells in a flow-through device.

[0043] FIG. 17. Higher rate of aspiration of the substrate solution improves sealing with silicone oil.

[0044] FIG. 18. Illustration of effect of pooling and the optimal sealing where substantial pooling does not occur.

[0045] FIG. 19 provides bead count (indicative of seeding efficiency) when using a substrate solution comprising no additive (no surfactant or anti-foam), surfactant Tween 20, or anti-foam, Korasilon®.

[0046] FIG. 20. Structure of Korasilon®® EMAI 19.

[0047] FIG. 21. Structure of Pluronic®17R4.

[0048] FIG. 22. The effect of Pooling reduction by the substrate solution containing the antifoam Pluronic on pTaul81 assay (A: image data, B: analyzed result data).

[0049] FIG. 23. The effect of Pooling reduction by the substrate solution containing the antifoam Korasilon® on pTaul81 assay. (A: image data, B: analyzed result data).

[0050] FIG. 24. The effect of Pooling reduction by the substrate solution containing the antifoam Pluronic on A[342 assay. (A: image data, B: analyzed result data).

[0051] FIG. 25. The effect of Pooling reduction by the substrate solution containing the antifoam Korasilon® on HBsAg assay. (A: image data, B: analyzed result data).

[0052] DETAILED DESCRIPTION

[0053] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to a particular embodiment described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be defined by the appended claims.

[0054] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The present disclosure is controlling to the extent there is a contradiction between the present disclosure and a publication incorporated by reference.

[0055] Definitions

[0056] Before the embodiments of the present disclosure are described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0057] It will be understood by those within the ail that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).

[0058] It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. It must be noted that as used herein and in the appended claims, the singular forms “a”, “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, refer to “a capture particle” includes plurality of such capture particles and reference to “the well” includes reference to one or more wells and equivalents thereof known to those skilled in the art, and so forth. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0059] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0060] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0061] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. When used in the context of a range, the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of from about “2 to about 10” also discloses the range “from 2 to 10.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1.

[0062] It should be noted that many of the terms used herein are relative terms. For example, the terms “upper” and “lower” are relative to each other in location, i.e., an upper component is located at a higher elevation than a lower component in a given orientation, but these terms can change if the component is flipped. The terms “inlet” and “outlet” are relative to a fluid flowing through them with respect to a given structure, e.g., a fluid flows through the inlet into the structure and flows through the outlet out of the structure. An inlet and an outlet may refer to the same structure that can be used to flow a liquid in and out.

[0063] The terms “horizontal” and “vertical” are used to indicate direction relative to an absolute reference, i.e., ground level. However, these terms should not be construed to require structures to be absolutely parallel or absolutely perpendicular to each other. For example, a first vertical structure and a second vertical structure are not necessarily parallel to each other. The terms “top” and “bottom” are used to refer to surfaces where the top is always higher than the bottom relative to an absolute reference, i.e., the surface of the earth. The terms “upwards” and “downwards” are also relative to an absolute reference; upwards is always against the gravity of the earth while downwards is always towards the gravity of the earth.

[0064] “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.

[0065] 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 number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0066] “Bead” and “particle” are used herein interchangeably and refer to a substantially spherical solid support. “Magnetic bead” and “Magnetic particle” and “paramagnetic particles” are used herein interchangeably and refer to a substantially spherical solid support that is influenced by a magnetic field such that the magnetic field can attract or repulse the magnetic bead or magnetic particle.

[0067] An “amount” as used herein refers to a quantity specified (e.g., high or low) or a number e.g., where the number is a level, such as a position on a real or imaginary’ scale of amount or quantity, or a concentration, such as, for example, a relative amount of a given substance contained within a solution or in a particular volume of space, e.g., the amount of solute per unit volume of solution.

[0068] “Component,” “components,” or “at least one component,” refer generally to a capture antibody, a detection reagent or conjugate, a calibrator, a control, a sensitivity panel, a container, a buffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, a detection reagent, a pretreatment reagent / solution, a substrate (e.g., as a solution), a stop solution, and the like that can be included in a kit for assay of a test sample, such as a patient urine, serum, whole blood, tissue aspirate, or plasma sample, in accordance with the methods described herein and other methods known in the art. Some components can be in solution or lyophilized for reconstitution for use in an assay.

[0069] “Antibody” and “antibodies” as used herein refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), and functionally active epitope-binding fragments of any of the above. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. For the sake of simplicity, an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody” or merely an “analyte antibody”.

[0070] “Antibody fragment” as used herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, singlechain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0071] “Label” or “detectable label” as used interchangeably herein refers to a moiety attached to a specific binding member or analyte to render the reaction between the specific binding member and the analyte detectable, and the specific binding member or analyte so labeled is referred to as “detectably labeled.” A label can produce a signal that is detectable by visual or instrumental means. Various labels include: (i) a tag attached to a specific binding member or analyte; or (ii) signal-producing substance, such as chromogens, fluorescent compounds, enzymes, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moictics that produce light, c.g., acridinium compounds, and moictics that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety, itself, may not be detectable but may become detectable upon reaction with yet another moiety. Use of the term “detectably labeled” is intended to encompass such labeling.

[0072] An “analog assay” as used herein refers to an assay in which the presence of and / or amount of an analyte in a test sample is determined by measuring the total signal produced (e.g., fluorescence, color, etc.) by the analyte in an entire reaction mixture (e.g., a single reaction vessel). An example of an analog assay is an assay in which the presence of and / or amount of an analyte is determined by measuring the total signal produced from a plurality of beads or microparticles contained in a single reaction vessel.

[0073] “Digital assay” as used herein refers to an assay in which an analyte is captured and a molecule of the analyte segregated and interrogated (e.g., to detect the presence and / or amount of the analyte in a sample). In a digital assay, presence of the analyte is assigned a value of 1 and absence of the analyte is assigned the value 0. Examples of digital assays include one or more of the following (which may overlap but are not mutually exclusive): single molecule detection assay, a nanowell assay, a single molecule enzyme linked immunosorbent assay, a direct capture counting assay, etc.

[0074] “Dynamic range” as used herein refers to range over which an assay readout is proportional to the amount of target molecule or analyte in the sample being analyzed.

[0075] “Microparticle(s)(s)” and “microbead(s)” are used interchangeably herein and refer to a microbead or microparticle that is allowed to occupy or settle in an array of wells, such as, for example, in an array of wells. The microparticle may include at least one specific binding member that binds to an analyte of interest.

[0076] “Specific binding partner” or “specific binding member” as used interchangeably herein refer to one of two different molecules that specifically recognizes the other molecule compared to substantially less recognition of other molecules. The one of two different molecules has an area on the surface or in a cavity, which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The molecules may be members of a specific binding pair. For example, a specific binding member may include, but not limited to, a protein, such as a receptor, an enzyme, an antibody and an aptamer, a peptide, a nucleotide, oligonucleotide, a polynucleotide, a ligand, and combinations thereof.

[0077] By "specifically bind" or "binding specificity," it is meant that the binding member binds the analyte molecule with specificity sufficient to differentiate between the analyte molecule and other components or contaminants of the test sample. For example, the binding member, may be an antibody that binds specifically to an epitope on an analyte. The antibody can be any antibody capable of binding specifically to an analyte of interest. For example, appropriate antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies (dAbs) (e.g., such as described in Holt et al. (2014) Trends in Biotechnology 21:484- 490), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), synthetic antibodies (sometimes referred to as antibody mimetics), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as antibody conjugates), and fragments of each, respectively. As another example, the analyte molecule may be an antibody and the first binding member may be an antigen and the second binding member may be a secondary antibody that specifically binds to the target antibody or the first binding member may be a secondary antibody that specifically binds to the target antibody and the second binding member may be an antigen.

[0078] “Aptamer” as used herein refers to an oligonucleotide or peptide molecule that can bind to pre-selected targets including small molecules, proteins, and peptides among others with high affinity and specificity. Aptamers may assume a variety of shapes due to their propensity to form helices and single- stranded loops. An oligonucleotide or nucleic acid aptamer can be a singlestranded DNA or RNA (ssDNA or ssRNA) molecule. A peptide aptamer can include a short variable peptide domain, attached at both ends to a protein scaffold.

[0079] “Control” as used herein refers to a reference standard for an analyte such as is known or accepted in the art, or determined empirically using acceptable means such as are commonly employed. A “reference standard” is a standardized substance which is used as a measurement base for a similar substance. For example, there arc documented reference standards published in the U.S. Pharmacopeial Convention (USP-NF), Food Chemicals Codex, and Dietary Supplements Compendium (all of which are available at http: / / www.usp.org), and other well-known sources. Methods for standardizing references are described in the literature. Also well-known are means for quantifying the amounts of analyte present by use of a calibration curve for analyte or by comparison to an alternate reference standard. A standard curve can be generated using serial dilutions or solutions of known concentrations of analyte, by mass spectroscopy, gravimetric methods, and by other techniques known in the art. Alternate reference standards that have been described in the literature include standard addition (also known as the method of standard addition), or digital polymerase chain reaction.

[0080] “Digital microfluidics (DMF),” “digital microfluidic module (DMF module),” or “digital microfluidic device (DMF device)” as used interchangeably herein refer to a module or device that utilizes digital or droplet-based microfluidic techniques to provide for manipulation of discrete and small volumes of liquids in the form of droplets. Digital microfluidic s uses the principles of emulsion science to create fluid-fluid dispersion into channels (principally water-in-oil emulsion). It allows the production of monodisperse drops / bubbles or with a very low poly dispersity. Digital microfluidics is based upon the micromanipulation of discontinuous fluid droplets within a reconfigurable network. Complex instructions can be programmed by combining the basic operations of droplet formation, translocation, splitting, and merging.

[0081] Digital microfluidics operates on discrete volumes of fluids that can be manipulated by binary electrical signals. By using discrete unit-volume droplets, a microfluidic operation may be defined as a set of repeated basic operations, i.e., moving one unit of fluid over one unit of distance. Droplets may be formed using surface tension properties of the liquid. Actuation of a droplet is based on the presence of electrostatic forces generated by electrodes placed beneath the bottom surface on which the droplet is located. Different types of electrostatic forces can be used to control the shape and motion of the droplets. One technique that can be used to create the foregoing electrostatic forces is based on dielectrophoresis which relies on the difference of electrical permittivity between the droplet and surrounding medium and may utilize high-frequency AC electric fields. Another technique that can be used to create the foregoing electrostatic forces is based on electrowetting, which relies on the dependence of surface tension between a liquid droplet present on a surface and the surface on the electric field applied to the surface.

[0082] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer including purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double- stranded polynucleotides.

[0083] By "hybridizable" or “complementary” or “substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) contains a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence- specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine / adenosine (A) pairing with thymine / thymidine (T), A pairing with uracil / uridine (U), and guanine / guanosine) (G) pairing with cytosine / cytidine (C). Inosine (I) bases pair with cytosine / cytidine. In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): G can also base pair with U. For example, G / U basepairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a G (e.g., of a protein-binding segment (e.g., dsRNA duplex) of a guide RNA molecule; of a target nucleic acid (e.g., target DNA or RNA) base pairing with a sensor RNA) is considered complementary to both a U and to C. For example, when a G / U base-pair can be made at a given nucleotide position of a protein-binding segment (e.g., dsRNA duplex) of a sensor RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary. Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases arc possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

[0084] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, and the like). A polynucleotide can include 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. The remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0085] The terms “peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0086] “Threshold” as used herein refers to an empirically determined and subjective cutoff level above which acquired data is considered “signal”, and below which acquired data is considered “noise”. A computer program based on CUSUM (Cumulative Sums Algorithm) is employed to process acquired data and detect events based on threshold input from the user. Variation between users is avoided by detection of as many events as possible followed by filtering the data afterwards for specific purposes. With a “loose” threshold a lesser number of events will be counted as signal. With a “tight” threshold a greater number of events will be counted as signal. Setting the threshold as loose or tight is a subjective choice based on the desired sensitivity or specificity for an assay, and whether in a given assessment false positives or false negatives would be preferred.

[0087] As used herein, “analyte”, “target analyte”, “analyte of interest” are used interchangeably and refer to a substance, material or chemical constituent the presence, absence and / or amount of which is being analyzed in a sample being measured in the methods and devices disclosed herein. In some aspects, the analyte is a biomolecule. Non-limiting examples of biomolecules include macromolecules such as, proteins, lipids, and carbohydrates. Analytes of interest are further described below.

[0088] “Contacting” and grammatical equivalents thereof as used herein refer to any type of combining action which brings a binding member into sufficiently close proximity with the analyte of interest in the sample such that a binding interaction will occur if the analyte of interest specific for the binding member is present in the sample. Contacting may be achieved in a variety of different ways, including combining the sample with a binding member, exposing a target analyte to a binding member by introducing the binding member in close proximity to the analyte, and the like.

[0089] As used herein, “immobilized” refers to a stable association of the analyte with a surface of a capture particle. By “stable association” is meant a physical association between two entities in which the mean half-life of association is at least 3 hours, 10 hours, 18 hours, one day or more, e.g., under physiological conditions. The stable association may arise from a covalent bond between the two entities, a non-covalent bond between the two entities (e.g., an ionic or metallic bond), or other forms of chemical attraction, such as hydrogen bonding, Van der Waals forces, and the like, or a combination thereof.

[0090] As used herein, a “positive well” refers to a well that has a signal related to presence of a nanobead / nanoparticle / analyte molecule, which signal is above a threshold value. As used herein, a “negative well” refers to a well that may not have a signal related to presence of a nanobead / nanoparticle / analyte molecule.

[0091] As used herein the term “hydrophobic”, refers to lacking an affinity for water, being substantially insoluble in water, or repelling water. Examples of hydrophobic molecules include alkanes, oils, and fats.

[0092] As used herein the term “hydrophilic”, refers to having an affinity for water, being substantially soluble in water, or attracting water. Pure water and water containing dissolved substances are both aqueous. A liquid that has water as the major constituent is hydrophilic. Examples of aqueous liquids include buffers and substrate solutions. The terms “aqueous liquid” and “hydrophilic liquid” are used interchangeably in the present disclosure.

[0093] 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. Preferred methods and materials arc described herein, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety to disclose and describe the methods and / or materials in connection with which the publications are cited. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0094] DEVICES

[0095] A device for sealing capture particles in an array of wells with a hydrophobic liquid is provided. The device may include a first plate comprising an upper surface and a lower surface and a second plate comprising an upper surface and a lower surface, the first plate positioned in a spaced-apart manner from the upper surface of the second plate, an array of wells disposed on the upper surface of the second plate, the lower surface of the first plate and the upper surface of the second plate defining a liquid holding region (c.g. wall-less chamber) comprising the array of wells; a first opening extending between the upper and lower surfaces in the first plate, wherein first the opening is positioned adjacent to a first side of the array of wells, wherein the first opening is configured for introducing an aqueous liquid over the array of wells; and a second opening extending between the upper and lower surfaces in the first plate, the second opening is positioned adjacent to a second side of the array of wells, directly opposite to the first side.

[0096] The size of the liquid holding region (e.g. wall-less chamber) is defined by a sealing pad formed in a region of the first plate. The sealing pad decreases the space between the lower surface of the first plate and the upper surface of the second plate such that the height of the liquid holding region (e.g. wall-less chamber) is lower than the space surrounding the sealing pad (e.g., such that the gap between the sealing pad and the upper surface of the second plate is smaller than the surrounding gap between the lower surface of the first plate and the upper surface of the second plate). The sealing pad may be formed by an indentation through the upper surface of the first plate which brings the lower surface of the first plate closer to the second plate in a defined area. Where the sealing pad is formed by an indentation, the thickness of the sealing pad may be substantially identical to the thickness of surrounding areas of the first plate. Alternatively, or in addition, the sealing pad is demarcated by a decreased thickness of the first plate around the sealing pad such that the space between the lower surface of the first plate and the upper surface of the second plate increases outside of the sealing pad region. For instance, the sealing pad may be defined by having a greater thickness (measured to the upper surface of the first plate) than areas of the first plate immediately outside of the sealing pad region. This increase in thickness may occur gradually or it may occur suddenly, e.g. as a step-change. It is possible that the sealing pad may be formed by both an indentation and a reduced thickness of the first plate around the sealing pad.

[0097] The sealing pad may be formed by embossing the first plate to create an indentation of a desired size, said indentation being made through the upper surface of the first plate such that the lower surface extends or protrudes towards the second plate in the region of the sealing pad. A sealing pad having a lower surface area is advantageous as it needs smaller volumes of consumables, such as, aqueous liquid and hydrophobic liquid. However, the surface area of the sealing pad can vary depending on assay requirements. The step of embossing may create an indentation having substantial vertical imprint. The step of embossing can be manipulated to create a gradually sloping indentation. FIG. 1H and II illustrate scaling pads created by an indentation in the first plate.

[0098] The sealing pad may have any suitable shape. For example, the sealing pad may be substantially rectangular in shape. The sealing pad may be rectangular with rounded corners. The array of wells may be positioned substantially centrally in the chamber. The first and second openings may be positioned closer to the shorter edges of the sealing pad on either side of the array of wells on the second plate.

[0099] The lower surface of the first plate may include a plurality of protrusions positioned around the circumference of the first opening, wherein an aqueous liquid (e.g. an assay buffer comprising a substrate solution) enters the chamber through the first opening, flows over the array of wells, and is removed through the first opening and wherein the protrusions are configured to prevent the hydrophobic liquid from reaching the opening before the aqueous liquid is removed, and wherein the second opening is configured for introducing the hydrophobic liquid over the array of wells.

[0100] The protrusions may be rectangular, square-like, circular, oval, triangular, sector-shaped, or cone shaped in shape. The protrusions may be arranged symmetrically around the circumference of the first opening. The protrusions may decrease in dimension towards the center of the first opening. The protrusions may extend partially into the first opening (e.g. when looking through the first opening, the profile of at least one of the protrusions overlaps the profile of the first opening). The protrusions may extend partially into the first opening and are configured to support the first apparatus for aspirating the hydrophilic liquid out of the chamber. For example, the first apparatus may be a hollow tube that enters the first opening and contacts the protrusions so that the hollow tube is at least partially prevented from contacting the upper surface of the second plate. There may be two protrusions arranged symmetrically around the circumference of the first opening. There may be four protrusions arranged symmetrically around the circumference of the first opening. There may be six protrusions arranged symmetrically around the circumference of the first opening. There may be eight protrusions arranged symmetrically around the circumference of the first opening. The number, shape, and size of the protrusions may be chosen based on the flow characterisitics of the hydrophilic and the hydrophobic liquids being used for assaying the capture particles. For example, the number and / or size of the protrusions may be reduced when the viscosity of the hydrophilic liquid is significantly lower than the viscosity of the hydrophobic liquid. Alternatively, when the viscosity of the hydrophilic and the hydrophobic liquids is similar, the number and / or size of the protrusions may be increased.

[0101] The protrusions may be sized to provide a contact area with the first apparatus, for introducing an aqueous liquid into the chamber, which contact area is sufficiently large to support the hollow tube to facilitate introduction of and removal of the aqueous liquid. The hollow tube may have an outer diameter of about 1 mm (e.g., 0.93 mm), the opening may have a diameter of about 1.5 mm (e.g., 1.3 mm) and the protrusion may occlude the opening such that the hollow tube stops, after being inserted through the first opening, upon contact with the protrusions. For example, the protrusion may decrease the effective diameter of the first opening to about 0.7 mm. Other combinations of opening and protrusion dimensions can also provide this function, provided the protrusions at least partially occlude the opening. As explained in the Examples, the protrusions aid in removal of the aqueous liquid from the chamber and aid in covering the array of wells with the hydrophobic liquid such that the hydrophobic liquid spreads over the array of wells by displacing the aqueous liquid and the hydrophobic liquid is not aspirated from the first opening prior to removal of the aqueous liquid. In other words, the protrusions increase aspiration rate of the aqueous liquid which aids in spread of the hydrophobic liquid but prevent aspiration of the hydrophobic liquid at least until the aquoeous liquid has been removed. This ensures complete coverage of the array of wells by the hydrophobic liquid and prevents formation of bubbles of the aqueous liquid trapped over the array of wells.

[0102] Fig. 1A shows a schematic of a device for seeding capture particles into an array of wells and for providing an aqueous solution that contacts the capture particles. The array of wells is sealed by a layer of hydrophobic liquid. A sideview of an exemplary device is shown. The device includes a liquid holding region (e.g. a wall-less chamber) defined between two surfaces, the lower surface of a top plate (1) and the upper surface of a bottom plate (2). The top plate includes a pad region (3), also referred to herein as sealing pad, which may have a thickness greater than the regions of the top plate surrounding it. The pad region (3) defines a chamber in conjunction with the upper surface of the bottom plate (2). The pad region (3) and the lower plate (2) hold a liquid present in the liquid holding region (e.g. wall-less chamber), e.g., by surface tension. The bottom plate may be referred to as the second plate and the top plate may be referred to as the first plate.

[0103] The top and bottom plates are substantially planar and may include one or more regions having different thickness. For example, the pad region is thicker than the region adjacent the pad region. The difference in thickness delineates the pad region from the surrounding lower surface of the top plate and helps limit the spread of the liquids from the pad region to the surrounding regions. See Figs. ID and IE.

[0104] Fig. IB is another schematic showing a space or liquid holding region (also referred to herein as a chamber or a wall-less chamber) defined between the pad region (3) and the upper surface of the second plate (2). The pad region and the upper surface of the lower plate may cooperate to hold liquid within the liquid holding region (e.g. wall-less chamber) by, e.g., surface tension. Thus, liquid may be held in place within the liquid holding region even in the absence of, e.g., walls partially or entirely enclosing the region. Liquid, which may be a hydrophobic liquid, hydrophilic liquid or both is held in the space defined between the lower surface of the pad region and the upper surface of the second plate. Only the pad region of the first plate is illustrated. The pad region may be made from a polymer such as polymethyl methacrylate (PMMA).

[0105] The liquid holding region (e.g. wall-less chamber) may only be defined by a lower surface and an upper surface and may not include any walls e.g. sidewalls. The liquid holding region (chamber) may include a lower surface and an upper surface and may include partial sidewalls. The partial sidewalls may not extend from the lower surface to the upper surface.

[0106] Fig. 1C is a see-through depiction of the pad region in the first plate of the device. The array of wells (4) is located on the upper surface of the bottom plate (bottom plate not shown). The array of wells is located centrally in the chamber, mid-way between the first and second openings. The first opening in the first plate is connected to a well (5) that can contain a hydrophilic liquid (e.g., a substrate solution or an assay buffer) or is connectable to a first apparatus for introducing the hydrophilic liquid into the space between the lower surface of the pad region and the upper surface of the second plate. Also depicted are protrusions (6) placed at the lower surface around the first opening. These protrusions are also referred to as substrate retention features (6). A second opening in the first plate is located directly across the array of wells from the first opening. The second opening (7) can contain a hydrophobic liquid (e.g., oil) or is connectable to a second apparatus for introducing the hydrophobic liquid into the space between the lower surface of the pad region and the upper surface of the second plate. The first plate or the second plate can include an additional inlet for introducing a liquid into the space between the lower surface of the pad region and the upper surface of the second plate. The liquid can be an assay buffer or a solution containing capture particles. The capture particles may be magnetic and may be moved into the chamber via a magnetic field. For example, paramagnetic capture particles may be immobilized by a magnet positioned under the second plate and the magnet may be moved to a location under the array of wells resulting in movement of the capture particles immobilized by the magnet into the wells.

[0107] The first opening may be connectable to a first apparatus for introducing the aqueous liquid into the chamber and the second opening is connectable to a second apparatus for introducing hydrophobic liquid into the chamber. The first apparatus and / or the second apparatus may comprise a pneumatic instrument, bulk reagent dispenser, a hollow tube connected to a dispenser bottle, etc. The first apparatus may comprise a pneumatic instrument. The pneumatic instrument may be a pipette. The second apparatus may comprise a pneumatic instrument. The pneumatic instrument may be a pipette. The apparatus for introducing the aqueous or hydrophobic liquid into the space may be an injection apparatus. The injection apparatus may be connected to a pump that regulates the rate of flow of the aqueous or hydrophobic liquid into the chamber. The injection apparatus may include a hollow tube (e.g., a needle) that establishes an water-tight or an air-tight seal with the circumference of the first and second openings. The inner diameter of the hollow tubes may be the same. The inner diameter of the hollow tubes may be different. The inner diameter of the hollow tube that injects the hyrophobic liquid into the chamber via the second opening may have a diameter larger than that of the hollow tube that injects the hyrophilic liquid into the chamber via the first opening. The flow rate for introducing the aqueous liquid and the hydrophobic liquid into the space may be the same. The flow rate for introducing the aqueous liquid and the hydrophobic liquid into the chamber may be different. The second apparatus for introducing the hydrophobic liquid into the chamber may be positioned at a distance from the second plate that is greater than the distance at which the first apparatus is positioned. The difference between the height at which the second apparatus is positioned and the height at which the first apparatus is positioned, with reference to the second plate, may be increased to decrease the flow rate of the hydrophobic liquid into the chamber.

[0108] The first apparatus for introducing the hydrophilic liquid into chamber via the first opening may be used for aspirating the hydrophilic liquid out of the chamber. The first apparatus may remove the aqueous liquid from the chamber at a rate that is faster than the rate at which the hydrophobic liquid is introduced into the chamber.

[0109] FIG. ID shows the lower surface of the first plate. The pad region (3) is rectangular in shape with rounded ends. In this example, the rounded ends bulge out relative to the width of the remainder of the pad region, resembling a dumbbell shape. In other example, the comers of the rectangular pad region may be rounded without increasing the width relative to the remainder of the pad region. The first opening (10) and the second opening (11) are located on opposite sides of the pad region closer to the short edges. The protrusions (6) disposed on the lower surface of the first plate around the first opening (10) are shown. The pad region (3) delineates the area of the lower surface of the first plate that defines the chamber for holding liquids over the array of wells (not shown).

[0110] FIG IE shows the lower surface of the first plate. The pad region is rectangular in shape with rounded ends where the rounded ends increase in width as compared to the rectangular region, resembling a dumbbell shape. The protrusions (6) disposed on the lower surface of the first plate around the first opening arc shown. The pad region (3) delineates the area of the lower surface of the second plate that defines the chamber for holding liquids over the array of wells (not shown). The pad region includes a pinning wall (15) for facilitating aspiration of the aqueous liquid. The pad region also includes an aqueous liquid stopper feature (20) for limiting the spread of the aqueous liquid to the second opening (11).

[0111] The aqueous liquid stopper feature (20) may not be included. For example, the spread of the aqueous liquid can be controlled by reducing the volume dispensed into the chamber and / or by dispensing the hydrophobic liquid from the second opening before the aqueous liquid reaches the second opening. When included, the aqueous liquid stopper feature can be a thin film disposed on the pad region. The film may extend from the first opening towards the second opening and stop before the second opening. The film can have a thickness of less than 100 m, e.g., less than 50 pm, less than 30 pm, less than 10 pm, less than 5 pm to as low as 1 pm.

[0112] The pad region of the present disclosure may have a thickness, measured as the distance between the upper surface of the first plate and the lower surface of the pad region, of about 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or about 1.0 mm. The pad may have a thickness between 0.3 mm - 0.8 mm.

[0113] The pad region may be substantially rectangular with rounded ends and may have a length of about 16 mm and a width of about 4 mm or a length of about 17 mm and a width of about 4.3 mm. In the pad regions shown in FIG. IF, as compared to the top panel, in the lower panel, the pad region width is increased (4.27 mm vs 4.2 mm in the top panel) . The wider pad region increases the area of the array that can be imaged while excluding inclusion of the side edges of the pad in the image. See, also, FIG. 1H and FIG. II. In FIG. II as compared to FIG. 1H, the width of the pad region is increased which avoids the side edge shadow that is imaged in FIG. 1H.

[0114] The sealing pad may have a length in the range of 3 mm to 20 mm and a width in the range of 1 mm to 20 mm, e.g., the sealing pad dimensions is in the range of about 20 mm x 20 mm, 20 mm x 10 mm, 20 mm x 5 mm, 15 mm x 5 mm, 12 mm x 5 mm, or 12 mm x 4.5 mm.

[0115] The diameter of the first and second openings may be similar. The diameter of the first and second openings may be about 0.5 mm-4 mm, e.g., about 1 mm-2.5 mm, 1.5 mm-2.3 mm, 0.5 mm- 1 mm, 1 mm- 1.5 mm, 1.5 mm- 2 mm, 0.5 mm-1 mm, 0.8, 0.9, 1.0, 1.1. 1.2, 1.3, 1.4, or about 1.5 mm. The diameter of the first and second openings may be different. The diameter of the first opening may be smaller than the second opening. The first and second openings may be spaced apart by about 8 mm-20 mm, e.g., 8 mm-10 mm, 10 mm-12 mm, 12 mm-14 mm, 14 mm-16 mm, 16 mm- 18 mm, or 18 mm-20mm. Distance between the two openings may be measured from the center of the first opening to the center of the second opening.

[0116] The first and second openings may be substantially cylindrical or frustoconical in shape. The first opening may be smaller than the second opening. The first opening and the second opening may decrease in diameter from the upper surface to the lower surface of the first plate. The first opening may decrease in diameter from the upper surface to the lower surface of the first plate and the second opening increases in diameter from the upper surface to the lower surface of the first plate. In the schematics shown in FIG. 1G, in the pad region shown in top panel, the first opening ( 1 ) is narrower than the second opening (11) and both decrease in diameter from the upper surface towards the lower surface. In the schematics shown in FIG. 1G, in the pad region shown in bottom panel, the first opening (1) is narrower than the second opening (11) and decrease in diameter from the upper surface towards the lower surface while the second opening increases in diameter from the upper surface towards the lower surface. Reference to the diameter of an opening that has a non-uniform width is reference to the diameter at the narrowest region.

[0117] The distance between the sealing pad and the upper surface of the second plate may be about 2 mm - 50 pm, e.g., about 1 mm - 100 m, 1 mm - 200 pm, 1 mm - 300 pm, 1 mm - 400 pm, or 0.5 mm - 400 pm. The surface area of the sealing pad may be about 3 mm2to 400 mm2, 5 mm2to 300 mm2, 5 mm2to 200 mm2, 10 mm2to 100 mm2, 20 mm2to 100 mm2, or 40 mm2to 80 mm 2.

[0118] The liquid capacity of the liquid holding region may be 1 mL or less, 750 pL or less, 500 pL or less, 400 pL or less, 300 pL or less, 250 pL or less, 200 pL or less, 150 pL or less, 100 pL or less, 50 pL or less, 25 pL or less, e.g., 25-10 pL or 25-15 pL.

[0119] The array of wells may a length of about 3-15 mm and a width of about 2- 10 mm, e.g., the dimensions of the array of wells is in the range of about 10 mm x 7.5 mm, 4.0 mm x 3.0 mm, 7 mm x 3.5 mm or 6.2 mm x 3.5 mm.

[0120] The sealing pad (also referred to herein as “pad region”) may comprise a pinning wall, for controlling movement of aqueous and / or hydrophobic liquid in the chamber. The pinning wall may extend from the pad region towards the second plate. The pinning wall is relatively short and does not extend to the second plate. The pinning wall can have a width of about 100 -500 pm, e.g., 200 -400 pm. The pinning wall can have a height of about 50-250 pm e.g., 150 -250 pm. Height refers to the distance the pinning wall extends, from the lower surface of the sealing pad, in a direction perpendicular to the lower surface of the sealing pad. The pinning wall may be positioned in a spaced apart manner from the first opening and may surround the first opening and extend towards the second opening. The pinning wall may extend downwards from the periphery of the sealing pad and extend along the periphery till the region of the sealing pad positioned over the array of wells. The pinning wall may extend downwards from the periphery of the scaling pad and extend along the periphery and stop short of the area where the second opening is positioned.

[0121] FIGS. 1J-1K, and IM illustrate the pinning wall (15). The pinning wall (15) extends from the periphery of the pad region. The extent to which the pinning wall surrounds the pad region can be determined empirically. In this illustration, the pinning wall extends around the periphery of the pad region except the area in which the second opening is located. In other examples, the pinning wall may be shorter and stop before the area of the pad region above the array of wells. FIG. IK depicts a side view of the pad region across one short edge of the pad region to the opposite short edge. The height (H) and width (W) of the pinning wall (15) is demarcated. The pinning wall can have a height of about 200 m and a width of about 300 pm.

[0122] FIG. IM depicts a side view of the chamber defined between the pad region and the upper surface of the second plate. A cross-section of the chamber across a region where a pinning wall is present is depicted. The cross-section is perpendicular to the longer edge and parallel to the shorter edge of the chamber. Liquid present in the chamber and curvature of the liquid are illustrated. The curvature of the hydrophobic liquid in absence of the pinning wall (see FIG. IL, where a pinning wall does not extend from the pad region) is much lower than the curvature in the presence of the pinning wall (see FIG. IM). This increase in curvature may help stabilize the hydrophobic liquid in the chamber and minimizes the chance that the hydrophobic liquid overtakes the aqueous liquid and reaches the first opening when the aqueous liquid is removed through the first opening and the hydrophobic liquid is simultaneously introduced through the second opening. The horizontal force vector of the surface tension is larger in FIG. IM since the L-shape created by the pinning wall pins the air-liquid interface when liquid moves inwards during aspiration. The apparent contact angle could be close to zero with the assistance of the pinning effect by the comer. To summarize, the presence of a pinning wall may help stabilize the hydrophobic liquid within the chamber and may thereby reduce the risk that the hydrophobic liquid reaches the first opening before the aqueous liquid is aspirated from the first opening.

[0123] The lower surface of the first plate may have a uniform hydrophobicity such that the entire lower surface of the first plate is hydrophobic or hydrophilic. The upper surface of the second plate may have uniform hydrophobicity such that the entire upper surface of the second plate is hydrophobic or hydrophilic. The lower surface of the first plate may be more hydrophobic than the upper surface of the second plate. The lower surface of the first plate may be less hydrophobic than the upper surface of the second plate.

[0124] The wells in the array of wells may have sub-femtoliter volume, femtoliter volume, sub- nanoliter volume, nanoliter volume, sub-microliter volume, or microliter volume. For example, wells may be femtoliter wells, nanoliter wells, or microliter wells. The wells in an array may all have substantially the same volume. The array of wells may have a volume up to about 100 microliter, e.g., about, 0.1 femtoliter- 1 femtoliter, 1 femtoliter - 10 femtoliter, 10 femtoliter - 25 femtoliter, 25 femtoliter - 50 femtoliter, 50 femtoliter - 100 femtoliter, 100 femtoliter - 0.1 pL, 0.1 pL - 1 pL, 1 pL - 10 pL, 10 pL - 25 pL, 25 pL - 50 pL, 50 pL - 100 pL, 100 pL - 0.1 nL, 0.1 nL - 1 nL, 1 nL - 10 nL, 10 nL - 25 nL, 25 nL -50 nL, 50 nL - 100 nL, 100 nL - 0.1 microliter, 0.1 microliter - 1 microliter, 1 microliter - 10 microliter, 10 microliter - 25 microliter, 25 microliter - 50 microliter, or 50 microliter - 100 microliter.

[0125] The wells in the array of wells may be micro- wells. The size and shape of the micro- wells is compatible with the size and shape of capture particles. For example, the micro-wells may have a substantially circular opening having a diameter larger than the diameter of the capture particles. The depth of the micro-wells may be more than the diameter of the capture particles. The number of micro-wells in an array may be selected based on the number of capture particles being analyzed.

[0126] The wells in the array of wells may be nano-wells. The size and shape of the nano-wells is compatible with the capture particles. For example, the nano-wells may have a substantially circular opening having a diameter larger than the diameter of the capture particles. The depth of the nano-wells may be more than the diameter of the capture particles. The number of nano-wells in an array may be selected based on the number of capture particles being analyzed.

[0127] The wells may be an array of wells that include a plurality of individual wells. The array of wells may include a plurality of wells that may range from about 109to 10 in number per 1 2 mm . An array of about 100,000 to 500,000 wells (e.g., femtoliter wells) covering an area approximately 12 mm2may be fabricated. The array of wells may range in size from about 3.0 x 4.0mm to about 7.0 x 4.0 mm, e.g., about 6.2 x 3.5 mm. Each well may measure about 4.2 m wide X 3.2 pm deep (volume approximately 50 femtoliters), and may be capable of holding a single bead / particle (about 3 pm diameter). At this density, the femtoliter wells are spaced at a distance of approx. 7.4 pm from each other. In some examples, the well array may be fabricated to have individual wells with a diameter of about 10 nm to 10,000 nm.

[0128] An array of wells may have about 100,000 to 500,000 wells, e.g., about 300,000 wells in an area of about 6.2 x 3.5 mm. An array of wells may have about 170,000 wells in an area of about 4 x 3 mm.

[0129] Each capture particle may have 1 or 0 copy of the analyte immobilized on the capture particle. Each capture particle may have a predetermined number of binding members immobilized on its surface. The predetermined number of binding members immobilized on the surface of each capture particle may range from about 1-10, e.g., about 1 -2, 2-3, 3-4, 4-5, 5-6, 6- 7, 7-8, 8-9, or 9-10.

[0130] The capture particle having a surface on which the analyte is immobilized may be any convenient surface, such an exterior surface of a bead or an interior and exterior surface of a porous bead. For example, the analyte may be attached covalently or non-covalently to a bead, e.g., latex, agarose, sepharose, streptavidin, tosylactivated, epoxy, polystyrene, amino bead, amine bead, carboxyl bead, silica bead, or the like. The capture particle may be a microparticle, also referred to as microbead. The microparticle may be between about 0.1 nm and about 10 microns, between about 50 nm and about 5 microns, between about 100 nm and about 1 micron, between about 0.1 nm and about 700 nm, between about 500 nm and about 10 microns, between about 500 nm and about 5 microns, between about 500 nm and about 3 microns, between about 100 nm and 700 nm, or between about 500 nm and 700 nm in diameter. For example, the microparticle may be about 4-6 microns, about 2-3 microns, or about 0.5- 1.5 microns in diameter. Particles less than about 500 nm diameter are sometimes considered nanoparticles. Thus, the microparticle optionally may be a nanoparticle between about 0.1 nm and about 500 nm, between about 10 nm and about 500 nm, between about 50 nm and about 500 nm, between about 100 nm and about 500 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, or about 500 nm in diameter. The particle may be roughly spherical in shape and the diameter may be the diameter across the largest dimension of the particle. While the size of the capture particles are described in terms of diameter, the particles may not necessarily be spherical in size and can be amorphous in shape. For amorphous particles, the diameter is the largest distance from one side to the diametrically opposite side. The capture particles may be substantially spherical.

[0131] The bead may be a magnetic bead, also referred to as a magnetic particle. Magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or ferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, C1O2. MnAs, MnBi, EuO, NiO / Fe. Examples of ferrimagnetic materials include NiFe^Or, CoFe^Or, FeaCE (or FeO.Fe Oa). Beads can have a solid core portion that is magnetic and is surrounded by one or more non-magnetic layers. Alternately, the magnetic portion can be a layer around a non-magnetic core. Exemplary magnetic particles include those available from commercial sources, such as, Dynabeads® Magnetic beads provided by Invitrogen, Estapor® SuperParamagnetic Microspheres and PureProteome™ Magnetic Beads by Merck Millipore, BcMag™ by Bioclone Inc., ProMag™ and BioMag® from Bangslabs, SupraMag™ by Polymicrospheres Inc., TurboBeads® by Turbobeads Lie., and SPHERO™ Polystyrene Magnetic Particles by Spherotech and the like. Superparamagnetic beads are available from Sigma-Aldrich and Thermo Scientific. The capture particle on which the analyte is immobilized may be stored in dry form or in a liquid. The magnetic beads may be subjected to a magnetic field prior to or after contacting with the sample with a magnetic bead on which the first binding member is immobilized.

[0132] The capture particles may be coated with one or more molecules of a binding member that specifically binds to the analyte of interest. In a multiplex assay, a population of first capture particles coated with one or more molecules of a first binding member that specifically binds to a first analyte of interest and a population of second capture particles coated with one or more molecules of a second binding member that specifically binds to a second analyte of interest may be used. The first capture particles and / or second capture particles may be barcoded to aid in identification of first and / or second capture particles. If an analyte is present in the sample, the capture particles bind to the analyte via the binding member. An optional wash step may be performed to dislodge non-specifically bound analytes. The capture particles may then be contacted with a second binding member that binds to the analyte bound to the capture particles. The second binding member may be conjugated to a phosphatase enzyme. The contacting steps may be in a binding buffer that facilitates the specific binding interaction. The capture particles may then be contacted with the fluorogenic substrate.

[0133] The placement of single nanobeads / nanoparticles / analyte molecules in the wells allows for either a digital readout or analog readout. For example, for a low number of positive wells (<~70% positive) Poisson statistics can be used to quantitate the analyte concentration in a digital format; for high numbers of positive wells (>~70%) the relative intensities of signal-bearing wells are compared to the signal intensity generated from a single nanobead / nanoparticle / analyte molecule, respectively, and used to generate an analog signal. A digital signal may be used for lower analyte concentrations, whereas an analog signal may be used for higher analyte concentrations. A combination of digital and analog quantitation may be used, which may expand the linear dynamic range. The signal intensity of a well increases overtime which may indicate the presence of more than analyte in the well. The signal intensity in a well may be stagnant and of higher intensity than wells containing a single analyte which may indicate the presence of more than analyte in the well. The signal from a negative well may be at a background level, i.e., below a threshold value.

[0134] The wells may be any of a variety of shapes, such as, cylindrical with a flat bottom surface, cylindrical with a rounded bottom surface, cubical, cuboidal, frustoconical, inverted frustoconical, or conical. , The wells may include a sidewall that may be oriented to facilitate the receiving and retaining of a nanobead or nanoparticle present liquid droplets that have been moved over the well array. , The wells may include a side wall that may be oriented to facilitate the receiving and retaining of a nanobead or nanoparticle that is not present in a liquid droplet that have been moved over the well array. The wells may include a first sidewall and a second sidewall, where the first sidewall may be opposite the second side wall. The first sidewall may be oriented at an obtuse angle with reference to the bottom of the wells and the second sidewall may be oriented at an acute angle with reference to the bottom of the wells. The movement of the capture particles is in a direction parallel to the bottom of the wells and from the first sidewall to the second sidewall. The movement of the capture particles may be in a direction parallel to the bottom of the wells and from the first sidewall to the second sidewall. The space (also referred to as a chamber or liquid holding region) defined between the lower surface of the pad region and the upper surface of the bottom plate may be a range of different sizes and shapes such that the chamber is suitable for the detection of the analyte. The chamber may accommodate a volume of liquid from about 5 microliters to 1 milliliter. For example, the reaction chamber may be sized to contain a liquid having a volume of from about 5 microliters to 500 microliters. The chamber may be sized to contain a liquid having a volume of from about 5 microliters to 100 microliters. The fluid capacity of the chamber may be about 1 mL or less, 750 pL or less, 500 pL or less, 400 pL or less, 300 pL or less, 250 pL or less, 200 pL or less, 150 pL or less, 100 pL or less, 50 pL or less, or 25 pL or less.

[0135] The chamber may be a range of different shapes to retain the capture beads, assay buffer, and substrate solution. The shape of the chamber may be a cube. The shape of the chamber may be a cylinder. The shape of the chamber may be hexagonal. The shape of the chamber may be a sphere. The shape of the chamber may be octagonal. The shape of the chamber may be conical. The shape of the chamber may be cuboidal.

[0136] The bottom or top of the chamber may be optical transparent such that the analyte immobilized on the capture particles may be detected through optical means. All sides of the chamber may be optically transparent such that the analyte may be detected through optical means. Alternatively, only the top and the bottom of the chamber may be optically transparent such that the analyte may be detected through optical means.

[0137] The fluid capacity of the chamber may be about 500 mL or less, 450 pL or less, 400 pL or less, 350 pL or less, 300 pL or less, 250 pL or less, 200 pL or less, 150 pL or less, 100 pL or less, 50 pL or less, 25 pL or less, 20 pL or less, 15 pL or less, 10 pL or less, or 5 pL or less.

[0138] FIG. 3 shows examples of shapes of the wells in an array for depositing capture particles. The shape of the wells may be selected based on a variety of considerations. One consideration may be the direction used for seeding, substrate filling, and sealing. Exemplary directions used for seeding, substrate filling, and sealing is depicted for one of the array of wells in FIG. 3.

[0139] The direction of seeding of the capture particles into the wells is selected to maximize entrapment of the seeded capture particles in the wells. For example, the direction for seeding the wells may be selected such that the capture particles enter the wells at a region that is the larger dimension of the well and exit at a region that has a smaller dimension. In case of wells with straight edges and corners, the capture particles are directed into the wells across the straight edges. The magnetic capture particles may be immobilized using a magnet and the magnet may pull the magnetic capture particles across a straight edge of the wells and into the wells. The magnet may continue to traverse across the array to fill all of the wells with the capture particles which may cause magnetic particles to move out of wells. However, movement of the magnet such that it moves from a straight edge (or another larger area) to a comer (or another smaller are) of the wells, minimizes exit of magnetic capture particles from the wells.

[0140] Filling of the wells with a substrate solution may be performed in a direction opposite to the direction of filling of the wells with a hydrophobic liquid such as an oil.

[0141] FIG. 4 shows examples of shapes of the pad region of the second plate for optimizing simultaneous aspiration of the hydrophilic liquid and introduction of hydrophobic liquid. The shape may also help in retaining the liquids in the chamber defined by the pad region and the bottom plate. The size of the pad region may be increased while retaining the original size of the array of wells. The area of the pad region around the first opening may be increased relative to the area of the pad region around the second opening to increase retention of the hydrophilic liquid in the chamber defined between the pad region and the bottom plate. The area of the pad region relative to the region occupied by the array of wells may be increased to increase retention of the hydrophobic liquid and the hydrophilic liquid in the chamber defined between the pad region and the bottom plate.

[0142] The sealing pad may be defined by a rectangle with at least one rounded end; the rounded end may define a circle having a diameter greater than the width of the rectangle. Both ends of the rectangle may be rounded and may each define circles having diameters greater than the width of the rectangle; one circle may have a greater diameter than the other, or the diameters may be the same. The circles may have the respective first or second opening as their center. The width of the sealing pad may also taper or suddenly change from one side to the other. For instance, the one rounded end may be larger than the other, and the width of the sealing pad may taper to join the larger circle to the smaller circle. FIG. 5 depicts exemplary protrusions that aid in aspiration of the hydrophilic liquid. These protrusions arc referred to as substrate retention features. In addition to the triangular or semi- triangular shape of the substrate retention features, other shapes and alternative position of the substrate retention features are also disclosed. These substrate retention features may extend partially or completely towards the bottom plate. The protrusions may partially occlude the first opening and extend to the bottom plate. The protrusions may support the first apparatus (e.g., a hollow tube).

[0143] FIG. 6 depicts exemplary substrate retention features that aid in aspiration of the hydrophilic liquid and / or limit aspiration of the hydrophobic liquid. For example, protrusions may be positioned to block flow of the hydrophobic liquid towards the first opening. Protrusions may be positioned to block flow of the hydrophobic liquid out of the chamber. These protrusions may be combined with the protrusions disposed at the first opening.

[0144] FIG. 7 depicts exemplary retention features and shapes of pad region for optimal aspiration of hydrophilic liquid and / or limit aspiration of the hydrophobic liquid and / or egress of the hydrophobic liquid from the chamber.

[0145] FIGS. 8A-8D show different paths for guiding the magnetic capture particles over the array of wells for reducing debris deposited over the array. In one embodiment depicted in FIG. 8A, the magnet guides the magnetic particles in a first direction straight across the array. In one embodiment FIG. 8B, the magnet guides the magnetic particles across a region of the chamber where the array is not present and positions the magnetic particles adjacent the array prior to guiding the magnetic particles over the array followed by removing any magnetic particles not deposited in the wells of the array in a direction perpendicular to which the magnetic particles were moved across the array. In one embodiment FIG. 8C, two side walls may also be included on either side of the array to increase retention of the liquids in the chamber. In another example FIG. 8D, the magnet drags the magnetic particles to a location under the first opening, then across the array of wells to a location under the second opening, and finally to a location outside the sealing pad.

[0146] FIG. 9A provides a schematic of flow rate of hydrophilic liquid (e.g., a substrate solution) during aspiration and flow rate of hydrophobic liquid (e.g., oil) during introduction of the hydrophobic liquid into the chamber. FIG. 9B shows position of the probe for injecting the hydrophobic liquid (e.g., oil) and position of the probe for aspirating the hydrophilic liquid (e.g., a substrate solution) relative to the distance from the upper surface of the second plate. This schematic also shows that the diameter of the probe for dispensing the hydrophobic liquid (e.g., oil) is smaller than the diameter of the probe for dispensing the hydrophilic liquid (e.g., a substrate solution).

[0147] FIG. 10A is a schematic of contact angle of the hydrophobic liquid relative to the surface of the lower plate. A hydrophobic liquid with an obtuse contact angle (due to low affinity for the surface) tends to seal the wells without displacing the hydrophilic liquid (e.g., a substrate solution) present in the wells. A hydrophobic liquid with an acute contact angle (due to higher affinity for the surface) tends to displace the hydrophilic liquid (e.g., a substrate solution) present in the wells.

[0148] FIG. 10B shows volume of hydrophilic liquid (e.g., a substrate solution) remaining in the wells over time. Sealing of wells with a hydrophobic liquid having a low affinity for the surface and thus having an obtuse contact angle relative to the surface results in more retention of the hydrophilic liquid in the wells as compared to the retention of the hydrophilic liquid in the wells upon sealing with a hydrophobic liquid having a high affinity for the surface and thus having an acute contact angle relative to the surface.

[0149] FIG. IOC shows the relationship between contact angle and fluorescence detected from the wells for fluorinated hydrophobic liquids (Novcc 7500, FC-40, and Galdcn HT200) and nonfluorinated hydrophobic liquids (soybean oil, Silicone 317667, Silicone 378356). Fluorinated hydrophobic liquids do not displace the substrate solution from the wells.

[0150] FIG. 11A shows submerged contact angle (SCA) for a hydrophobic liquid suitable as a sealing liquid. FIG. 11B shows SCA for a hydrophobic liquid unsuitable as a sealing liquid. FIG. 11C provides an illustration of relationship between contact angle and sealing of a well.

[0151] Systems and Kits

[0152] A system and a kit for seeding and sealing capture particles in wells of an array are disclosed. The system may include the device as described herein, a first apparatus for introducing an aqueous liquid into the chamber and for removing the aqueous liquid from the chamber, and a second apparatus for introducing a hydrophobic liquid into the chamber. , The system may include a smart device for controlling the first and the second apparatus for simultaneous removal of the aqueous liquid from the chamber and introduction of the hydrophobic liquid into the chamber. The small device may be a local computer or a remote computer or another means for controlling the operations of the first and second apparatus. For example, one or more pneumatic instrument for controlling the first apparatus, the second apparatus, or both may be connected to a computer chip designed to operate the first apparatus to release the aqueous liquid into the chamber of the device, aspirate the released aqueous liquid after a period of time and release the hydrophobic liquid into chamber of the device. The pneumatic instrument may also control the flow rate of the hydrophobic liquid and the aqueous liquid.

[0153] The system may be configured into an instrument, e.g., a tabletop instrument. The instrument may be configured to house additional components for conducting seeding and sealing, e.g., capture particles, aqueous liquid, hydrophobic liquid, etc. The instrument may additionally include components for processing a sample and generating the capture particles that include a target analyte, if the analyte is present in the sample. The device for seeding and sealing the capture particles may include a region for processing a sample such that a target analyte if present is immobilized by the capture particles.

[0154] The system may include a source for magnetic force that is used for moving paramagnetic capture particles into the array of wells. The source may be a movable magnet or electric coils that generate magnetic field or another means for generating magnetic force.

[0155] The system may include an imaging device for imaging the array of wells to detect presence or absence of the target analyte. The system may include components for reducing air bubbles in the wells, e.g., a heater, a vacuum generator, a cooling unit, a degasser, etc.

[0156] A kit for seeding and sealing capture particles in wells of an array is disclosed. The kit may include disposable components for performing a method of seeding and sealing capture particles in wells of an array. The kit may include the device comprising the array of wells, as described herein. The kit may also include one or more of capture particles, hydrophobic liquid, and aqueous liquid, etc. Method for Seeding and Sealing Capture Particles

[0157] A method for seeding and sealing wells of an array in the device of the present disclosure and using the system or kit of the present disclosure is provided. The device of the present disclosure is referred to as the subject device and the system and kit of the present disclosure are referred to as subject system and subject kit, respectively. The method may comprise guiding capture particles to the array located in the subject device; flowing an aqueous liquid over the array to fdl the wells; simultaneously aspirating the aqueous liquid and flowing the hydrophobic liquid over the array, wherein aspirating the aqueous liquid comprises positioning a first hollow tube in the first opening and applying suction pressure, wherein flowing the hydrophobic liquid comprises positioning a second hollow tube in the second opening and introducing the hydrophobic liquid in the chamber. The aspiration of the aqueous liquid facilitates spreading of the hydrophobic liquid over the array, and wherein the protrusions positioned around the first opening facilitate removal of the aqueous liquid while substantially preventing removal of the hydrophobic liquid. FIG. 16 provides a schematic for a method of seeding and sealing wells of an array in the device. Step 1 may involve filling the wells with a substrate solution. The substrate solution may be a hydrophilic liquid, e.g., an assay buffer. The substrate solution may be an assay buffer with the substrate added to the assay buffer. Step 2 may involve guiding capture particles (e.g., PMPs) into the array of wells. In FIG. 16, the PMPs comprise a mixture of two types of PMPs. Functionalized PMPs that can bind to a target analyte and helper PMPs that are not functionalized and do not bind to the target analyte. The helper PMPs facilitate movement of the functionalized PMPs. Step 3 may involve pushing away substrate solution and PMPs not disposed in the wells by flowing a hydrophobic liquid over the array of wells and sealing the wells with the hydrophobic liquid.

[0158] An embodiment of the method is depicted in FIG. 2A. Actual images taken during filling of the chamber in the device show introduction of an aqueous liquid into the chamber which liquid has spread over the array of wells (see top panel, a drawing of the image is shown on the left). The 2ndfrom the top panel shows aspiration of the aqueous liquid and introduction of the hydrophobic liquid. The hydrophobic liquid overtakes the aqueous liquid and surrounds the aqueous liquid (see 3rdpanel). The substrate retention features facilitate removal of the remainder of the aqueous liquid while limiting removal of the hydrophobic liquid (bottom panel). Fig. 9A shows that there is partial overlap in aspiration of the aqueous liquid (e.g., substrate solution) and introduction of the hydrophobic liquid (e.g., oil). The time period of overlap may be determined empirically based on the type of aqueous liquid and / or hydrophobic liquid and the aspiration rate and / or dispense rate.

[0159] The aqueous liquid may be an assay buffer. The assay buffer may include a buffer that facilitates generation of a signal from the capture particles. The assay buffer may include a substrate for an enzyme immobilized on the capture particles.

[0160] The capture particles may be paramagnetic particles and guiding capture particles to the array may comprise immobilizing the capture particles with a magnet positioned underneath the second plate and moving the magnet to the array of wells. The capture particles may be nonmagnetic and guiding capture particles to the array may comprise flowing the capture particles over the array of wells and allowing the capture particles to settle into the wells under gravitational force.

[0161] The first hollow tube may contact the protrusions extending into the first opening. The protrusions may be sized to support the hollow tube and prevent it from going through the opening.

[0162] Methods for Reducing Air Bubbles in Wells

[0163] The methods of seeding and sealing as described herein, e.g., in the preceding section, may be modified to reduce air bubbles in the device, e.g., in the wells. Such methods may include, after guiding the capture particles to the array and prior to flowing an aqueous liquid over the array to fill the wells, heating the device to temperature higher than room temperature such that the device has a temperature higher than the temperature of the aqueous liquid for filling the wells. Alternatively, or in addition, the method comprises after guiding the capture particles to the array and prior to flowing an aqueous liquid over the array to fill the wells, heating the aqueous liquid to a temperature higher than room temperature. These steps aid in reducing air bubbles in the wells. Heating the device and / or the aqueous liquid reduces surface tension of the liquid and contact angle formed between the air-liquid interface and the device. This change in liquid- surface interaction makes it easier for the liquid to fill individual wells, preventing the likelihood of trapped air. The device may be heated to a temperature of about up to 30°C, up to 50°C, up to 60°C, up to 70°C, up to 80°C, up to 90°C, or up to 100°C. The aqueous liquid may be heated to a temperature of about up to 30°C, up to 50°C, up to 60°C, up to 70°C, up to 80°C, up to 90°C, or up to 100°C.

[0164] The method may comprise degassing the aqueous liquid prior to flowing the aqueous liquid over the array to fill the wells. Once the liquid fills the wells, it is less likely to outgas (produce bubbles) in wells. A degassed liquid is more soluble to air. Thus, beginning with a degassed liquid allows for a greater level of air absorption after filling the reaction chamber. This reduces the prevalence of air bubbles in wells. This step of reducing bubbles in the wells may be combined with one or more of the above-listed steps for reducing air bubbles or may be performed alone.

[0165] Degassing the aqueous liquid may comprise subjecting the aqueous liquid to vacuum for a time period sufficient to reduce air dissolved in the aqueous liquid. For example, the aqueous liquid may be degassed under vacuum for about at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, and up to 30 minutes. The assay buffer may be held in vacuum for a period of time sufficient to reduce the amount of dissolved air by about at least 30%, at least 50%, or at least 75%. The degassed liquid may be held under vacuum up till the point of introducing it into the device.

[0166] Degassing the aqueous liquid may comprise holding it at a high temperature for a period of time. The high temperature may range from about 35°C-95°C, c.g., about 40°C-90°C, 45°C- 85°C, 50°C-80°C, or 50°C-70°C. The period of time may be varied depending on the volume of the assay buffer and may vary from about 30 seconds to 1 hour. The degassed liquid may be held under vacuum and / or a higher than room temperature up till the point of introducing it into the device.

[0167] The method may comprise following the steps of guiding capture particles to the array and flowing the aqueous liquid over the array to fill the wells, cooling the array of wells to a temperature below room temperature. This is another method for reducing the air bubbles in the wells. Cooling the device after filling the wells with the aqueous liquid can increase the likelihood that the air dissolves into the liquid. Reducing the temperature of the liquid increases its solubility to air, making it more likely to absorb the air trapped in wells. Cooling the device can be implemented by using a thermoelectric cooling instrument. The device may be cooled to a temperature of down to about 15°C, to 10°C, to 5°C, to 0°C. In some cases, lowering the temperature of the array of wells may include placing the array in a cooled environment or on a cooled surface or both. The cooled environment may be a refrigerator, a cold room, thermoelectric cooling instrument and the like. The cooled surface may be a metal surface or ice. The cooled environment or on a cooled surface may have a temperature lower than about 20°C, e.g., about 20°C-10°C, 20°C-4°C, or 20°C-0°C. The array may be placed in the cooled environment or on the cooled surface for a period of time sufficient to lower the temperature of the wells to lower than 20°C-10°C 20°C, e.g., 20°C-10°C 20°C-0°C. For example, the period of time may be 20°C-10°C up to 35 min, e.g., 10 seconds to 25 minutes, 30 seconds to 20 minutes, 1 minute to 15 minutes, 10 minutes to 15 minutes.

[0168] One or more of these methods for reducing air bubbles in the wells may be performed to improve detection of signal indicative of presence of an analyte of interest.

[0169] The subject devices, subject systems, and subject kits may be used in a method for detecting presence of an analyte immobilized on capture particles, the method comprising: seeding and sealing the capture particles by a method as described herein and detecting presence of a signal from the array of wells, where the presence of the signal is indicative of the presence of an analyte immobilized on capture particles. The signal may be any detectable signal, e.g., a fluorescent signal.

[0170] Methods for Pre-filling, Seeding and Sealing Capture particles

[0171] Also provided herein are alternate methods for seeding and sealing the array of wells in the devices disclosed herein. The sealed wells may be imaged for determining presence or absence of a signal from the wells and determining presence or absence of an analyte.

[0172] Methods for detecting presence of an analyte immobilized on capture particles is provided. The method may include flowing an assay buffer over an array of wells; depositing the capture particles over the array of wells, wherein the wells in the array are sized to hold a single capture particle per well; flowing a substrate solution over the array of wells; removing the substrate solution and flowing a hydrophobic liquid over the array of wells to cover the array of wells with the hydrophobic liquid; and detecting presence of a signal from the array of wells, wherein the presence of the signal is indicative of the presence of an analyte immobilized on capture particles. Removing the substrate solution and flowing the hydrophobic liquid over the array of wells to cover the array of wells with the hydrophobic liquid may be performed simultaneously.

[0173] The capture particles may be magnetic particles. The capture particles may be as set forth in the preceding section.

[0174] The analyte may be associated with an enzyme that converts the substrate into a product that emits a detectable signal. The analyte may be associated with an alkaline phosphatase enzyme. The assay buffer may not include the substrate. The assay buffer may include diethylamine (DEA). The assay buffer includes diethylamine-HCl. The assay buffer may include IM-30 mM DEA- HC1, e.g., IM-750 mM DEA-HC1, 750 mM -500 mM DEA-HC1, 500 nM-300 mM DEA-HC1, 300 mM-250 mM DEA-HC1, 250 mM -200 mM DEA-HC1, 200 mM-150 mM DEA-HC1, 150 m-100 mM DEA-HC1, 100 mM-75 mM, 75 mM-50 mM, or 50 mM-30 mM. The assay buffer may include IM-30 mM DEA-HC1 and MgCh. For example, the assay buffer may include 10 mM-0.1 mM MgCh, e.g., 10 mM-5 mM MgCh, 5 mM- 2 mM MgCh, 2 mM-1 mM MgCh, 1 mM-0.5 mM MgCh, or 0.5 mM-0.1 mM MgCh. The pH of the buffer may be alkaline, e.g., 8.5-11, 9-10.5, or 9-10.

[0175] Flowing the assay buffer over the array of wells may comprise passively flowing the assay buffer in a space over the array of wells, wherein the space may be defined by a plate positioned above the array of wells in a spaced-apart manner, as described with reference to the device in the preceding section.

[0176] Flowing the assay buffer over the array of wells may comprise injecting the assay buffer in a space over the array of wells, wherein the space may be defined by a plate positioned above the array of wells in a spaced-apart manner, as described with reference to the device in the preceding section.

[0177] Depositing the capture particles over the array of wells may comprise passively flowing the capture particles in a space over the array of wells, wherein the space may be defined by a plate positioned above the array of wells in a spaced-apart manner. Depositing the capture particles over the array of wells may comprise injecting the capture particles in a space over the array of wells, wherein the space may be defined by a plate positioned above the array of wells in a spaced-apart manner.

[0178] The capture particles may be magnetic and depositing the capture particles over the array of wells may comprise applying a magnetic force to the magnetic capture particles to pull them into the wells in the array.

[0179] The capture particles are magnetic and wherein depositing the capture particles over the array of wells may comprise passively flowing or injecting the capture particles in a space over the array of wells, wherein the space may be defined by a plate positioned above the array of wells in a spaced-apart manner and applying a magnetic force to the magnetic capture particles to guide them into the wells in the array.

[0180] Flowing the substrate solution over the array of wells may comprise passively flowing or injecting the substrate solution in a space over the array of wells, wherein the space may be defined by a plate positioned above the array of wells in a spaced-apart manner.

[0181] Simultaneously removing the substrate solution and flowing the hydrophobic liquid over the array of wells to cover the array of wells with the hydrophobic liquid may comprise aspirating the substrate solution and injecting the hydrophobic liquid.

[0182] The array of wells are present in a bottom plate of an assay device, wherein the device may comprise a top plate positioned in a spaced-apart manner from the array of wells, wherein the top plate defines a space between openings of the array of wells and a lower surface of the top plate and wherein the space may be open on all other sides other than the upper and lower sides.

[0183] The top plate may comprise a first opening positioned adjacent to a first side of the array of wells, wherein the first opening extends from the upper surface to the lower surface of the top plate and wherein the lower surface of the top plate may comprise a plurality of protrusions positioned around the circumference of the first opening, wherein the substrate solution enters the space over the array of wells through the first opening and is removed through the first opening and wherein the protrusions are configured to prevent the hydrophobic liquid from reaching the opening before the substrate solution is removed; and a second opening positioned adjacent to a second side of the array of wells, wherein the second side is opposite to the first side, wherein the hydrophobic liquid is flowed over the array of wells through the second opening.

[0184] The top plate may comprise another opening positioned adjacent to the array of wells, wherein the opening extends from the upper surface to the lower surface of the top plate and wherein the lower surface of the top plate may comprise a plurality of protrusions positioned around the circumference of the opening, wherein the substrate solution enters the space over the array of wells through the opening and is removed through the opening and wherein the protrusions are configured to prevent the hydrophobic liquid from reaching the opening before the substrate solution is removed.

[0185] The method described herein may be performed using the device, means for introducing and aspirating liquids, capture particles, etc., as described in the preceding section.

[0186] Method for reducing air bubbles in the subject device are discussed in the foregoing section. These methods can also be used for reducing formation of air bubbles in the wells of an array in any device. Air bubbles can interfere with imaging of the wells by creating artifacts that can lead to erroneous conclusions regarding presence or absence of the target analyte or erroneous calculation of analyte concentration.

[0187] A method for reducing air bubbles in the wells may include treating liquids that will be introduced into the wells. For example, the assay buffer may be subjected to a treatment to reduce the amount of air present in the assay buffer. Air may be dissolved in the assay buffer and can be removed to substantially reduce the amount of air in the assay buffer by subjecting the assay buffer to a degassing procedure prior to introducing into a device comprising an array of wells. Degassed assay buffer can absorb the air present in the wells at a faster rate than assay buffer not subjected to degassing, thereby removing air from the wells and thus reducing generation of air bubbles in the wells when the wells are filled with the assay buffer as well as removing any air bubbles present in the wells prior to filling the wells with the assay buffer. The degassing procedure can include holding the assay buffer at a high temperature for a period of time. The degassing procedure can include subjecting the assay buffer to vacuum prior to introducing it into a device comprising an array of wells. The assay buffer may be held in vacuum for a period of time sufficient to reduce the amount of dissolved air by at least 30%, at least 50%, or at least 75%. A method for reducing air bubbles in the wells may include lowering the temperature of the array of wells.

[0188] Exemplary Hydrophobic Liquids

[0189] Any inert hydrophobic liquid which does not substantially interfere with generation of detectable signal from the capture particles if analytes are immobilized on the capture particles may be used in the devices and methods provided herein. Any inert hydrophobic liquid which does not mix with the hydrophilic liquids used in the devices and methods of the present disclosure may be used.

[0190] The hydrophobic liquid may be selected based on its contact angle with a glass, Cyclic olefin copolymer, polypropylene and polyethylene or PDMS surface. , The hydrophobic liquid may be selected for having a contact angle with the material used for the first and second plates of the device disclosed herein. The hydrophobic liquid that has a low affinity for the surface of the first and second plates may be selected. For example, the contact angle of a hydrophobic liquid used for sealing the wells may be obtuse, e.g., greater than 80 degrees. , The hydrophobic liquid may be selected based on its low affinity for water to decrease mixing of the hydrophobic liquid with the substrate solution. The hydrophobic liquid may be an oil. The hydrophobic liquid may be 3M FC-40 oil, a hydrocarbon oil, a vegetable oil, or silicone liquids (e.g., a silicone oil). The oil may be a fluorocarbon oil. The oil may be Novcc 7500, FC-40, or Galdcn HT200. The hydrophobic liquid may be selected from Table 1:

[0191] Table 1: Hydrophobic liquid

[0192] A hydrophobic liquid for sealing the array of wells may be selected based on its submerged contact angle (SC A). Use of SCA is more suitable for identifying a sealing liquid that has properties that enable it to displace a hydrophilic liquid remaining on top of the array of wells and outside the wells but to not displace the hydrophilic liquid present inside the wells. SCA may be calculated by covering a substrate (e.g., a substrate with an array of wells) with a hydrophilic liquid (e.g., an assay buffer), depositing a droplet of the hydrophobic liquid over a substrate, and measuring the angle between the substrate and curvature of the droplet. SCA may be measured by any suitable device for measuring contact angles, e.g., optical tensiometer.

[0193] The hydrophobic liquids described in the present disclosure are commercially available. For example, silicone oil may be obtained from Sigma, Dow Chemicals, Gelest Inc., etc.

[0194] FIG. 11 A shows SCA for a hydrophobic liquid suitable as a sealing liquid. FIG. 1 IB shows SCA for a hydrophobic liquid unsuitable as a sealing liquid. FIG. 11C provides an illustration of relationship between contact angle and sealing of a well.

[0195] Based on SCA, the sealing liquid may be an oil with a SCA of 35° or higher. Such oils include, fluorinated oil, such as, FC-40. While FC-40 has a high SCA, it is potentially environmentally hazardous. As described in the Examples section, siloxane-based or silicone- based oils, such as, the following types of oils have been discovered as replacements for fluorinated oils: Polydimethylsiloxane (PDMS), Disiloxane, Triphenylnonamethylpentasiloxane, Phenyltris(trimethylxiloxy)silane, and Diphenyl-dimethylsiloxane copolymer. In certain examples, the oil may be silicone oil. In certain examples, the SCA may be further increased by changing the composition of the assay buffer, as discussed in the next section.

[0196] Exemplary Aqueous Liquids

[0197] Any aqueous / hydrophilic liquid that is compatible with the generation and / or detection of the detectable signal from the capture analyte may be used in the devices, system, kits, and methods described herein. In certain cases, the aqueous liquid has sufficient hydrophilicity for wetting the array of wells such that the aqueous liquid fills the wells.

[0198] The aqueous liquid may be an assay buffer that does not include a substrate for an enzyme associated with the capture particles. In certain cases, the aqueous liquid may be a substrate solution that includes the substrate that the enzyme, bound to the analyte bound to the capture particles, converts into a detectable product. Formulations of assay buffer and substrate solution may be as known in the field.

[0199] The substrate solution may include twice the concentration of the substrate as compared to seeding and sealing methods that do not involve pre- wetting of the array of wells with the assay buffer.

[0200] The aqueous liquid may be an assay buffer with or without a substrate and may be formulated to increase the SCA of the hydrophobic liquid for sealing the wells and / or decrease aggregation of the capture particles. Effect of formulation of assay buffer on SCA of the hydrophobic liquid for sealing the array of wells is shown in FIGS. 12A-12C.

[0201] The aqueous liquid may be an assay buffer with or without a substrate and may be formulated to decrease aggregation of the capture particles. For example, the assay buffer may include a defoaming agent, e.g., a silicone-based defoamer. Examples of silicone-based defoamers include silicone emulsion or polysiloxane emulsion. Silicone emulsion or polysiloxane emulsion may have 45% silicone. Silicone emulsion or polysiloxane emulsion may have 45% silicone and may be water soluble. Silicone emulsion or poly siloxane emulsion may have 45% silicone, specifically, a mixture of octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5) and dodecamethylcyclohexasiloxane (D6). Silicone emulsion or polysiloxane emulsion may be present in the assay buffer at a concentration of about 1 to about 500 parts per million (ppm) or mg / L, e.g., about 5 to about 250 ppm or mg / L, about 10 to about200 ppm or mg / L, about 5 to about200 ppm or mg / L, or about 5 to about 100 ppm or mg / L. Korasilon® may be Korasilon® EMA 119 (available from Kurt Obermeier GmBH). Korasilon® EMA 119 is an known emulsified antifoaming agent comprising about 45% silicone with a viscosity between about 1,500 to about 2,000 mPa s (millipascal second) at 25°C, and a pH of approximately 8. As mentioned, Korasilon® EMA 119 comprises about 45% silicone, specifically, a mixture of octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5) and dodecamethylcyclohexasiloxane (D6) as shown in FIG. 20. Korasilon® may be another Korasilon® defoamer available from Kurt Obermeier GmBH, e.g., Korasilon® FG 10, FG 20, FG 30, FT 10N, FT 20N, FT 30N, GD, GD 20, GD 30, CFT, VP 70, VP 90, EMA 124, AF 9010E, AF 9020E, AF 9030E, or SAG 7133.

[0202] The polysiloxane emulsion may comprises Korasilon®. Korasilon® may be present in the assay buffer at a concentration of from about 1 to about 500 parts per million (ppm) or mg / L, e.g., from about 5 to about 250 ppm or mg / L, from about 10 to about200 ppm or mg / L, from about 5 to about 200 ppm or mg / L, or from about 5 to about 100 ppm or mg / L. Korasilon® may be Korasilon® EMA 119.

[0203] Korasilon® EMA 119 may be added at a concentration effective to increase the SC A of the hydrophobic liquid for sealing the wells, e.g., silicone oil. For example, Korasilon® EMA 1 19 may be present in the assay buffer at a concentration of from about 1 to aboutlOO parts per million (ppm) or mg / L, e.g., from about 1 to about 50 ppm or mg / L, from about 1 to about 30 ppm or mg / L, from about 1 to about 20 ppm or mg / L, or from about 5 to about 10 ppm or mg / L. In certain cases, the assay buffer may include, in addition to Korasilon® EMA 119, a substrate for an enzyme associated with the capture particle when the capture particle is bound to an analyte. The assay buffer additionally includes a buffer suitable for assaying enzymatic activity of the enzyme.

[0204] Korasilon® EMA 119 may be added to the assay buffer to reduce the aggregation of the capture particles, e.g., PMPs. Aggregation of PMPs may be reduced by decreasing the energy required to dissipate the aggregated particles. The dissipated PMPs may then be deposited into the wells or may be removed when the sealing oil flows over the array of wells. In certain cases, Korasilon® EMA 119 may be added to the assay buffer, e.g., substrate solution at from about 10 ppm to about 500 ppm, e.g., from about 10 to about 250 ppm, from about 10 to about 200 ppm, from about 10 to aboutl50 ppm, or from about 10 to aboutlOO ppm.

[0205] The analyte bound to the capture particles may be associated with an enzyme that converts the substrate into a product that emits a detectable signal. The analyte may be associated with an alkaline phosphatase enzyme. The analyte may be associated with a calf alkaline phosphatase enzyme (CIAP). The pH of the buffer may be alkaline, e.g., 8.5-11, 9-10.5, or 9-10. The assay buffer may include from about 10 mM to about 0.1 mM MgCh, e.g., from about 10 mM to about 5 mM MgCh, from about 5 mM to about 2 mM MgCh, from about 2 mM to about 1 mM MgCh, from about 1 mM to about 0.5 mM MgCh, or from about 0.5 mM to about 0.1 mM MgCh.

[0206] The assay buffer may not include the substrate. The assay buffer may include diethylamine (DEA). The assay buffer may include diethylamine-HCl. The assay buffer may include from about IM to about30 mM DEA-HC1, e.g., from about IM to about 750 mM DEA-HC1, from about 750 mM to about 500 mM DEA-HC1, from about 500 nM to about 300 mM DEA-HC1, from about 300 mM to about250 mM DEA-HC1, from about 250 mM to about 200 mM DEA-HC1, from about 200 mM to about 150 mM DEA-HC1, from about 150 mM to about 100 mM DEA-HC1, from about 100 mM to about75 mM, from about 75 mM to about50 mM, or from about 50 mM to about 30 mM. The assay buffer may include from about IM to about30 mM DEA-HC1 and MgCh. For example, the assay buffer may include from about 10 mM to about 0.1 mM MgCh, e.g., from about 10 mM to about 5 mM MgCh, from about 5 mM to about 2 mM MgCh, from about 2 mM to aboutl mM MgCh, from about 1 mM to about 0.5 mM MgCh, or from about 0.5 mM to about 0.1 mM MgCh. The assay buffer may include from about IM to about 30 mM DEA-HC1, from about 10 mM to aboutO.l mM MgCh, and from about 1 to about50 ppm or mg / L, from about 1 to about30 ppm or mg / L, from about 1 to about 20 ppm or mg / L, or from about 5 to about 10 ppm or mg / L Karosilon®.

[0207] An exemplary assay buffer may include triethylamine (TEA), tris(hydroxymethyl)aminomethane (Tris) base, Tris HO, MgCh, NaCl, and Korasilon®. For example, in the assay buffer, the TEA may have a concentration of from about 0.5 mM to about 5mM, e.g., about 2 mM, Tris base may have a concentration of from about 100 to about300 mM, e.g., from about 200 to about250 mM, Tris HO may have a concentration of from about 20 to about30mM, MgCh may have a concentration of from about 0.5 mM to about 4 mM, e.g., 2-3 mM. Korasilon® may be Korasilon® EMA 119. Korasilon® may have a concentration of from about 1 to about50 ppm, e.g., from about 5 to about 20 ppm, such as, about 10 ppm. The assay buffer may include preservatives, e.g., sodium azide and / or levamisole. The assay buffer may include from about 10 to about 20 mM sodium azide and from about 1 to aboutlOmM levamisole.

[0208] Another exemplary assay buffer may include triethylamine (TEA), Tris base, Tris HC1, MgCh, and Korasilon®. For example, in the assay buffer, the TEA may have a concentration of from about 0.5 mM to about 5mM, e.g., about 2 mM, Tris base may have a concentration of from bout 50 to about200 mM, e.g., from about 70 to about 100 mM, Tris HC1 may have a concentration of from about 1 to about 20mM, MgCh may have a concentration of from about 0.5 mM to about 10 mM, e.g., about 5 mM and Korasilon® EMA 119. Korasilon® EMA 119 may have a concentration of from about 1 to about50 ppm, e.g., about 10 ppm. The assay buffer may include preservatives, e.g., sodium azide and / or levamisole. The assay buffer may include from aboutlO to about20 mM sodium azide and from about 1 to about lOmM levamisole.

[0209] Another exemplary assay buffer may include triethylamine (TEA), Tris base, Tris HC1, MgCh, and Korasilon®. For example, in the assay buffer, the TEA may have a concentration of from about 0.5 mM to about 5mM, e.g., about 2 mM, Tris base may have a concentration of from about 50 to about 200 mM, e.g., from about 70 to about 100 mM, Tris HC1 may have a concentration of from about 1 to about 20mM, MgCh may have a concentration of from about 0.5 mM to about 10 mM, e.g., about 5 mM and Korasilon® EMA 119. Korasilon® EMA 119 may have a concentration of from about 1 to about 500 ppm, e.g., from about 10 to about 100 ppm. The assay buffer may include Proclin 950 (e.g., from about 0.1 to about 0.2%). The assay buffer may include preservatives, e.g., sodium azide and / or levamisole. The assay buffer may include from about 10 to about 20 mM sodium azide.

[0210] Another exemplary assay buffer may include tris(hydroxymethyl)aminomethane (Tris), magnesium chloride (MgCh), and Korasilon® EMA 119 (an emulsified antifoaming agent comprising about 45% silicone with a viscosity between about 1,500 to about 2,000 rnPa s (millipascal second) at 25 °C, and a pH of approximately 8. As mentioned, Korasilon® EMA 119 comprises about 45% silicone, specifically, a mixture of octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5) and dodecamethylcyclohexasiloxane (D6) as shown in FIG. 20.). The buffer may have a pH from about 8.5 to about 9.5 and may contain from about 100 to about 250 mM of Tris, from about 5 to about 10 mM of MgCh, and from about 1 pp to about 10 ppm of Korasilon® EMA 119. The diluent may have a pH from about 9.0 to about 9.25. The diluent may further comprise from about 0.001 to about 5 mM of levamisole. The diluent may further comprise from 0.001 to about 400 nM of sodium chloride (NaCl). The diluent may further comprise levamisole and sodium chloride. The diluent may not comprise any levamisole. The diluent may not comprise any sodium chloride. The diluent may not comprise any levamisole or any sodium chloride.

[0211] The effect of the formulation of the assay buffer on SCA of the hydrophobic liquid for sealing the array of wells is shown in FIGS. 12A-12C. FIG. 12 A. SCA of the listed oils was measured using LC assay buffer. LC assay buffer comprises 2 mM TEA, 89.498 mM Tris Base, 10.51 Tris HC1, 5mM MgCh, 0.1% Sodium Azide, 10 ppm Korasilon®. FIG. 12 B. Effect of assay buffer formulation on SCA of silicone oil. ABJ assay buffer, “ABJ substrate with Korasilon® (24- 0560A)” substantially increased SCA of silicone oil. ABJ assay buffer comprises: 2 mM TEA, 223.72 mM Tris base, 26.28 mM Tris HCL (250 mM Tris), 1 mM MgCh, 12.9 mM sodium azide, 50 mM NaCl, 5 mM levamisole, 10 ppm Korasilon®. LC assay buffer “Original LC substrate (23- 139D)” comprises 2 mM TEA, 89.498 mM Tris Base, 10.51 Tris HC1, 5mM MgCh, 0.1% (w / v) sodium azide, 10 ppm Korasilon®. New LC buffer “Newer LC substrate+antimicrobials (099-B)” may comprise 2 mM TEA, 89.498 mM Tris Base, 10.51 Tris HO, 5mM MgCh, 0.15% Proclin 950, 10 ppm Korasilon® and 0.1% sodium azide. FIG. 12C shows that when using ABJ assay buffer, the performance of silicone oil is better than FC-40. Thus, silicone oil can replace FC-40 without negatively impacting assay performance.

[0212] The assay buffers of the present disclosure may include a substrate that fluoresces upon contact with an enzyme. The substrate may be pyranine and / or pyranine phosphate. The substrate, e.g., pyranine or pyranine phosphate, may be present at a concentration of from about 1 to about 5 mM, e.g., from about 1 to about 3 mM or about 2 mM.

[0213] Exemplary Target Analytes As will be appreciated by those in the art, any analyte that can be specifically bound by a first binding member and a second binding member may be detected and, optionally, quantified using methods and devices of the present disclosure.

[0214] The analyte may be a biomolecule. Non-limiting examples of biomolecules include macromolecules such as, proteins, lipids, and carbohydrates. In certain instances, the analyte may be hormones, antibodies, growth factors, cytokines, enzymes, receptors (e.g., neural, hormonal, nutrient, and cell surface receptors) or their ligands, cancer markers (e.g., PSA, TNF-alpha), markers of myocardial infarction (e.g., troponin, creatine kinase, and the like), toxins, drugs (e.g., drugs of addiction), metabolic agents (e.g., including vitamins), and the like. Non-limiting embodiments of protein analytes include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, or the like.

[0215] The analyte may be a post-translationally modified protein (e.g., phosphorylated, methylated, glycosylated protein) and the first or the second binding member may be an antibody specific to a post-translational modification. A modified protein may be bound to a first binding member immobilized on a solid support where the first binding member binds to the modified protein but not the unmodified protein. In other embodiments, the first binding member may bind to both the unmodified and the modified protein, and the second binding member may be specific to the post-translationally modified protein.

[0216] The analyte may be a cell, such as, circulating tumor cell, pathogenic bacteria, viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, Filoviruses (Ebola), hepatitis viruses (e.g., A, B, C, D, and E); HPV, etc.; spores, etc.

[0217] A non-limiting list of analytes that may be analyzed by the methods presented herein include B-type natriuretic peptide (BNP), N-terminal pro B-type natriuretic peptide (proBNP), A 42 amyloid beta-protein, fetuin-A, tau, secretogranin II, prion protein, Alpha-synuclein, tau protein, neurofilament light chain, parkin, PTEN induced putative kinase 1, DJ-1, leucine-rich repeat kinase 2, mutated ATP13A2, Apo H, ceruloplasmin, Peroxisome proliferator- activated receptor gamma coactivator- 1 alpha (PGC-la), transthyretin, Vitamin D-binding Protein, proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR), CXCL13, IL-12p40, CXCL13, IL-8, Dkk-3 (semen), pl 4 endocan fragment, Serum, ACE2, autoantibody to CD25, hTERT, CAI25 (MUC 16), VEGF, sIL-2, Ostcopontin, Human epididymis protein 4 (HE4), Alpha- Fetoprotein , Albumin, albuminuria, microalbuminuria, neutrophil gelatinase-associated lipocalin (NGAL) , interleukin 18 (IL- 18) , Kidney Injury Molecule -1 (KIM-1) , Liver Fatty Acid Binding Protein (L-FABP) , LMP1, BARF1, IL-8, carcinoembryonic antigen (CEA), BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, and LZTS1, alpha-amylase, carcinoembryonic antigen, CA 125, IL8 , thioredoxin, beta-2 microglobulin levels - monitor activity of the virus, tumor necrosis factor-alpha receptors - monitor activity of the virus, CA15-3, follicle-stimulating hormone (FSH), leutinizing hormone (LH), T-cell lymphoma invasion and metastasis 1 (TIAM1), N-cadherin, EC39, amphiregulin, dUTPase, secretory gelsolin (pGSN), PSA (prostate specific antigen), thymosin pi5, insulin, plasma C-peptide, glycosylated hemoglobin (HBAlc), C-Reactive Protein (CRP), Interleukin-6 (IL-6), ARHGDIB (Rho GDP-dissociation inhibitor 2), CFL1 (Cofilin-1), PFN1 (profilin-1), GSTP1 (Glutathione S-transferase P), S100A11 (Protein S100- All), PRDX6 (Peroxiredoxin-6), HSPE1 (10 kDa heat shock protein, mitochondrial), LYZ (Lysozyme C precursor), GPI (Glucose-6-phosphate isomerase), HIST2H2AA (Histone H2A type 2-A), GAPDH (Glyceraldehyde-3- phosphate dehydrogenase), HSPG2 (Basement membrane- specific heparan sulfate proteoglycan core protein precursor), LGALS3BP (Galectin-3-binding protein precursor), CTSD (Cathepsin D precursor), APOE (Apolipoprotein E precursor), IQGAP1 (Ras GTPase-activating-like protein IQGAP1), CP (Ceruloplasmin precursor), and IGLC2 (IGLC1 protein), PCDGF / GP88, EGFR, HER2, MUC4, IGF-IR, p27(kipl), Akt, HER3, HER4, PTEN, PIK3CA, SHIP, Grb2, Gab2, PDK-1 (3-phosphoinositide dependent protein kinase-1), TSC1, TSC2, mTOR, MIG-6 (ERBB receptor feedback inhibitor 1), S6K, src, KRAS, MEK mitogen- activated protein kinase 1, cMYC, TOPO II topoisomerase (DNA) II alpha 170 kDa, FRAP1, NRG1, ESRI, ESR2, PGR, CDKN1B, MAP2K1, NEDD4-1, FOXO3A, PPP1R1B, PXN, ELA2, CTNNB1, AR, EPHB2, KLF6, ANXA7, NKX3-1, PITX2, MKI67, PHLPP, adiponectin (ADIPOQ), fibrinogen alpha chain (FGA), leptin (LEP), advanced glycosylation end productspecific receptor (AGER aka RAGE), alpha-2-HS-glycoprotein (AHSG), angiogenin (ANG), CD14 molecule (CD14), ferritin (FTH1), insulin-like growth factor binding protein 1 (IGFBP1), interleukin 2 receptor, alpha (IL2RA), vascular cell adhesion molecule 1 (VCAM1) and Von Willebrand factor (VWF), myeloperoxidase (MPO), ILla, TNFa, perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA), lactoferrin, calprotectin, Wilm’s Tumor- 1 protein, Aquaporin- 1 , MLL3, AMBP, VDAC1 , E. coli enterotoxins (heat-labile exotoxin, heat-stable enterotoxin), influenza HA antigen, tetanus toxin, diphtheria toxin, botulinum toxins, Shiga toxin, Shiga-likc toxin I, Shiga-like toxin II, Clostridium difficile toxins A and B, Glial Fibrillary Acidic Protein (GFAP), Ubiquitin C-terminal hydrolase LI (UCH-L1), Hepatitis B e-antigen (HBeAg), Hepatitis B surface antigen (HBsAg), Hepatitis B core antigen (HBeAg), phosphorylated Hepatitis B core antigen (P-HBcAg), Hepatitis B core-related antigen (HBcrAg), etc.

[0218] Exemplary targets of nucleic acid aptamers that may be measured in a sample such as an environmental sample, a biological sample obtained from a patient or subject in need using the subject methods and devices include: drugs of abuse (e.g. cocaine), protein biomarkers (including, but not limited to, Nucleolin, nuclear factor-kB essential modulator (NEMO), CD-30, protein tyrosine kinase 7 (PTK7), vascular endothelial growth factor (VEGF), MUC1 glycoform, immunoglobulin p Heavy Chains (IGHM), Immunoglobulin E, avP3 integrin, a-thrombin, HIV gp!20, NF-KB, E2F transcription factor, HER3, Plasminogen activator inhibitor , Tenascin C,CXCL12 / SDF-1, prostate specific membrane antigen (PSMA), gastric cancer cells, HGC-27); cells (including, but not limited to, non-small cell lung cancer (NSCLC), colorectal cancer cells, (DLD-1), H23 lung adenocarcinoma cells, Ramos cells, T-cell acute lymphoblastic leukemia (T- ALL) cells, CCRF-CEM, acute myeloid leukemia (AML) cells (HL60), small-cell lung cancer (SCLC) cells, NCIH69, human glioblastoma cells, U118-MG, PC-3 cells, HER-2-overexpressing human breast cancer cells, SK-BR-3, pancreatic cancer cell line (Mia-PaCa-2)); and infectious agents (including, but not limited to, Mycobacterium tuberculosis, Staphylococcus aureus, Shigella dysenteriae, Escherichia coli O157:H7, Campylobacter jejuni, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella 08, Salmonella enteritidis).

[0219] Exemplary targets of protein or peptide aptamers that may be measured in a sample obtained from a patient or subject in need using the subject methods and devices include, but are not limited to: HBV core capsid protein, CDK2, E2F transcription factor, Thymidylate synthase, Ras, EB1, and Receptor for Advanced Glycated End products (RAGE). Aptamers, and use and methods of production thereof are reviewed in e.g., Shum et al., J Cancer Ther. 2013 4:872; Zhang et al., Curr Med Chem. 2011;18:4185; Zhu et al., Chem Commun (Camb). 2012 48: 10472; Crawford et al., Brief Funct Genomic Proteomic. 2003 2:72; Reverdatto et al., PLoS One. 2013 8:e6518O. The analyte may be present in a sample, such as, a liquid, fluent particulate solid, or fluid suspension of solid particles. The sample may be processed prior to the analysis described herein. For example, the analyte may be separated or purified from its source prior to analysis; however, an unprocessed analyte may be assayed directly. The source of the analyte molecule may be synthetic (e.g., produced in a laboratory), the environment (e.g., air, soil, fluid samples, e.g., water supplies, etc.), an animal, e.g., a mammal, a plant, or any combination thereof. In a particular example, the source of an analyte may be a human bodily substance (e.g., bodily fluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal fluid, feces, tissue, organ, or the like). Tissues may include, but are not limited to skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, myocardial tissue, brain tissue, bone marrow, cervix tissue, skin, etc. The source of the analyte may be an organ or tissue, such as a biopsy sample, which may be solubilized by tissue disintegration / cell lysis.

[0220] A wide range of volumes of the fluid samples may be analyzed. The sample volume may be about 0.5 nL, about 1 nL, about 3 nL, about 0.01 pL, about 0.1 pL, about 1 pL, about 5 pL, about 10 pL, about 100 pL, about 1 mL, about 5 mL, about 10 mL, or the like. The volume of the fluid analyte may be between about 0.01 pL and about 10 mL, between about 0.01 pL and about 1 mL, between about 0.01 pL and about 100 pL, or between about 0.1 pL and about 10 pL.

[0221] The fluid sample may be diluted prior to use in an assay. For example, in embodiments where the source of an analyte molecule is a human body fluid (e.g., blood, serum), the fluid sample may be diluted with an appropriate solvent (e.g., a buffer such as PBS buffer). A fluid sample may be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater, prior to use.

[0222] The sample may undergo pre-analytical processing. Pre-analytical processing may offer additional functionality such as nonspecific protein removal and / or effective yet cheaply implementable mixing functionality. General methods of pre-analytical processing may include the use of electrokinetic trapping, AC electrokinetics, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other pre-concentration techniques known in the ail. The fluid analyte may be concentrated prior to use in an assay. For example, in embodiments where the source of an analyte molecule is a human body fluid (e.g., blood, serum), the fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. A fluid sample may be concentrated about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5- fold, about 6-fold, about 10-fold, about 100-fold, or greater, prior to use.

[0223] The analyte may not be amplified (i.e., the copy number of the analyte is not increased) prior to the measurement of the analyte. For example, in cases where the analyte is DNA or RNA, the analyte is not replicated to increase copy numbers of the analyte. , The analyte may be a protein or a small molecule.

[0224] Binding Members

[0225] As will be appreciated by those in the art, the binding members will be determined by the analyte to be analyzed. Binding members for a wide variety of target molecules are known or can be readily found or developed using known techniques. For example, when the target analyte is a protein, the binding members may include proteins, particularly antibodies or fragments thereof (e.g., antigen-binding fragments (Fabs), Fab' fragments, F(ab')2 fragments, recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, such as variable heavy chain domains (“VHH”; also known as “VHH fragments”) derived from animals in the Camelidae family (VHH and methods of making them are described in Gottlin et al., Journal of Biomolecular Screening, 14:77-85 (2009)), recombinant VHH singledomain antibodies, and VNAR fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti- id”) antibodies, and functionally active epitope-binding fragments of any of the above, full-length polyclonal or monoclonal antibodies, antibody-like fragments, etc.), other proteins, such as receptor proteins, Protein A, Protein C, or the like. In case where the analyte is a small molecule, such as, steroids, bilins, retinoids, and lipids, the first and / or the second binding member may be a scaffold protein (e.g., lipocalins) or a receptor, the binding member for protein analytes may be a peptide. For example, when the target analyte is an enzyme, suitable binding members may include enzyme substrates and / or enzyme inhibitors which may be a peptide, a small molecule and the like. When the target analyte is a phosphorylated species, the binding members may comprise a phosphate-binding agent. For example, the phosphate-binding agent may comprise metal-ion affinity media such as those describe in U.S. Pat. No. 7,070,921 and U.S. Patent Application No. 20060121544. At least one of the binding members may be an aptamer, such as those described in U.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337. Nucleic acid aptamers (e.g., single-stranded DNA molecules or single-stranded RNA molecules) may be developed for capturing virtually any target molecule. Aptamers bind target molecules in a highly specific, conformation-dependent manner, typically with very high affinity, although aptamers with lower binding affinity can be selected. Aptamers may distinguish between target analyte molecules based on very small structural differences such as the presence or absence of a methyl or hydroxyl group and certain aptamers can distinguish between D- and L-enantiomers and diastereomers. Aptamers may bind small molecular targets, including drugs, metal ions, and organic dyes, peptides, biotin, and proteins. Aptamers can retain functional activity after biotinylation, fluorescein labeling, and when attached to glass surfaces and microspheres.

[0226] Nucleic acid aptamers are oligonucleotides that may be single stranded oligodeoxynucleotides, oligoribonucleotides, or modified oligodeoxynucleotide or oligoribonucleotides. Modified nucleotides encompass nucleotides with a covalently modified base and / or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus, modified nucleotides may also include 2' substituted sugars such as 2'-O-methyl-; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'- fluoro-; 2'-halo or 2-azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.

[0227] Peptide aptamers may be designed to interfere with protein interactions. Peptide aptamers may be based on a protein scaffold onto which a variable peptide loop is attached, thereby constraining the conformation of the aptamer. The scaffold portion of the peptide aptamer may be derived from Bacterial Thioredoxin A (TrxA).

[0228] When the target analyte is a carbohydrate, potentially suitable capture particles (as defined herein) include, for example, antibodies, lectins, and selectins. As will be appreciated by those of ordinary skill in the art, any molecule that can specifically associate with a target molecule of interest may potentially be used as a binding member. Suitable target analyte / binding member complexes can include, but are not limited to, antibodics / antigcns, antigcns / antibodics, rcccptors / ligands, ligands / rcccptors, protcins / nuclcic acid, enzymes / substrates and / or inhibitors, carbohydrates (including glycoproteins and glycolipids) / lectins and / or selectins, proteins / proteins, proteins / small molecules, etc.

[0229] The first binding member may be attached to a capture particle via a linkage, which may comprise any moiety, functionalization, or modification of the capture particle and / or binding member that facilitates the attachment of the binding member to the capture particle. The linkage between the binding member and the capture particle may include one or more chemical or physical (e.g., non-specific attachment via van der Waals forces, hydrogen bonding, electrostatic interactions, hydrophobic / hydrophilic interactions; etc.) bonds and / or chemical spacers providing such bond(s).

[0230] A capture particle may also comprise a protective, blocking, or passivating layer that can eliminate or minimize non-specific attachment of non-capture components (e.g., analyte molecules, binding members) to the binding surface during the assay which may lead to false positive signals during detection or to loss of signal. Examples of materials that may be utilized to form passivating layers include, but are not limited to: polymers, such as poly(ethylene glycol), that repel the non-specific binding of proteins; naturally occurring proteins with this property, such as serum albumin and casein; surfactants, e.g., zwitterionic surfactants, such as sulfobetaines; naturally occurring long-chain lipids; polymer brushes, and nucleic acids, such as salmon sperm DNA.

[0231] Certain embodiments utilize binding members that are proteins or polypeptides. As is known in the art, any number of techniques may be used to attach a polypeptide to a wide variety of solid supports. A wide variety of techniques are known to add reactive moieties to proteins, for example, the method outlined in U.S. Pat. No. 5,620,850. Further, methods for attachment of proteins to surfaces are known, for example, see Heller, Acc. Chem. Res. 23:128 (1990).

[0232] As explained herein, binding between the binding members and the analyte, is specific, e.g., as when the binding member and the analyte are complementary parts of a binding pair. The binding member may bind specifically to the analyte. The binding member may be chemically programmed antibodies (cpAbs) (described in Rader (2014) Trends in Biotechnology 32:186-197), bispccific cpAbs, antibody-recruiting molecules (ARMs) (described in McEnaney et al. (2012) ACS Chem. Biol. 7:1139-1151), branched capture agents, such as a triligand capture agent (described in Millward et al. (2011) J. Am. Chem. Soc. 133:18280-18288), engineered binding proteins derived from non-antibody scaffolds, such as monobodies (derived from the tenth fibronectin type III domain of human fibronectin), affibodies (derived from the immunoglobulin binding protein A), DARPins (based on Ankyrin repeat modules), anticalins (derived from the lipocalins bilin-binding protein and human lipocalin 2), and cysteine knot peptides (knottins) (described in Gilbreth and Koide, (2012) Current Opinion in Structural Biology 22:1-8; Banta et al. (2013) Annu. Rev. Biomed. Eng. 15:93- 113),WW domains (described in Patel et al. (2013) Protein Engineering, Design & Selection 26(4):307-314), repurposed receptor ligands, affitins (described in Behar et al. (2013) 26:267-275), and / or Adhirons (described in Tiede et al. (2014) Protein Engineering, Design & Selection 27 : 145- 155).

[0233] The binding affinity between analyte molecules and binding members may be sufficient to remain bound under the conditions of the assay, including wash steps to remove molecules or particles that are non-specifically bound. In some cases, for example in the detection of certain biomolecules, the binding constant of the analyte molecule to its complementary binding member may be between at least about 104and about 106M’1, at least about 105and about 109M’1, at least about 107and about 109M’1, greater than about 109M’1, or greater.

[0234] Assay Formats

[0235] The devices and methods of the present disclosure may be used for any assay suitable for detecting an analyte. The different types of assays include, without limitation, immunoassays, nucleic acid analysis, metabolite analysis, clinical chemistry, complete blood count (CBC), etc.

[0236] The device may be used to perform immunoassays. Any immunoassay may be utilized. The immunoassay may be an enzyme - linked immunoassay (ELISA), a competitive inhibition assay, such as forward or reverse competitive inhibition assays, or a competitive binding assay, for example. A detectable label (e.g., such as one or more fluorescent labels, one or more tags attached by a cleavable linker (which can be cleaved chemically or by photocleavage)) may be attached to the capture antibody and / or the detection antibody.

[0237] The device may be used to perform nucleic acid analysis. The device may employ various forms of nucleic acid analysis to detect analytes of interest, e.g., a nucleic acid, a non-nucleic acid containing a nucleic acid tag, or a nucleic acid produced from the analyte, including, without limitation, PCR, isothermal amplification, etc.

[0238] The device may be used to perform clinical chemistry. Clinical chemistry may involve detection of electrochemical species or chromogenic reaction product generated by action of an enzyme on a substrate. For example, the substrate may be an analyte present in a sample and the enzyme may be specific for the analyte and may catalytically react with the analyte to generate an electrochemical species or a colored reaction product. In other cases, clinical chemistry may involve capturing the analyte using a first binding member to generate a first complex comprising the analyte and the first binding member; contacting the complex with a second binding member, that binds to the analyte, to generate a second complex comprising the analyte, the first binding member, and the second binding member. The second binding member is conjugated to an enzyme that generates an electrochemical species or chromogenic reaction product upon exposure to a suitable substrate.

[0239] Silicone Oil as a Sealing Agent

[0240] While the devices, systems, and kits described herein may be used with a variety of hydrophobic liquids, fluorinated hydrocarbons, such as, FC-40 are routinely used. However, in some cases, it may be desirable to use non-fluorinated oils. An example, of a replacement oil for fluorinated oils as described herein a silicone oil. In some cases, one or more aspects of the devices, systems, and kits described herein may be modified to improve performance of silicone oil to match or even exceed that of fluorinated oils, such as, FC-40.

[0241] In some cases, the volume of the wells of the array of wells may be increased relative to the well volume when using fluorinated oils, such as, FC-40. Volume could be increased via increased diameter and / or depth. In certain cases, the wells may be about conical or frustoconical in shape and have a depth of about 4 pm and an upper diameter (diameter at well opening) of about 6 pm.

[0242] In some cases, the substrate solution used for filling the wells of the array may be formulated to increase the SCA of the silicone oil, as described in the section titled “Exemplary Aqueous Liquids”. For example, the substrate solution may be formulated in an assay buffer that includes a polysiloxane emulsion (e.g., Korasilon®), salts (e.g., NaCl, MgCh), sugar (e.g., sucrose), and / or concentration of substrate (e.g., pyranine phosphate).

[0243] In some cases, the performance of silicone oil may be optimized by conducting the enzymatic reaction for conversion of the substrate into a detectable product (e.g., a fluorogenic product) at a lower temperature as compared to when using fluorinated oils, such as, FC-40 for sealing the wells. Enzymatic reaction at a lower temperature results in a lower decrease in surface tension of the substrate solution thereby maintaining the silicone oil as a layer over the wells.

[0244] In some cases, the performance of silicone oil may be optimized by conducting the enzymatic reaction for conversion of the substrate into a detectable product (e.g., a fluorogenic product) for a shorter time as compared to the reaction time when using fluorinated oils, such as, FC-40. The shorter period of enzymatic reaction results in detection of signal from the enzymatic reaction product before the surface tension of the substrate solution is reduced and the silicone oil displaces the substrate solution in the wells. In some cases, the signal from the wells may be measured within 4 mins, within 3 mins, within 2 mins, within 1 min, e.g., within 2-4 mins after the sealing step.

[0245] In some cases, the performance of silicone oil may be optimized by aspirating the substrate solution at a higher rate as compared to aspiration rate when using fluorinated oils, such as, FC- 40. As shown in FIG. 17, a higher rate of aspiration of the substrate solution improves sealing with silicone oil. Suitable aspiration rate includes, at least 1 pL / s, at least 2 pL / s, at least 10 pL / s, at least 20 pL / s, at least 30 pL / s, at least 100 pL / s, at least 200 pL / s, e.g., 1- 50pL / s, 2- 25pL / s, or 2- 20pL / s. Silicone oil offers improved clearance of artifacts (e.g., debris, aggregates, etc.) and reduction of double-seeding (e.g., a bead inside a well and another bead partially inside the well) as compared to fluorinated oils, such as, FC-40.

[0246] Method for Improving Sealing of Array of Wells

[0247] Also provided herein are methods, aqueous liquids, systems, and kits for reducing pooling of aqueous liquid over the array of wells during the sealing step. The pooled aqueous liquid interferes with imaging of the wells and detection of signal from the wells. While the pooled aqueous liquid may be excluded from imaging or detection, this results in lower number of wells inteiTogated. FIG. 18 provides an illustration of effect of pooling and the optimal sealing where no pooling occurs. As used herein, the term “pooling” or “pooled” in the context of an aqueous liquid (e.g., a substrate solution) refers to presence of a layer of aqueous liquid over an array of wells, where the array is covered with a layer of hydrophobic liquid (e.g., oil), where the layer of aqueous liquid prevents sealing of the wells by the hydrophobic liquid. In some cases, the layer of aqueous liquid is surrounded by the layer of hydrophobic liquid. The amount of pooling may be estimated visually and / or computationally. For example, the amount of pooling may be estimated as the percentage of the area of the array of wells covered by the layer of aqueous liquid. A substantial decrease in pooling refers to a statistically significant decrease as compared to a negative control. A statistically significant decrease in pooling is a decrease in pooling of at least 30% or more as compared to pooling in absence of the anti-foam reagent as disclosed herein.

[0248] The aqueous liquid may be any aqueous liquid described herein, e.g., an assay buffer or a substrate solution (i.e., an assay buffer comprising a substrate) described herein, see, e.g., section titled “Exemplary Aqueous Liquids”. The device and method for seeding and sealing may be as described in herein, see, e.g., section titled “Devices”. In some cases, the device comprises two planar plates disposed in a spaced apart manner. The lower plate comprises an array of wells on the upper surface of the lower plate. The upper surface of the lower plate and the lower surface of the upper plate defining a chamber comprising the array of wells. The method involves seeding the wells by guiding PMPs (capture particles or mixture of functional beads (i.e., capture particles) and helper beads) to the array of wells filled with the substrate solution and sealing the array of wells by aspirating any substrate solution not disposed in the wells and simultaneously introducing the hydrophobic liquid over the wells.

[0249] As described herein, pooling of the aqueous liquid may be reduced by including an antifoam reagent in the aqueous liquid, e.g., in the substrate solution. In some cases, the anti-foam reagent may be a polysiloxane emulsion such as Korasilon® or Pluronic. Pluronic is made up of hydrophilic poly(ethylene oxide) (PEO) and hydrophobic poly(propylene oxide) (PPO) blocks in a triblock structure. In some cases, the anti-foam reagent may be a polydimethyl-siloxane. In some cases, the substrate solution does not include a surfactant such as Tween 20 which reduces seeding of wells. A surfactant can also reduce pooling but at the expense of seeding. A substrate solution provided herein comprises an anti-foam reagent which reduces pooling without reducing seeding. FIG. 19 provides bead count (indicative of seeding efficiency) when using a substrate solution comprising no additive (no surfactant or anti-foam), surfactant Tween 20, or anti-foam Korasilon®. While Tween 20 reduced the number of beads, Korasilon® almost doubled the number of seeded beads. Both Tween 20 and Korasilon® reduced pooling. Both Tween 20 and Korasilon® were added to the substrate solution to a concentration of 100 ppm.

[0250] EXAMPLES

[0251] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.

[0252] Example 1: Device for sealing of capture particles

[0253] Fig. 1A is a schematic of a device for seeding and sealing capture particles into an array of wells. A magnetic force may be used to seed the capture particles into the wells and an aqueous solution may be introduced into the wells to contact the capture particles. The array of wells is sealed by a layer of hydrophobic liquid. A sideview of an exemplary device is shown. The device includes a chamber defined between two surfaces: the lower surface of a top plate (1) and the upper surface of a bottom plate (2). The lower surface of the top plate includes a first region that is closer to the upper surface of the bottom plate than the regions surrounding it. In this figure, this first region is depicted as a pad (3) that extends away from the surrounding surface. Alternatively, or in addition, the pad may be formed by stamping the top plate to create an indention such that the lower surface of the top plate in the indented region is closer to the upper surface of the bottom plate, creating a chamber. The pad (3) defines a chamber in conjunction with the upper surface of the bottom plate (2). The pad (3) and the upper surface of the bottom plate (2) form a capillary space defining a chamber where a liquid present in the chamber is retained by surface tension in absence of side walls.

[0254] Fig. IB is another schematic showing a chamber defined between the pad (3) and the upper surface of the bottom plate (2). Liquid which may be a hydrophobic liquid, hydrophilic liquid or combination of both is held in the space defined between the lower surface of the pad and the upper surface of the second plate. Only the pad region of the top plate is illustrated. A first opening 10 is used to remove an aqueous liquid with a substrate (referred to as “substrate solution”) and a second opening 11 is used to add an oil for sealing the array of wells (not shown).

[0255] Fig. 1C is a see-through depiction of a top view of the pad region (3) in the top plate of the device. The microwell array (4) is located on the upper surface of the bottom plate. A first opening (5) in the first plate is connected to a well that can contain a hydrophilic liquid (e.g., a substrate solution or an assay buffer) or can be connected to a first apparatus for introducing the hydrophilic liquid into the space between the lower surface of the pad region and the upper surface of the second plate. Also depicted are four protrusions (6) placed at the lower surface around the first opening. These protrusions are referred to as substrate retention design in this embodiment. A second opening (7) in the first plate is located directly across the array of wells from the first opening (5). The second opening can contain a hydrophobic liquid (e.g., oil) or can be connected to a second apparatus for introducing the hydrophobic liquid into the space between the lower surface of the pad region and the upper surface of the second plate to cover the array of wells. The first plate can also include an inlet for introducing a liquid into or removing a liquid from the space between the lower surface of the pad region and the upper surface of the second plate. The liquid can be an assay buffer or a solution containing capture particles or waste liquid.

[0256] Fig. ID shows the lower surface of the first plate (1). The rectangular pad region (3) with rounded edges, resembling a dumbbell shape, is depicted. Four protrusions (6) disposed on the lower surface of the first plate (1) are visible. The pad (3) delineates the area of the lower surface of the first plate (1) that defines, in conjunction with the second plate, the chamber for holding liquids over the array of wells (not shown).

[0257] Fig. IE shows another embodiment of the device where the pad (3) includes a pinning wall (15) that extends from the pad (3). The pinning wall (15) is positioned along the periphery of the pad (3) and extends towards the bottom plate and stops short of the second opening. The pinning wall (15) improves the aspiration of the aqueous liquid.

[0258] FIG. 13 illustrates an issue that may be improved through use of a pinning wall. The device shown in this image does not include a pinning wall. During aspiration of the aqueous liquid and flowing of the hydrophobic liquid over the array of wells, the hydrophobic liquid can sometimes overtake the aqueous liquid and reach and surround the first opening cutting off the flow of the aqueous liquid to the first opening. The remaining aqueous liquid, visible in the lower panel, can interfere with sealing of the array of wells with the hydrophobic liquid and / or imaging of the array of wells. Including a pinning wall, such as, the pinning wall shown in FIGS. IE, 1J, IK, IM, 7 may significantly reduce pinching off the aqueous liquid.

[0259] Fig. 2A shows the filling of the device shown in Figs. 1A-1D with an aqueous liquid, followed by simultaneous aspiration of the hydrophilic liquid and introduction of hydrophobic liquid. The protrusions facilitate removal of the hydrophilic liquid without aspiration of the hydrophobic liquid.

[0260] Fig. 2B - 2C depict the effect of aqueous liquid stopper feature on extent of spread of substrate solution. Fig. 2D depicts the extent of spread of substrate solution in absence of the aqueous liquid stopper feature.

[0261] Example 2: Assay Buffer and SCA of Silicone oil

[0262] Study 1:

[0263] Addition of Substrate. Pyranine was added directly into the assay buffer. This assay buffer was then combined with pyranine phosphate to a total of 2 mM substrate in the buffer. Silicone oil was used for sealing the array of wells.

[0264] FIG. 14 shows that addition of proclin 950 and pyranine phosphate increase the SCA. LC assay buffer comprises: 2 mM TEA, 89.498 mM Tris Base, 10.51 Tris HC1, 5mM MgC12, 0.1% Sodium Azide, 10 ppm Korasilon®. Study 2:

[0265] Korasilon® EMA 119 titration: 0, 5, 10, 50, and 100 ppm Korasilon® EMA 119 was added to the ABJ assay buffer.

[0266] Addition of Korasilon® elevated the SCA. See FIG. 15. AB J comprises: 2 mM TEA, 223.72 mM Tris base, 26.28 mM Tris HCL (250 mM Tris), 1 mM MgC12, 12.9 mM Sodium Azide, 50 mM NaCl, 5 mM Levamisole.

[0267] Example 3: Reduction of Pooling of Substrate Solution

[0268] An aqueous thin layer can remain on the surface of digital nano well array. This effect is referred to as pooling. As shown by the data presented, pooling is completed eliminated by addition of antifoam (Pluronic or Korasilon®) into the substrate solution.

[0269] In the assay scheme, the substrate solution on the detection zone (nanowell “NW”-array) is isolated in single nanowells respectively by water-immiscible oil-flow, in other words, oilsealing makes the nano-reactor (chamber) array. If enzyme exists in the nano-reactor, the fluorogenic signal is generated making the nano well brighter fluorescently. The bright wells / dots are counted by image analysis.

[0270] Method:

[0271] ELISA: following materials (i-iv) were used: (i) pTaul81 antigen and (ii) detection conjugate of pTau81- specific antibody and nCIAP (native Calf Intestinal Alkaline Phosphatase, from BB1 Solution company), (iii) pTaul81 assay microparticles (2.7 pm, magnetic microparticle). Incubation time for immuno-reaction mixture (i, ii and iii) was 20 mins. The mixture was added (iv) 45,000 particles / test of the helper beads (10 pm magnetic microparticle, from Sigma- Aldrich-Merck) and loaded into the flowthrough chip with the NW array. The chip is configured for automated- washing, -seeding, -sealing, and -microscopic imaging of nano wells. At the washing step, additional helper beads were added to the mixture (total 180,000 beads). After washing, the pellet of beads transferred into the fluorogenic substrate solution which had been loaded on nanowell-array zone. Seeding of functional beads in single nanowells occurs here. Flow of 80pL of oil (FC-40, from 3M company) resulted in formation of the nanochamber. Images of nanowells were analyzed to measure the signal% (-ratio of bright dot per 100 beads) and the degree of pooling.

[0272] In addition to pTaul81 assay, HBsAg assay and A^42 assay were performed using the same method. Each assay used the specific antigen and the specific antibody conjugated with nCIAP. HBsAg assay takes about 5min and A042 assay takes about 30min.

[0273] The effect of Pooling reduction by the substrate solution including various concentrations of antifoams (Pluronicl7R4 or Korasilon®® EMA119) was evaluated. Structure of Korasilon®® EMA119 is shown in FIG. 20. Structure of Pluronic®17R4 is shown in FIG. 21.

[0274] FIG. 22. The effect of Pooling reduction by the substrate solution containing the antifoam Pluronic on pTaul81 assay (A: image data, B: analyzed result data).

[0275] FIG. 23. The effect of Pooling reduction by the substrate solution containing the antifoam Korasilon® on pTaul81 assay. (A: image data, B: analyzed result data).

[0276] FIG. 24. The effect of Pooling reduction by the substrate solution containing the antifoam Pluronic on A|342 assay. (A: image data, B: analyzed result data).

[0277] FIG. 25. The effect of Pooling reduction by the substrate solution containing the antifoam Korasilon® on HBsAg assay. (A: image data, B: analyzed result data).

[0278] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0279] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0280] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0281] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §1 12(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. § 112(6) is not invoked.

[0282] The following list of embodiments is envisaged:

[0283] 1. A device for sealing capture particles in an array of wells with a hydrophobic liquid, the device comprising: a first plate comprising an upper surface and a lower surface and a second plate comprising an upper surface and a lower surface, the first plate positioned in a spacedapart manner from the upper surface of the second plate, an array of wells disposed on the upper surface of the second plate, the lower surface of the first plate and the upper surface of the second plate defining a liquid holding region comprising the array of wells; a first opening extending between the upper and lower surfaces in the first plate and positioned adjacent to a first side of the array of wells, wherein the first opening is configured for introducing an aqueous liquid over the array of wells and removing the aqueous liquid; and a second opening extending between the upper and lower surfaces in the first plate and positioned adjacent to a second side of the array of wells wherein the second opening is configured for introducing the hydrophobic liquid over the array of wells.

[0284] 2. The device of embodiment 1, wherein the size of the liquid holding region is defined by a sealing pad on the lower surface of the first plate, wherein the sealing pad decreases the space between the lower surface of the first plate and the upper surface of the second plate such that the height of the liquid holding region is lower than the space surrounding the sealing pad.

[0285] 3. The device of embodiment 2, wherein the sealing pad is formed by an indentation in the first plate which brings the lower surface of the first plate closer to the second plate in a defined area.

[0286] 4. The device of embodiment 2 or 3, wherein the sealing pad is demarcated by a decrease in thickness of the first plate around the sealing pad such that the space between the lower surface of the first plate and the upper surface of the second plate increases outside of the scaling pad region.

[0287] 5. The device of any of embodiments 2-4, wherein the distance between the sealing pad and the upper surface of the second plate is about 1 mm - 300 p.m or 0.5 mm - 400 m.

[0288] 6. The device of any of embodiments 2-5, wherein the sealing pad dimensions is in the range of 14 mm x 5 mm to 11 mm x 4 mm.

[0289] 7. The device of any of embodiments 2-6, wherein the surface area of the sealing pad is about 20 mm2to 100 mm2or 40 mm2to 80 mm2.

[0290] 8. The device of any of embodiments 2-7, wherein the sealing pad is rectangular or is rectangular with rounded comers.

[0291] 9. The device of any of embodiments 2-8, wherein the sealing pad is substantially rectangular in shape and wherein the array of wells is positioned substantially centrally in the liquid holding region and the first and second openings are positioned closer to the shorter edges of the sealing pad.

[0292] 10. The device of any of embodiments 2-9, wherein the sealing pad is formed integrally with the first plate.

[0293] 11. The device of any preceding embodiment, wherein the liquid capacity of the liquid holding region is 25-10 pL or 25-15 pL.

[0294] 12. The device of any preceding embodiment, wherein the dimensions of the array of wells is in the range of about 10 mm x 7.5 mm to 6.2 mm x 3.5 mm.

[0295] 13. The device of any preceding embodiment, wherein the wells are micro wells.

[0296] 14. The device of any of embodiments 1-13, wherein the wells are nanowells. 15. The device of any preceding embodiment, wherein the wells all have substantially the same volume.

[0297] 16. The device of any preceding embodiment, wherein the array of wells has a volume up to 100 microliter, e.g., about 0.1 femtoliter- 1 femtoliter, 1 femtoliter - 10 femtoliter, 10 femtoliter - 25 femtoliter, 25 femtoliter - 50 femtoliter, 50 femtoliter - 100 femtoliter, 100 femtoliter - 0.1 pL, 0.1 pL - 1 pL, 1 pL - 10 pL, 10 pL - 25 pL, 25 pL - 50 pL, 50 pL - 100 pL, 100 pL - 0.1 nL, 0.1 nL - 1 nL, 1 nL - 10 nL, 10 nL - 25 nL, 25 nL -50 nL, 50 nL - 100 nL, 100 nL - 0.1 microliter, 0.1 microliter - 1 microliter, 1 microliter - 10 microliter, 10 microliter - 25 microliter, 25 microliter - 50 microliter, or 50 microliter - 100 microliter.

[0298] 17. The device of any preceding embodiment, wherein the first plate has thickness in range of 3 mm to 1 mm.

[0299] 18. The device of any preceding embodiment, wherein the second plate has thickness in range of 0.5 mm to 0.075 mm, e.g., 0.5 mm to 0.1 mm, 0.5 mm to 0.2 mm, 0.5 mm to 0.3 mm.

[0300] 19. The device of any preceding embodiment, wherein the first plate is formed from at least one of glass, cyclic olefin copolymer, polypropylene, polyethylene, and PMMA.

[0301] 20. The device of any preceding embodiment, wherein the second plate is formed from at least one of glass, cyclic olefin copolymer, polypropylene, polyethylene, and PMMA.

[0302] 21. The device of any preceding embodiment, wherein the lower surface of the first plate has a uniform hydrophobicity such that the entire lower surface of the first plate is hydrophobic or hydrophilic.

[0303] 22. The device of any preceding embodiment, wherein the upper surface of the second plate has a uniform hydrophobicity such that the entire upper surface of the second plate is hydrophobic or hydrophilic. 23. The device of any preceding embodiment, wherein the lower surface of the first plate is more hydrophobic than the upper surface of the second plate, or wherein the lower surface of the first plate is less hydrophobic than the upper surface of the second plate.

[0304] 24. The device of any preceding embodiments, wherein the lower surface of the first plate comprises a plurality of protrusions positioned around a circumference of the first opening, wherein the aqueous liquid enters the liquid holding region through the first opening and is removed through the first opening and wherein the protrusions are configured to prevent the hydrophobic liquid from reaching the first opening during the removal of the aqueous liquid.

[0305] 25. The device of embodiment 24, wherein the protrusions are circular, semi-circular, triangular, semi-triangular, wedge-shaped, sector- shaped, or arched in shape.

[0306] 26. The device of embodiment 24 or embodiment 25, wherein the protrusions are arranged symmetrically around the circumference of the first opening.

[0307] 27. The device of any one of embodiment 24-26, wherein the protrusions decrease in dimension towards the center of the first opening.

[0308] 28. The device of any one of embodiments 24-27, wherein the protrusions extend into the first opening, partially occluding the first opening.

[0309] 29. The device of any one of embodiments 2-28, when dependent on embodiment 2, wherein the sealing pad comprises a pinning wall, for controlling movement of aqueous and / or hydrophobic liquid in the liquid holding region, extending towards the second plate, wherein the pinning wall does not extend to the second plate, wherein the pinning wall is positioned in a space apart manner from the first opening and surrounds the first opening and extends towards the second opening. 30. The device of embodiment 29, wherein the pinning wall extends downwards from the periphery of the scaling pad and extends along the periphery till the region of the scaling pad positioned over the array of wells.

[0310] 31. The device of embodiment 29, wherein the pinning wall extends downwards from the periphery of the sealing pad and extends along the periphery and stops short of the area where the second opening is positioned.

[0311] 32. The device of any of embodiments 29-31, wherein the pinning wall is integral with the sealing pad and optionally integral with the first plate.

[0312] 33. The device of any of embodiments 29-32, wherein the pinning wall extends between 50- 250pm, optionally 150 -250pm, perpendicular from the sealing pad.

[0313] 34. The device of any of embodiments 29-33, wherein the pinning wall has width of 100 - 500 pm, optionally 200 - 400pm.

[0314] 35. The device of any one of embodiments 2-34, when dependent on embodiment 2, wherein the sealing pad comprises an aqueous liquid stopper feature disposed on the sealing pad and extending from the first opening towards the second opening and terminating before to the second opening, the aqueous liquid stopper feature configured to reduce the flow of aqueous liquid beyond the stopper feature.

[0315] 36. The device of embodiment 35, wherein the aqueous liquid stopper feature is a thin film disposed on the pad region. The film may extend from the first opening towards the second opening and stop before the second opening. The film can have a thickness of less than 100 pm, e.g., less than 50 pm, less than 30 pm, less than 10 pm, less than 5 pm to as low as 1 pm.

[0316] 37. The device of any preceding embodiment, wherein the first opening is dimensioned to accommodate a first apparatus for introducing the aqueous liquid into the liquid holding region and for removing the aqueous liquid from the liquid holding region and the second opening is dimensioned to accommodate a second apparatus for introducing the hydrophobic liquid into the liquid holding region.

[0317] 38. The device of any preceding embodiment, wherein the first opening has a diameter of about 1.5 mm-2 mm.

[0318] 39. The device of any preceding embodiment, wherein the second opening has a diameter of about 1.5 mm-2.5 mm.

[0319] 40. The device of any preceding embodiment, wherein the first and second openings may be spaced apart by about 8 mm- 14 mm or 10 mm- 12 mm.

[0320] 41. The device of any preceding embodiment, wherein the first opening and second opening have the same diameter or wherein the first opening and second opening have different diameters.

[0321] 42. The device of any preceding embodiment, wherein the device is configured for simultaneous removal of the aqueous liquid from the liquid holding region and introduction of the hydrophobic liquid into the liquid holding region.

[0322] 43. The device of any of embodiments 37-42, when dependent from embodiment 37, wherein the first apparatus comprises a hollow tube connected to a pneumatic instrument, wherein the hollow tube is insertable into the first opening.

[0323] 44. The device of embodiment 43, wherein the protrusions in the first opening are positioned to contact the hollow tube and prevent the hollow tube from contacting the second plate.

[0324] 45. The device of any of embodiments 37-44, when dependent from embodiment 37, wherein the second apparatus comprises a hollow tube connected to a pneumatic instrument.

[0325] 46. The device of embodiment 45, wherein the second opening is dimensioned to allow the hollow tube of the second apparatus to pass through the second opening. 47. The device of any one of embodiments 37-46, when dependent from embodiment 37, wherein the first apparatus is configured for introducing the aqueous liquid into the liquid holding region via the first opening and removing the aqueous liquid from the liquid holding region via the first opening and the second apparatus is configured for introducing the hydrophobic liquid into the liquid holding region via the second opening, wherein the device is configured for simultaneously removing the aqueous liquid from the liquid holding region and introducing the hydrophobic liquid into the liquid holding region.

[0326] 48. The device of any preceding embodiment, wherein the second side to which the second opening is positioned adjacent is opposite to the first side.

[0327] 49. A system for seeding and sealing capture particles in an array of wells for detecting presence of an analyte of interest bound to the capture particles, the system comprising: the device of any one of embodiments 1-48; a first apparatus for introducing the aqueous liquid into the liquid holding region and for removing the aqueous liquid from the liquid holding region and a second apparatus for introducing the hydrophobic liquid into the liquid holding region; a hydrophobic liquid; and an aqueous liquid, or a kit for seeding and sealing capture particles in an array of wells for detecting presence of an analyte of interest bound to the capture particles, the system comprising: the device of any one of embodiments 1-48; a hydrophobic liquid; and an aqueous liquid.

[0328] 50. The system or the kit of embodiment 49, wherein the hydrophobic liquid is a nonfluorinated oil.

[0329] 51. The system or the kit of embodiment 50, wherein the non-Huorinated oil is a siloxane-based oil, optionally, wherein the siloxane-based oil is silicone oil. 52. The system or the kit of any one of embodiments 49-51 , wherein the aqueous liquid is an assay buffer.

[0330] 53. The system or the kit of embodiment 52, wherein the assay buffer comprises a substrate for an enzyme immobilized on the capture particles, optionally, wherein the substrate is pyrophosphate, further optionally, wherein the pyrophosphate is present at a concentration of 2- 10 mM.

[0331] 54. The system or the kit of embodiment 52 or 53, wherein the aqueous liquid comprises a silicone emulsion or polysiloxane emulsion, optionally, wherein the polysiloxane emulsion comprises Korasilon®, further optionally, wherein the Korasilon® is Korasilon® EMA 119 (which is an emulsion comprising about 45% of silicone, a viscosity of between about 1,500 to about 2,000 mPa s (millipascal second) at 25°C, and a pH of about 8).

[0332] 55. The system or the kit of any one of embodiments 49-54, wherein the aqueous liquid MgCh and / or sucrose.

[0333] 56. The system or the kit of embodiment 55, wherein the aqueous liquid comprises 5- 100 mM, 100-400 mM, or 400-800 mM MgCl2.

[0334] 57. The system or the kit of embodiment 55 or embodiment 56, wherein the aqueous liquid comprises 5-100 mM, 100-400 mM, or 400-800 mM.

[0335] 58. The system or the kit of any one of embodiments 49-57, further comprising capture particles.

[0336] 59. The system or the kit of any one of embodiments 49-58, wherein the capture particles are paramagnetic particles.

[0337] 60. A method for seeding and sealing wells of an array in the device of any one of embodiments 1-48 or using the system or kit of any one of embodiments 49-59, the method comprising: aspirating the aqueous liquid and flowing the hydrophobic liquid over the array, wherein aspirating the aqueous liquid comprises positioning a first hollow tube in the first opening and applying suction pressure, wherein flowing the hydrophobic liquid comprises positioning a second hollow tube in the second opening and introducing the hydrophobic liquid in the liquid holding region, wherein the aspiration of the aqueous liquid facilitates spreading of the hydrophobic liquid over the array, and wherein the protrusions positioned around the first opening facilitate removal of the aqueous liquid while substantially preventing removal of the hydrophobic liquid.

[0338] 61. The method of embodiment 60, wherein aspirating the aqueous liquid and flowing the hydrophobic liquid over the array occurs simultaneously.

[0339] 62. The method of embodiment 60 or 61, wherein the aqueous liquid is an assay buffer, optionally, wherein the assay buffer comprises a substrate for an enzyme immobilized on the capture particles.

[0340] 63. The method of any of embodiments 60-62, wherein the capture particles are paramagnetic particles and guiding capture particles to the array comprises immobilizing the capture particles with a magnet positioned underneath the second plate and moving the magnet to the array of wells.

[0341] 64. The method of any one of embodiments 60-63, wherein the first hollow tube contacts the protrusions extending into the first opening.

[0342] 65. The method of any one of embodiments 60-64, comprising after guiding the capture particles to the array and prior to flowing an aqueous liquid over the array to fill the wells, heating the device to temperature higher than room temperature such that the device has a temperature higher than the temperature of the aqueous liquid for filling the wells.

[0343] 66. The method of any one of embodiments 60-65, comprising after guiding the capture particles to the array and prior to flowing an aqueous liquid over the array to fill the wells, heating the aqueous liquid to a temperature higher than room temperature. 67. The method of any one of embodiments 60-66, wherein the method comprises degassing the aqueous liquid prior to flowing the aqueous liquid over the array to fill the wells.

[0344] 68. The method of embodiment 67, wherein degassing the aqueous liquid comprises subjecting the aqueous liquid to vacuum for a time period sufficient to reduce air dissolved in the aqueous liquid in any one of embodiments 60-68, wherein the method comprises following the steps of guiding capture particles to the array and flowing the aqueous liquid over the array to fill the wells, cooling the array of wells to a temperature below room temperature.

[0345] 69. A method for detecting presence of an analyte immobilized on capture particles, the method comprising: seeding and scaling the capture particles by a method of any one of embodiments 60-69 and detecting presence of a signal from the array of wells, wherein the presence of the signal is indicative of the presence of an analyte immobilized on capture particles.

[0346] 70. The method of embodiment 70, wherein the signal is a fluorescent signal.

[0347] 71. A method for reducing air bubbles in an array of wells in a device upon filling the wells with an aqueous liquid, the method comprising:

[0348] (i) heating the device to temperature higher than room temperature such that the device has a temperature higher than the temperature of the aqueous liquid for filling the wells;

[0349] (ii) heating the aqueous liquid for filling the wells to a temperature higher than room temperature;

[0350] (iii) degassing the aqueous liquid prior to filling the wells with the aqueous liquid; and / or

[0351] (iv) filling the wells with the aqueous liquid and cooling the array of wells to a temperature below room temperature.

[0352] 72. The method of embodiment 72 (i), embodiment 72 (ii), or embodiment 72 (iii), further comprising filling the wells with the aqueous liquid. 73. The method of embodiment 72 or embodiment 73, further comprising covering the array of wells with a hydrophobic liquid.

[0353] 74. The method of any of embodiments 72-74, wherein the device is heated to a temperature of up to 30°C, up to 50°C, up to 60°C, up to 70°C, up to 80°C, up to 90°C, or up to 100°C.

[0354] 75. The method of any of embodiments 72-75, wherein aqueous liquid is heated to a temperature of up to 30°C, up to 50°C, up to 60°C, up to 70°C, up to 80°C, up to 90°C, or up to 100°C.

[0355] 76. The method of any of embodiments 72-76, wherein the method further comprises degassing the aqueous liquid by subjecting the aqueous liquid to vacuum for a time period sufficient to reduce air dissolved in the aqueous liquid, optionally wherein, the aqueous liquid is degassed under vacuum for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, and up to 30 minutes and / or the degassed liquid is held under vacuum up till the point of introducing it into the device.

[0356] 77. The method of any of embodiments 72-77, wherein the array of wells comprises cooling the device using a thermoelectric cooling instrument and / or wherein the device is cooled to a temperature of down to 15°C, to 10°C, to 5°C, to 0°C

[0357] 78. A method for detecting presence of an analyte immobilized on capture particles, the method comprising: flowing an assay buffer over an array of wells; depositing the capture particles over the array of wells, wherein the wells in the array are sized to hold a single capture particle per well; flowing a substrate solution over the array of wells; removing the substrate solution and flowing a hydrophobic liquid over the array of wells to seal the capture particles and substrate solution in the array of wells with the hydrophobic liquid; and detecting presence of a signal from the array of wells, wherein the presence of the signal is indicative of the presence of an analyte immobilized on capture particles.

[0358] 79. The method of embodiment 79, wherein the steps of removing the substrate solution and flowing a hydrophobic liquid over the array of wells to seal the capture particles and substrate solution in the array of wells with the hydrophobic liquids occur simultaneously.

[0359] 80. The method of embodiment 79 or 80, wherein the capture particles are magnetic particles.

[0360] 81. The method of any of embodiments 79-81, wherein the analyte is associated with an enzyme that converts the substrate into a product that emits a detectable signal.

[0361] 82. The method of any of embodiments 79-82, wherein the substrate solution includes the substrate and the assay buffer does not include the substrate, and wherein the substrate solution and the assay buffer include diethylamine (DEA).

[0362] 83. The method of any one of embodiments 79-83, wherein flowing the assay buffer over the array of wells comprises passively flowing the assay buffer over the array of wells, wherein the array of wells is positioned in a liquid holding region defined by a first plate and a second plate, wherein the array is positioned on the second plate, and a first plate is positioned above the array of wells a spaced-apart manner.

[0363] 84. The method of any one of embodiments 79-84, wherein flowing the assay buffer over the array of wells comprises injecting the assay buffer in a liquid holding region defined by a first plate and a second plate, wherein the array of wells is positioned on the second plate and the first plate is positioned above the array of wells a spaced-apart manner.

[0364] 85. The method of any one of embodiments 79-85, wherein depositing the capture particles over the array of wells comprises passively flowing the capture particles over the array of wells, wherein the array of wells is positioned in a liquid holding region defined by a first plate and a second plate, wherein the array of wells is positioned on the second plate and the first plate is positioned above the array of wells a spaccd-apart manner.

[0365] 86. The method of any one of embodiments 79-86, wherein depositing the capture particles over the array of wells comprises injecting the capture particles in a liquid holding region defined a first plate and a second plate, wherein the array of wells is positioned on the second plate and the first plate is positioned above the array of wells a spaced-apart manner.

[0366] 87. The method of any one of embodiments 79-87, wherein the capture particles are magnetic and wherein depositing the capture particles over the array of wells comprises applying a magnetic force to the magnetic capture particles to guide them into the wells in the array.

[0367] 88. The method of any one of embodiments 79-88, wherein the capture particles are magnetic and wherein depositing the capture particles over the array of wells comprises passively flowing or injecting the capture particles in a liquid holding region defined by a first plate and a second plate, wherein the array of wells is positioned on the second plate and the first plate is positioned above the array of wells a spaced-apart manner and applying a magnetic force to the magnetic capture particles to guide them into the wells in the array.

[0368] 89. The method of any one of embodiments 79-89, wherein flowing the substrate solution over the array of wells comprises passively flowing or injecting the substrate solution in a liquid holding region defined by a first plate and a second plate, wherein the array of wells is positioned on the second plate and the first plate is positioned above the array of wells a spaced- apart manner.

[0369] 90. The method of any one of embodiments 79-90, wherein simultaneously removing the substrate solution and flowing the hydrophobic liquid over the array of wells to cover the array of wells with the hydrophobic liquid comprises aspirating the substrate solution and injecting the hydrophobic liquid.

[0370] 91. The method of embodiment 91, wherein the top plate comprises: a first opening positioned adjacent to a first side of the array of wells, wherein the first opening extends from the upper surface to the lower surface of the top plate and wherein the lower surface of the top plate comprises a plurality of protrusions positioned around the circumference of the first opening, wherein the substrate solution enters the liquid holding region over the array of wells through the first opening and is removed through the first opening and wherein the protrusions are configured to prevent the hydrophobic liquid from reaching the opening before the substrate solution is removed; and a second opening positioned adjacent to a second side of the array of wells, wherein the second side is opposite to the first side, wherein the hydrophobic liquid is flowed over the array of wells through the second opening.

[0371] 92. The device or method of any preceding embodiment, wherein the liquid holding region is a wall-less chamber.

Claims

CLAIMS:

1. A device for sealing capture particles in an array of wells with a hydrophobic liquid, the device comprising: a first plate comprising an upper surface and a lower surface and a second plate comprising an upper surface and a lower surface, the first plate positioned in a spaced-apart manner from the upper surface of the second plate, an array of wells disposed on the upper surface of the second plate, the lower surface of the first plate and the upper surface of the second plate defining a liquid holding region comprising the array of wells; a first opening extending between the upper and lower surfaces in the first plate and positioned adjacent to a first side of the array of wells, wherein the first opening is configured for introducing an aqueous liquid over the array of wells and removing the aqueous liquid; and a second opening extending between the upper and lower surfaces in the first plate and positioned adjacent to a second side of the array of wells, wherein the second opening is configured for introducing the hydrophobic liquid over the array of wells.

2. The device of claim 1 , wherein the size of the liquid holding region is defined by a sealing pad on the lower surface of the first plate and the distance between the sealing pad and the upper surface of the second plate, wherein the sealing pad decreases the space between the lower surface of the first plate and the upper surface of the second plate such that the height of the chamber is lower than the space surrounding the sealing pad, optionally, wherein the sealing pad is formed by an indentation in the first plate which brings the first plate closer to the second plate in a defined area, or wherein the sealing pad is demarcated by a decrease in thickness of the first plate around the sealing pad such that the space between the lower surface of the first plate and the upper surface of the second plate increases outside of the sealing pad region.

3. The device of claim 2, wherein the sealing pad is substantially rectangular in shape and wherein the array of wells is positioned substantially centrally in the chamber and the first and second openings are positioned closer to the shorter edges of the sealing pad.

4. The device of any one of claims 1-3, wherein the lower surface of the first plate comprises a plurality of protrusions positioned around a circumference of the first opening, wherein the aqueous liquid enters the chamber through the first opening and is removed through the first opening and wherein the protrusions are configured to prevent the hydrophobic liquid from reaching the first opening during the removal of the aqueous liquid.

5. The device of claim 4, wherein the protrusions are circular, semi-circular, triangular, semi-triangular, wedge-shaped, sector-shaped, or arched in shape.

6. The device of claim 4 or claim 5, wherein the protrusions are arranged symmetrically around the circumference of the first opening.

7. The device of any one of claim 4-6, wherein the protrusions decrease in dimension towards the center of the first opening.

8. The device of any one of claim 4-7, wherein the protrusions extend into the first opening, partially occluding the first opening.

9. The device of any one of claims 2-8, wherein the sealing pad comprises a pinning wall, for controlling movement of aqueous and / or hydrophobic liquid in the chamber, extending towards the second plate, wherein the pinning wall does not extend to the second plate, wherein the pinning wall is positioned in a space apart manner from the first opening and surrounds the first opening and extends towards the second opening.

10. The device of claim 9, wherein the pinning wall extends downwards from the periphery of the sealing pad and extends along the periphery up to the region of the sealing pad positioned over the array of wells.

11. The device of claim 9, wherein the pinning wall extends downwards from the periphery of the sealing pad and extends along the periphery and stops short of the area where the second opening is positioned.

12. The device of any one of claims 2-11, wherein the sealing pad comprises an aqueous liquid stopper feature disposed on the sealing pad and extending from the first opening towardsthe second opening and terminating before the second opening, the aqueous liquid stopper feature configured to reduce the flow of aqueous liquid beyond the stopper feature.

13. The device of any one of claim 1-12, wherein the first opening is dimensioned to accommodate a first apparatus for introducing the aqueous liquid into the chamber and for removing the aqueous liquid from the chamber and the second opening is dimensioned to accommodate a second apparatus for introducing the hydrophobic liquid into the chamber, optionally wherein the device is configured for simultaneous removal of the aqueous liquid from the chamber and introduction of the hydrophobic liquid into the chamber.

14. The device of claim 13, wherein the first apparatus comprises a hollow tube connected to a pneumatic instrument, wherein the hollow tube is insertable into the first opening.

15. The device of claim 14, wherein the protrusions in the first opening are positioned to contact the hollow tube and prevent the hollow tube from contacting the second plate.

16. The device of any one of claims 13-15, wherein the second apparatus comprises a hollow tube connected to a pneumatic instrument.

17. The device of claim 16, wherein the second opening is dimensioned to allow the hollow tube of the second apparatus to pass through the second opening.

18. The device of any one of claims 13-17, wherein the first apparatus is configured for introducing the aqueous liquid into the chamber via the first opening and removing the aqueous liquid from the chamber via the first opening and the second apparatus is configured for introducing the hydrophobic liquid into the chamber via the second opening, wherein the device is configured for simultaneously removing the aqueous liquid from the chamber and introducing the hydrophobic liquid into the chamber.

19. A system for seeding and sealing capture particles in an array of wells for detecting presence of an analyte of interest bound to the capture particles, the system comprising: the device of any one of claims 1-18; the first apparatus and the second apparatus; a hydrophobic liquid; and an aqueous liquid,or a kit for seeding and sealing capture particles in an array of wells for detecting presence of an analyte of interest bound to the capture particles, the system comprising: the device of any one of claims 1-18; a hydrophobic liquid; and an aqueous liquid.

20. The system or the kit of claim 19, wherein the hydrophobic liquid is a non-fluorinated oil.

21. The system or the kit of claim 20, wherein the non-fluorinated oil is a siloxane-based oil, optionally, wherein the siloxane-based oil is silicone oil.

22. The system or the kit of any one of claims 19-21 , wherein the aqueous liquid is an assay buffer.

23. The system or the kit of claim 22, wherein the assay buffer is a substrate solution comprising a substrate for an enzyme immobilized on the capture particles, optionally, wherein the substrate is pyranine phosphate, further optionally, wherein the pyranine phosphate is present at a concentration of 2-10 mM.

24. The system or the kit of claim 22 or 23, wherein the aqueous liquid comprises a silicone emulsion or polysiloxane emulsion, optionally, wherein the polysiloxane emulsion comprises Korasilon®, further optionally, wherein the Korasilon® is Korasilon® EMA 119 (which is an emulsion comprising about 45% of silicone, a viscosity of between about 1,500 to about 2,000 mPa s (millipascal second) at 25°C, and a pH of about 8).

25. The system or the kit of any one of claims 19-24, wherein the aqueous liquid comprises MgCb and / or sucrose.

26. The system or the kit of claim 25, wherein the aqueous liquid comprises 5-100 mM, 100- 400 mM, or 400-800 mM MgCh.

27. The system or the kit of claim 25 or claim 26, wherein the aqueous liquid comprises 5- 100 mM, 100-400 mM, or 400-800 mM sucrose.

28. The system or the kit of any one of claims 19-27, further comprising capture particles.

29. The system or the kit of any one of claims 19-28, wherein the capture particles are paramagnetic particles.

30. A method for seeding and sealing wells of an array in the device of any one of claims 1- 18 or using the system or kit of any one of claims 19-29, the method comprising: guiding capture particles to the array; flowing an aqueous liquid over the array to fill the wells; simultaneously aspirating the aqueous liquid and flowing the hydrophobic liquid over the array, wherein aspirating the aqueous liquid comprises positioning a first hollow tube in the first opening and applying suction pressure, wherein flowing the hydrophobic liquid comprises positioning a second hollow tube in the second opening and introducing the hydrophobic liquid in the chamber, wherein the aspiration of the aqueous liquid facilitates spreading of the hydrophobic liquid over the array, and wherein the protrusions positioned around the first opening facilitate removal of the aqueous liquid while substantially preventing removal of the hydrophobic liquid.

31. The method of claim 30, wherein the aqueous liquid is an assay buffer, optionally, wherein the assay buffer is a substrate solution comprising a substrate for an enzyme immobilized on the capture particles.

32. The method of claim 30 or 31, wherein the capture particles are paramagnetic particles and guiding capture particles to the array comprises immobilizing the capture particles with a magnet positioned underneath the second plate and moving the magnet to the array of wells.

33. The method of any one of claims 30-32, wherein the first hollow tube contacts the protrusions extending into the first opening.

34. The method of any one of claims 30-33, comprising after guiding the capture particles to the array and prior to flowing an aqueous liquid over the array to fill the wells, heating the device to temperature higher than room temperature such that the device has a temperature higher than the temperature of the aqueous liquid for filling the wells.

35. The method of any one of claims 30-34, comprising after guiding the capture particles to the array and prior to flowing an aqueous liquid over the array to fill the wells, heating the aqueous liquid to a temperature higher than room temperature.

36. The method of any one of claims 30-35, wherein the method comprises degassing the aqueous liquid prior to flowing the aqueous liquid over the array to fill the wells.

37. The method of claim 36, wherein degassing the aqueous liquid comprises subjecting the aqueous liquid to (i) vacuum and / or (ii) a higher temperature for a time period sufficient to reduce air dissolved in the aqueous liquid.

38. The method of any one of claims 30-37, wherein the method comprises following the steps of guiding capture particles to the array and flowing the aqueous liquid over the array to fill the wells, cooling the array of wells to a temperature below room temperature prior to the step of introducing the hydrophobic liquid into the chamber.

39. A method for detecting presence of an analyte immobilized on capture particles, the method comprising: seeding and sealing the capture particles by a method of any one of claims 30-38 and detecting presence of a signal from the array of wells, wherein the presence of the signal is indicative of the presence of an analyte immobilized on capture particles.

40. The method of claim 39, wherein the signal is a fluorescent signal.

41. A method for reducing air bubbles in an array of wells in a device upon filling the wells with an aqueous liquid, the method comprising:(i) heating the device to temperature higher than room temperature such that the device has a temperature higher than the temperature of the aqueous liquid for filling the wells;(ii) heating the aqueous liquid for filling the wells to a temperature higher than room temperature;(iii) degassing the aqueous liquid prior to filling the wells wi th the aqueous liquid; and / or(i v) filling the wells with the aqueous liquid and cooling the array of wells to a temperature below room temperature.

42. The method of claim 41 (i), claim 41 (ii), or claim 41 (iii), further comprising filling the wells with the aqueous liquid.

43. The method of claim 41 or claim 42, further comprising covering the array of wells with a hydrophobic liquid.

44. A method for detecting presence of an analyte immobilized on capture particles, the method comprising: flowing an assay buffer over an array of wells; depositing the capture particles over the array of wells, wherein the wells in the array are sized to hold a single capture particle per well; flowing a substrate solution over the array of wells; simultaneously removing the substrate solution and flowing a hydrophobic liquid over the array of wells to seal the capture particles and substrate solution in the array of wells with the hydrophobic liquid; and detecting presence of a signal from the array of wells, wherein the presence of the signal is indicative of the presence of an analyte immobilized on capture particles.

45. The method of claim 44, wherein the capture particles are magnetic particles.

46. The method of claim 44 or 45, wherein the analyte is associated with an enzyme that converts the substrate into a product that emits a detectable signal.

47. The method of claim 44, wherein the substrate solution includes the substrate and the assay buffer does not include the substrate, and wherein the substrate solution and the assay buffer include diethylamine (DEA).

48. The method of any one of claims 44-47, wherein flowing the assay buffer over the array of wells comprises passively flowing the assay buffer over the array of wells, wherein the array of wells is positioned in a chamber defined by a first plate and a second plate, wherein the array is positioned on the second plate, and a first plate is positioned above the array of wells a spaced-apart manner.

49. The method of any one of claims 44-48, wherein flowing the assay buffer over the array of wells comprises injecting the assay buffer in a chamber defined by a first plate and a second plate, wherein the array of wells is positioned on the second plate and the first plate is positioned above the array of wells a spaced-apart manner.

50. The method of any one of claims 44-49, wherein depositing the capture particles over the array of wells comprises passively flowing the capture particles over the array of wells, wherein the array of wells is positioned in a chamber defined by a first plate and a second plate, wherein the array of wells is positioned on the second plate and the first plate is positioned above the array of wells a spaced-apart manner.

51. The method of any one of claims 44-50, wherein depositing the capture particles over the array of wells comprises injecting the capture particles in a chamber defined a first plate and a second plate, wherein the array of wells is positioned on the second plate and the first plate is positioned above the array of wells a spaced-apart manner.

52. The method of any one of claims 44-51, wherein the capture particles are magnetic and wherein depositing the capture particles over the array of wells comprises applying a magnetic force to the magnetic capture particles to guide them into the wells in the array.

53. The method of any one of claims 44-52, wherein the capture particles are magnetic and wherein depositing the capture particles over the array of wells comprises passively flowing or injecting the capture particles in a chamber defined by a first plate and a second plate, wherein the array of wells is positioned on the second plate and the first plate is positioned above the array of wells a spaced-apart manner and applying a magnetic force to the magnetic capture particles to guide them into the wells in the array.

54. The method of any one of claims 44-53, wherein flowing the substrate solution over the array of wells comprises passively flowing or injecting the substrate solution in a chamber defined by a first plate and a second plate, wherein the array of wells is positioned on the second plate and the first plate is positioned above the array of wells a spaced-apart manner.

55. The method of any one of claims 44-54, wherein simultaneously removing the substrate solution and flowing the hydrophobic liquid over the array of wells to cover the array of wells with the hydrophobic liquid comprises aspirating the substrate solution and injecting the hydrophobic liquid.

56. The method of claim 55, wherein the top plate comprises: a first opening positioned adjacent to a first side of the array of wells, wherein the first opening extends from the upper surface to the lower surface of the top plate and wherein the lower surface of the top plate comprises a plurality of protrusions positioned around the circumference of the first opening, wherein the substrate solution enters the chamber over the array of wells through the first opening and is removed through the first opening and wherein the protrusions are configured to prevent the hydrophobic liquid from reaching the opening before the substrate solution is removed; and a second opening positioned adjacent to a second side of the array of wells, wherein the second side is opposite to the first side, wherein the hydrophobic liquid is flowed over the array of wells through the second opening.

57. A method for reducing the pooling of substrate solution in a device for seeding and sealing capture particles, the method comprising: guiding capture particles to an array of wells positioned in the device; prior to or after the guiding, flowing a substrate solution over the array of wells such that the substrate solution enters the well and covers the wells; after flowing the substrate solution over the array of wells, flowing a hydrophobic liquid over the array of wells, wherein the hydrophobic liquid displaces capture particles and substrate solution not present inside the wells, optionally, aspirating the substrate solution while flowing the hydrophobic liquid over the array of wells,wherein the substrate solution comprises an anti-foam reagent that substantially decreases pooling of the substrate solution over the array of wells as compared to the pooling observed in absence of the anti-foam reagent.

58. The method of claim 57, wherein the anti-foam reagent is a polysiloxane or a polymer comprising hydrophilic poly(ethylene oxide) (PEO) and hydrophobic polypropylene oxide) (PPO) blocks.

59. The method of claim 58, wherein the anti-foam reagent is present at a concentration of 10-1000ppm, e.g., 10-500ppm, 10-250ppm, or 10-100ppm.

60. The method of any one of claims 57-59, wherein the polysiloxane is Korasilon®, optionally, wherein the Korasilon® is Korasilon® EMA 119 (an emulsion comprising about 45% of silicone, a viscosity of between about 1,500 to about 2,000 mPA s (millipascal second) at 25°C, and a pH of about 8).

61. The method of claim 58, wherein the polymer comprising hydrophilic poly(ethylene oxide) (PEO) and hydrophobic polypropylene oxide) (PPO) blocks comprises hydrophilic poly(ethylene oxide) (PEO) and hydrophobic polypropylene oxide) (PPO) blocks in a triblock structure.

62. The method of claim 58, wherein the polymer is Pluronic, e.g., Pluronic®17R4.

63. The method of any one of claims 57-62, wherein the device is the device of any one of claims 1-18 or wherein the device comprises a first plate and a second plate positioned in a spaced-apart manner, a lower surface of the second plate comprising the array of wells, wherein the space between the first plate and the second plate is configured as a flow-through channel.

64. A method for seeding and sealing an array of wells in the device of any one of claims 1 - 18 or using the system or kit of any one of claims 19-29, the method comprising: guiding capture particles to the array; flowing an aqueous liquid over the array to fill the wells; and simultaneously aspirating the aqueous liquid and flowing a hydrophobic liquid over the array to seal the array of wells, wherein the hydrophobic liquid is a non-fluorinated oil.

65. The method of claim 64, wherein the hydrophobic liquid is silicone oil.

66. The method of claim 64 or 65, wherein the aqueous liquid is an assay buffer comprising a salt, e.g., NaCl and / or MgCh and / or a sugar, e.g., sucrose.

67. The method of any one of claims 64-66, wherein the aqueous liquid is a substrate solution comprising an assay buffer and an enzyme substrate.

68. The method of claim 67, wherein the enzyme substrate is a substrate for a phosphatase, optionally, wherein the phosphatase is an alkaline phosphatase.

69. The method of claim 67 or 68, wherein the enzyme substrate is pyranine phosphate, optionally, wherein the pyranine phosphate is present at a concentration of 2-20 mM.

70. The method of any one of claims 64-69, further comprising incubating the device at a temperature suitable for enzymatic activity of an enzyme immobilized on the capture particles, wherein the temperature is lower than the ideal temperature for the enzymatic activity of an enzyme, wherein the lower temperature facilitates maintenance of a barrier between the aqueous liquid in the wells and the hydrophobic liquid on the array of wells thereby preventing the hydrophobic liquid from entering the wells and displacing the aqueous liquid and / or the capture particle present in the wells.

71. The method of any one of claims 64-70, further comprising incubating the device at a temperature suitable for enzymatic activity of an enzyme immobilized on the capture particles for a period of time that is less than 4 minutes, wherein the period of time is shorter than the period of time for maximal enzymatic activity of an enzyme, wherein the shorter period enables detection of enzymatic activity before any disruption of the barrier between the aqueous liquid in the wells and the hydrophobic liquid on the array of wells.

72. The method of any one of claims 64-71, wherein the aspiration rate of the aqueous liquid is higher than the aspiration rate of the aqueous liquid when using a fluorinated oil such as FC- 40 and wherein the higher aspiration rate reduces the likelihood of the hydrophobic liquidentering the wells, optionally, wherein the aspiration rate is 1 pL / s to 200 pL / s, e.g., 2 pL / s-20 pL / s.

73. The method of any one of claims 64-72, wherein the wells have a volume of about 90- 150 pm3, e.g., 90- 100 pm3, 95- 100 pm3, or 99- 100 pm3.

74. An assay buffer for detecting presence of capture particles bound to a target analyte of interest, the assay buffer comprising: a buffering agent, e.g.. Tris; andMgCh and / or NaCl.

75. The assay buffer of claim 74, comprising the buffering agent, MgCh and sodium azide,76. The assay buffer of claim 74 or 75, comprising the buffering agent, NaCl, MgCh and sodium azide.

77. The assay buffer of any one of claims 74-76, comprising the buffering agent, NaCl, MgCh, sodium azide, and Levamisole.

78. The assay buffer of any one of claims 74-77, further comprising triethylamine (TEA).

79. The assay buffer of any one of claims 74-78, further comprising an anti-foaming reagent.

80. The assay buffer of claim 79, wherein the anti-foaming reagent is Korasilon® or plutonic.

81. The assay buffer of any one of claims 74-80, comprising 0.5 mM-5mM TEA, wherein the buffering agent comprise Tris base and Tris HC1, Tris base having a concentration of 50- 200 mM, Tris HC1 having a concentration of l-20mM, MgCh having a concentration of 0.5 mM - 10 mM, and optionally, Korasilon® EMA 119 (an emulsion comprising about 45% of silicone, a viscosity of between about 1,500 to about 2,000 mPA s at 25 °C, and a pH of about 8) having a concentration of 1-500 ppm, e.g., 10-100 ppm.

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