Optical imaging system using lateral illumination for digital assay

Abstract

To provide a simplified optical imaging system for digital immunoassay which provides a small and low-cost digital immunoassay device.SOLUTION: A small optical imaging system 100 includes a single filter 132 for detecting a bead in a digital assay and light sources 112 and 116 for lateral irradiation. The light sources radiate light to a detection container. The single filter is arranged to receive light emitted from the light sources and reflected from a sample in the detection container and receive output from a sample in the detection container. A detector 136 receives a part of reflected light and a part of output which passes through the single filter.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] This application is a non-provisional application of U.S. Provisional Patent Application No. 62 / 437,534, filed on December 21, 2016, and claims the benefit thereof, the entire content of which is incorporated herein by reference.

[0002] The present disclosure relates to an optical imaging system for analyte analysis in digital assays, including a single filter and a light source providing lateral illumination.

Background Art

[0003] Devices and methods capable of accurately analyzing analytes of interest in a sample are essential, for example, in diagnostics such as the diagnosis of diseases, disorders, or health conditions, prognosis, environmental assessment, food safety, detection of chemical or biological warfare agents, etc. Most current techniques for quantifying low levels of analyte molecules in a sample provide a measurable signal by using an amplification procedure that increases the number of reporter molecules. Examples of current techniques include enzyme-linked immunosorbent assay (ELISA) for amplifying signals in antibody-based assays, as well as polymerase chain reaction (PCR) for amplifying target DNA strands in DNA-based assays. Most detection methods require the presence of a large number of molecules in the whole in order for the signal ensemble to exceed the detection threshold. This requirement limits the sensitivity and dynamic range (i.e., the range of concentrations that can be detected) of most detection techniques. Many of the known methods and techniques are further troubled by the problem of non-specific binding, which leads to an increase in background signal and limits the lowest concentration that can be accurately or reproducibly detected.

[0004] Digital ELISA is a candidate for the next-generation immunoassay because it can detect one molecule of enzyme using a conjugate. See FIGS. 1 and 2. In digital ELISA, the target molecule is captured on beads between a capture antibody and a detection antibody, and the detection antibody is conjugated to an enzyme. At this time, the beads are captured in a droplet chamber together with the enzyme substrate, and the aqueous phase is replaced by heavy oil, enabling removal of the aqueous phase before analysis.

[0005] In bead-based digital ELISA, single beads are encapsulated within the microchambers of an array. Some of the chambers in which the beads have captured immunoreactive species present bright spots (i.e., bright chambers) during fluorescence imaging. The percentage of chambers in which beads are present correlates with the concentration of the antigen. Therefore, it is necessary to identify the positions of the beads and the enzyme within the microchambers of the array.

[0006] Optical imaging is a method for determining the location of beads during an assay. Optical imaging can also detect data from a large number (more than 10,000) of microchambers simultaneously using bead / enzyme dual-channel imaging. However, conventional optical systems for dual-channel imaging are large and costly to attach to existing products due to the complex optics for fluorescence / fluorescence type bead / enzyme dual-channel imaging. Conventional optical systems require multiple optical filters for each channel and an external actuator for mode switching.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Document

[0008]

Non-Patent Document 1

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Summary of the Invention

Problems to be Solved by the Invention

[0009] Therefore, it is desirable to provide a simplified optical imaging system for digital immunoassay that provides a small and low-cost digital immunoassay device. Embodiments of the present invention provide an imaging system using light scattering optics for digital immunoassay, the size and cost of which are significantly reduced compared to conventional optical imaging systems.

Means for Solving the Problems

[0010] Embodiments of the present invention simplify the optical imaging system by using a light source that applies light scattering to the assay for bead detection. This optical imaging system utilizes the light scattering / fluorescence type and is simplified by removing the optical components and the corresponding actuator structure for mode switching used in conventional systems. To demonstrate the power of the optical imaging system according to embodiments of the present system, a small device for digital ELISA was designed and tested. Embodiments of this optical imaging system demonstrated good performance and were comparable to or better than conventional optical imaging systems. (Notable specifications are summarized in FIG. 1.) The detector is approximately one tenth and one hundredth in volume and cost, respectively.

[0011] In one embodiment, the present invention provides a miniaturized digital assay device that includes a detection vessel, a light source configured to emit light toward the detection vessel, a single filter, and a detector. The single filter is arranged to receive light emitted from the light source and reflected from a sample within the detection vessel, and to receive an output from the sample within the detection vessel. The detector is configured to receive a portion of the reflected light and a portion of the output passing through the single filter.

[0012] In another embodiment, the present invention provides a miniaturized digital assay device that includes a detection vessel, a first light source configured to emit light toward the detection vessel at an angle with respect to a sample array, a second light source configured to emit light toward the detection vessel, a single filter, and a detector. The single filter is arranged to receive light emitted from the first light source and reflected from a sample within the detection vessel, and to receive a fluorescence output from the sample within the detection vessel. The detector is configured to receive a portion of the reflected light and a portion of the fluorescence passing through the single filter.

[0013] In a further embodiment, the present invention provides a miniaturized digital assay device that includes a sample array having a plurality of wells, a first light source configured to emit light toward the sample array at an angle with respect to the sample array to irradiate a sample within the sample array, a second light source configured to emit light toward the sample array without using a mirror or other reflective object, and further configured to activate a sample within the sample array to emit an output, a filter, and a detector. The filter is arranged to receive light emitted from the first light source and reflected from the sample array, and to receive an output from the sample within the sample array. The detector is configured to receive the reflected light and the output from the sample passing through the filter, and to generate optical data for identifying which wells contain beads and which wells contain labels.

[0014] In yet another embodiment, the present invention provides a miniaturized digital assay device comprising a sample array including a plurality of samples disposed within a plurality of nanowells, a light source configured to emit light towards the sample array at an angle with respect to the sample array to irradiate the samples within the sample array and having a wavelength between 450 nm and 550 nm, and a detector configured to receive a portion of the light reflected from the samples.

[0015] Other aspects of the invention will become apparent by considering the detailed description and the accompanying drawings.

[0016] Details of the subject matter described herein may be apparent, both as to its structure and operation, by considering the accompanying drawings, in which like reference numerals refer to like parts. The components in the drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the subject matter. Further, all the figures are intended to convey concepts, and relative sizes, shapes, and other detailed attributes may be shown schematically, not literally or precisely.

Brief Description of the Drawings

[0017]

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DETAILED DESCRIPTION OF THE INVENTION

[0018] 1. Definitions Before describing the embodiments of the present disclosure, it should be understood that the present invention is not limited to the specific embodiments described, and thus can be naturally modified. It should also be understood that the terms used in this specification are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0019] As used herein, the terms "comprise", "include", "having", "has", "can", "contain", and variations thereof are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure contemplates other embodiments that "comprise", "consist of", and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly described.

[0020] In the description of numerical ranges herein, each intervening number therebetween is specifically contemplated with the same degree of precision. For example, in the range of 6 - 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range of 6.0 - 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are specifically contemplated.

[0021] As used herein, "affinity" and "binding affinity", as used interchangeably herein, refer to the tendency or strength of binding of a binding member to an analyte. For example, binding affinity may be represented by an equilibrium dissociation constant (KD), a dissociation rate (kd), or an association rate (ka).

[0022] As used herein, "analog" refers to a molecule having a structure similar to the molecule of interest (e.g., nucleoside analogs, nucleotide analogs, sugar phosphate analogs, analyte analogs, etc.). An analyte analog is a molecule that is structurally similar to an analyte but has a different affinity for a binding member.

[0023] As used interchangeably herein, "analyte", "target analyte", and "analyte of interest" refer to the analyte being measured in the methods and devices disclosed herein. The analyte of interest is further described herein.

[0024] As used herein, "antibody" and "antibodies" refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), and, without limitation, animal antibodies such as avian (e.g., duck or goose), shark, whale, and mammalian including non-primates (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.) or non-human primates (e.g., monkey, chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fv ("scFv"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-bonded Fv ("sdFv"), anti-idiotype ("anti-Id") antibodies, dual-domain antibodies, antibodies with dual variable domains (DVD) or triple variable domains (TVD) (dual variable domain immunoglobulins and methods for generating them are described in Wu, C. et al., Nature Biotechnology, 25(11), pp. 1290-1297 (2007) and PCT International Patent Application WO2001 / 058956, the contents of each of which are incorporated herein by reference), and functionally active epitope-binding fragments of any of the foregoing. In particular, an antibody includes an immunoglobulin molecule and an immunologically active fragment of the immunoglobulin molecule, i.e., a molecule that includes an analyte-binding site. The immunoglobulin molecule can have any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For simplicity, an antibody to an analyte is often referred to herein as either "anti-analyte antibody" or simply "analyte antibody".

[0025] As used herein, "antibody fragment" refers to a portion of an intact antibody that includes an antigen-binding site or variable region. This portion does not include the constant heavy-chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, or CH4 depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragment, Fab' fragment, Fab'-SH fragment, F(ab')2 fragment, Fd fragment, Fv fragment, bispecific antibody, single-chain Fv (scFv) molecule, single-chain polypeptide containing only one light-chain variable domain, single-chain polypeptide containing three CDRs of the light-chain variable domain, single-chain polypeptide containing only one heavy-chain variable region, and single-chain polypeptide containing three CDRs of the heavy-chain variable region.

[0026] "Beads" and "particles" are used interchangeably herein and refer to a substantially spherical solid-phase support.

[0027] "Binding protein" is used herein to refer to a monomeric or multimeric protein that binds to a binding partner, such as, for example, a polypeptide, antigen, compound or other molecule, or any type of substrate, and forms a complex with the binding partner. A binding protein binds specifically to a binding partner. Binding proteins include antibodies and antigen-binding fragments thereof and various other forms and derivatives thereof known in the art and described herein below, and other molecules that include one or more antigen-binding domains that bind to an antigen molecule or a specific site (epitope) on an antigen molecule. Thus, binding proteins include, but are not limited to, antibodies, tetrameric immunoglobulins, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity-matured antibodies, and fragments of any such antibody that maintain the ability to bind to an antigen.

[0028] As used herein, "capture molecule" refers to a specific binding partner or specific binding member that is used to capture or immobilize an analyte of interest in a biological sample. A capture molecule is often one component of a complex that also includes one or more detection molecules, in addition to the analyte of interest. The complex may optionally be bound to a solid support.

[0029] "Component", "a plurality of components", or "at least one component" generally refers 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 cofactor for the enzyme, a detection reagent, a pretreatment reagent / solution, a substrate (e.g., as a solution), a stop solution, etc., which may be included in a kit for assaying a test sample such as a patient's urine, serum, whole blood, tissue aspirate, or plasma sample, by the methods described herein and other methods known in the art. Some components may be in solution or lyophilized for reconstitution for use in the assay.

[0030] As used herein, "contact" and its grammatical synonyms refer to any type of binding action that brings a binding member sufficiently close to an analyte of interest in a sample such that a binding interaction occurs if the analyte of interest specific to the binding member is present in the sample. Contact may be achieved in a variety of ways, including binding the sample to the binding member, exposing the target analyte to the binding member by bringing the binding member close to the analyte.

[0031] As used herein, "control" refers to a standard sample of an analyte that is known in the art, or is acceptable or generally used, or is experimentally determined using acceptable means known in the art. A "standard sample" is a standard substance used as a measurement basis for similar substances. For example, documented standard samples are 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 reference substances are described in the literature. Means for quantifying the amount of analyte present by using a calibration curve for the analyte or by comparison to an alternative standard sample are also well known. Standard curves can be generated using serial dilutions or solutions of an analyte of known concentration by mass spectrometry, gravimetry, and other techniques known in the art. Alternative standard samples that have been described in the literature include standard addition (also known as the standard addition method) or digital polymerase chain reaction.

[0032] As used herein, "detection molecule" refers to a specific binding partner or specific binding member used to detect the presence of an analyte of interest in a biological sample and / or to quantify or measure the amount of that analyte. A detection molecule is often one component of a complex that may include one or more capture molecules and an analyte of interest. The complex may optionally be bound to a solid support.

[0033] As used herein, a “detection” container or a “reaction” container refers to a container or other device that can contain a reaction mixture that may or may not contain an analyte of interest. Examples of suitable “detection” containers or “reaction” containers include cuvettes, tubes, individual tubes of a tube plate, wells or holes in a microtiter plate, individual reaction wells (such as an array of wells, such as a microarray or nanoarray), or pits in a test slide plate or assay array plate.

[0034] As used herein, “immobilization” refers to a stable association between a first specific binding member and the surface of a solid support. “Stable association” means, for example, a physical association between two entities where the average half-life of the association is greater than 1 day under physiological conditions. In some embodiments, the physical association between two entities has an average half-life of greater than 2 days, greater than 1 week, greater than 1 month, including greater than 6 months, such as greater than 1 year in PBS at 4°C. According to some embodiments, the stable association results from a covalent bond between two entities, a non-covalent bond between two entities (such as an ionic or metal bond), or other forms of chemical attraction such as hydrogen bonds or van der Waals forces.

[0035] As used herein, “label” and “detectable label” refer to a moiety attached to an antibody or an analyte that provides for a reaction between the antibody and a detectable analyte, and an antibody or analyte so labeled is said to be “detectably labeled”. A label can generate a signal that is detectable by visual or instrumental means. Various labels include signal generating substances such as chromogens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, etc. Other labels are described herein. In this regard, a moiety may become detectable by reacting with yet another moiety even if it is not itself detectable. The use of the term “detectably labeled” is intended to include such labeling.

[0036] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutants that may be present in minor proportions. Monoclonal antibodies are highly specific and are directed against a single antigen. In contrast to polyclonal antibody preparations, which typically include various antibodies directed against various determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains is from antibodies obtained from a particular species or belongs to a particular antibody class or subclass and is identical or homologous to the corresponding sequences of antibodies of that class or subclass, while the remaining portions of these chains are from antibodies obtained from another species or belong to another antibody class or subclass and are identical or homologous to the corresponding sequences of antibodies of such other class or subclass and fragments of such antibodies, as long as they exhibit the desired biological characteristics.

[0037] "Polynucleotide" or "oligonucleotide" refers to a polymer or oligomer of nucleobases, wherein the nucleobases are connected by sugar-phosphate linkages (sugar-phosphate backbone). Exemplary polynucleotides and oligonucleotides include polymers of 2'-deoxyribonucleotides (DNA) and polymers of ribonucleotides (RNA). A polynucleotide may consist entirely of ribonucleotides, 2'-deoxyribonucleotides, or combinations thereof. "Nucleic acid" includes "polynucleotide" and "oligonucleotide" and includes single-stranded and double-stranded polymers of nucleotide monomers.

[0038] As used herein, "predetermined cut-off" and "predetermined level" refer to assay cut-off values used to evaluate diagnostic results, prognostic results, or treatment efficacy results by comparing assay results to a predetermined cut-off / level, although the predetermined cut-off / level is already linked or associated with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement of disease, etc.). The present disclosure provides exemplary predetermined levels. However, it is well known that cut-off values can vary depending on the nature of the immunoassay (e.g., antibodies used, reaction conditions, sample purity, etc.). Further, it is well known to those skilled in the art to adapt the disclosure herein to these other immunoassays to obtain immunoassay-specific cut-off values for these other immunoassays based on the description provided by the present disclosure. The exact value of the predetermined cut-off / level may vary between assays, but the correlations described herein should generally be applicable.

[0039] "Pretreatment reagents" used in the diagnostic assays described herein, such as lysis reagents, precipitation reagents, and / or solubilization reagents, lyse any cells present in the test sample and / or make any analyte present in the test sample soluble. Pretreatment is not necessary for all samples, as further described herein. In particular, making the analyte soluble involves releasing the analyte from any endogenous binding proteins present in the sample. The pretreatment reagent may be homogeneous (not requiring a separation step) or heterogeneous (requiring a separation step). When using a heterogeneous pretreatment reagent, any precipitated analyte-binding protein is removed from the test sample before proceeding to the next step of the assay. The pretreatment reagent can optionally comprise: (a) one or more solvents and salts, (b) one or more solvents, salts, and surfactants, (c) surfactants, (d) surfactants and salts, or (e) any reagent or combination of reagents suitable for cell lysis and / or solubilization of the analyte.

[0040] "Quality control reagents" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity panels. A "calibrator" or "standard" is typically used to construct a calibration (standard) curve (e.g., one or more, such as a number) for interpolation of the concentration of an analyte, such as an antibody or analyte. Alternatively, a single calibrator close to a predetermined positive / negative cutoff can be used. Multiple calibrators (i.e., two or more calibrators or calibrators in varying amounts) can be used together to include a "sensitivity panel".

[0041] As used herein, "receptor" refers to a protein molecule that recognizes and responds to an endogenous chemical signal. Such an endogenous chemical signal, when bound to the receptor, causes some form of cell / tissue response. Examples of receptors include, but are not limited to, nerve receptors, hormone receptors, nutrient receptors, and cell surface receptors.

[0042] "Recombinant antibody" and "plural recombinant antibodies" refer to antibodies prepared by one or more steps including cloning nucleic acid sequences encoding all or part of one or more monoclonal antibodies into a suitable expression vector, and subsequently expressing the antibody in a suitable host cell. These terms include, but are not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized), multispecific or multivalent structures formed from antibody fragments, bifunctional antibodies, heteroconjugate Abs, [Math.1]DVD-Ig(R), and (i) other antibodies described herein. (Dual variable domain immunoglobulins and methods for generating them are described in Wu, C. et al., Nature Biotechnology, 25, 1290-1297 (2007).) As used herein, the term "bifunctional antibody" refers to an antibody that includes a first arm having specificity for one antigen site and a second arm having specificity for a different antigen site, i.e., a bifunctional antibody has dual specificity.

[0043] As used herein, "sample", "test sample", "biological sample", "sample from a subject", "fluid biological sample", and "patient sample" are used interchangeably and may refer to a fluid sample that contains or is suspected of containing an analyte of interest.

[0044] As used herein, "sample array" refers to a collection of one or more (or plural) detection vessels or reaction vessels.

[0045] As used herein, "signal generating compound" refers to any molecule, compound, protein, etc. that can be converted into a detectable product or detectable label when exposed to a suitable or appropriate converter such as a signal generating substrate. A "detectable product" or "detectable label" is any molecule, particle, etc. that facilitates detection by acting as an entity that is detected using a selected technique known in the art. Examples of signal generating compounds are enzymes such as amylase, polynucleotidase, arginase, adenase, aminopolypeptidase, pepsin, lipase, catalase, tyrosinase, alcohol dehydrogenase, succinate dehydrogenase, diaphorase, glyoxalase, aldolase, glucose oxidase, horseradish peroxidase, galactosidase (such as beta-galactosidase), phosphatase, phosphorylase and hexokinase, or combinations thereof.

[0046] As used herein, "signal generating substrate" refers to any molecule, compound, protein, substrate, particle, etc. that can be converted into a signal generating compound that is converted into a detectable product or detectable label when exposed to a suitable or appropriate converter such as a signal generating compound, or that can give rise to this signal generating compound. A "detectable compound" or "detectable label" is any molecule, particle, etc. that facilitates detection by acting as an entity that is detected using a selected technique known in the art. The signal generating substrate may be colorimetric, chemiluminescent, or chemifluorescent. Examples of signal generating substrates are [Math.2]CDP-Star(R) (disodium 4-chloro-3-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1 3,7 decane}-4-yl)phenyl phosphate), [Math.3]CSPD(R) i.e. (disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.1 3,7Chemiluminescent substrates such as {decan}-4-yl)phenyl phosphate), and luminescent substrates such as p-nitrophenyl phosphate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 4-nitroblue tetrazolium chloride (NBT), or iodonitrotetrazolium (INT), and fluorescent substrates such as 4-methylumbelliferyl phosphate (4-MUP), and chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium 5-bromo-6-chloro-indolyl phosphate, or p-nitrophenyl phosphate are enzyme substrates.

[0047] As used interchangeably herein, a "specific binding partner" or "specific binding member" refers to one of two or more different molecules that specifically recognize a molecule as compared to substantially less recognition of other molecules. One of the two different molecules specifically binds to a spatially and polar-specific configuration of the other molecule and thereby has a region on its surface or within a cavity that is defined as complementary to that configuration. These molecules may be members of a specific binding pair. For example, specific binding members may include, but are not limited to, proteins such as receptors, enzymes, and antibodies.

[0048] In addition to the specific binding pairs of antigen and antibody in common immunoassays, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrate and lectin, complementary nucleotide sequences, effector molecule and receptor molecule, cofactor and enzyme, enzyme and enzyme inhibitor, etc. Further, a specific binding pair can include members that are analogs of the original specific binding member, such as analyte analogs. Immunoreactive specific binding members include antigens, antigen fragments, and antibodies, which can be produced singly or recombinantly, and include monoclonal and polyclonal antibodies and their conjugates and fragments.

[0049] "Solid support" refers to any material that is insoluble or may be rendered insoluble by subsequent reactions. The solid support may be selected due to its inherent ability to attract and immobilize the capture agent. Alternatively, the solid support may include a binder attached to the solid support, and this binder has the ability to attract and immobilize the capture agent. For example, the binder can include a charged substance that is oppositely charged with respect to the capture agent itself or a charged substance conjugated with the capture agent. Generally, the binder may be any binding partner (preferably specific) that is immobilized (attached) on the solid support and has the ability to immobilize the capture agent through a binding reaction. The binder enables the indirect binding of the capture agent to the solid support material before or during the execution of the assay. For example, the solid support can be plastic, derivatized plastic, magnetic or non-magnetic metal, glass, or silicon, including, for example, test tubes, microtiter wells, sheets, beads, microparticles, chips, and other configurations known to those skilled in the art.

[0050] As used interchangeably herein, "subject" and "patient" refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice), non-human primates (e.g., monkeys such as cynomolgus monkeys or rhesus monkeys, chimpanzees, etc.), and humans. In some embodiments, the subject may be human or non-human. The subject or patient may be undergoing other forms of treatment.

[0051] As used herein, "threshold" refers to an experimentally determined subjective cut-off level above which acquired data is considered a "signal" and below which acquired data is considered "noise". A computer program based on CUSUM (cumulative sum algorithm) is used to process acquired data and detect events based on a threshold input from the user. Variation between users is avoided by detecting as many events as possible and subsequently filtering the data later for a particular purpose. Using a "loose" threshold will result in a smaller number of events being counted as signals. Using a "strict" threshold will result in a larger number of events being counted as signals. Setting the threshold loose or strict is a subjective choice based on the desired sensitivity or specificity for the assay and whether false positives or false negatives are preferred in a given assessment.

[0052] "Treat", "treating", or "treatment" are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progression of a disease, or one or more symptoms of such a disease to which such terms apply. Depending on the condition of the subject, the term also refers to preventing a disease, including preventing the occurrence of a disease or preventing symptoms associated with a disease. Treatment may be effected by either acute or chronic means. The term also refers to reducing the severity of a disease or symptoms associated with such a disease before the subject suffers from the disease. Such prevention or reduction of the severity of the disease prior to pain refers to administration of an antibody or pharmaceutical composition of the invention to the subject, who does not suffer from the disease at the time of administration. "Preventing" also refers to preventing recurrence of a disease or one or more symptoms associated with such a disease. "Treatment" and "therapeutically" refer to the act of treating as "treating" is defined above.

[0053] Unless defined otherwise, 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 are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for the purpose of disclosing and describing the methods and / or materials related to the cited publications. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0054] 1. Optical Imaging System The present invention provides a small and relatively low-cost device for performing digital assays, such as digital immunoassays, to detect and / or quantify an analyte of interest in a sample. The device may be hand-held, or placed on a support surface, or connected to and supported by an adjacent processing device. The devices described herein provide several advantages over conventional optical imaging systems known in the art. For example, the devices of the present disclosure are small, provide a larger imaging area, and include a simplified structure, among other things. As depicted in FIG. 1, one embodiment of the present invention is compared to a conventional system (e.g., an optical microscope system). The size of the device of the present invention is much smaller than that of a conventional system. For example, one configuration of the device is 12 cm × 10 cm × 10 cm, while a conventional system is 70 cm × 50 cm × 50 cm. In this configuration, the device is approximately one-fifth the size of a conventional system. Additionally, the imaging area of the device of the present invention is approximately 9 mm for a conventional system 2 for a certain 30,000 sample chambers, approximately 30 mm 2There is a sample chamber of 100,000. Image acquisition can occur under the ambient conditions of the embodiments of the present invention, while conventional systems require a darkroom. An additional comparison between the embodiments of the present invention and the embodiments of conventional systems is shown in FIG. 1.

[0055] The structure of the apparatus of the present invention is also not more complex than the structure of conventional systems. Conventional systems that utilize fluorescence imaging techniques for the detection of one or more analytes of interest in a sample require multiple optical filters for each of two channels, along with an actuator for filter replacement. The present apparatus utilizes light scattering imaging techniques for analyte detection and a light source directed at an angle to a sample array (including one or more detection or reaction vessels), thus enabling the use of a single filter and the elimination of the multiple filters and actuators used in conventional systems. Additionally, the color of the light source can be selected based on the emission filter used for analyte detection, thereby enabling the use of a single filter for analyte detection.

[0056] According to one embodiment, the apparatus includes several components, including one or more detection or reaction vessels, a light source configured to emit light towards the detection vessel, a single filter arranged to receive light emitted from the light source and reflected from a sample within the one or more detection vessels and to receive an output from the sample within the detection vessel, and a detector configured to receive a portion of the reflected light and a portion of the output passing through the single filter.

[0057] FIGS. 2-5 show an optical imaging system 100 according to an embodiment of the present invention. Referring to FIG. 4, the optical imaging system 100 is configured to detect a solid support (such as beads) that may contain an analyte of interest in a detection vessel 104 (such as a sample array including a plurality of microwells or nanowells) and to detect an output from the sample within the detection vessel when the sample is activated (such as by using a detectable label).

[0058] The optical imaging system 100 may include a support 108 for receiving a detection container 104. The system 100 also includes a first light source 112 and may include a second light source 116. The first light source 112 may be composed of a single light source or a plurality of light sources. The first light source 112 is disposed above or below the detection container 104 and is directed at an angle θ with respect to an axis 120 that extends substantially perpendicular to the detection container 104. For example, substantially perpendicular includes 90 degrees + / - about 2 degrees (i.e., from 88 degrees to 92 degrees) with respect to the detection container 104. The angle θ is between about 0 degrees and about 90 degrees. In other embodiments, the angle θ is between about 45 degrees and about 90 degrees. In another embodiment, the angle θ is 80 degrees.

[0059] The first light source 112 emits scattered light 124 toward the detection container 104. The first light source 112 can include a light emitting diode (LED) that emits light at a specific wavelength. For example, the first light source 112 can include a green LED that emits light at about 520 nm. The scattered light 124 is reflected by the sample in the detection container 104 as reflected light 128, and the reflected light 128 is received by a filter 132. The filter 132 allows a portion of the reflected light 128 to pass to a detector or camera 136. The camera 136 generates an image, and the image shows bright pixels that visualize or determine the position of any solid phase support (e.g., beads) that contains the analyte of interest in the image. The image helps determine the number of detection containers that contain solid phase supports that contain the analyte of interest and / or provides spatial information regarding the location of these sites. See, for example, FIG. 6, which shows images (A) and (D) generated by the first light source 112 described above. In other configurations, the color of the LED of the first light source 112 may be selected based on the filter 132 used in the device.

[0060] Continuing to refer to FIGS. 4 and 5, the second light source 116 is disposed above or below the detection container 104 and is directed substantially perpendicular to the detection container 104. For example, substantially perpendicular includes 90 degrees and + or - about 2 degrees (i.e., 88 degrees to 92 degrees with respect to the detection container 104). In some embodiments, the second light source 116 is disposed along the axis 120, while in other embodiments, the second light source 116 is disposed adjacent to the axis 120. For example, as shown in FIG. 4 (Type A), the second light source 116 is laterally offset from the axis 120 so as not to interfere with the scattered light reflected from the sample and the fluorescence emitted from the sample (described below).

[0061] The second light source 116 emits excitation light 140 toward the detection container 104. The second light source 116 can include an LED that emits excitation light at a specific wavelength. For example, the second light source 116 can include a blue LED that emits light at about 450 nm. The excitation light 140 excites or activates the sample within the detection container 104. For example, if a specific analyte is present in the sample, the sample emits an output 144 such as fluorescence. If a specific analyte is not present in the sample, the output 144 is not produced. The filter 132 (the same filter that receives the reflected light 128) receives the output 144 and allows it to pass to the camera 136. The camera 136 generates an image, and the image shows bright pixels that visualize or determine which detection containers contain the specific analyte of interest. For example, refer to FIG. 6, which shows images (B) and (E) generated by the second light source 116 as described above. The second light source 116 (e.g., the color of the LED) may be specifically selected based on the filter 132 used for analyte detection in the sample.

[0062] The camera 136 may be a CCD camera used to capture an image. Other examples of cameras include charge injection devices (CID), complementary metal oxide semiconductor (CMOS) devices, scientific CMOS (sCMOS) devices, and time delay integration (TDI) devices.

[0063] Camera 136 may be electronically or communicatively coupled to computer 148. Computer 148 includes an electronic processor (e.g., a microprocessor, an application specific integrated circuit (ASIC), or another suitable electronic device), a memory device (e.g., a non-transitory computer-readable storage medium), and a communication interface, such as a communication network (e.g., wireless) and, optionally, a transceiver for communicating over one or more additional communication networks or connections. The electronic processor, the memory device, and the communication interface communicate over one or more communication lines or buses. It should be understood that the computer may include additional components for the components described above in various configurations and may perform additional functions for the functions described in this application. For example, in some embodiments, the functions described herein performed by the computer may be distributed among multiple devices such as multiple computers and servers.

[0064] The electronic processor executes instructions stored in the memory device. In particular, the memory device stores an image analyzer. The image analyzer is a software application executable by the electronic processor. As described below, when executed by the electronic processor, the image analyzer communicates with camera 136 over a communication network (through the communication interface) to manage the transfer of data locally stored on camera 136 to one or more remote storage locations (e.g., a memory device within a computer).

[0065] The image analyzer receives an input of an image generated by camera 136. The image analyzer can process the images and combine them into a merged image indicating the location of the beads and enzymes of the sample within detection vessel 104. For example, see FIG. 6 showing images (C) and (F) with the analyzed and merged images. In particular, image (C) is the analyzed and merged image of images (A) and (B) in FIG. 6. Similarly, image (F) is the analyzed and merged image of images (D) and (E) in FIG. 6.

[0066] 2. Method for analyte analysis A method for analyte analysis is also provided herein. The method may include counting single molecules. In some embodiments, the method for analyte analysis may include evaluating an analyte of interest present in a sample. In some embodiments, this evaluating may be used to determine the presence and / or concentration of the analyte of interest in the sample. In some embodiments, the method may also be used to determine the presence and / or concentration of a plurality of different analytes of interest present in the sample.

[0067] A method for detecting an analyte of interest in a droplet is provided herein, where the analyte of interest is from a test sample or a biological sample. The method includes providing a first droplet containing the analyte of interest, providing a second droplet containing at least one solid support (such as a magnetic solid support (e.g., beads)) that includes a specific binding member that binds to the analyte of interest, using energy to exert a force to manipulate the first droplet (containing the analyte of interest) with the second droplet (containing at least one solid support) to produce a mixture (also referred to herein as the "reaction mixture"), moving all or at least a portion of the mixture to an array of wells, where one or more wells of the array have a size sufficient to accommodate at least one solid support, adding at least one detectable label to the mixture before, after, or both before and after moving a portion of the mixture to the array of wells, and detecting the analyte of interest within the wells. The array of wells is also referred to herein as the "detection container".

[0068] In some embodiments, the wells can be a micro-well array or a nano-well array. In some embodiments, the micro-well array or nano-well array has a diameter of at least about 4 mm, at least about 5 mm, at least about 6 mm, at least about 7 mm, at least about 8 mm, at least about 9 mm, or at least about 10 mm. In some embodiments, the micro-well array has a diameter of 6 mm. In some embodiments, the micro-well array or nano-well array contains from about 100,000 to about 1,000,000 wells, from about 200,000 to about 750,000 wells, or from about 300,000 to about 500,000 wells. In some embodiments, the micro-well array contains about 100,000, about 200,000, about 300,000, about 350,000, about 375,000, about 400,000, about 425,000, about 450,000, about 475,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, or about 1,000,000 wells. In some embodiments, the micro-well array contains 400,000 wells. In some embodiments, the wells can have a diameter of at least about 1 μm, at least about 2 μm, at least about 3 μm, at least about 4 μm, at least about 5 μm, at least about 6 μm, at least about 7 μm, at least about 8 μm, at least about 9 μm, or at least about 10 μm at the bottom of the well. In some embodiments, the plurality of reaction vessels can be a micro-well array or a nano-well array having a diameter of 6 mm and containing about 400,000 wells having a diameter of 5 μm at the bottom of the wells.

[0069] In some embodiments, the step of "using energy to exert a force on the first droplet using the second droplet" refers to providing or exerting a force that manipulates (e.g., combines or joins) at least the first and second droplets (and optionally additional droplets) into a mixture, using a non-mechanical force (i.e., energy generated without using, for example, pumps and / or valves). Examples of non-mechanical forces that may be used in the methods described herein include electric driving forces (such as droplet driving, electrophoresis, electro-wetting, dielectrophoresis, electrostatic driving, mediating electric fields, mediating electrodes, capillary forces, chromatography, centrifugation, or suction) and / or acoustic forces (such as surface acoustic waves (i.e., "SAW")). In some embodiments, the electric driving force generated is an alternating current. For example, the alternating current can have a root mean square (rms) voltage of 10V, 15V, 20V, 25V, 30V, 35V, or more than these. For example, such an alternating current can have an rms voltage of 10V or more, 15V or more, 20V or more, 25V or more, 30V or more, or 35V or more. Alternatively, the alternating current can have a frequency in the radio frequency range.

[0070] In some embodiments, when a magnetic solid support is used, the electric driving force and the magnetic field can be applied from opposite directions to at least a portion of the mixture. In some other embodiments, the mixture is mixed by moving the mixture in a circumferential direction, back and forth, or by separating the mixture into two or more sub-mixtures and then integrating these sub-mixtures. In some other embodiments, the electric driving force is used to move the mixture into an array of wells (each loaded with at least one solid support) to seal the wells, and can be generated using a series or plurality (i.e., at least two or more, at least three or more, at least four or more, at least five or more, at least six or more, at least seven or more, at least eight or more, at least nine or more, at least ten or more, at least eleven or more, at least twelve or more, at least thirteen or more, at least fourteen or more, at least fifteen or more, etc.) of electrodes.

[0071] In some embodiments, the step of moving all or at least a portion of the mixture to the array of wells results in the loading (filling and / or placement) of at least one solid support onto the array of wells. In some embodiments, a magnetic field is used to facilitate the movement of the mixture and thus at least one solid support to one or more wells of the array. In some embodiments, after at least one solid support has been loaded into a well, any solid support that has not been loaded into the well may be removed using defined techniques known in the art. For example, such removal may involve generating an electrical driving force (as described earlier herein) using a series or plurality of electrodes to move at least a portion of the mixture away from the array of wells (to a non-critical distance), such as by moving fluid droplets (such as polarized fluid droplets) to the array of wells. In some embodiments, an aqueous wash liquid may be used to remove solid supports that are not bound to any analyte of interest. In such embodiments, this removal involves generating an electrical driving force using a series or plurality of electrodes to move aqueous wash (or washing) droplets (a third droplet) across the array of wells. The amount and type of aqueous liquid used for said washing is not critical.

[0072] In some embodiments, the mixture in the method is an aqueous liquid. In other embodiments, the mixture is an immiscible liquid. In other embodiments, the droplets are hydrophobic droplets. In other embodiments, the droplets are hydrophilic droplets. In some embodiments, the array of wells used in the method has a hydrophobic surface. In other embodiments, the array of wells has a hydrophilic surface.

[0073] In some embodiments, the first droplet used in the method is a polar liquid. In some embodiments, the second droplet used in the method is a polar liquid. In some embodiments, the first and second droplets used in the method are polar liquids. In some embodiments, the mixture is a polar liquid. In some embodiments, one or more of the first droplet, the second droplet, and the mixture is a polar liquid.

[0074] In some embodiments, at least one solid support comprises at least one specific binding member that specifically binds to the analyte of interest. In some embodiments, the detectable label is added to the mixture before moving at least a portion of the mixture to an array of wells. In some other embodiments, the detectable label is added to the mixture after moving at least a portion of the analyte of interest. In some embodiments, the detectable label comprises at least one specific binding member that specifically binds to the analyte of interest. In some embodiments, the detectable label comprises a chromophore, a fluorescent compound, an enzyme, a chemiluminescent compound, or a radioactive compound. In some embodiments, the specific binding member is a receptor, an aptamer, or an antibody. In some embodiments, the method further comprises the step of disposing at least a portion of the mixture across an array of wells using a capillary element configured to facilitate movement of the mixture to the array of wells.

[0075] In some embodiments, when the mixture is a liquid and after the mixture has moved into the well array, an aqueous phase is formed within the wells. In some embodiments, the wells may be sealed by the addition of one or more solvents ("solvent well sealing"). Hydrophilic or hydrophobic solvents may be used. Hydrophilic solvents that may be used include hydrophilic alcohols, hydrophilic ethers, ketones, nitrile solvents, dimethyl sulfoxide, and N,N-dimethylformamide, or mixtures thereof. Examples of hydrophilic alcohols include ethanol, methanol, propanol, and glycerin. Examples of hydrophilic ethers include tetrahydrofuran, polyethylene oxide, and 1,4-dioxane. Examples of ketones include acetone and methyl ethyl ketone. Examples of nitrile solvents include acetonitrile. Hydrophobic solvents that may be used include hydrocarbons, unsaturated hydrocarbons, aromatic hydrocarbons, silicone oils, perfluorocarbons, halogenated solvents, hydrophobic ionic solutions, and mixtures thereof. Examples of saturated hydrocarbons include alkanes such as decane and hexadecane. Examples of unsaturated hydrocarbons include squalene. Examples of aromatic hydrocarbons include benzene and toluene. Examples of perfluorocarbons include [Math.4]Fluorinert(R) (FC-40, FC-72, FC-84, FC-77, FC-3255, FC-3283, FC-43, FC-7), 3M Novec4200, 3M Novec4300, 3M FC-4432, 3M FC-4430, or 3M FC-4434. Examples of halogenated solvents include chloroform, methylene chloride, and chlorobenzene. Hydrophobic ionic solutions refer to ionic solutions that do not dissociate, at least in water. Examples of ionic solutions include 1-butyl-3-methylimidazolium hexafluorophosphate.

[0076] Since the hydrophilic or hydrophobic solvent has a density greater than that of the aqueous phase, after the solvent is added, the solvent moves towards the bottom of the well and displaces the aqueous phase, pushing the aqueous phase to the surface and forming a distinct separation between the upper aqueous phase and the lower solvent phase. The upper aqueous phase can be removed using established techniques known in the art.

[0077] In some embodiments, the methods described herein are performed using the optical imaging system described in Section 1. In some embodiments, the methods described herein are performed using a microfluidic device. In some embodiments, the methods described herein are performed using a digital microfluidic device (DMF). In some embodiments, the methods described herein are performed using a surface acoustic wave-based microfluidic device (SAW). In some embodiments, the methods described herein are performed using an integrated DMF and analyte detection device. In some embodiments, the methods described herein are performed using an integrated surface acoustic wave-based microfluidic device and analyte detection device. In some embodiments, the methods described herein are performed using a robotics-based assay processing unit.

[0078] A method for detecting an analyte of interest in a droplet is provided herein (wherein the analyte of interest is from a test sample or a biological sample). The method includes providing a first droplet containing the analyte of interest, providing a second droplet containing at least one detectable label including a specific binding member that binds to the analyte of interest, using energy to exert a force to manipulate the first droplet (containing the analyte of interest) with the second droplet (containing at least one solid support) to produce a mixture (i.e., a reaction mixture containing an analyte / detectable label-specific binding member complex), moving all or at least a portion of the mixture to an array of wells (where one or more wells of the array have a size sufficient to accommodate at least one solid support), optionally sealing the wells using one or more solvents, and detecting the analyte of interest within the wells. In some embodiments, the step of "using energy to exert a force to manipulate the first droplet with the second droplet" provides or exerts a force to manipulate (e.g., integrate or combine) at least the first and second droplets (and optionally additional droplets) into a mixture, or uses a non-mechanical force (i.e., energy generated without using, for example, pumps and / or valves) to exert such a force. Examples of non-mechanical forces that may be used in the methods described herein include electric driving forces (such as droplet driving, electrophoresis, electro-wetting, dielectrophoresis, electrostatic driving, mediated electric fields, mediated electrodes, capillary forces, chromatography, centrifugation, or suction, etc.) and / or acoustic forces (such as surface acoustic waves (i.e., "SAW"), etc.). In some embodiments, the generated electric driving force is an alternating current. For example, the alternating current can have a root mean square (rms) voltage of 10V, 15V, 20V, 25V, 30V, 35V, or more than those. For example, such an alternating current can have an rms voltage of 10V or more, 15V or more, 20V or more, 25V or more, 30V or more, or 35V or more. Alternatively, the alternating current can have a frequency in the radio frequency range.

[0079] In some other embodiments, the mixture is mixed by moving the mixture circumferentially, going back and forth, or by separating the mixture into two or more sub-mixtures and then integrating these sub-mixtures. In some other embodiments, the electric driving force is used to move the mixture into an array of wells (in which at least one solid support is loaded) to seal the well, and a series or plurality (i.e., at least two or more, at least three or more, at least four or more, at least five or more, at least six or more, at least seven or more, at least eight or more, at least nine or more, at least ten or more, at least eleven or more, at least twelve or more, at least thirteen or more, at least fourteen or more, at least fifteen or more, etc.) of electrodes may be used to generate it.

[0080] In some embodiments, the step of moving all or at least a portion of the mixture into an array of wells results in loading (filling and / or disposing) of the analyte / detectable label-specific binding member complex into the array of wells. In some embodiments, a magnetic field is used to facilitate the movement of the mixture and thus at least one analyte / detectable label-specific binding member complex into one or more wells of the array. For example, such removal may involve generating an electric driving force (described earlier herein) using a series or plurality of electrodes to move a fluid droplet (such as a polarized fluid droplet) into the array of wells to move at least a portion of the mixture away (at an insignificant distance) from the array of wells. In some embodiments, an aqueous wash liquid may be used to remove any detectable label-specific binding member not bound to any analyte. In such embodiments, this removal involves generating an electric driving force using a series or plurality of electrodes to move an aqueous wash (or washing) droplet (a third droplet) across the array of wells. The amount and type of the aqueous liquid used for the washing are not critical.

[0081] In some embodiments, the mixture in the method is an aqueous liquid. In other embodiments, the mixture is an immiscible liquid. In other embodiments, the droplets are hydrophobic droplets. In other embodiments, the droplets are hydrophilic droplets. In some embodiments, the array of wells used in the method has a hydrophobic surface. In other embodiments, the array of wells has a hydrophilic surface.

[0082] In some embodiments, the first droplet used in the method is a polar liquid. In some embodiments, the second droplet used in the method is a polar liquid. In some embodiments, the first and second droplets used in the method are polar liquids. In some embodiments, the mixture is a polar liquid. In some embodiments, one or more of the first droplet, the second droplet, and the mixture is a polar liquid.

[0083] In some embodiments, the detectable label is bound to at least one solid support. In some embodiments, the detectable label includes a chromophore, a fluorescent compound, an enzyme, a chemiluminescent compound, or a radioactive compound. In some embodiments, the specific binding member is a receptor, an aptamer, or an antibody. In some embodiments, the method further includes the step of disposing at least a portion of the mixture across the array of wells using a capillary element configured to facilitate movement of the mixture to the array of wells.

[0084] In some embodiments, when the mixture is a liquid and after the mixture has moved to the array of wells, an aqueous phase is formed within the wells. In some embodiments, the wells may be sealed by the addition of one or more solvents as previously described herein.

[0085] In some embodiments, the methods described herein are performed using the small digital immunoassay devices described herein. In some embodiments, the methods described herein are performed using a microfluidic device. In some embodiments, the methods described herein are performed using a digital microfluidic (DMF) device. In some embodiments, the methods described herein are performed using a surface acoustic wave (SAW)-based microfluidic device. In some embodiments, the methods described herein are performed using an integrated DMF and analyte detection device. In some embodiments, the methods described herein are performed using an integrated SAW-based microfluidic device and analyte detection device. In some embodiments, the methods described herein are performed using a robotics-based assay processing unit.

[0086] A method for measuring an analyte of interest in a droplet is provided herein, where the analyte of interest is from a test sample or a biological sample. The method includes providing a first droplet containing the analyte of interest; providing a second droplet containing at least one solid support (such as a magnetic solid support (such as beads)) that includes a specific binding member that binds to the analyte of interest; using energy that exerts a force to manipulate the first droplet (containing the analyte of interest) with the second droplet (containing at least one solid support) to produce a mixture (also referred to herein as a "reaction mixture"); moving all or at least a portion of the mixture to an array of wells, where one or more wells of the array have a size sufficient to accommodate at least one solid support; adding at least one detectable label to the mixture before, after, or both before and after moving a portion of the mixture to the array of wells; optionally, sealing the array of wells using one or more solvents; and measuring the analyte of interest within the wells. In some embodiments, the step of "using energy that exerts a force to manipulate the first droplet with the second droplet" provides or exerts a force to manipulate (such as integrate or combine) at least the first and second droplets (and optionally additional droplets) into a mixture, or uses a non-mechanical force (i.e., energy generated, for example, without using pumps and / or valves). Examples of non-mechanical forces that may be used in the methods described herein include electric driving forces (such as droplet driving, electrophoresis, electro-wetting, dielectrophoresis, electrostatic driving, mediating electric fields, mediating electrodes, capillary forces, chromatography, centrifugation, or suction) and / or acoustic forces (such as surface acoustic waves (i.e., "SAW")). In some embodiments, the electric driving force generated is an alternating current. For example, the alternating current can have a root mean square (rms) voltage of 10V, 15V, 20V, 25V, 30V, 35V, or more.For example, such an alternating current can have an rms voltage of 10 V or more, 15 V or more, 20 V or more, 25 V or more, 30 V or more, or 35 V or more. Alternatively, the alternating current can have a frequency in the radio frequency range.

[0087] In some embodiments, when a magnetic solid support is used, the electric driving force and the magnetic field can be applied from opposite directions to at least a portion of the mixture. In some other embodiments, the mixture is mixed by moving the mixture in a circumferential direction, back and forth, or by separating the mixture into two or more sub-mixtures and then integrating these sub-mixtures. In some other embodiments, the electric driving force is used to move the mixture into an array of wells (loaded with at least one solid support) to seal the wells, and can be generated using a series or plurality (i.e., at least two or more, at least three or more, at least four or more, at least five or more, at least six or more, at least seven or more, at least eight or more, at least nine or more, at least ten or more, at least eleven or more, at least twelve or more, at least thirteen or more, at least fourteen or more, at least fifteen or more, etc.) of electrodes.

[0088] In some embodiments, the step of moving all or at least a portion of the mixture to the array of wells results in the loading (filling and / or placement) of at least one solid support into the array of wells. In some embodiments, a magnetic field is used to facilitate the movement of the mixture and thus at least one solid support into one or more wells of the array. In some embodiments, after at least one solid support has been loaded into a well, any solid support that was not loaded into the well may be removed using defined techniques known in the art. For example, such removal may involve generating an electrokinetic force (as described earlier herein) using a series or plurality of electrodes to move at least a portion of the mixture away from the array of wells (to a non-critical distance), such as by moving fluid droplets (such as polarized fluid droplets) to the array of wells. In some embodiments, an aqueous wash liquid may be used to remove solid supports that are not bound to any analyte of interest. In such embodiments, this removal involves generating an electrokinetic force using a series or plurality of electrodes to move aqueous wash (or rinsing) droplets (a third droplet) across the array of wells. The amount and type of aqueous liquid used for the wash is not critical.

[0089] In some embodiments, the mixture in the method is an aqueous liquid. In other embodiments, the mixture is an immiscible liquid. In other embodiments, the droplets are hydrophobic droplets. In other embodiments, the droplets are hydrophilic droplets. In some embodiments, the array of wells used in the method has a hydrophobic surface. In other embodiments, the array of wells has a hydrophilic surface.

[0090] In some embodiments, the first droplet used in the method is a polar liquid. In some embodiments, the second droplet used in the method is a polar liquid. In some embodiments, the first and second droplets used in the method are polar liquids. In some embodiments, the mixture is a polar liquid. In some embodiments, one or more of the first droplet, the second droplet, and the mixture is a polar liquid.

[0091] In some embodiments, at least one solid support comprises at least one specific binding member that specifically binds to the analyte of interest. In some embodiments, the detectable label is added to the mixture before moving at least a portion of the mixture to an array of wells. In some other embodiments, the detectable label is added to the mixture after moving at least a portion of the analyte of interest to an array of wells. In some embodiments, the detectable label comprises at least one specific binding member that specifically binds to the analyte of interest. In some embodiments, the detectable label comprises a chromophore, a fluorescent compound, an enzyme, a chemiluminescent compound, or a radioactive compound. In some embodiments, the specific binding member is a receptor, an aptamer, or an antibody. In some embodiments, the method further comprises the step of disposing at least a portion of the mixture across an array of wells using a capillary element configured to facilitate movement of the mixture to the array of wells.

[0092] In some embodiments, when the mixture is a liquid and after the mixture has moved to an array of wells, an aqueous phase is formed within the wells. In some embodiments, the wells may be sealed by the addition of one or more solvents as previously described herein.

[0093] In some embodiments, the methods described herein are performed using the optical imaging system described in Section 1. In some embodiments, the methods described herein are performed using a microfluidic device. In some embodiments, the methods described herein are performed using a digital microfluidic device (DMF). In some embodiments, the methods described herein are performed using a surface acoustic wave-based microfluidic device (SAW). In some embodiments, the methods described herein are performed using an integrated DMF and analyte detection device. In some embodiments, the methods described herein are performed using an integrated surface acoustic wave-based microfluidic device and analyte detection device. In some embodiments, the methods described herein are performed using a robotics-based assay processing unit.

[0094] In some embodiments, the measuring step initially includes determining the total number of solid phase supports (the "total solid phase support number") within the wells of the array. Next, the number of solid phase supports (the "positives") within the wells of the array that contain a detectable label is determined, such as by determining the intensity of a signal generated by the detectable label. The positives are subtracted from the total solid phase support number to provide the number of solid phase supports (the "negatives") within the wells of the array that do not contain or are not detected by the detectable label. Then, the ratio of positives to negatives within the wells of the array is determined and can then be compared to a calibration curve. Alternatively, digital quantification using the Poisson equation P(x;μ) shown below: P(x;μ)=(e -μ )(μ x ) / x! where: e: a constant equal to approximately 2.71828 μ: the average number of successes occurring in a specified region, and x: the actual number of successes occurring in a specified region.

[0095] The sample used in the methods described herein can be any test sample that contains or is suspected of containing an analyte of interest. As used herein, the terms "analyte", "target analyte", and "analyte of interest" are used interchangeably and refer to the analyte that is measured in the methods and devices disclosed herein. The analyte of interest is further described below.

[0096] As used herein, "contact" and its grammatical synonyms refer to any type of binding interaction that brings a specific binding member sufficiently close to an analyte of interest in a sample such that a binding interaction occurs if a specific analyte of interest for the specific binding member is present in the sample. Contact can be achieved in a variety of ways, including binding the sample to a specific binding member, exposing the target analyte to the specific binding member by bringing the binding member close to the analyte.

[0097] In some cases, the first specific binding member may be immobilized on a solid support. As used herein, the term "immobilized" refers to a stable association between the first specific binding member and the surface of the solid support. "Stable association" means a physical association between two entities such that, for example, under physiological conditions, the average half-life of the complex is greater than one day. In some embodiments, the physical association between two entities has an average half-life of greater than two days, greater than one week, greater than one month, including greater than six months, for example greater than one year in PBS at 4°C. According to some embodiments, the stable association results from a covalent bond between the two entities, a non-covalent bond between the two entities (e.g., an ionic or metal bond), or other forms of chemical attraction such as hydrogen bonds, van der Waals forces.

[0098] A solid support having a surface to which a specific binding member is immobilized can be any convenient surface of a planar or non-planar structure, such as the surface of a microfluidic chip, the inner surface of a chamber, the outer surface of a bead (as defined herein), or the inner and / or outer surfaces of a porous bead. For example, the first specific binding member may be covalently or non-covalently attached to beads such as latex, agarose, sepharose, streptavidin, tosyl active, epoxy, polystyrene, amino beads, amine beads, carboxyl beads, etc. In some embodiments, the beads may be particles such as microparticles. In some embodiments, the microparticles 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 about 700 nm, or between about 500 nm and about 700 nm. For example, the microparticles may be about 4 - 6 microns, about 2 - 3 microns, or about 0.5 - 1.5 microns. Particles smaller than about 500 nm may be considered nanoparticles. Thus, the microparticles may optionally be nanoparticles 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.

[0099] In some embodiments, the beads may be magnetic beads or magnetic particles. In some embodiments, the beads may be magnetic nanobeads, nanoparticles, microbeads, or microparticles. The magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or a magnetic fluid. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (or FeO.Fe2O3). The beads can have a solid core portion that is magnetic and is surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion can be a layer around a non-magnetic core. The solid support to which the first specific binding member is immobilized may be stored in a dry state or in a liquid. These magnetic beads may be subjected to a magnetic field before or after contact with the sample together with the magnetic beads to which the first specific binding member is immobilized.

[0100] After the contacting step, the sample and the first specific binding member may be incubated for a time sufficient to allow a binding interaction to occur between the specific binding member and the analyte. Additionally, the incubation may occur in a binding buffer that facilitates the specific binding interaction. The binding affinity and / or specificity of the first specific binding member and / or the second specific binding member may be manipulated or varied in the assay by changing the binding buffer. In some embodiments, the binding affinity and / or specificity may be increased by changing the binding buffer. In some embodiments, the binding affinity and / or specificity may be decreased by changing the binding buffer.

[0101] The binding affinity and / or specificity of the first specific binding member and / or the second specific binding member may be measured using the disclosed methods and devices described below. In some embodiments, one aliquot of a sample is assayed using one set of conditions and compared to another aliquot of the sample assayed using a different set of conditions, thereby determining the effect of the conditions on binding affinity and / or specificity. For example, changing or varying the conditions can be one or more of removing the target analyte from the sample, adding molecules that compete with the target analyte or the ligand for binding, and changing the pH, salt concentration, or temperature. In addition or alternatively, the duration may be variable and changing the conditions may include waiting for a period of time before re-running the detection method.

[0102] The binding buffer may contain standard molecules of antigen-antibody binding buffers such as albumin (e.g., BSA), non-ionic surfactants (Tween-20, Triton X-100), and / or protease inhibitors (e.g., PMSF). In some cases, the binding buffer may be added to a microfluidic chip, chamber, etc. either before or after adding the sample. In some cases, the first specific binding member may be present in the binding buffer before contacting the sample. The length of time during which the binding interaction between the binding member and the analyte occurs may be determined experimentally and may depend on the binding affinity and binding avidity between the binding member and the analyte. In some embodiments, the contacting or incubating may occur for a period of 5 seconds to 1 hour, such as 10 seconds to 30 minutes, or 1 minute to 15 minutes, or 5 minutes to 10 minutes, e.g., 10 seconds, 15 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours. Other conditions regarding the binding interaction, such as temperature, salt concentration, etc., may also be determined experimentally or based on the manufacturer's instructions. For example, the contacting may be performed at room temperature (21°C to 28°C, e.g., 23°C to 25°C), 37°C, or 4°C. In some embodiments, any mixing of the sample and the first specific binding member may be performed during the contacting step.

[0103] Following complex formation between the immobilized first specific binding member and the analyte, any unbound analyte may be removed from the vicinity of the first specific binding member along with the sample, but the complex of the first specific binding member and the analyte may be retained by its association with the solid support. Optionally, the solid support may be contacted with a wash buffer to remove any molecules that non-specifically bind to the solid support.

[0104] After the first contacting step and any removal and / or any washing steps of the sample, the complex of the first specific binding member and the analyte contacts the second specific binding member, thereby leading to the formation of a sandwich complex in which the analyte is bound by the two specific binding members. Any mixing of the second member with the first specific binding member-analyte complex may be carried out during the second contacting step. In some embodiments, immobilization of the analyte molecules to the surface may assist in removing any excess second specific binding member from the solution without fear of removing the analyte molecules from the surface. In some embodiments, the second specific binding member may comprise a detectable label that includes one or more signal generating substances such as a chromophore, a fluorescent compound, a chemiluminescent compound, an enzyme, a radioactive compound, etc.

[0105] As described above, the second contact step may be carried out under conditions sufficient for the binding interaction between the analyte and the second specific binding member. Following the second contact step, any unbound second specific binding member may be removed, followed by any washing step. Any unbound second specific binding member may be separated from the first specific binding member-analyte-second specific binding member complex by suitable means such as droplet driving, electrophoresis, electro-wetting, dielectrophoresis, electrostatic driving, a mediating electric field, a mediating electrode, capillary force, chromatography, centrifugation, suction, or SAW. When any unbound second specific binding member is removed from the vicinity of the first specific binding member-analyte-second specific binding member complex, the detectable label attached to the second specific binding member present in the first specific binding member-analyte-second specific binding member complex may be separated by suitable means or detected using techniques known in the art. In some embodiments, the detectable label comprises one or more signal generating substances such as chromophores, fluorescent compounds, enzymes, chemiluminescent compounds, radioactive compounds, etc., including the detectable label. Alternatively, in some embodiments, if the detectable label comprises a tag, the tag may be cleaved or dissociated from the complex remaining after removal of the unbound reagent. For example, the tag may be attached to the second specific binding member via a cleavable linker (a "cleavable linker" as described herein). The first specific binding member-analyte-second specific binding member complex may be exposed to a cleaving agent that mediates cleavage of the cleavable linker.

[0106] As described above, tags may contain nucleic acids. In some embodiments, quantification of an analyte does not include determining the identification information of the tag by determining the identification information of at least a portion of the nucleic acid sequence present in the tag. For example, the counting step does not include determining the sequence of the tag. In other embodiments, the tag is not sequenced, but the identification information of the tag is determined to the extent that it can distinguish one tag from another based on a distinguishable signal associated with the tag, such as the size, structure, charge, amount of charge, etc. of the tag. Identification of tags may be useful in methods that include simultaneous analysis of multiple different analytes in a sample, such as two, three, four, or more different analytes in a sample.

[0107] In some embodiments, simultaneous analysis of multiple analytes in a single sample may be performed by using multiple different first and second specific binding members, where a pair of first and second specific binding members is specific for a single analyte in the sample. In these embodiments, the detectable label associated with the second specific binding member of the first pair of first and second specific binding members that is specific for a single analyte may be distinguishable from the detectable label associated with the second specific binding member of the second pair of first and second specific binding members that is specific for a different analyte. As described above, the first detectable label may be distinguishable from the second detectable label based on differences such as signal generating substances.

[0108] In some embodiments, the concentration of the analyte in the fluid sample that can be determined with near accuracy is less than about 5000 fM (femtomolar), less than about 3000 fM, less than about 2000 fM, less than about 1000 fM, less than about 500 fM, less than about 300 fM, less than about 200 fM, less than about 100 fM, less than about 50 fM, less than about 25 fM, less than about 10 fM, less than about 5 fM, less than about 2 fM, less than about 1 fM, less than about 500 aM (attomolar), less than about 100 aM, less than about 10 aM, less than about 5 aM, less than about 1 aM, less than about 0.1 aM, less than about 500 zM (zeptomolar), less than about 100 zM, less than about 10 zM, less than about 5 zM, less than about 1 zM, less than about 0.1 zM, or less.

[0109] In some cases, the limit of detection (e.g., the lower limit concentration of the analyte that may be determined in solution) is about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, about 1 fM, about 500 aM (attomolar), about 100 aM, about 50 aM, about 10 aM, about 5 aM, about 1 aM, about 0.1 aM, about 500 zM (zeptomolar), about 100 zM, about 50 zM, about 10 zM, about 5 zM, about 1 zM, about 0.1 zM, or less. In some embodiments, the concentration of the analyte in the fluid sample that may be determined with near accuracy is between about 5000 fM and about 0.1 fM, between about 3000 fM and about 0.1 fM, between about 1000 fM and about 0.1 fM, between about 1000 fM and about 0.1 zM, between about 100 fM and about 1 zM, between about 100 fM and about 0.1 zM, or less.

[0110] The upper limit of detection (e.g., the upper limit concentration of the analyte that may be determined in solution) is at least about 100 fM, at least about 1000 fM, at least about 10 pM (picomolar), at least about 100 pM, at least about 100 pM, at least about 10 nM (nanomolar), at least about 100 nM, at least about 1000 nM, at least about 10 μM, at least about 100 μM, at least about 1000 μM, at least about 10 mM, at least about 100 mM, at least about 1000 mM, or higher.

[0111] In some cases, the presence and / or concentration of the analyte in the sample may typically be detected rapidly in less than about one hour, such as 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute, or 30 seconds.

[0112] In some embodiments, at least some of the methods described herein may be performed using the optical imaging system described in Section 1. In some embodiments, at least some steps of the methods described herein may be performed on a digital integrated microfluidic and analyte detection device such as the devices described herein. In some embodiments, the methods of the present disclosure are performed using a digital integrated microfluidic device in combination with an analyte detection device. For example, the digital microfluidic device and the analyte detection device may be separate devices, and droplets containing detectable labels may be generated within the microfluidic device and transported to the analyte detection device.

[0113] In some embodiments, the methods of the present disclosure are performed using a device in which the digital microfluidic module is integrated with an analyte detection device such as the devices described below. In some embodiments, the digital integrated microfluidic module and the analyte detection device may be reversibly integrated. For example, the two modules may be physically coupled to form an integrated device, which may then be separated into individual modules. In some embodiments, the methods of the present disclosure are performed using a disposable cartridge that includes a microfluidic module having a built-in analyte detection device. Exemplary embodiments of the devices used to perform the methods provided herein are further described in the following section.

[0114] Exemplary embodiments of the method include integrating a sample droplet containing an analyte of interest with a droplet containing a first specific binding member that may be immobilized on a solid support (such as magnetic particles or beads) that binds to the analyte of interest. The single integrated droplet can be incubated for a time sufficient to allow the first specific binding member to bind to the analyte of interest. Optionally, the single droplet may be agitated to facilitate mixing the sample with the first specific binding member. Mixing may be achieved by moving the single droplet back and forth, moving the single droplet around a plurality of electrodes, separating the droplets and then integrating these droplets, or using SAW, among other things. Next, the single droplet may be subjected to a magnetic force that holds the beads in place within the device, while the droplet may be pulled away and replaced with a droplet containing a second specific binding member, which can optionally include a detectable label. Any washing step may be performed prior to adding the second specific binding member by moving a droplet of washing buffer to the location where the beads are held using magnetic force. After a time sufficient for the second specific binding member to bind to the analyte bound to the first specific binding member, the droplet containing the second specific binding member may be pulled away, while the beads are held in the first location. The beads may be washed using a droplet of washing buffer. Following the washing step, the magnetic force may be removed, and the droplet containing the labeled beads (including the first specific binding member / analyte / second specific binding member - including any detectable label) moves to the detection module described herein. The labeled beads can be made to settle within an array of wells of the detection module. The beads may settle via gravity or by applying an electric or magnetic force. Following a washing step to remove any beads not located within the wells, the wells may be sealed using a solvent (such as a hydrophobic liquid such as oil).In the above embodiments, optionally, after the binding, the droplets are manipulated (e.g., moved back and forth, moved circumferentially, vibrated, separated / merged, exposed to SAW, etc.) to facilitate mixing of the assay reagents and the sample, such as the first specific binding member, the second specific binding member, etc. In embodiments where the detectable label is an enzyme, the substrate may be added either before or after the complex is moved to the well array.

[0115] The movement of droplets within an integrated microfluidic and analyte detection device can be performed using electrokinetic forces (e.g., electro-wetting, dielectrophoresis, mediating electrodes, opto-electro-wetting, mediating electric fields, and electrostatic actuation), pressure, surface acoustic waves, etc. The forces used to move the droplets may be determined based on the specifications of the device, and these specifications are described in the next section with respect to the specific devices described herein.

[0116] In some embodiments, one or more detection signals correspond to the event of a specific binding member binding to an analyte. In some embodiments, one detection signal corresponds to the event of a specific binding member binding to an analyte. In some embodiments, two or more detection signals correspond to the event of a specific binding member binding to an analyte.

[0117] In some embodiments, a solid support comprising a first specific binding member and a second specific binding member is added to the sample continuously or simultaneously.

[0118] The detection of an analyte is associated with a detectable product or a detectable label, i.e., a signal generated by at least one signal-generating compound and at least one signal-generating substrate. In some embodiments, the at least one signal-generating compound is an enzyme and the at least one signal-generating substrate is a substrate for the enzyme. In some embodiments, the substrate for the enzyme is a colorimetric fluorogenic (non-fluorescent) substrate or a chromogenic substrate. In some embodiments, the detectable signal is a fluorescent signal. For example, the enzyme may be polynucleotidase, arginase, adenase, aminopolypeptidase, pepsin, lipase, catalase, tyrosinase, alcohol dehydrogenase, succinate dehydrogenase, diaphorase, glyoxalase, aldolase, glucose oxidase, horseradish peroxidase, galactosidase (such as beta-galactosidase), phosphatase, phosphorylase and hexokinase, or a combination thereof. Examples of enzyme substrates that may be used are chemiluminescent substrates such as [Math.5]CDP-Star(R) (disodium 4-chloro-3-(methoxypyro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1 3,7 decane}-4-yl)phenyl phosphate), [Math.6]CSPD(R) i.e., (disodium 3-(4-methoxypyro{1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.1 3,7 decane}-4-yl)phenyl phosphate), and luminescent substrates such as p-nitrophenyl phosphate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 4-nitroblue tetrazolium chloride (NBT), or iodonitrotetrazolium (INT), and fluorescent substrates such as 4-methylumbelliferyl phosphate (4-MUP), and chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium 5-bromo-6-chloro-indolyl phosphate, or p-nitrophenyl phosphate.

[0119] In some embodiments, the enzyme that may be used comprises an enzyme that includes an inhibitor molecule (protein, peptide, etc.) bound to a site other than the active binding site of the enzyme. Such inhibitor molecules alter the structure of the active binding site of the enzyme and prevent the enzyme from binding to the substrate. Examples of inhibitor molecules include protease inhibitors. The inhibitor can be removed from the enzyme using defined techniques known in the art in order to enable the enzyme to bind to the substrate and thus to enable the signal generation reaction to occur.

[0120] In some embodiments, the enzyme can convert a non-fluorescent substrate to a fluorescent substrate. In some embodiments, the enzyme can generate color using a chromogenic substrate.

[0121] 3. Specific binding members As will be appreciated by those skilled in the art, the specific binding member is determined by the analyte to be analyzed. Specific binding members for a wide variety of target molecules may be known or may be readily found or developed using known techniques. For example, when the target analyte is a protein, the specific binding member may be a protein and, in particular, its antibody or fragment (e.g., antigen-binding fragment (Fab), Fab’ fragment, F(ab’)2 fragment, recombinant antibody, chimeric antibody, single-chain Fv (“scFv”), single-chain antibody, heavy-chain variable region obtained from camelids (“VHH,” also known as “VHH fragment”) (VHH and methods for generating them are described in Gottlin et al., Journal of Biomolecular Screening, 14, pp. 77-85 (2009)), single-domain antibodies such as those, recombinant VHH single-domain antibodies, and VNAR fragments, disulfide-bonded Fv (“sdFv”), and anti-idiotype (“anti-Id”) antibodies, and functionally active epitope-binding fragments of any of the above, full-length polyclonal antibodies or full-length monoclonal antibodies, antibody-like fragments, etc.), other proteins such as receptor proteins, protein A, protein C, etc. When the analyte is a small molecule such as a steroid, bilin, retinoid, and lipid, the first and / or second specific binding member may be a scaffold protein (e.g., lipocalin) or a receptor. In some cases, the specific binding member for a protein analyte may be a peptide. For example, when the target analyte is an enzyme, the appropriate specific binding member may include an enzyme substrate and / or an enzyme inhibitor, which may be a peptide, a small molecule, etc. In some cases, when the target analyte is a phosphate species, the specific binding member may include a phosphate binder. For example, the phosphate binder may include a metal-ion affinity medium such as those described in U.S. Patent No. 7,070,921 and U.S. Patent Application No. 2006 / 0121544.

[0122] When the target molecule is a carbohydrate, capture components (as defined herein) that may be appropriate include, for example, antibodies, lectins, and selectins. As will be appreciated by those skilled in the art, any molecule that can specifically associate with the target molecule of interest can potentially be used as a specific binding member.

[0123] In some embodiments, suitable target analyte / specific binding member complexes can include, but are not limited to, antibody / antigen, antigen / antibody, receptor / ligand, ligand / receptor, protein / nucleic acid, enzyme / substrate and / or inhibitor, carbohydrate (including glycoprotein and glycolipid) / lectin and / or selectin, protein / protein, protein / small molecule, and the like.

[0124] In certain embodiments, the first specific binding member and / or the second specific binding member may be attached to a solid support via a linkage, which may include any moiety, functionality, or modification of the support and / or the specific binding member that facilitates attachment of the specific binding member to the support. The linkage between the specific binding member and the support 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 a chemical spacer that provides such a bond.

[0125] In some embodiments, the solid support can also include a protective, blocking, or passivating layer that can exclude or minimize non-specific attachment of non-capturing components (e.g., analyte molecules, specific binding members) in the assay to the binding surface that can lead to false positive signals or loss of signal during detection. Examples of materials that may be utilized to form a passivating layer in some embodiments include, but are not limited to, polymers such as poly(ethylene glycol) that avoid non-specific binding of proteins, naturally occurring proteins having this property such as serum albumin and casein, surfactants such as zwitterionic surfactants like sulfo betaine, naturally occurring long-chain lipids, polymer brushes, and nucleic acids such as salmon sperm DNA.

[0126] Some embodiments utilize a specific binding member that is a protein or polypeptide. As is known in the art, numerous techniques may be used to attach polypeptides to a wide variety of solid supports. A variety of techniques for adding reactive moieties to proteins are known, such as the method outlined in U.S. Patent No. 5,620,850. Additionally, methods for attaching proteins to surfaces are known, see, for example, Heller, Acc. Chem. Res. 23, 128 (1990).

[0127] As described herein, the binding between a specific binding member and an analyte is specific, for example, when the specific binding member and the analyte are complementary parts of a binding pair. In some embodiments, the specific binding member specifically binds to the analyte. "Specifically binds" or "binding specificity" means that the specific binding member binds to an analyte molecule with sufficient specificity to distinguish the analyte molecule from other components or contaminants of the test sample. For example, a specific binding member according to one embodiment may be an antibody that specifically binds to an epitope of the analyte. An antibody according to one embodiment may be any antibody capable of specifically binding to an analyte of interest. Suitable antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies (dAbs) (e.g., Holt et al. (2014) Trends in Biotechnology 21, pp. 484-490), such as those that occur naturally, e.g., within cartilaginous fish and camels, or are synthetic, e.g., single domain antibodies sdAbs, which are nanobodies, VHH, or other domain structures, synthetic antibodies (sometimes referred to as antibody mimetics), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each. As another example, the analyte molecule may be an antibody, the first specific binding member may be an antigen, and the second specific binding member may be a secondary antibody that specifically binds to the target antibody, or the first specific binding member may be a secondary antibody that specifically binds to the target antibody, and the second specific binding member may be an antigen.

[0128] In some embodiments, the specific binding member is a chemically programmed antibody (cpAb) (Rader (2014) Trends in Biotechnology 32, 186-197), a bispecific cpAb, an antibody recruitment molecule (ARM) (described in McEnaney et al., (2012) ACS Chem. Biol. 7, 1139-1151), a branched capture agent such as a triligand capture agent (described in Millward et al. (2011) J. Am. Chem. Soc. 133, 18280-18288), an engineered binding protein obtained from a non-antibody scaffold such as a monobody (obtained from the 10th fibronectin type III domain of human fibronectin), an affibody (obtained from immunoglobulin-binding protein A), a DARPin (based on ankyrin repeat molecules), an anticalin (obtained from lipocalin, bilin-binding protein, and human lipocalin 2), a cystine knot peptide (knottin) (described in Gilbreth and Koide, (2012) Current Opinion in Structural Biology 22, 1-8, and Banta et al. (2013) Annu. Rev. Biomed. Eng. 15, 93-113), a WW domain (described in Patel et al. (2013) Protein Engineering, Design & Selection 26(4), 307-314), a diverted receptor ligand, an affitin (described in Behar et al. (2013) 26, 267-275), and / or an adhirons (Tiede et al. (2014) Protein Engineering, Design & Selection 27, 145-155).

[0129] According to one embodiment, where the analyte is a biological cell (e.g., cells such as mammalian, avian, worm, other vertebrate, insect, yeast, bacterial cells), the specific binding member may be a ligand having a specific affinity for a cell surface antigen (e.g., a cell surface receptor). In one embodiment, the specific binding member may be an adhesion molecule receptor or a portion thereof having binding specificity for a cell adhesion molecule expressed on the surface of the target cell type. In use, the adhesion molecule receptor binds to the adhesion molecule on the extracellular surface of the target cell, thereby immobilizing or capturing the cell, and the bound cell is then detected by using a second specific binding member that may be the same as the first specific binding member, or may bind to a different molecule expressed on the surface of the cell.

[0130] In some embodiments, the binding affinity between the analyte molecule and the specific binding member should be sufficient to remain bound under the assay conditions, including a washing step to remove non-specifically bound molecules or particles. In some cases, for example, in the detection of some biomolecules, the binding constant between the analyte molecule and its complementary specific binding member is at least about 10 4 to about 10 6 M -1 between, at least about 10 5 to about 10 9 M -1 between, at least about 10 7 to about 10 9 M -1 between, about 10 9 M -1 or above them, or above them.

[0131] 4. Exemplary Target Analytes As will be appreciated by those skilled in the art, any analyte that may be specifically bound by the first specific binding member and the second specific binding member may be detected, and optionally quantified, using the methods and devices of the present disclosure.

[0132] In some embodiments, the analyte may be a biomolecule or a biological molecule. Non-limiting examples of biomolecules and biological molecules include macromolecules such as proteins, lipids, and carbohydrates. In some cases, the analyte may be a hormone, an antibody, a growth factor, a cytokine, an enzyme, a receptor (e.g., a neurotransmitter receptor, a hormone receptor, a nutrient receptor, and a cell surface receptor) or its ligand, a cancer marker (e.g., PSA, TNF-alpha), a marker for myocardial infarction (e.g., troponin, creatine, kinase, BNP, pro-BNP, NT-ProBNP, CK-MB, galectin-3, etc.), a thyroid marker (e.g., anti-Tg, anti-TPO, free T3, free T4, T uptake rate, total T3, total T4, TSH), a toxin, a drug (e.g., a toxic drug), a metabolic agent (e.g., including vitamins), etc. Non-limiting embodiments of protein analytes include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphorylated proteins, glycoproteins, lipoproteins, etc. In some embodiments, the analyte may be a biomarker such as a biomarker for traumatic brain injury, sepsis, or coagulation, an analyte related to general chemistry (e.g., ammonia, AST, cholesterol, etc.), a protein (e.g., transferrin, CRP, etc.), an analyte for therapeutic drug monitoring (e.g., methotrexate), an analyte for transplantation (e.g., tacrolimus), a drug of dependence, or a biomarker for a genetic disorder.

[0133] In some embodiments, the analyte may be a post-translationally modified protein (e.g., a phosphorylated, methylated, or glycosylated protein), and the first or second specific binding member may be an antibody specific for the post-translational modification. The modified protein may bind to the first specific binding member immobilized on a solid support, and the first specific binding member binds to the modified protein but not to the unmodified protein. In other embodiments, the first specific binding member may bind to both the unmodified and modified proteins, and the second specific binding member may be specific for the post-translationally modified protein.

[0134] In some embodiments, the analyte may be a cell such as a circulating tumor cell, a pathogen, a virus (including retroviruses, herpesviruses, adenoviruses, lentiviruses, filoviruses (e.g., West Nile virus, Ebola virus, and Zika virus), hepatitis viruses (e.g., A, B, C, D, and E), HPV, parvovirus, etc.), a spore, etc.

[0135] A non-limiting list of analytes that may be analyzed by the methods provided herein includes Aβ42 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, mutant ATP13A2, apo H, ceruloplasmin, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), transthyretin, vitamin D binding protein, active B12, B12, cortisol, folic acid, fructosamine, homocysteine, intact PTH, pepsinogen I&II, DHEA-S, estradiol, hCG, progesterone, prolactin, SHBG, testosterone, apoptosis-promoting kinase R (PKR) and phosphorylated PKR (pPKR), IL-12p40, CXCL13, IL-8, Dkk-3 (semen), p14 endocan fragment, serum, ACE2, autoantibody to CD25, hTERT, CAI25 (MUC16), VEGF, sIL-2, osteopontin, human epididymal protein 4 (HE4), alpha-fetoprotein, albumin, albuminuria, microalbuminuria, neutrophil gelatinase-associated lipocalin (NGAL), cystatin C, interleukin 18 (IL-18), kidney injury molecule-1 (KIM-1), liver-type fatty acid binding protein (L-FABP), LMP1, BARF1, IL-8, BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, and LZTS1, alpha-amylase, carcinoembryonic antigen (CEA), CA125, thioredoxin, beta-2 microglobulin levels - active monitoring of virus, active monitoring of tumor necrosis factor-alpha receptor - virus, alpha-fetoprotein (AFP), CA15-3, CA19-9, CYFRA21-1, HE-4, PIVKA-II, ProGRP, SCC, follicle-stimulating hormone (FSH), luteinizing hormone (LH), T cell invasion and metastasis 1 (TIAM1), N-cadherin, EC39, amphiregulin, dUTPase, secreted gelsolin (pGSN), PSA (prostate-specific antigen), thymosin beta15, insulin, plasma C-peptide,Glycated hemoglobin (HBA1c), 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-A11), PRDX6 (Peroxiredoxin-6), HSPE1 (10 kDa heat shock protein, mitochondrial 1), 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(kip1), 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, TOPOII Topoisomerase (DNA) II alpha 170 kDa, FRAP1, NRG1, ESR1, 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), Receptor for advanced glycation end products (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,It includes alpha (IL2RA), vascular cell adhesion molecule 1 (VCAM1) and von Willebrand factor (VWF), myeloperoxidase (MPO), IL1α, TNFα, perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA), lactoferrin, calprotectin, Wilms tumor-1 protein, aquaporin-1, MLL3, AMBP, VDAC1, Escherichia coli enterotoxin (heat-labile enterotoxin, heat-stable enterotoxin), influenza HA antigen, tetanus toxin, diphtheria toxin, botulinum toxin, Shiga toxin, Shiga-like toxin I, Shiga-like toxin II, Clostridium difficile toxins A and B, etc.

[0136] Exemplary targets that may be measured in samples such as environmental and biological samples obtained from a patient or subject in need using the methods of the subject are: abused drugs (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 μ heavy chain (IGHM), immunoglobulin E, αvβ3 integrin, α-thrombin, HIV gp120, NF-κB, 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 pathogens (including but not limited to Mycobacterium tuberculosis, Staphylococcus aureus, Shigella dysenteriae, Escherichia coli O157:H7, Campylobacter jejuni, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella O8, and Enterobacteriaceae).

[0137] Exemplary targets that may be measured in a sample obtained from a patient or subject in need using the subject methods include, but are not limited to, HBV core capsid protein, CDK2, E2F transcription factor, thymidylate synthase, Ras, EB1, and receptor for advanced glycation end products (RAGE).

[0138] 5. Sample As used herein, "sample", "test sample", "biological sample" refers to a fluid sample that contains or is suspected of containing an analyte of interest. The sample may be obtained from any suitable source. In some cases, the sample may contain a liquid, a fluid particulate solid, or a suspension of solid particles. In some cases, the sample may be processed prior to the analysis described herein. For example, the sample may be separated or purified from its source prior to analysis, although in some embodiments, an unprocessed sample containing the analyte may be assayed directly. The source of the analyte molecule may be synthetic (e.g., generated in a laboratory), environmental (e.g., fluid samples such as air, soil, aqueous replenishment, etc.), an animal such as a mammal, a plant, or any combination thereof. In a specific example, the source of the analyte is a substance of the human body (e.g., body fluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tear fluid, lymph fluid, amniotic fluid, interstitial fluid, lung lavage fluid, cerebrospinal fluid, feces, tissue, organ, etc.). Tissues include, but are not limited to, skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, myocardial tissue, brain tissue, bone marrow, cervical tissue, skin, etc. The sample may be an extract of a liquid sample or a solid sample. In some cases, the source of the sample may be an organ or tissue such as a biopsy sample that may be solubilized by tissue decomposition / cell lysis.

[0139] A variety of volumes of fluid samples may be analyzed. In some exemplary embodiments, the sample volume may be about 0.5 nL, about 1 nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL, about 10 μL, about 100 μL, about 1 mL, about 5 mL, about 10 mL, etc. In some cases, the volume of the liquid sample is between about 0.01 μL and about 10 mL, between about 0.01 μL and about 1 mL, between about 0.01 μL and about 100 μL, or between about 0.1 μL and about 10 μL.

[0140] In some cases, the fluid sample may be diluted before use in the assay. For example, in embodiments where the source of the analyte molecules is a body fluid (e.g., blood, serum), the fluid may be diluted with a suitable solvent (e.g., a buffer such as PBS buffer). The 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 more before use.

[0141] In some cases, the sample may be subjected to pre-analysis processing. The pre-analysis processing may provide additional functions such as non-specific protein removal and / or an effective and more inexpensive mixing function. General methods of pre-analysis processing may include the use of electrokinetic capture, AC electrokinetics, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other preconcentration techniques known in the art. In some cases, the fluid sample may be concentrated before use in the assay. For example, in embodiments where the source of the analyte molecules is a body fluid (e.g., blood, serum), the fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. The 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 more before use.

[0142] In some embodiments, the analyte is not amplified prior to measurement of the analyte (i.e., the copy number of the analyte is not increased). For example, if the analyte is DNA or RNA, the analyte is not replicated to increase the copy number of the analyte. In some cases, the analyte is a protein or a small molecule.

[0143] 6. Variations of the Method The disclosed methods for determining the presence or amount of an analyte of interest present in a sample may be as described above. These methods may be adapted in view of other methods for analyzing analytes. Examples of well-known variations include, but are not limited to, immunoassays such as enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA)), competitive inhibition immunoassays (e.g., forward and reverse), enzyme amplified immunoassay techniques (EMIT: enzyme multiplied immunoassay technique), competitive binding assays, bioluminescence resonance energy transfer (BRET), one-step antibody detection assays, homogeneous assays, heterogeneous assays, sandwich immunoassays (e.g., monoclonal - polyclonal sandwich immunoassays) including capture-on-the-fly assays, etc. In some instances, the following description may overlap with the methods described above, while on the other hand, the following description may provide alternatives.

[0144] (a) Immunoassay The analyte of interest, and / or the peptide or fragment thereof, may be analyzed using an immunoassay. The presence or amount of the analyte of interest can be determined by detecting specific binding of the antibody described herein to the analyte of interest. Any immunoassay may be utilized. The immunoassay may be, for example, an enzyme-linked immunosorbent assay (ELISA), a competitive inhibition assay such as a forward or reverse competitive inhibition assay, or a competitive binding test. In some embodiments, one signal generating compound or signal generating substrate is attached to the capture antibody and the detection antibody. Alternatively, the microparticles used for capture may also function for detection.

[0145] A homogeneous format may be used. For example, after a test sample is obtained from a subject, a mixture is prepared. This mixture includes the test sample to be evaluated with respect to the analyte, the first specific binding partner, and the second specific binding partner. The order in which the test sample, the first specific binding partner, and the second specific binding partner are added to generate the mixture is not important. The test sample is contacted simultaneously with the first specific binding partner and the second specific binding partner. In some embodiments, the first specific binding partner and any analyte of interest included in the test sample may form a first specific binding partner-analyte of interest-antigen complex, and the second specific binding partner may form a first specific binding partner-analyte of interest-second specific binding partner complex. In some embodiments, the second specific binding partner and any analyte of interest included in the test sample may form a second specific binding partner-analyte of interest-antigen complex, and the first specific binding partner may form a first specific binding partner-analyte of interest-second specific binding partner complex. Further, the second specific binding partner is labeled with a detectable label described herein or includes such a detectable label.

[0146] A uniform format may be used. For example, after a test sample is obtained from a subject, a first mixture is prepared. This mixture includes the test sample to be evaluated with respect to the analyte of interest and the first specific binding partner, and any analyte of interest contained in the first specific binding partner and the test sample forms a first specific binding partner-analyte of interest complex. Preferably, the first specific binding partner is an antibody or a fragment thereof that resists the analyte of interest. The order in which the test sample and the first specific binding partner are added to generate the mixture is not important. Preferably, the first specific binding partner is immobilized on a solid support. The solid supports (for the first specific binding partner and optionally for the second specific binding partner) used in immunoassays include, but are not limited to, magnetic particles, beads, nanobeads, microbeads, nanoparticles, microparticles, membranes, backbone molecules, thin films, filter paper, disks, or chips (e.g., microfluidic chips), and may be any solid support known in the art. In these embodiments where the solid support is beads, the beads may be magnetic beads or magnetic particles. The magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or magnetic fluids. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (or FeO·Fe2O3). The beads can have a solid core portion that is magnetic and be surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion can be a layer around a non-magnetic core. The solid support on which the first specific binding member is immobilized may be stored in a dry state or in a liquid. These magnetic beads may be subjected to a magnetic field before or after contacting the sample with the magnetic beads on which the first specific binding member is immobilized.

[0147] After a mixture containing a first specific binding partner - analyte complex of interest is generated, any unbound analyte of interest is removed from this complex using any technique known in the art. For example, the unbound analyte of interest may be removed by washing. However, it is desirable that the first specific binding partner be present in excess over any analyte of interest present in the test sample such that all analytes of interest present in the test sample are bound by the first specific binding partner.

[0148] After any unbound analyte of interest has been removed, a second specific binding partner is added to the mixture to form a first specific binding partner - analyte of interest - second specific binding partner complex. The second specific binding partner is preferably an antibody against the analyte of interest that binds to an epitope of the analyte of interest that is different from the epitope of the analyte of interest bound by the first specific binding partner. Further, the second specific binding partner is preferably labeled with a signal - generating compound or signal - generating substrate or contains these signal - generating compounds or signal - generating substrates as described above.

[0149] The use of immobilized antibodies or fragments thereof may be incorporated into immunoassays. The antibodies may be immobilized on various carriers such as magnetic or chromatographic matrix particles, latex particles or surface - modified latex particles, polymers or polymer thin films, plastics or plastic thin films, planar substrates, microfluidic surfaces, a portion of a solid substrate material, etc.

[0150] (b) Sandwich immunoassay A sandwich immunoassay measures the amount of antigen between two layers of antibodies (i.e., a capture antibody (i.e., at least one capture antibody) and a detection antibody (i.e., at least one detection antibody)). The capture antibody and the detection antibody bind to different epitopes of the antigen, such as the analyte of interest. It is desirable that the binding of the capture antibody to the epitope does not prevent the binding of the detection antibody to the epitope. Either monoclonal antibodies or polyclonal antibodies may be used as capture antibodies and detection antibodies in a sandwich immunoassay.

[0151] Generally, at least two antibodies are used to separate and quantify the analyte of interest in a test sample. More specifically, at least two antibodies bind to some epitopes of the analyte of interest or fragments of the analyte of interest to form an immune complex called a "sandwich". One or more antibodies may be used to capture the analyte of interest in the test sample (these antibodies are often referred to as one or more "capture" antibodies), and one or more antibodies having a signal generating compound or signal generating substrate that binds similarly to the analyte of interest (these antibodies are often referred to as one or more "detection" antibodies) may be used to complete the sandwich. In a sandwich assay, the binding of an antibody to its epitope is not decreased by the binding of any other antibody in the assay to its respective epitope. In other words, the antibody is selected such that one or more first antibodies that have contacted a test sample suspected of containing the analyte of interest bind to all or a portion of the epitope recognized by a second antibody or subsequent antibodies, thereby not preventing the ability of one or more second detection antibodies to bind to the analyte of interest. The capture antibody described above is an example of a capture molecule. The detection antibody described above is an example of a detection molecule.

[0152] In one embodiment, a test sample suspected of containing an analyte of interest may be contacted simultaneously or sequentially with at least one capture antibody (or antibodies) and at least one detection antibody. In a sandwich assay format, a test sample suspected of containing an analyte of interest (membrane-bound analyte of interest, soluble analyte of interest, fragment of membrane-bound analyte of interest, fragment of soluble analyte of interest, variant of analyte of interest (membrane-bound or soluble analyte of interest), or any combination thereof) first contacts at least one capture antibody that specifically binds to a particular epitope under conditions that permit the formation of an antibody-analyte of interest complex. If more than one capture antibody is used, multiple capture antibody-analyte of interest complexes are formed. In a sandwich assay, the antibody, preferably at least one capture antibody, is used in a molar amount that exceeds the maximum amount of analyte of interest or fragment of analyte of interest expected to be present in the test sample.

[0153] Optionally, prior to contacting the test sample with at least one first capture antibody, the at least one capture antibody may be bound to a solid support that facilitates the separation of antibody-analyte of interest complexes from the test sample. Any solid support known in the art may be used, including but not limited to a solid support made from a polymeric material in the form of a planar substrate or beads. The antibody (or antibodies) may be bound to the solid support by adsorption, by covalent bonding using a chemical coupling agent, or by other means known in the art, provided that such binding does not interfere with the ability of the antibody to bind to the analyte of interest or fragment of analyte of interest. Further, if desired, the solid support may be derivatized to enable reaction with various functional groups of the antibody. Such derivatization requires the use of several coupling agents, including but not limited to maleic anhydride, N-hydroxysuccinimide, azide, alkynyl, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0154] A test sample suspected of containing an analyte of interest is incubated after contact with at least one capture antibody to allow formation of one capture antibody (or capture antibodies)-analyte of interest complex. This incubation can be carried out at a pH from about 4.5 to about 10.0, a temperature from about 2 °C to about 45 °C, and for a period of at least about 1 minute to about 18 hours, about 2 - 6 minutes, or about 3 - 4 minutes.

[0155] After formation of one capture antibody (antibodies)-analyte of interest complex, this complex is contacted with at least one detection antibody (under conditions that allow formation of one capture antibody (antibodies)-analyte of interest-one detection antibody (antibodies) complex). When the capture antibody-analyte of interest complex contacts two or more detection antibodies, one capture antibody (antibodies)-analyte of interest-one detection antibody (antibodies) detection complex is formed. Similar to the capture antibody, when at least one detection (and subsequent) antibody contacts the capture antibody-analyte of interest complex, an incubation period under conditions similar to those described above is required for formation of one capture antibody (antibodies)-analyte of interest-one detection antibody (antibodies) complex. It is preferred that at least one detection antibody contains a signal generating compound or a signal generating substrate. The signal generating compound or signal generating substrate can be bound to at least one detection antibody before, simultaneously with, or after formation of one capture antibody (antibodies)-analyte of interest-one detection antibody (antibodies) complex.

[0156] For generating the assay mixture, the order in which the test sample and the specific binding partner are added is not important. When the first specific binding partner is attached to the signal generating compound or signal generating substrate, a signal generating compound or signal generating substrate-attached first specific binding partner-analyte of interest complex is formed. Alternatively, a second specific binding partner is used and when the second specific binding partner is attached to the signal generating compound or signal generating substrate, a signal generating compound or signal generating substrate-first specific binding partner-analyte of interest-second specific binding partner attached complex is formed. Any unbound specific binding partner, whether labeled or unlabeled, can be removed from the mixture using any technique known in the art, such as washing.

[0157] Next, a signal indicating the presence of the analyte of interest or a fragment thereof is generated. Based on the parameters of the generated signal, the amount of the analyte of interest in the sample can be quantified. Optionally, a standard curve can be generated using serial dilutions or solutions of known concentrations of the analyte of interest by mass spectrometry, gravimetry, and other techniques known in the art.

[0158] (c) Forward competitive inhibition In the forward competition format, aliquots of the labeled analyte of interest at known concentrations are used to compete with the analyte of interest in the test sample for binding to the antibody of interest.

[0159] In a forward competitive assay, the immobilized specific binding partner (such as an antibody) can be contacted continuously or simultaneously with the test sample and the labeled analyte of interest, a fragment of the analyte of interest, or a variant of the analyte of interest. The peptide of the analyte of interest, the fragment of the analyte of interest, or the variant of the analyte of interest may be attached to a signal generating compound or a signal generating substrate. In this assay, the antibody may be immobilized on a solid phase support. Alternatively, the antibody may be bound to an antibody, such as an anti-species antibody, immobilized on a solid phase support such as a microparticle or a planar substrate.

[0160] The labeled analyte of interest, test sample, and antibody are incubated under conditions similar to those described above in relation to the sandwich assay format. Two different species of antibody-analyte of interest complexes may then be generated. Specifically, one of the generated antibody-analyte of interest complexes contains a signal generating compound or signal generating substrate, while the other antibody-analyte of interest complex does not contain a signal generating compound or signal generating substrate. The antibody-analyte of interest complexes may, but are not required to be, separated from the remainder of the test sample prior to quantification of the detectable product or detectable label. Whether or not the antibody-analyte of interest complexes are separated from the remainder of the test sample, the amount of detectable product or detectable label (e.g., detectable signal) in the antibody-analyte of interest complexes is then quantified. The concentration of the analyte of interest in the test sample (membrane-bound analyte of interest, soluble analyte of interest, fragment of soluble analyte of interest, variant of analyte of interest (membrane-bound or soluble analyte of interest), or any combination thereof, etc.) may then be determined, for example, as described above. Advantageously, the determination may be made by comparing the amount of detectable product or detectable label (e.g., detectable signal) in the antibody-analyte of interest complexes to a standard curve. The standard curve may be generated using serial dilutions of known concentrations of the analyte of interest (membrane-bound analyte of interest, soluble analyte of interest, fragment of soluble analyte of interest, variant of analyte of interest (membrane-bound or soluble analyte of interest), or any combination thereof, etc.), where the concentrations are determined by mass spectrometry, gravimetry, and other techniques known in the art.

[0161] Optionally, the antibody-analyte of interest complexes may be separated from the test sample by binding the antibody to a solid support, such as the solid support described above in relation to the sandwich assay format, and then excluding the remainder of the test sample from contact with the solid support.

[0162] (d) Reverse competitive assay In a reverse competitive assay, the immobilized analyte of interest can be contacted continuously or simultaneously with a test sample and at least one labeled antibody. The analyte of interest may be bound to a solid support such as the solid support described above in connection with the sandwich assay format.

[0163] The immobilized analyte of interest, the test sample, and at least one labeled antibody are incubated under conditions similar to those described above in connection with the sandwich assay format. Two different types of analyte - antibody complexes of interest are then generated. Specifically, one of the generated analyte - antibody complexes of interest is immobilized and contains a signal - generating compound or signal - generating substrate, while the other analyte - antibody complex of interest is not immobilized and contains a signal - generating compound or signal - generating substrate. The unimmobilized analyte - antibody complexes of interest and the remainder of the test sample are removed from the presence of the unimmobilized analyte - antibody complexes of interest via techniques known in the art, such as washing. Once the unimmobilized analyte - antibody complexes of interest are removed, the amount of the signal - generating compound or signal - generating substrate in the immobilized analyte - antibody complex of interest is quantified. The concentration of the analyte of interest in the test sample can then be determined by comparing the amount of the detectable signal described above. If helpful, this may be done by using a standard curve. The standard curve may be generated using serial dilutions of the analyte of interest or fragments of the analyte of interest of known concentration, where the concentration is determined by mass spectrometry, gravimetry, and other techniques known in the art.

[0164] (e) One - step immunoassay or capture - on - the - fly assay In a one-step immunoassay or a capture-on-the-fly assay, the solid substrate is pre-coated with an immobilizing agent. A capture agent, an analyte, and a detection agent are added together to the solid substrate, followed by a washing step prior to detection. The capture agent can bind the analyte and includes a ligand for the immobilizing agent. The capture agent and the detection agent may be antibodies, or any other moiety capable of capture or detection as described herein or known in the art. The ligand may include a peptide tag, and the immobilizing agent may include an antibody against the peptide tag. Alternatively, the ligand and the immobilizing agent may be any pair of agents (e.g., specific binding pairs and others known in the art) that are capable of binding together as used in a capture-on-the-fly assay. Two or more analytes may be measured. In some embodiments, the solid substrate may be coated with an antigen, and the analyte to be analyzed is an antibody.

[0165] In some embodiments, a solid phase support (such as microparticles) pre-coated with an immobilizing agent (such as biotin, streptavidin, etc.) and at least one first specific binding member and a second specific binding member (each functioning as a capture reagent and a detection reagent, respectively) is used. The first specific binding member includes a ligand for the immobilizing agent (for example, if the immobilizing agent on the solid phase support is streptavidin, the ligand of the first specific binding member may be biotin), and further binds to the analyte of interest. The second specific binding member includes a signal generating compound or a signal generating substrate and binds to the analyte of interest. The solid phase support and the first and second specific binding members may be added to the test sample (either continuously or simultaneously). The ligand of the first specific binding member binds to the immobilizing agent on the solid phase support to form a solid phase support / first specific binding member complex. Any analyte of interest present in the sample binds to the solid phase support / first specific binding member complex to form a solid phase support / first specific binding member / analyte complex. The second specific binding member binds to the solid phase support / first specific binding member / analyte complex and the signal generating compound or signal generating substrate to be detected. Optional washing steps may be used prior to detection. In some embodiments, in a one-step assay, two or more analytes may be measured. In some other embodiments, three or more specific binding members may be used. In some other embodiments, multiple signal generating compounds or signal generating substrates may be added. In some other embodiments, multiple analytes of interest may be detected.

[0166] The use of one-step immunoassays or capture-on-the-fly assays can be performed in various formats as described herein and known in the art. For example, this format can be a sandwich assay as described above, but alternatively, it can be a competitive assay, using a single specific binding member or other known variations.

[0167] (f) Combination assay (co - coating of Ag / Ab on microparticles) In a combination assay, solid substrates such as microparticles are co - coated with antigen and antibody, respectively, to capture antibody and antigen from the sample. This solid support may be co - coated with two or more different antigens to capture two or more different antibodies from the sample. This solid support may be co - coated with two or more different antibodies to capture two or more different antigens from the sample.

[0168] In addition, the methods described herein may use blocking agents to prevent either specific or non - specific binding reactions (e.g., HAMA interference) between assay compounds. When this agent (and optionally, any controls) is immobilized on a carrier, the remaining binding sites of this agent may be blocked on the carrier. Any suitable blocking agent known to those skilled in the art may be used. For example, bovine serum albumin (“BSA”), a PBS solution of casein in phosphate - buffered saline (“PBS”), Tween20 (TM) (Sigma Chemical Company, St. Louis, Mo.), or other suitable surfactants, as well as other blocking agents may be used.

[0169] As is apparent from the present disclosure, the methods disclosed herein, including a plurality of variations, may be used to diagnose the disease, disorder, or health condition of a subject suspected of having a disease, disorder, or health condition. For example, sample analysis may be useful for detecting markers of disease, such as cancer markers, markers of cardiac status, toxins, viruses, bacteria, or markers for pathogens such as parts thereof. These methods may also be used to measure analytes present in a biological sample. These methods may also be used in blood screening assays to detect a target analyte. Blood screening assays may be used to screen blood for transfusion.

[0170] 7. Multiplexing These methods may include one or more (or alternatively two or more) specific binding members for detecting one or more (or alternatively two or more) target analytes in a sample in a multiplex assay. Each of the one or more (or alternatively two or more) specific binding members binds to a different target analyte, and each specific binding member is bound to a different signal generating compound or signal generating substrate. For example, a first specific binding member binds to a first target analyte, a second specific binding member binds to a second target analyte, a third specific binding member binds to a third target analyte, etc., and the first specific binding member is labeled with a first signal generating compound or a first signal generating substrate, the second specific binding member is labeled with a second signal generating compound or a second signal generating substrate, the third specific binding member is labeled with a third signal generating compound or a third signal generating substrate, etc. In some embodiments, the state of the sample can change at various times during the assay, enabling detection of the first signal generating compound or the first signal generating substrate, the second signal generating compound or the second signal generating substrate, the third signal generating compound or the third signal generating substrate, etc., thereby detecting one or more (or alternatively two or more) target analytes. In some embodiments, one or more (or alternatively two or more) signal generating compounds or signal generating substrates are detected simultaneously. In some embodiments, one or more (or alternatively two or more) signal generating compounds or signal generating substrates are detected sequentially. In some embodiments, the one or more (or alternatively two or more) signal generating compounds or signal generating substrates generate different detectable signals, such as fluorescence signals of different wavelengths.

[0171] Alternatively, each of one or more (or instead two or more) specific binding members binds to a different target analyte, and each specific binding member is bound to a different solid support such as a different fluorophore bead. For example, a first specific binding member binds to a first target analyte, a second specific binding member binds to a second target analyte, a third specific binding member binds to a third target analyte, etc., and the first specific binding member is labeled with a first signal generating compound or a first signal generating substrate, the second specific binding member is labeled with a second signal generating compound or a second signal generating substrate, the third specific binding member is labeled with a third signal generating compound or a third signal generating substrate, etc., and the first specific binding member is immobilized on a first solid support, the second specific binding member is immobilized on a second solid support, the third specific binding member is immobilized on a third solid support, etc. In some embodiments, one or more (or instead two or more) signal generating compounds or signal generating substrates generate different detectable signals such as fluorescence signals of different wavelengths, and the different solid supports are detected simultaneously or sequentially.

[0172] In some embodiments, a first specific binding member binds to a first target analyte, a second specific binding member binds to a second target analyte, a third specific binding member binds to a third target analyte, etc., and the first specific binding member, the second specific binding member, the third specific binding member, etc. are labeled with a signal generating compound or a signal generating substrate, the first specific binding member is immobilized on a first solid support, the second specific binding member is immobilized on a second solid support, the third specific binding member is immobilized on a third solid support, etc. In some embodiments, the signal generating compound or the signal generating substrate generates a detectable signal such as a fluorescence signal of a different wavelength, and the different solid supports are detected simultaneously or sequentially.

[0173] 8. Kit Also provided herein is a kit for use in performing the methods described above. The kit may include instructions for analyzing an analyte using the disclosed methods. The instructions included in the kit may be attached to the packaging material or included as a package insert. The instructions may be written or printed materials, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROMs), etc. As used herein, "instructions" may include the address of an Internet site that provides the instructions.

[0174] Alternatively or in addition, the kit may include a calibrator or control (e.g., an analyte of interest that is purified, optionally lyophilized, or in liquid, gel, or other form), and / or at least one container (e.g., a tube, microtiter plate, or strip) for use with the methods described above, and / or a buffer such as an assay buffer or wash buffer, any of which may be provided as a concentrate. In some embodiments, the kit includes all of the components necessary to perform the assay, i.e., reagents, standards, buffers, diluents, etc. The instructions can also include instructions for generating a standard curve.

[0175] The kit may further include a standard sample for quantifying an analyte of interest. The standard sample may be used to construct a standard curve for interpolation and / or extrapolation of the concentration of the analyte of interest. The kit may include standard samples that vary with respect to concentration levels. For example, the kit may include one or more standard samples having any of a high concentration level, a medium concentration level, or a low concentration level. With respect to the range of concentrations of the standard sample, this may be optimized for each assay. Exemplary concentration ranges of the standard sample include, but are not limited to, for example, about 10 fg / mL, about 20 fg / mL, about 50 fg / mL, about 75 fg / mL, about 100 fg / mL, about 150 fg / mL, about 200 fg / mL, about 250 fg / mL, about 500 fg / mL, about 750 fg / mL, about 1000 fg / mL, about 10 pg / mL, about 20 pg / mL, about 50 pg / mL, about 75 pg / mL, about 100 pg / mL, about 150 pg / mL, about 200 pg / mL, about 250 pg / mL, about 500 pg / mL, about 750 pg / mL, about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 12.It includes 5 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 165 ng / mL, about 175 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 275 ng / mL, about 300 ng / mL, about 400 ng / mL, about 425 ng / mL, about 450 ng / mL, about 465 ng / mL, about 475 ng / mL, about 500 ng / mL, about 525 ng / mL, about 550 ng / mL, about 575 ng / mL, about 600 ng / mL, about 700 ng / mL, about 725 ng / mL, about 750 ng / mL, about 765 ng / mL, about 775 ng / mL, about 800 ng / mL, about 825 ng / mL, about 850 ng / mL, about 875 ng / mL, about 900 ng / mL, about 925 ng / mL, about 950 ng / mL, about 975 ng / mL, about 1000 ng / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 20 μg / mL, about 30 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 70 μg / mL, about 80 μg / mL, about 90 μg / mL, about 100 μg / mL, about 200 μg / mL, about 300 μg / mL, about 400 μg / mL, about 500 μg / mL, about 600 μg / mL, about 700 μg / mL, about 800 μg / mL, about 900 μg / mL, about 1000 μg / mL, about 2000 μg / mL, about 3000 μg / mL, about 4000 μg / mL, about 5000 μg / mL, about 6000 μg / mL, about 7000 μg / mL, about 8000 μg / mL, about 9000 μg / mL, or about 10000 μg / mL.

[0176] Any specific binding member provided in the kit may include at least one signal generating compound, one or more signal generating substrates, etc., or the kit can include reagents for labeling the specific binding member or reagents for detecting the specific binding member, and / or reagents for labeling the analyte or reagents for detecting the analyte. Optionally, the kit can include one or more different signal generating compounds and / or signal generating substrates. The specific binding member, calibrator, and / or control can be provided in separate containers or may be pre-dispensed in a suitable assay format.

[0177] The kit may include one or more specific binding members, for example, to detect one or more target analytes in a sample in a multiplex assay. The number of different types of specific binding members in the kit may vary widely depending on the intended use of the kit. The number of specific binding members in the kit may range from 1 to about 10 or more.For example, the kit may include from 1 to 10 specific binding members, from 1 to 9 specific binding members, from 1 to 8 specific binding members, from 1 to 7 specific binding members, from 1 to 6 specific binding members, from 1 to 5 specific binding members, from 1 to 4 specific binding members, from 1 to 3 specific binding members, from 1 to 2 specific binding members, from 2 to 10 specific binding members, from 2 to 9 specific binding members, from 2 to 8 specific binding members, from 2 to 7 specific binding members, from 2 to 6 specific binding members, from 2 to 5 specific binding members, from 2 to 4 specific binding members, from 3 to 10 specific binding members, from 3 to 9 specific binding members, from 3 to 8 specific binding members, from 3 to 7 specific binding members, from 3 to 6 specific binding members, from 3 to 5 specific binding members, from 3 to 4 specific binding members, from 4 to 10 specific binding members, from 4 to 9 specific binding members, from 4 to 8 specific binding members, from 4 to 7 specific binding members, from 4 to 6 specific binding members, from 5 to 10 specific binding members, from 5 to 9 specific binding members, from 5 to 8 specific binding members, from 5 to 7 specific binding members, from 5 to 6 specific binding members, from 6 to 10 specific binding members, from 6 to 9 specific binding members, from 6 to 8 specific binding members, from 6 to 7 specific binding members, from 7 to 10 specific binding members, from 7 to 9 specific binding members, from 7 to 8 specific binding members, from 8 to 10 specific binding members, from 8 to 9 specific binding members, or from 9 to 10 specific binding members. Each of the one or more specific binding members may bind to a different target analyte, and each specific binding member may associate with a different signal generating compound and / or signal generating substrate.For example, the kit may include a first specific binding member that binds to a first target analyte, a second specific binding member that binds to a second target analyte, a third specific binding member that binds to a third target analyte, etc., where the first specific binding member associates with a first signal generating compound and / or a first signal generating substrate, the second specific binding member associates with a second signal generating compound and / or a second signal generating substrate, the third specific binding member associates with a third signal generating compound and / or a third signal generating substrate, and so on. In addition to one or more specific binding members, the kit may further include one or more additional assay components, such as a suitable buffer medium. The kit may also include a device for detecting and measuring the signal generating compound and / or signal generating substrate, such as those described above. Finally, the kit may include instructions for using the specific binding members in a method of analyte detection according to the subject invention, and these instructions for use may be present on the kit package and / or on a package insert.

[0178] Optionally, the kit includes quality control components (e.g., a sensitivity panel, a calibrator, and a positive control). Preparation of quality control reagents is well known in the art and is described in the insert sheets for various immunodiagnostic products. Sensitivity panel members are used to construct assay performance characteristics and, further optionally, serve as an indicator of the integrity of the kit reagents and the standardization of the assay.

[0179] The kit can optionally also include other reagents necessary to perform a diagnostic assay or facilitate quality control evaluation, such as buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents, etc. Other components (e.g., pretreatment reagents) such as buffers and solutions for the separation and / or processing of test samples may also be included in the kit. The kit can further include one or more other controls. One or more of the components of the kit are lyophilized, and in that case, the kit can further include reagents suitable for the reconstitution of the lyophilized components. One or more of these components may be in liquid form.

[0180] The various components of the kit are provided in suitable containers as needed. The kit can further include a container for holding or storing a sample (e.g., a container for a urine, saliva, plasma, cerebrospinal fluid, or serum sample, or a suitable container for storing, transporting, or processing tissue to generate a tissue aspirate). If appropriate, the kit can optionally also include reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or test samples. The kit can also include one or more sample collection / acquisition instruments to assist in obtaining a test sample, such as microsampling devices, micro needles, or other minimally invasive painless blood collection methods, blood collection tubes, lancets, capillary blood collection tubes, other single finger prick blood collection methods, oral swabs, nasal / throat swabs, 16-gauge or other sized needles, circular blades for punch biopsies (e.g., 1 - 8 mm, or other suitable sizes), scalpels or lasers (e.g., particularly hand-held types), syringes, sterile containers, or cannulas. The kit can include one or more instruments to assist in arthrocentesis, cone biopsy, punch biopsy, fine needle aspiration biopsy, image-guided percutaneous needle aspiration biopsy, bronchoalveolar lavage, endoscopic biopsy, and laparoscopic biopsy.

[0181] Optionally, the kit can include a solid-phase support such as magnetic particles, beads, membranes, scaffold molecules, thin films, filter paper, disks, or chips.

[0182] Optionally, the kit can further include one or more components, alone or in combination with instructions, for assaying test samples of other analytes that may be biomarkers, such as biomarkers of medical conditions or disorders such as infectious diseases, heart diseases, metabolic diseases, thyroid diseases, etc.

[0183] 9. Example (Example 1) FIG. 4 shows a microchamber array for digital immunoassay and the mechanism of fluorescence signal amplification by enzymatic reaction. In this example, the bead diameter is about 2.7 μm, the well diameter / depth is about 4 μm / about 4 μm, the enzyme is alkaline phosphatase, the fluorescent generating substrate is fluorescein diphosphate, and the signal amplification was 90 minutes at RT.

[0184] To evaluate the usefulness of the digital ELISA immunoassay, a handheld (about 10×10×12 cm) optical imaging system based on an embodiment of the present invention was developed. In the demonstration, antigen solutions (recombinant HBsAg) of 0 and 10 femto M were applied. The first light sources applied to the detection containers resulted in images (A) and (D) shown in FIG. 6, respectively. The second light sources applied to the detection containers resulted in images (B) and (E) shown in FIG. 6, respectively. As shown in FIG. 6, the image analyzer combined image (A) and (B) to result in image (C), and combined image (D) and (E) to result in image (F). Images (C) and (F) show the detection positions of "beads only" and "beads and enzyme" plotted by gray and white dots, respectively, where the white dots are identified as signals of immune complexes. The performance of the handheld detector showed good performance of capturing 100,000 chamber regions in one image of the digital assay.

[0185] (Example 2) Digital Immunoassay Optical Detection of HBsAg (Hepatitis B Virus Surface Antigen) Anti-HBsAg mouse monoclonal antibody (prepared in-house) was coated with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) on the surface of 3-μm diameter paramagnetic microparticles (Agilent Technologies) to prepare the capture antibody. After washing, the coated paramagnetic microparticles were added to a buffer solution containing protein.

[0186] Anti-HBsAg goat polyclonal antibody (prepared in-house) was conjugated to alkaline phosphatase (Abbott) using standard chemical reactions known in the art to prepare the detection antibody. The conjugated antibody was purified using a determined gel filtration method known in the art. The purified conjugated antibody was diluted in a buffer solution containing protein.

[0187] 50 μL of the anti-HBsAg antibody microparticle solution and 50 μL of the alkaline phosphatase-conjugated anti-HBsAg antibody solution were incubated with 75 μL of human serum (with or without HBsAg) at 37 °C for 18 minutes. After incubation, the beads were washed with buffer solution, mixed with MUP or FDP solution, and then loaded into wells (6 mm in diameter) having a micro-well array (about 400,000 wells with a diameter of 5 μm (femtoliter chambers)) at the bottom. Fluorinated oil (e.g., FC-40, etc.) was added on top of the wells, and the aqueous and oil phases were exchanged. The aqueous phase (upper phase) was removed, and the wells were set up with the handheld optical imaging system described herein.

[0188] The images (photos) taken using this optical system are shown in FIGS. 7 - 11. Micro - particles were detected by scattered - image detection. A femtoliter chamber having a complex of antibody - coated micro - particles - HBsAg - alkaline - phosphate - conjugated antibody (HBsAg immune complex) was detected by fluorescence - image detection. The detected images were analyzed using Image J software to count the number of micro - particles and the number of femtoliter chambers having the HBsAg immune complex.

[0189] (Example 3) FIG. 4 shows a micro - chamber array for digital immunoassay and the mechanism of fluorescence - signal amplification by enzymatic reaction. In this example, the bead diameter was about 2.7 μm, the beads included a mixture of Qdot625 (Ex / Em: blue / red) - coated and non - coated ones (the ratio was about 10%), and the well diameter / depth was about 4 μm / about 4 μm. The red fluorescence of all the beads and the Qdot - coated beads was visualized by light scattering and fluorescence imaging, respectively.

[0190] To evaluate the usefulness of fluorescence imaging for digital ELISA immunoassay, a handheld - type (about 10×10×12 cm) optical imaging system based on an embodiment of the present invention was utilized. In this example, a single emission filter was a 515 nm long - pass - type filter (e.g., Semrok 515LP). In the demonstration, a binary mixture of 10% red - fluorescent beads and non - fluorescent beads was applied.

[0191] The demonstrated imaging method showed that the radiation filter did not need to be changed. This imaging method utilized a first autofocus step using a first light source including a green LED (525 nm), and then the first light source was applied to the detection container for scattering and bead imaging. This resulted in the image (A) shown in FIG. 12. In the next step, a second light source including a blue LED (450 nm) was applied to the detection container to excite the sample therein. This resulted in the fluorescence image (B) shown in FIG. 12. Next, the chromatic aberration was refreshed to include a red LED (625 nm) as the first light source. Autofocus was performed using this refreshed first light source and then applied to the detection container for scattering. This resulted in the image (C) shown in FIG. 12. To excite the sample, the second light source (blue LED) was applied to the detection container, which resulted in the image (D) shown in FIG. 12.

[0192] The image analyzer combined image (A) with (D), resulting in image (E) as shown in FIG. 12. ImageJ software was used to merge images (A) and (D). Image (E) shows the detected positions of "beads and fluorescent species" plotted by green and red dots.

[0193] The various features and advantages of the present invention are set forth in the following claims.

Claims

1. A method for detecting an analyte of interest in a well, the method comprising: (a) contacting a suspect sample containing the analyte with (i) a detection vessel comprising a plurality of wells each having at least one well containing at least one bead, wherein at least one bead comprises a specific binding member that binds to the analyte of interest, the detection vessel comprising an axis extending therethrough at approximately 90 degrees, (ii) a first light source arranged at an angle between 45 degrees and 90 degrees with respect to the axis, configured to emit scattered light towards the detection vessel, (iii) a second light source configured to emit excitation light substantially perpendicularly above or below the detection vessel from a position laterally offset from the axis with respect to the detection vessel, (iv) a single filter arranged to receive and pass a portion of the light reflected from at least one bead resulting from the first light source and a portion of the fluorescence output from at least one bead within the detection vessel, (v) a detector configured to receive a portion of the reflected light and a portion of the fluorescence output passing through the single filter, and (b) using information from the reflected light and the fluorescence output to determine which wells contain beads. A method having the above steps.

2. The method according to claim 1, wherein the angle is 80 degrees.

3. The method according to claim 1, wherein the first light source comprises a plurality of light emitting diodes.

4. The method according to claim 1, wherein the first light source is a light emitting diode.

5. The method according to claim 1, wherein the first light source is composed of two or more light sources.

6. The method according to claim 1, wherein the first light source is configured to change color.

7. The method according to claim 6, wherein the first light source is configured to change between blue and green.

8. The method according to claim 7, wherein the green first light source reflects at least one bead within the detection vessel such that the detector generates optical data for identifying whether beads are present in the sample.

9. The method according to claim 7, wherein the blue first light source excites the beads within the detection vessel such that the detector generates optical data for identifying whether an enzyme is present in the sample.

10. The method according to claim 1, wherein the fluorescence output is fluorescence generated after excitation of the beads within the detection vessel by the second light source.

Images