Multiplex assay plate and method of manufacture

Abstract

Described herein are methods for preparing bifunctional assay surfaces. In particular, methods are provided for preparing assay surfaces that include a primary reagent and a secondary reagent. In one aspect, the primary reagent and the secondary reagent are immobilized on the assay surface by different surface chemistries. In one aspect, methods are provided for preparing assay surfaces that include a proteinaceous primary reagent and a thiol-containing secondary reagent. In one aspect, methods are provided for preparing assay surfaces that include a capture-target hybrid and a thiol-containing secondary reagent.

Description

[Technical field]

[0001] Described herein are methods for preparing a bifunctional assay surface. [Background technology]

[0002] Numerous methods and systems have been developed for performing chemical, biochemical and / or biological assays in which the presence and / or amount of one or more analytes in a sample is determined by directly or indirectly detecting the participation of the analyte in a binding reaction, such as an antigen-antibody reaction, a nucleic acid hybridization or a receptor-ligand reaction. In some approaches, the participation of the analyte in the binding reaction is indicated by measuring an observable label attached to one or more binding materials. These methods and systems are used in a wide range of applications, including, but not limited to, medical diagnostics, food and beverage testing, environmental monitoring, manufacturing quality control, drug discovery, and basic science research.

[0003] Assays that measure the level of a single analyte (singleplex assays) have been the mainstay of biological research for decades. Assays that measure multiple analytes in parallel (multiplex assays) can also be performed to reduce workflow and sample volume requirements. Multiplex assays typically come in two formats: immobilization on a solid surface containing one or more binding domains, and immobilization on beads or particles, where the binding domain for each analyte is on a different bead or particle.

[0004] In some assays, it may be desirable to have a bifunctional assay surface in which one or more binding domains contain one or more different reagents. However, there remains a need for methods to create bifunctional assay surfaces for the analysis of one or more target analytes in a sample. Summary of the Invention

[0005] Provided herein are methods for preparing a bifunctional assay surface. In one aspect, the method includes a direct coating method in which a coating solution comprising a primary reagent and a secondary reagent is dispensed onto an assay surface to form a coated assay surface. In one aspect, the method includes an overcoating method in which an overcoating solution comprising a secondary reagent is dispensed onto an assay surface having a primary reagent immobilized thereon to form a coated assay surface. In one aspect, the primary and secondary reagents are immobilized on the assay surface via different surface chemistries.

[0006] In one aspect, a straightforward method for preparing a bifunctional assay surface is provided in which a coating solution comprising a primary reagent and a secondary reagent is dispensed onto the assay surface to form a coated assay surface. In one aspect, the primary reagent is a proteinaceous primary reagent. In one aspect, the primary reagent is a capture target hybrid. In one aspect, the secondary reagent is a thiol-containing secondary reagent. In one aspect, the secondary reagent is a thiolated oligonucleotide. In one aspect, the assay surface is a carbon-containing assay surface. In one aspect, the method includes incubating the coated assay surface under conditions in which the primary and secondary reagents are immobilized on the carbon-containing assay surface. In one aspect, the thiol-containing secondary reagent is immobilized on the carbon-containing assay surface via a thiol group. In one aspect, the thiol-containing secondary reagent is immobilized on the carbon-containing assay surface through the formation of a covalent bond between the thiol group and a reactive functional group on the assay surface. In one aspect, the method comprises dispensing about 10 nl to about 100 nl, about 25 nl to about 75 nl, about 40 nl to about 60 nl, or about 50 nl of coating solution onto the assay surface.

[0007] In one embodiment, the coating solution comprises about 100 μg / ml to 750 μg / ml, about 500 μg / ml to 750 μg / ml, or about 100 μg / ml to about 400 μg / ml of proteinaceous primary reagent or capture-target hybrid. In one embodiment, the coating solution comprises about 15 nM to about 1500 nM of thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 1.5 nM to about 1500 nM of thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 1.5 nM to about 10 nM of thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 1.5 nM to about 15 nM of thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 15 nM to about 30 nM of thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 30 nM to about 50 nM of thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 50 nM to about 100 nM of the thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 100 nM to about 150 nM of the thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 150 nM to about 300 nM of the thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 300 nM to about 500 nM of the thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 500 nM to about 750 nM of the thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 750 nM to about 1000 nM of the thiol-containing secondary reagent. In one embodiment, the coating solution comprises about 1000 nM to about 1500 nM of the thiol-containing secondary reagent.

[0008] In one aspect, the coating solution comprises streptavidin at about 100 μg / ml to about 500 μg / ml, 500 μg / ml to about 750 μg / ml, or 750 μg / ml to about 1000 μg / ml, a thiol-containing secondary reagent at about 15 nM to about 1500 nM, TRITON X-100 at about 0.01% to about 0.1%, optionally trehalose at about 0.1% to about 0.5% in phosphate buffered saline, optionally additional proteinaceous components including, but not limited to, bovine serum albumin (BSA), human serum albumin (HSA), protein A, and protein G at about 400 μg / ml to about 750 μg / ml, and EDTA at about 1 mM to about 20 mM. In one aspect, the coating solution comprises streptavidin at about 100 μg / ml to about 400 μg / ml, 400 μg / ml to about 750 μg / ml, or 750 μg / ml to about 1000 μg / ml, a thiol-containing secondary reagent at about 15 nM to about 1500 nM, about 0.03% TRITON X-100, optionally about 0.4% trehalose in phosphate buffered saline, optionally additional proteinaceous components including, but not limited to, about 400 μg / ml to about 750 μg / ml of BSA, HSA, protein A, and protein G, and about 10 mM EDTA. In one aspect, the coating solution comprises streptavidin at about 100 μg / ml to about 500 μg / ml, 500 μg / ml to about 750 μg / ml, or 750 μg / ml to about 1000 μg / ml, a thiol-containing secondary reagent at about 1500 nM to about 2500 nM, about 0.03% TRITON X-100, optionally about 0.4% trehalose in phosphate buffered saline, optionally additional proteinaceous components including, but not limited to, about 400 μg / ml to about 750 μg / ml of BSA, HSA, protein A, and protein G, and about 10 mM EDTA. In one embodiment, the phosphate buffered saline comprises Dulbecco's Phosphate Buffered Saline (DPBS) containing 2.67 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, and 138 mM NaCl. In one embodiment, the method comprises drying the coated assay surface overnight at room temperature.In one embodiment, the method comprises drying the coated assay surface at a controlled humidity of about 40%.

[0009] In one aspect, the coating solution comprises about 100 μg / ml to about 400 μg / ml, 400 μg / ml to about 750 μg / ml, or 750 μg / ml to about 1000 μg / ml of a proteinaceous primary reagent or capture-target hybrid, about 15 nM to about 1500 nM of a thiol-containing secondary reagent, about 0.01% to about 0.1% TRITON X-100, optionally about 0.1% to about 0.5% trehalose in phosphate buffered saline, optionally additional proteinaceous components including, but not limited to, about 400 μg / ml to about 750 μg / ml of BSA, HSA, Protein A, and Protein G, and about 1 mM EDTA to about 20 mM EDTA. In one aspect, the coating solution comprises about 100 μg / ml to about 400 μg / ml, 400 μg / ml to about 750 μg / ml, or 750 μg / ml to about 1000 μg / ml of a proteinaceous primary reagent or capture-target hybrid, about 15 nM to about 1500 nM of a thiol-containing secondary reagent, about 0.03% TRITON X-100, about 0.4% trehalose in phosphate buffered saline, optionally additional proteinaceous components including, but not limited to, about 400 μg / ml to about 750 μg / ml of BSA, HSA, Protein A, and Protein G, and about 10 mM EDTA. In one embodiment, the phosphate buffered saline comprises Dulbecco's Phosphate Buffered Saline (DPBS) containing 2.67 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, and 138 mM NaCl. In one embodiment, the method comprises drying the coated assay surface overnight at room temperature. In one embodiment, the method comprises drying the coated assay surface at a controlled humidity of about 40%.

[0010] In one aspect, an overcoating method for preparing a bifunctional assay surface is provided in which an overcoating solution containing a secondary reagent is dispensed onto an assay surface having one or more primary reagents immobilized thereon to form a coated assay surface. In one aspect, the assay surface is a carbon-containing assay surface. In one aspect, the primary reagent is a proteinaceous primary reagent. In one aspect, the primary reagent is a capture-target hybrid. In one aspect, the secondary reagent is a thiol-containing secondary reagent. In one aspect, the secondary reagent is a thiolated oligonucleotide. In one aspect, the method comprises incubating the coated assay surface under conditions in which the thiol-containing secondary reagent is immobilized onto the carbon-containing assay surface via the thiol group. In one aspect, the thiol-containing secondary reagent is immobilized onto the carbon-containing assay surface through the formation of a covalent bond between the thiol group and a reactive functional group on the assay surface. In one aspect, the method comprises dispensing about 10 μL to about 100 μL, about 25 μL to about 75 μL, or about 30 μL to about 50 μL of the overcoating solution onto the assay surface. In one embodiment, the overcoating solution comprises about 0.01 μM to about 20 μM of the thiol-containing secondary reagent. In one embodiment, the overcoating solution comprises a buffer selected from deprotection conjugation buffer (10 mM phosphate, pH 7.4, 150 mM NaCl, 10 mM EDTA), 1×PBS (phosphate buffered saline) / 10 mM EDTA (ethylenediaminetetraacetic acid) or Diluent 100 (Meso Scale Diagnostics, LLC). In one embodiment, the method comprises incubating the coated assay surface at room temperature for about 1 hour to about 5 hours, about 1 hour to about 3 hours, or about 2 hours with shaking at about 500 rpm to about 725 rpm. In one embodiment, the method comprises incubating the coated assay surface at room temperature with shaking at about 705 rpm.

[0011] In one aspect, the assay surface is part of a multi-well plate.In one aspect, the carbon-containing assay surface is part of a multi-well plate.

[0012] In one embodiment, one or more primary reagents are immobilized on the assay surface at distinct binding domains. In one embodiment, one or more proteinaceous primary reagents or capture target hybrids are immobilized on the carbon-containing assay surface at distinct binding domains. In one embodiment, a plurality of proteinaceous primary reagents or capture target hybrids are immobilized on the carbon-containing assay surface. In one embodiment, a plurality of primary reagents comprises primary reagents that bind to different targets. In one embodiment, the primary reagents are immobilized on the assay surface in an array. In one embodiment, one or more proteinaceous primary reagents or capture target hybrids are immobilized on the carbon-containing assay surface in an array. In one embodiment, one or more secondary reagents are immobilized on the assay surface. In one embodiment, one or more thiol-containing secondary reagents are immobilized on the assay surface.

[0013] In one embodiment, a primary reagent is immobilized to a first binding domain and a secondary reagent is immobilized to a second binding domain. In one embodiment, a plurality of proteinaceous primary reagents or capture target hybrids are immobilized to a plurality of primary binding domains and a plurality of thiol-containing secondary reagents are immobilized to a plurality of secondary binding domains. In one embodiment, each primary binding domain contains a unique primary reagent. In one embodiment, each secondary binding domain contains a unique secondary reagent. In one embodiment, each secondary binding domain contains a common secondary reagent.

[0014] In one aspect, each binding domain comprises a primary reagent and a secondary reagent. In one aspect, each binding domain comprises a proteinaceous primary reagent and a thiol-containing secondary reagent. In one aspect, each binding domain comprises a capture target hybrid and a thiol-containing secondary reagent. In one aspect, each binding domain comprises a unique primary reagent. In one aspect, each binding domain comprises a unique secondary reagent. In one aspect, each binding domain comprises a common secondary reagent. In one aspect, each binding domain comprises a unique primary reagent and a unique secondary reagent. In one aspect, each binding domain comprises a unique primary reagent and a common secondary reagent.

[0015] In one aspect, the primary reagent is non-covalently immobilized on the carbon-containing assay surface. In one aspect, the primary reagent is a proteinaceous primary reagent or capture target hybrid non-covalently immobilized on the carbon-containing assay surface. In one aspect, the primary reagent is covalently immobilized on the carbon-containing assay surface. In one aspect, the primary reagent is a proteinaceous primary reagent or capture target hybrid covalently immobilized on the carbon-containing assay surface. In one aspect, the primary reagent or capture target hybrid is immobilized on the assay surface via a binding pair. In one aspect, the primary reagent is a proteinaceous primary reagent or capture target hybrid immobilized on the carbon-containing assay surface via a binding pair. In one aspect, the primary reagent comprises a first member of a binding pair and the assay surface comprises a second member of the binding pair. In one aspect, the primary reagent comprises biotin and the assay surface comprises avidin or streptavidin. In one aspect, the primary reagent comprises a proteinaceous capture reagent. In one aspect, the proteinaceous capture reagent comprises an antibody, an antigen-binding fragment of an antibody, or a receptor. In one aspect, the secondary reagent is a thiol-containing secondary reagent. In one aspect, the thiol-containing secondary reagent comprises a thiolated oligonucleotide.

[0016] In one embodiment, the assay surface is a carbon-containing assay surface that is treated to introduce one or more maleimide groups before the thiol-containing secondary reagent is dispensed onto the carbon-containing assay surface. In one embodiment, the carbon-containing assay surface is a SM (PEG) n (n=2-24). In one embodiment, n=4.

[0017] In one embodiment, one or more secondary reagents are immobilized on the assay surface. In one embodiment, a plurality of secondary reagents are immobilized on the assay surface. In one embodiment, a plurality of thiol-containing secondary reagents are immobilized on the carbon-containing assay surface. In one embodiment, the secondary reagent comprises a thiolated oligonucleotide. In one embodiment, the thiolated oligonucleotide has a length of about 1 to about 100, about 5 to about 50, about 5 to about 10 nucleotides, or about 10 to about 30 nucleotides. In one embodiment, the thiolated oligonucleotide comprises an oligonucleotide attached to a thiol group via a linker. In one embodiment, the linker comprises about 3 to about 20 atoms or molecules or units. In one embodiment, the thiolated oligonucleotide comprises an oligonucleotide sequence having a 5' and a 3' end and a thiol group incorporated at the 5' end (5' end thiolated oligonucleotide), the 3' end (3' end thiolated oligonucleotide), an internal position of the oligonucleotide, or a combination thereof. In one embodiment, the thiolated oligonucleotide comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), 2'-O-methyl nucleotide (2'-OMe), 2'-O-methoxyethyl nucleotide (2'-MOE), 2'-deoxy-2'-fluororibonucleotide (2'-F-RNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or a combination thereof. In one embodiment, the thiolated oligonucleotide comprises one or more non-natural nucleotide bases. In one embodiment, the non-natural nucleotide bases are selected from 2,6-diaminopurine (2-amino-dA); 5-methyldeoxycytidine, Super T (5-hydroxybutynyl-2'-deoxyuridine), or a combination thereof.

[0018] In one aspect, the secondary reagent is a thiol-containing secondary reagent that comprises a thiolated member of a binding pair. In one aspect, the secondary reagent comprises thiolated biotin (thiol-biotin). In one aspect, the secondary reagent comprises thiolated polyethylene glycol (thiol-PEG). In one aspect, the thiolated polyethylene glycol further comprises biotin (thiol-PEG-biotin).

[0019] In one embodiment, the secondary reagent is a thiol-containing secondary reagent that comprises thiolated fluorescein isothiocyanate (FITC).

[0020] In one embodiment, the primary reagent comprises a capture reagent that specifically binds to or comprises a target molecule or target analyte. In one embodiment, the primary reagent comprises a proteinaceous capture reagent that specifically binds to a target molecule. In one embodiment, the primary reagent comprises a proteinaceous primary reagent selected from an antibody, an antigen, a receptor, an enzyme, or a combination thereof. In one embodiment, the primary reagent comprises a capture target hybrid that comprises a target molecule or a target analyte. In one embodiment, the primary reagent comprises a capture target hybrid selected from a cell, a viral derivative, an organelle, a subcellular structure, a vesicle, a therapeutic molecule, or a combination thereof. In one embodiment, the primary reagent comprises an antigen-binding agent. In one embodiment, the primary reagent comprises an antibody or an antigen-binding fragment thereof. In one embodiment, the primary reagent comprises a first member of a binding pair and the target molecule comprises a second member of the binding pair. In one embodiment, the binding pair comprises streptavidin or avidin and biotin.

[0021] In one embodiment, the assay surface is part of a multi-well plate. In one embodiment, the assay surface is a carbon-containing assay surface that is part of a multi-well plate. In one embodiment, the carbon-containing assay surface comprises an electrode. In one embodiment, the carbon-containing assay surface is part of a multi-well plate, and one or more wells of the multi-well plate comprise one or more electrodes. In one embodiment, the carbon-containing assay surface is part of a multi-well plate, and each well of the plate comprises one or more electrodes. In one embodiment, the carbon-containing assay surface comprises a carbon-containing electrode. In one embodiment, the carbon-containing assay surface comprises a carbon ink electrode.

[0022] In one embodiment, the primary reagent is a proteinaceous primary reagent comprising an antibody or an antigen-binding antibody fragment, and the secondary reagent comprises a thiolated oligonucleotide. In one embodiment, the primary reagent is a proteinaceous primary reagent that binds to a target that is a surface protein of an extracellular vesicle (EV). In one embodiment, the extracellular vesicle is an exosome. In one embodiment, the target is an exosome-associated protein. In one embodiment, the target is encapsulated by an exosome. In one embodiment, the secondary reagent binds to a target that is an exosome-associated protein. In one embodiment, the secondary reagent binds to a target encapsulated by an exosome. [Brief description of the drawings]

[0023] [Figure 1] 1 is a graph showing the assay sensitivity of a bifunctional assay surface prepared using the direct coating method described herein. [Diagram 2] 1 is a graph showing the assay sensitivity of a bifunctional assay surface prepared using the overcoating method described herein. [Figure 3A] Assay sensitivity of assay plates with different types of anchor oligonucleotides: biotinylated and pegylated anchor oligonucleotides attached using the direct coating method described herein (Bio-Peg-SH / Anchor); thiolated anchor oligonucleotides (Anchor-SH); and no anchor oligonucleotides are compared. [Figure 3B] Non-specific binding (NSB) of assay plates with different types of anchor oligonucleotides: biotinylated and pegylated anchor oligonucleotides attached using the direct coating method described herein (Bio-Peg-SH / Anchor); thiolated anchor oligonucleotide (Anchor-SH); and no anchor oligonucleotide are compared. [Figure 3C]The signal to non-specific binding ratios of assay plates with different types of anchor oligonucleotides: biotinylated and pegylated anchor oligonucleotides attached using the direct coating method described herein (Bio-Peg-SH / Anchor); thiolated anchor oligonucleotides (Anchor-SH); and no anchor oligonucleotide are compared. [Figure 4] 1 shows signal generation of FITC-Peg-SH immobilized by the overcoating method described herein. [Figure 5A] Non-specific binding (NSB) in assays for IFNg using PEG oligomer modified assay surfaces. Five Peg-SH reagents (MW 350, 550, 750, 1000, and 2000) were immobilized via overcoating and tested in a standard sandwich format (10-plex). [Figure 5B] Non-specific binding (NSB) in assays for TNFα using PEG oligomer modified assay surfaces. Five Peg-SH reagents (MW 350, 550, 750, 1000, and 2000) were immobilized via overcoating and tested in a standard sandwich format (10-plex). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, the singular shall include the plural and the plural shall include the singular. The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0025] As used herein, the term "about" is used to indicate that a value includes the inherent variation of error for the device or the method being used to determine the value.

[0026] As used herein, "between" refers to a range that includes the ends of the range, e.g., a number between x and y specifically includes the numbers x and y, as well as any number between x and y.

[0027] As used herein, "room temperature" refers to an indoor temperature suitable for long-term storage of biological materials and laboratory experiments, typically ranging from 15 to 28° C. In various embodiments, room temperature is 20 to 25° C.

[0028] In the context of an analyte to be measured in an assay, or a reagent used in an assay, the term "multiple" refers to two or more structurally and / or functionally different analytes or reagents (e.g., reagent A and reagent B), rather than simply two or more copies of an analyte or reagent (e.g., reagent A and another copy of reagent A). For example, the term "multiple immobilized reagents" refers to two or more structurally or functionally different reagents being immobilized, and does not describe a situation in which there are multiple copies of one reagent. However, the use of the term "multiple" in this context does not exclude the possibility that there are multiple copies of either the multiple analytes or reagents. For example, multiple immobilized reagents can refer to an immobilized reagent that includes one or more copies of reagent A and one or more copies of reagent B. In the context of an assay surface, the term "bifunctional" or "multifunctional" refers to a surface on which two or more different types of reagents are immobilized. In one aspect, the different types of reagents are immobilized on the surface by different surface chemistries. As used herein, "surface chemistry" refers to the chemical reactions that result in the immobilization of the reagents on the assay surface. Surface chemistry includes covalent and non-covalent interactions. Covalent immobilization refers to immobilization by forming one or more covalent bonds between a reagent and a reactive functional group on the assay surface. Non-covalent interactions include, for example, hydrogen bonds, electrostatic or ionic interactions, and van der Waals forces. Passive adsorption refers to immobilization of biomolecules on a surface via hydrophobic interactions or hydrophobic and ionic interactions between the biomolecule and the surface.

[0029] As used herein, the term "polypeptide" is intended to encompass multiple polypeptides as well as single polypeptides and refers to a molecule composed of amino acid monomers linked by peptide bonds. The term polypeptide refers to any chain or chains of amino acids having a peptide backbone and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, proteins, amino acid chains, or any other term used to refer to a chain or chains of amino acids are included within the definition of a polypeptide, and the term polypeptide can be used in place of or synonymously with any of these terms. The term polypeptide also includes products of post-expression modifications of polypeptides, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology. A polypeptide is not necessarily translated from a specified nucleic acid sequence. A polypeptide can be generated in any manner, including chemical synthesis. In the context of a polypeptide, "sequence" refers to the amino-terminal to carboxyl-terminal order of amino acids in a polypeptide, where adjacent residues in a sequence are contiguous in the primary structure of the polypeptide. As used herein, the term "proteinaceous" refers to a macromolecule of natural or non-natural origin comprising one or more polypeptide chains, including, but not limited to, peptides, proteins, antibodies, antigens, enzymes, receptors, or fragments or portions thereof.

[0030] The term "oligonucleotide" as used herein refers to a short polymer of nucleic acid having a phosphate backbone, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or combinations thereof. In one aspect, the oligonucleotide has a length of about 5, 10, 15, 20, or 25 nucleotides up to about 50, 75, 100, or 150 nucleotides, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or 150 nucleotides. Oligonucleotides can be designed to specifically hybridize to DNA or RNA sequences, for example, to detect target nucleotides having a sequence complementary to the nucleotide sequence of the oligonucleotide probe. Oligonucleotides can be single-stranded or double-stranded and can be obtained by methods including, but not limited to, isolation from biological samples, recombinant synthesis, and chemical synthesis. The term "oligonucleotide" can include structural analogs, including modifications of non-natural origin. For example, an oligonucleotide can include a chemical modification that links the oligonucleotide to another entity, such as a label, or that provides a reactive functional group that can be linked to another entity. An oligonucleotide can also include one or more non-natural nucleotide bases.

[0031] The term "reactive functional group" refers to an atom or associated group of atoms that can undergo further chemical reaction, for example, to form a covalent bond with another functional group. Examples of reactive functional groups include, but are not limited to, amino, thiol, hydroxy, and carbonyl groups. In one aspect, the reactive functional group comprises a reactive thiol group. In one aspect, the reactive functional group is used to immobilize a biomolecule on a surface. In one aspect, the reactive functional group is used to add a label to a biomolecule. Labels that can be linked to nucleotides or nucleic acids via these chemical modifications include, but are not limited to, detectable moieties such as biotin, haptens, fluorophores, and electrochemiluminescence (ECL) labels.

[0032] The terms "antibody" and "immunoglobulin" can be used interchangeably to refer to biological molecules capable of specifically binding to an antigen. In most vertebrates, antibodies exist as dimers of two heavy (H) chains, each paired with one light (L) chain. The N-terminus of the heavy and light chains contains variable domains (V and V, respectively) that provide the antibody with its unique antigen-binding specificity. H and V L ). As used herein, the term "antibody" refers to a whole antibody molecule or an antigen-binding fragment thereof. Antibodies or fragments thereof may be naturally produced or partially or wholly synthetically or recombinantly produced. An antigen-binding fragment refers to any antibody fragment that contains at least a portion of the variable region of an immunoglobulin molecule and retains the binding specificity of the full-length immunoglobulin. The term "antibody" includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, including but not limited to Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single chain Fv (scFv), single chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. A "binding reagent" refers to a reagent characterized by its ability to preferentially bind to another substance, sometimes referred to as a "binding partner." The binding reagent and the binding partner are sometimes referred to as a "binding pair." Examples of binding pairs include, but are not limited to, biotin and streptavidin or avidin; complementary oligonucleotides; a hapten and a hapten-binding partner; an antibody / antigen binding pair; a receptor / ligand binding pair; and an enzyme / substrate binding pair. In one embodiment, a first member of the binding pair is immobilized to a binding domain on the assay surface and a second member of the binding pair is the target.

[0033] The term "specifically bind" refers to a preferential interaction between a binding reagent and its target compared to the interaction of the binding reagent with other molecules or components in a sample. The specific binding between a binding reagent and a target is due to the complementarity between the binding domain of the binding reagent and the target, as well as non-covalent binding forces such as electrostatic forces, hydrogen bonds, van der Waals forces, and hydrophobic forces. As used herein, the term "preferentially bind" means that one member of a binding pair binds to its binding partner under appropriate conditions without any significant binding, e.g., any statistically significant binding, to other compounds present in the sample. In one embodiment, the members of a binding pair, e.g., a binding reagent and its target, have an affinity for each other that is at least about 50 times, 75 times, or 100 times greater than the affinity between either member of the binding pair and other compounds present in the sample.

[0034] The term "target" refers to a substance in a sample that can be specifically bound, for example, by a reagent, and retained at a specific location on the assay surface. In one aspect, the target is retained at a specific location on the assay surface so that the presence or amount of the target in the sample can be determined, in which case the target may be referred to as a target analyte. In one aspect, the target is retained at a specific location on the assay surface to facilitate detection of different target analytes. In one aspect, the target is covalently bound to a reagent immobilized on the assay surface. In one aspect, the target is non-covalently bound to a reagent immobilized on the assay surface. In one aspect, the target is directly bound to a reagent immobilized on the assay surface. In one aspect, the target is indirectly bound to a reagent immobilized on the assay surface, for example, via a binding member that binds to the target and is also bound by the reagent immobilized on the assay surface. Examples of targets include, but are not limited to, cells; cell membranes; organelles; receptors, such as vesicles, lipids, or receptors on cell membranes; ligands, agonists, or antagonists that bind to receptors; antibodies; antigens; proteins; peptides; polysaccharides; oligonucleotides or polynucleotides (including, for example, mRNA, tRNA, rRNA, DNA, cDNA, or amplification products or amplicons), or therapeutic molecules such as drugs. In one embodiment, the target comprises a member of a binding pair, such as biotin, avidin, or streptavidin.

[0035] "Complementary" refers to nucleic acid molecules or sequences of nucleic acid molecules that interact by forming hydrogen bonds, e.g., according to the Watson-Crick base pairing model, e.g., A pairs with T or U, and C pairs with G. For example, hybridization can occur between two complementary DNA molecules (DNA-DNA hybridization), between two RNA molecules (RNA-RNA hybridization), or between complementary DNA and RNA molecules (DNA-RNA hybridization). Hybridization can occur between a short nucleotide sequence that is complementary to a portion of a longer nucleotide sequence. Full complementarity or 100% complementarity refers to a situation in which each nucleotide of one oligonucleotide sequence or region can hydrogen bond with each nucleotide of at least a portion of a second oligonucleotide strand or region. Hybridization can occur between sequences that do not have 100% "sequence complementarity" (i.e., sequences in which less than 100% of the nucleotides are aligned based on a base pairing model, such as the Watson-Crick base pairing model). Generally, sequences with low sequence complementarity are less stable and less likely to hybridize than sequences with high sequence complementarity.In one embodiment, the nucleotides of complementary sequences have 100% sequence complementarity based on the Watson-Crick model.In another embodiment, the nucleotides of complementary sequences have at least about 90%, 95%, 96%, 97%, 98% or 99% sequence complementarity based on the Watson-Crick model and can hybridize under stringent hybridization conditions.It is understood that complementary sequences do not have to hybridize along their entire length, i.e., a shorter oligonucleotide sequence can hybridize to a portion of the longer oligonucleotide sequence to which it is complementary.

[0036] Whether two complementary sequences hybridize may depend on the stringency of the hybridization conditions, which may vary depending on conditions such as temperature, solvent, ionic strength, and other parameters. The stringency of the hybridization conditions can be selected to provide the selective formation or maintenance of the desired hybridization product of two complementary nucleic acid sequences in the presence of other potentially cross-reactive or interfering sequences. Stringent conditions are sequence-dependent, and typically, longer complementary sequences hybridize selectively at higher temperatures than shorter complementary sequences.

[0037] As used herein, a "capture molecule" refers to a molecule, or a complex or combination thereof, that can specifically bind to a target. In one aspect, the capture molecule is a peptide or protein, including, for example, an antibody, an antigen-binding antibody fragment, or a receptor. In another aspect, the capture molecule is an oligonucleotide or a nucleic acid that hybridizes to a complementary oligonucleotide sequence of the target under stringent hybridization conditions. In one aspect, the capture molecule comprises a vitamin, an oligosaccharide, a carbohydrate, a lipid, a small molecule, or a complex thereof. In one aspect, the primary reagent comprises a primary capture molecule that specifically binds to the primary target. In one aspect, the secondary reagent comprises a secondary capture molecule that specifically binds to the secondary target. In one aspect, the secondary target is associated with the primary target. In one aspect, the secondary target is encapsulated by the primary target.

[0038] The term "biomolecule" as used herein refers to any compound or substance produced by a living organism, e.g., a cell. In one aspect, biomolecules are isolated from biological material by solvent, thermal, and / or physical methods of extraction, separation, purification known in the art.

[0039] As used herein, a "capture target hybrid" is a biomolecule, molecule or complex, or combination thereof, that includes a region that allows it to be immobilized to a binding domain on an assay surface and that includes a target analyte. In one aspect, the target analyte is on the surface of the capture target hybrid. In one aspect, the target analyte is on the surface of a biomolecule, e.g., a cell or organelle. In another aspect, the target is on the surface of an artificial or engineered capture molecule. In one aspect, the capture target hybrid includes an antigen binding agent.

[0040] In one aspect, the capture target hybrid is a cell, a viral derivative, a cellular organelle, a subcellular structure, a vesicle, or a therapeutic molecule. In one aspect, the capture target hybrid is a cell, such as a bacterial cell, an archaeal cell, a mammalian cell, an insect cell, or a plant cell. In one aspect, the capture target hybrid is a cell engineered to produce a surface protein analyte or other desired target analyte. In one aspect, the capture target hybrid is a cellular organelle, such as a nucleus, an endoplasmic reticulum, a Golgi apparatus, a mitochondria, a vacuole, a chloroplast, an inflammasome, a cellular translation complex, and combinations thereof. In one aspect, the capture target hybrid is a viral derivative, such as a virus, a viral particle, a virion, a virion membrane, a virion membrane fragment, and combinations thereof. In one aspect, the capture target hybrid is a subcellular structure, such as a micelle, a membrane preparation, a membrane raft, a membrane ghost, a membrane vesicle, a membrane fragment, an artificial lipid membrane, or combinations thereof. In one aspect, the capture target hybrid is a vesicle, such as a lysosome, endosome, peroxisome, liposome, and combinations thereof.

[0041] As used herein, "viral derivatives" refers to particles or fragments obtained or derived from viruses. Viral derivatives can be formed by degradation via molecular mechanisms including mechanical manipulation, intracellular factors, and receptor-induced structural remodeling, low pH-activated conformational changes, protease-dependent proteolysis, reductase-catalyzed disulfide bond disruption, and chaperone-mediated unfolding / refolding reactions that viruses use to facilitate their degradation. In one aspect, viral derivatives include viruses, viral particles, virions, virion membranes, and virion membrane fragments.

[0042] "Subcellular structure" refers to a compartment or structure located in or present within a whole cell. In one aspect, the subcellular structure can be derived from a compartment or structure of a whole cell or can be artificially prepared by various means known in the art. The subcellular structure can be separated or removed from the cell by cell fractionation methods including extraction, homogenization and centrifugation techniques known in the art. In one aspect, the subcellular structure includes micelles, membrane preparations, membrane rafts, membrane ghosts, membrane vesicles, membrane fragments, artificial lipid membranes, or combinations thereof.

[0043] As used herein, the term "vesicle" refers to an intracellular or extracellular structure consisting of liquid or cytoplasm surrounded by a lipid bilayer. Vesicles can be separated or removed from cells by cell fractionation methods, including extraction, homogenization, and centrifugation techniques known in the art. In one aspect, the vesicles are artificial or synthetic vesicles prepared in vitro using biochemical and microfluidic synthesis techniques known in the art. In one aspect, the vesicles include lysosomes, endosomes, peroxisomes, liposomes, and combinations thereof.

[0044] overview Methods for immobilizing reagents on a surface are provided herein. In one aspect, the surface is an assay surface used to perform an assay for detecting and / or quantifying one or more analytes of interest in a sample. In one aspect, one or more reagents are immobilized on the assay surface. In one aspect, the surface is a bifunctional or multifunctional assay surface, where two or more reagents differing in structure and / or function are immobilized using different surface chemistries. In one aspect, the surface is a bifunctional or multifunctional assay surface, where a primary reagent and a secondary reagent are immobilized. In one aspect, the primary reagent and the secondary reagent have different structures and / or functions. For example, in one aspect, the primary reagent is a proteinaceous reagent and the secondary reagent is a thiol-containing reagent. In one aspect, the primary reagent is a proteinaceous reagent and the secondary reagent is a thiolated oligonucleotide. In one aspect, the primary reagent and the secondary reagent are immobilized using different surface chemistries. In one aspect, the assay surface is a multiwell plate. In one aspect, the assay surface comprises particles or beads.

[0045] Biomolecules can be attached to a surface by several mechanisms. Passive adsorption refers to immobilization via hydrophobic interactions or hydrophobic and ionic interactions between the biomolecule and the surface. In one aspect, one or more reagents are immobilized on the assay surface by passive adsorption. In one aspect, multiple reagents are immobilized on the assay surface by passive adsorption. In one aspect, one or more primary reagents are immobilized on the assay surface by passive adsorption. In one aspect, multiple primary reagents are immobilized on the assay surface by passive adsorption. In one aspect, one or more proteinaceous primary reagents or capture target hybrids are immobilized on the assay surface by passive adsorption. In one aspect, multiple proteinaceous primary reagents or capture target hybrids are immobilized on the assay surface by passive adsorption. In one aspect, the proteinaceous primary reagent is a capture molecule. In one aspect, the proteinaceous primary reagent comprises bovine serum albumin (BSA), e.g., the proteinaceous primary reagent may comprise an oligonucleotide conjugated to BSA. In one aspect, a member of a binding pair is immobilized on the assay surface by passive adsorption. In one embodiment, streptavidin is immobilized on the assay surface by passive adsorption, hi one embodiment, passive adsorption is enhanced by using a slightly ionic hydrophobic surface.

[0046] Covalent immobilization refers to immobilization by the formation of one or more covalent bonds between one or more reactive functional groups on the reagent and one or more reactive functional groups on the assay surface. The term "reactive functional group" refers to an atom or associated group of atoms that can undergo further chemical reaction, for example, to form a covalent bond with another functional group. Examples of reactive functional groups include, but are not limited to, amino, thiol, hydroxy, and carbonyl groups. In one aspect, the reactive functional group comprises a thiol group. In one aspect, the reagent is covalently immobilized on the assay surface via a covalent bond formed between a thiol moiety (-SH) on the reagent and a reactive functional group on the assay surface. In one aspect, the oligonucleotide is covalently immobilized on the assay surface via a covalent bond formed between a thiol moiety on the oligonucleotide and a reactive functional group on the assay surface.

[0047] In some instances, it may be desirable to prepare bifunctional or multifunctional assay surfaces on which two or more reagents of differing structure and / or function are immobilized. For example, PCT Publication No. 2014 / 165061, filed March 12, 2014 and entitled IMPROVED ASSAY METHODS, the disclosure of which is incorporated herein by reference in its entirety, describes a method for amplifying signals in immunoassays using a bifunctional assay surface on which a primary capture reagent and a secondary anchor reagent are immobilized. In one aspect, the primary capture reagent is a proteinaceous primary reagent, such as an antibody, an antigen-binding antibody fragment, or a receptor, and the secondary anchor reagent is an oligonucleotide. In one aspect, the primary capture reagent is a capture target hybrid, such as a cell, a virus, an organelle, a subcellular structure, a vesicle, or a therapeutic molecule, and the secondary anchor reagent is an oligonucleotide. In one aspect, the primary capture reagent and the secondary anchor reagent are dispensed onto the assay surface simultaneously and immobilized using the same surface chemistry for both reagents. For example, the secondary oligonucleotide reagent can be conjugated to a protein such as bovine serum albumin (BSA) such that both the proteinaceous primary reagent and the secondary oligonucleotide anchor reagent are immobilized by passive adsorption. Alternatively, the secondary oligonucleotide anchor reagent and the primary capture reagent can be conjugated to a hapten such as biotin and immobilized on a streptavidin-coated surface. The bifunctional assay surfaces described in PCT Publication No. 2014 / 165061 provide improved assay sensitivity, but the assay modules must be specially prepared for a particular application, which can increase manufacturing costs.

[0048] Provided herein is a method for preparing a bifunctional assay surface in which a primary reagent and a secondary reagent are immobilized on the assay surface. In one aspect, the primary reagent and the secondary reagent are immobilized on the assay surface using different surface chemistries. In one aspect, the primary reagent is immobilized on the assay surface by passive adsorption and the secondary reagent is immobilized by the formation of a covalent bond between a reactive functional group on the secondary reagent and a reactive functional group on the assay surface. In one aspect, the primary reagent is immobilized on the assay surface by a binding pair interaction. In one aspect, the primary reagent includes a biotin moiety and is immobilized on a streptavidin-coated surface via the interaction of a streptavidin / biotin binding pair. In one aspect, the primary reagent is a proteinaceous molecule, such as an antibody, an antigen-binding antibody fragment, or a receptor, and is immobilized on the assay surface by passive adsorption.

[0049] In one aspect, a "direct" coating method is provided in which the primary and secondary reagents are immobilized simultaneously on the assay surface using different surface chemistries. In another aspect, a "direct" coating method is provided in which proteinaceous streptavidin and thiol-modified anchor oligonucleotides are immobilized on the plate surface. Advantageously, the secondary reagent does not need to be modified to be immobilized using the same surface chemistry as the primary reagent. For example, the proteinaceous primary reagent or capture target hybrid can be immobilized simultaneously with the oligonucleotide secondary reagent, for example, a thiolated oligonucleotide secondary reagent, by passive adsorption or via a binding pair such as avidin / streptavidin and biotin, without the need to conjugate the oligonucleotide secondary reagent to a protein such as bovine serum albumin (BSA) or a member of a binding pair such as biotin.

[0050] In another aspect, an "overcoating" method is provided by which a secondary reagent can be immobilized onto an assay surface on which a primary reagent has already been immobilized. Advantageously, a secondary reagent can be immobilized onto an existing assay surface on which a primary reagent has previously been immobilized.

[0051] As shown herein, assay surfaces prepared by the overcoating and direct coating methods have comparable or superior performance compared to bifunctional surfaces in which different reagents are immobilized using the same surface chemistry.

[0052] Assay Module In one embodiment, a method for preparing a bifunctional surface is provided. In one embodiment, a method for preparing a bifunctional surface in an assay module is provided. In one embodiment, the assay module includes a plurality of surfaces, including, for example, particles or beads. In one embodiment, the assay module includes a unitary surface, such as a cartridge or plate. In one embodiment, the assay module includes one or more assay cells, such as a well, a compartment, a chamber, a conduit, or a flow cell. In one embodiment, the assay module is a multi-well assay plate. Multi-well assay plates are available in a variety of formats, sizes, and shapes with standard dimensions used for high throughput assays. In one embodiment, the assay module is a multi-well plate with a standard well configuration, such as a 6-well, 24-well, 96-well, 384-well, 1536-well, 6144-well, or 9600-well plate. In one embodiment, the assay module is a 96-well plate.

[0053] In one embodiment, the assay surface comprises a two-dimensional patterned array in which one or more reagents can be printed at known locations called binding domains. In one embodiment, the assay surface comprises a patterned array of discrete, non-overlapping, addressable binding domains on which multiple reagents are immobilized, and the identity of the reagent in each binding domain is known and can be correlated with a target. In one embodiment, at least two of the binding domains comprise reagents with different binding specificities from each other. In one embodiment, the array is arranged in a symmetric grid pattern. In other embodiments, the array is arranged in another pattern, including but not limited to radially distributed lines, spiral lines, or ordered clusters. In another embodiment, each binding domain is positioned on the surface of one or more microparticles or beads, and the microparticles or beads are coded to allow discrimination between different binding domains.

[0054] In one aspect, the primary reagents are immobilized on the assay surface in an array. In one aspect, the proteinaceous primary reagents or the capture target hybrids are immobilized on the assay surface in an array. In one aspect, the proteinaceous streptavidin and the thiol-modified anchor oligonucleotides are immobilized on the assay surface in an array. In one aspect, the primary reagents are immobilized on a carbon-containing assay surface in an array. In one aspect, the proteinaceous streptavidin and the thiol-modified anchor oligonucleotides are immobilized on a carbon-containing assay surface in an array. In one aspect, the primary reagents are immobilized on a carbon-containing electrode in an array. In one aspect, the proteinaceous streptavidin and the thiol-modified anchor oligonucleotides are immobilized on a carbon-containing electrode in an array. In one aspect, the secondary reagents are immobilized on an array. In one aspect, the assay surface is coated with the secondary reagent.

[0055] In one embodiment, the assay surface comprises one or more binding domains. In one embodiment, the assay surface comprises a plurality of binding domains. In one embodiment, the assay module is a multi-well plate, and one or more wells of the multi-well plate comprise one or more binding domains. In one embodiment, each well of the multi-well plate comprises a plurality of binding domains. In one embodiment, each well comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 binding domains and up to 25, 64, 100, or 250 binding domains. In one embodiment, each well comprises 4, 7, 10, 25, 64, or 100 binding domains. In one embodiment, at least two of the binding domains comprise two different reagents.

[0056] In one embodiment, the assay module comprises distinct binding domains on one or more solid surfaces. In one embodiment, the assay module comprises one or more particles or beads, the one or more particles or beads comprising one or more binding domains. In one embodiment, each binding domain is on a distinct surface, e.g., on the surface of a distinct bead. In one embodiment, the assay module comprises one or more particles or beads having one or more primary and one or more secondary reagents immobilized thereon. In one embodiment, the assay module comprises one or more particles or beads having one or more proteinaceous primary reagents and one or more thiol-containing secondary reagents immobilized thereon. In one embodiment, the assay module comprises one or more particles or beads having one or more capture-target hybrids and one or more thiol-containing secondary reagents immobilized thereon. In one embodiment, the binding domains are individual beads, such that a distinct assay signal is generated on and measured from each binding domain. In one embodiment, the assay module comprises one or more coded particles or beads having one or more primary capture reagents and one or more secondary reagents immobilized thereon, the coding identifying the capture reagent and target for a particular bead.

[0057] In one embodiment, one or more binding domains on the assay surface comprise one or more assay reagents. In one embodiment, each binding domain on the assay surface comprises one or more assay reagents. In one embodiment, one or more binding domains comprise a primary reagent. In one embodiment, all of the primary reagents in a particular binding domain have the same binding specificity, and the primary reagent in one binding domain has a different binding specificity than the primary reagent in another binding domain. In one embodiment, one or more binding domains comprise a secondary reagent. In one embodiment, all of the secondary reagents in a particular binding domain have the same binding specificity, and the secondary reagent in one binding domain has a different binding specificity than the secondary reagent in another binding domain. In one embodiment, the secondary reagents in two or more binding domains have the same binding specificity.

[0058] In one embodiment, a primary reagent is immobilized to a first binding domain and a secondary reagent is immobilized to a second binding domain. In one embodiment, a plurality of primary reagents with different binding specificities are immobilized to a plurality of first binding domains and a plurality of secondary reagents with different binding specificities are immobilized to a plurality of second binding domains.

[0059] In one embodiment, one binding domain in the array may contain a different primary reagent than another binding domain, such that some or all of the binding domains in the array contain a "unique" primary reagent. In one embodiment, one binding domain in the array may contain a different secondary reagent than another binding domain, such that some or all of the binding domains in the array contain a "unique" secondary reagent. In one embodiment, each binding domain in the array may contain the same secondary reagent, such that some or all of the binding domains in the array contain a "common secondary reagent". In one embodiment, one or more binding domains contain a primary reagent and a secondary reagent. In one embodiment, one or more binding domains contain a unique primary reagent and a unique secondary reagent. In one embodiment, one or more binding domains contain a unique primary reagent and a common secondary reagent.

[0060] In one embodiment, one or more binding domains of the assay surface comprise a proteinaceous primary reagent or a capture target hybrid. In one embodiment, one or more binding domains of the assay surface comprise a thiol-containing secondary reagent. In one embodiment, a proteinaceous primary reagent or a capture target hybrid is immobilized to a first binding domain that does not comprise a thiol-containing secondary reagent, and a thiol-containing secondary reagent is immobilized to a second binding domain that does not comprise a primary reagent. In one embodiment, each binding domain comprises a proteinaceous primary reagent or a capture target hybrid. In one embodiment, each binding domain comprises a thiol-containing secondary reagent. In one embodiment, one or more binding domains on the assay surface comprise a proteinaceous primary reagent and a thiol-containing secondary reagent. In one embodiment, one or more binding domains on the assay surface comprise a capture target hybrid and a thiol-containing secondary reagent. In one embodiment, each binding domain on the assay surface comprises a proteinaceous primary reagent and a thiol-containing secondary reagent. In one embodiment, each binding domain on the assay surface comprises a capture target hybrid and a thiol-containing secondary reagent. In one embodiment, each binding domain on the assay surface comprises a proteinaceous primary reagent and a thiol-containing secondary reagent. In one embodiment, each binding domain on the assay surface comprises a capture target hybrid and a thiol-containing secondary reagent. In one aspect, each binding domain comprises a unique proteinaceous primary reagent and a unique thiol-containing secondary reagent. In one aspect, each binding domain comprises a unique capture target hybrid and a unique thiol-containing secondary reagent. In one aspect, each binding domain comprises a unique proteinaceous primary reagent and a common thiol-containing secondary reagent. In one aspect, each binding domain comprises a unique capture target hybrid and a common thiol-containing secondary reagent.

[0061] In one embodiment, the assay surface comprises a multi-well plate, and the plurality of primary reagents are immobilized to one or more binding domains on the surface of the multi-well plate. In one embodiment, the one or more binding domains on the surface of the wells of the multi-well plate comprise one or more copies of a proteinaceous primary reagent or a capture target hybrid. In one embodiment, all of the proteinaceous primary reagents immobilized to a particular binding domain have the same binding specificity, and a proteinaceous primary reagent immobilized to one binding domain has a different binding specificity than a proteinaceous primary reagent immobilized to another binding domain. In one embodiment, all of the capture target hybrids immobilized to a particular binding domain have the same binding specificity, and a capture target hybrid immobilized to one binding domain has a different binding specificity than a capture target hybrid immobilized to another binding domain. In one embodiment, the assay surface comprises a multi-well plate, and the one or more thiol-containing reagents are immobilized to one or more binding domains on the surface of the multi-well plate. In one embodiment, each binding domain comprises one or more copies of a thiol-containing secondary reagent. In one embodiment, a proteinaceous primary reagent or capture target hybrid is immobilized to a first binding domain and a thiol-containing secondary reagent is immobilized to a second binding domain. In one embodiment, each binding domain on the surface of a well of a multiwell plate comprises a primary reagent and a secondary reagent. In one embodiment, each binding domain comprises one or more copies of a unique thiol-containing secondary reagent. It should be noted that a "unique" thiol-containing secondary reagent in one well of a multiwell plate may be contained in other wells of the multiwell plate. In one embodiment, all binding domains in a well of a multiwell plate comprise one or more copies of a common thiol-containing secondary reagent. It should be noted that other wells of a multiwell plate may comprise the same thiol-containing secondary reagent or different thiol-containing secondary reagent. In one embodiment, each binding domain comprises a unique proteinaceous primary reagent or capture target hybrid and a unique thiol-containing secondary reagent. In one embodiment, each binding domain comprises a unique proteinaceous primary reagent or capture target hybrid and a unique thiol-containing secondary reagent. In one embodiment, each binding domain comprises a unique proteinaceous primary reagent or capture target hybrid and a common thiol-containing secondary reagent.In one aspect, each binding domain comprises a unique capture antibody or antigen-binding antibody fragment and a unique thiolated oligonucleotide.In one aspect, each binding domain comprises a unique capture antibody or antigen-binding antibody fragment and a common thiolated oligonucleotide.

[0062] In one aspect, the proteinaceous streptavidin and the thiol-modified anchor oligonucleotide are immobilized on an assay surface in an array. In one aspect, the proteinaceous streptavidin is immobilized by passive adsorption of streptavidin to the plate surface, and the thiol-modified anchor oligonucleotide is immobilized by covalent attachment of thiol groups on the plate surface. In one aspect, the proteinaceous streptavidin and the thiol-modified anchor oligonucleotide are immobilized on a carbon-containing assay surface in an array. In one aspect, the proteinaceous streptavidin and the thiol-modified anchor oligonucleotide are immobilized on a carbon-containing electrode in an array.

[0063] In one aspect, the proteinaceous primary reagents or the capture target hybrids are immobilized on an assay surface in an array. In one aspect, the proteinaceous primary reagents or the capture target hybrids are immobilized on a carbon-containing assay surface in an array. In one aspect, the proteinaceous primary reagents or the capture target hybrids are immobilized on a carbon-containing electrode in an array.

[0064] In one aspect, the proteinaceous primary reagent and the thiol-containing secondary reagent are immobilized on an assay surface in an array. In one aspect, the proteinaceous primary reagent and the thiol-containing secondary reagent are immobilized on a carbon-containing assay surface in an array. In one aspect, the proteinaceous primary reagent and the thiol-containing secondary reagent are immobilized on a carbon-containing electrode in an array. In one aspect, the capture target hybrid and the thiol-containing secondary reagent are immobilized on an assay surface in an array. In one aspect, the capture target hybrid and the thiol-containing secondary reagent are immobilized on a carbon-containing assay surface in an array. In one aspect, the capture target hybrid and the thiol-containing secondary reagent are immobilized on a carbon-containing electrode in an array.

[0065] In one aspect, the primary reagent is immobilized on the assay surface via a different surface chemistry than the secondary reagent. In one aspect, the primary reagent is a proteinaceous primary reagent. In one aspect, the primary reagent is a proteinaceous capture reagent. In one aspect, the primary reagent is a capture target hybrid. In one aspect, the proteinaceous primary reagent or the capture target hybrid is immobilized on the assay surface by passive adsorption. In one aspect, the proteinaceous primary reagent or the capture target hybrid is immobilized on the assay surface via a binding partner such as streptavidin or avidin and biotin. In one aspect, the proteinaceous primary reagent or the capture target hybrid contains a biotin moiety and the assay surface is coated with streptavidin. In one aspect, the secondary reagent is a thiol-containing secondary reagent. In one aspect, the proteinaceous primary reagent or the capture target hybrid is immobilized on the assay surface via a different surface chemistry than the thiol-containing secondary reagent. In one aspect, the secondary reagent is immobilized on the assay surface through the interaction of a thiol group with a reactive functional group on the assay surface. In one aspect, the secondary reagent is immobilized on the assay surface by a covalent bond between a thiol group and a reactive functional group on the assay surface. In one aspect, the thiol-containing secondary reagent is immobilized on the assay surface through the formation of a disulfide bond or a maleimide bond. In one aspect, one or more thiol-containing secondary reagents are immobilized on a carbon-containing assay surface. In one aspect, the assay surface is modified with a thiol-reactive moiety, such as a maleimide, an iodosuccinimide, or an activated disulfide (e.g., a pyridyl disulfide).

[0066] In one embodiment, the proteinaceous primary reagent is a proteinaceous capture reagent. As used herein, a "capture reagent" is a reagent immobilized on an assay surface that binds to a target that may be present in a sample. In one embodiment, the capture reagent specifically binds to a target. In one embodiment, the primary capture reagent specifically binds to a primary target. In one embodiment, the proteinaceous primary reagent comprises an antigen-binding agent. In one embodiment, the proteinaceous primary reagent comprises an antibody or an antigen-binding antibody fragment. In one embodiment, the proteinaceous primary reagent is an enzyme. In one embodiment, the proteinaceous capture reagent is a receptor. In one embodiment, the proteinaceous capture reagent comprises a proteinaceous moiety and a member of a binding pair, where a first member of the binding pair is attached to the proteinaceous primary reagent and the other member of the binding pair is attached to the target. Non-limiting examples of binding pairs include biotin and streptavidin or avidin; complementary oligonucleotides; haptens and hapten-binding partners; receptor / ligand; enzyme / substrate and antibody / antigen binding pairs. In one embodiment, the proteinaceous primary reagent comprises a proteinaceous moiety, and the binding pair is selected from streptavidin or avidin and biotin. In one embodiment, the primary reagent comprises a target binding moiety and a proteinaceous moiety. In one embodiment, the proteinaceous moiety of the primary reagent comprises bovine serum albumin (BSA). In one embodiment, the target binding moiety of the primary reagent comprises a capture molecule. In one embodiment, the capture molecule is a proteinaceous molecule, such as a peptide or protein, including, for example, an antibody, an antigen-binding antibody fragment, or an antigen; a receptor or a ligand; or an enzyme or a substrate. In another embodiment, the capture molecule comprises an oligonucleotide or a nucleic acid that hybridizes to a complementary oligonucleotide sequence of the target under stringent hybridization conditions. In one embodiment, the capture molecule comprises a vitamin, an oligosaccharide, a carbohydrate, a lipid, a small molecule, or a complex thereof.

[0067] In one aspect, the reactive functional group is a thiol group. As used herein, "thiol-containing secondary reagent" refers to a reagent that contains a sulfhydryl moiety (-SH). In one aspect, the thiol-containing secondary reagent is covalently immobilized on a surface via a reactive functional group. In one aspect, the thiol-containing secondary reagent is an oligonucleotide, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, a mimotope, or a combination or complex thereof. In one aspect, the thiol-containing secondary reagent comprises a thiol-modified single- or double-stranded oligonucleotide, such as DNA or RNA. In one aspect, the thiol-containing secondary reagent comprises a DNA-binding protein.

[0068] In one aspect, the thiol-containing secondary reagent comprises a target binding moiety and a thiolated moiety, and the target binding moiety of the secondary reagent is immobilized on the assay surface via the thiolated moiety. In one aspect, the thiol-containing secondary reagent comprises a target binding moiety and a thiolated oligonucleotide, and the target binding moiety of the secondary reagent is immobilized on the assay surface via the thiolated oligonucleotide. In one aspect, the target binding moiety of the secondary reagent comprises a capture molecule. In one aspect, the capture molecule is a peptide or protein, including, for example, an antibody, an antigen-binding antibody fragment, or a receptor. In another aspect, the capture molecule is an oligonucleotide or a nucleic acid that hybridizes to a complementary oligonucleotide sequence of the target under stringent hybridization conditions. In one aspect, the capture molecule comprises a vitamin, an oligosaccharide, a carbohydrate, a lipid, a small molecule, or a complex thereof.

[0069] In one embodiment, the secondary reagent comprises an oligonucleotide moiety that hybridizes to a complementary oligonucleotide under stringent conditions. In one embodiment, the complementary oligonucleotide is the target analyte. In one embodiment, the complementary oligonucleotide is a targeting oligonucleotide that associates with the target analyte, for example, by covalent or non-covalent interactions, such that the target analyte can be immobilized on the assay surface via the interaction between the thiolated oligonucleotide and the targeting oligonucleotide. In one embodiment, the target is a complementary oligonucleotide that is an amplification product or amplicon. In one embodiment, the complementary oligonucleotide is an amplification product generated by amplification techniques, including, but not limited to, PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (sustained synthesis reaction), and isothermal amplification methods, such as helicase-dependent amplification and rolling circle amplification (RCA).

[0070] In one embodiment, the thiolated oligonucleotide has a length of about 5, 6, 7, 8, 9, or 10 nucleotides up to about 20, 30, 40, 50, 75, or 100 nucleotides, or about 5 to about 100, about 5 to about 50, or about 10 to about 30 nucleotides, or about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, or 100 nucleotides. In one embodiment, the thiolated oligonucleotide comprises an oligonucleotide attached to a thiol group via a linker. In one embodiment, the linker comprises about 3 to about 20 atoms or molecules or units, or at least about 3, 4, 5, 6, 7, 8, 9, 10 up to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms or molecules or units. In one embodiment, the thiolated oligonucleotide comprises an oligonucleotide sequence having a 5' and a 3' end, and a thiol group incorporated at the 5' end (5' terminal thiolated oligonucleotide), the 3' end (3' terminal thiolated oligonucleotide), an internal position of the oligonucleotide, or a combination thereof. In one embodiment, the thiolated oligonucleotide comprises a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a combination thereof. In one embodiment, the thiolated oligonucleotide comprises one or more non-natural nucleotide bases. In one embodiment, the non-natural nucleotide bases are selected from 2,6-diaminopurine (2-amino-dA); 5-methyldeoxycytidine, Super T (5-hydroxybutynyl-2'-deoxyuridine); or a combination thereof.

[0071] In one embodiment, the thiol-containing secondary reagent comprises a protein. In one embodiment, the thiol-containing secondary reagent comprises a protein, such as bovine serum albumin (BSA). In one embodiment, the thiol-containing secondary reagent comprises a protein, such as BSA, and is immobilized on an assay surface to reduce non-specific binding (NSB). Sulfhydryl groups, present in many proteins, can be generated by reduction of native disulfide bonds or can be introduced by reacting primary amines with sulfhydryl-adding reagents, such as 2-iminothiolane (Traut's Reagent), SATA, SATP, SAT(PEG)4, or combinations thereof.

[0072] In one aspect, the thiol-containing secondary reagent comprises a thiolated member of a binding pair. In one aspect, the thiol-containing secondary reagent comprises a thiolated biotin (thiol-biotin). In one aspect, the thiol-containing secondary reagent comprises a thiolated polyethylene glycol (thiol-PEG). In one aspect, the thiolated polyethylene glycol further comprises biotin (thiol-PEG-biotin). In one aspect, the thiolated biotin is immobilized on the assay surface such that the biotin moiety can be used to immobilize additional reagents on the assay surface. In one aspect, streptavidin or avidin is conjugated to one or more assay reagents and then immobilized on the assay surface via the immobilized biotin moiety.

[0073] In one aspect, the thiol-containing reagent comprises one or more reactive groups. In one aspect, the thiol-containing secondary reagent comprises an amine-reactive moiety. In one aspect, the thiol-containing secondary reagent comprises fluorescein (FITC). In one aspect, the thiol-containing secondary reagent comprises fluorescein and a thiol heterofunctionalized polyethylene glycol (FITC-PEG-SH).

[0074] In one embodiment, the assay module includes one or more electrodes. In one embodiment, the assay surface includes an electrode surface. In one embodiment, the electrode surface is a component of a multi-well plate. In one embodiment, the electrode surface is a component of a particle or bead. In one embodiment, the electrode is formed from a conductive material, including but not limited to metals such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, or conductive alloys. In one embodiment, the electrode includes a metal coated with an oxide, including but not limited to aluminum coated with aluminum oxide. In one embodiment, the electrode is constructed from a carbon-based material, such as carbon, carbon black, graphitic carbon, carbon nanotubes, carbon fibrils, graphite, graphene, carbon fiber, or mixtures thereof. In one embodiment, the electrode is formed from elemental carbon, such as graphite, carbon black, or carbon nanotubes. In one embodiment, the electrode includes conductive particles dispersed in a matrix, such as conductive carbon-polymer composites, carbon inks, carbon pastes, metal inks, and graphene inks, and / or conductive polymers. In one embodiment, the assay module is a multi-well plate having one or more carbon-containing electrodes, eg, one or more electrodes comprise a carbon layer and / or a screen-printed layer of carbon ink.

[0075] In one embodiment, the assay module is a multi-well plate. In one embodiment, the assay module is a multi-well plate suitable for electrode-induced luminescence-based assays. In one embodiment, the assay surface is located within one or more wells of the multi-well plate. In one embodiment, the assay module comprises a plurality of wells and one or more electrodes. In one embodiment, the assay module comprises one or more working electrodes and one or more counter electrodes. In one embodiment, one or more wells of the assay module comprise one or more electrodes. In one embodiment, each well of the multi-well plate comprises at least one working electrode and at least one counter electrode. In one embodiment, the one or more electrodes comprise one or more or multiple binding domains. In one embodiment, the method comprises detecting at least one electrochemiluminescent moiety to detect and / or quantify a target analyte in a sample. In one embodiment, the electrochemiluminescent moiety is an electrochemiluminescent label. In one embodiment, the assay module is used to detect and / or quantify multiple analytes in parallel. In one embodiment, the assay module is used in a simultaneous multiplex assay. Multiplexed measurement of analytes on surfaces containing multiple binding domains using electrochemiluminescence is known, see, e.g., Meso Scale Diagnostics, LLC, MULTI-ARRAY® and SECTOR® Imager product lines, and U.S. Patent Nos. 7,842,246 and 6,977,722, the disclosures of which are incorporated herein by reference in their entireties.

[0076] target Targets include, but are not limited to, proteins, toxins, nucleic acids, amplification products or amplicons, microorganisms, viruses, cells, fungi, spores, carbohydrates, lipids, glycoproteins, lipoproteins, liposomes, exosomes, polysaccharides, drugs, hormones, steroids, nutrients, metabolites, and any modified derivatives of the above molecules, or any complexes containing one or more of the above molecules, or combinations thereof. In one embodiment, the target is an analyte of interest in a sample that is indicative of a disease or disease state. In one embodiment, the target is an analyte of interest in a sample that is indicative of whether a patient has been exposed to the analyte. In one embodiment, the target is retained on an assay surface to facilitate detection of a different target that is an analyte of interest.

[0077] In one aspect, the primary reagent specifically binds to a target. In one aspect, the primary reagent specifically binds to a target analyte. In one aspect, the secondary reagent specifically binds to a target. In one aspect, the secondary reagent specifically binds to a target analyte. In one aspect, the primary and secondary reagents bind different targets. In one aspect, the primary reagent binds to a target to facilitate detection of a target analyte bound by the secondary reagent. In one aspect, the secondary reagent binds a target to facilitate detection of a target analyte bound by the primary reagent.

[0078] In one aspect, the primary reagent is a proteinaceous capture reagent, such as an antibody or an antigen-binding antibody fragment, and the thiol-containing secondary reagent comprises a thiolated anchor oligonucleotide. In one aspect, the primary reagent is a capture target hybrid, such as a cell, a subcellular structure, a virus derivative, a vesicle, or a therapeutic molecule, and the thiol-containing secondary reagent comprises a thiolated anchor oligonucleotide. In one aspect, the capture antibody specifically binds to a target analyte. In one aspect, the capture target hybrid comprises a target molecule or a target analyte. In one aspect, the anchor oligonucleotide reagent hybridizes to a target comprising an oligonucleotide. In one aspect, the anchor oligonucleotide reagent hybridizes to a target comprising an oligonucleotide amplification product. In one aspect, the oligonucleotide amplification product is generated by an amplification technique, including, but not limited to, PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (sustained synthesis reaction), and isothermal amplification methods, such as helicase-dependent amplification and rolling circle amplification (RCA). In one aspect, the amplification product comprises an RCA amplicon. See, e.g., PCT Publication No. 2014 / 165061, filed March 12, 2014, and entitled IMPROVED ASSAY METHODS, the disclosure of which is incorporated by reference in its entirety herein.

[0079] In one aspect, the primary reagent binds to a target associated with an extracellular vesicle (EV). In one aspect, the primary reagent binds to a surface protein of the extracellular vesicle. In one aspect, the primary reagent binds to an exosome. In one aspect, the exosome contains signaling molecules, including but not limited to surface-associated or cytoplasmic proteins, lipids, mRNA, miRNA, or combinations thereof. In one aspect, the identity and concentration of signaling molecules within an exosome is used to infer its cellular origin and function. See, for example, International Application No. PCT / US2020 / 20288, filed February 28, 2020, and entitled IMMUNOASSAY METHODS, the disclosure of which is incorporated herein by reference in its entirety.

[0080] In one embodiment, the primary reagent specifically binds to a primary target and the secondary reagent specifically binds to a secondary target, e.g., a target analyte associated with or encapsulated by the primary target. In one embodiment, the primary reagent specifically binds to a target comprising a surface protein of an extracellular vesicle (EV). In one embodiment, the primary reagent is a proteinaceous primary reagent that binds to a surface protein of an extracellular vesicle (EV). In one embodiment, the extracellular vesicle is an exosome. In one embodiment, the target analyte is an EV-associated protein. In one embodiment, the target analyte is encapsulated by an EV. In one embodiment, the secondary reagent binds to a target analyte that is an EV-associated protein. In one embodiment, the secondary reagent binds to a target analyte encapsulated by an EV.

[0081] Capture Target Hybrid In various embodiments, the capture target hybrid is a biological part, such as a cell, a virus derivative, an organelle, a subcellular structure, a vesicle, a therapeutic molecule, or a combination thereof, and the target and the capture are integrated and form a capture target hybrid together. An exemplary capture target hybrid is a whole cell, whereby a part of the cell is bound to the plate and the target is on the surface of the cell, such as a protein. In one aspect, the capture target hybrid is biotinylated and bound to a streptavidin plate. In one aspect, the capture target hybrid is coated with a thiol-containing compound and bound to a streptavidin plate. In one aspect, the capture target is uncoated and in its natural state or context and bound to a streptavidin plate. In one aspect, the capture target hybrid is immobilized with an anchor oligonucleotide. Non-limiting examples of anchor oligonucleotides include BSA-conjugated anchor oligonucleotides (BSA-oligo) or thiol-modified anchor oligonucleotides (thiol-oligo). In one aspect, the capture target hybrid and the anchor oligonucleotide are immobilized sequentially. Non-limiting examples of anchor oligonucleotides include BSA-conjugated anchor oligonucleotides (BSA-oligos) or thiol-modified anchor oligonucleotides (thiol-oligos).

[0082] In one aspect, the capture target hybrid immobilized on a carbon-containing assay surface is coated with a binding reagent that preferentially binds to a binding partner on the assay surface, hi one aspect, the capture target hybrid is immobilized on a carbon-containing assay surface coated with streptavidin, and the surface is coated with a solution containing biotinylated anchor oligonucleotides.

[0083] In one aspect, the use of a capture-target whole hybrid containing a target analyte on its surface limits off-target recognition of additional proteins released into the solution of lysed cells, hi one aspect, the native conformation of the target analyte on the surface of the capture-target whole hybrid is maintained.

[0084] Direct Coating Method In one aspect, a direct coating method is provided for preparing a bifunctional assay surface comprising a primary reagent and a secondary reagent. In one aspect, a direct coating method is provided for preparing a bifunctional assay surface comprising a proteinaceous primary reagent and a thiol-containing secondary reagent. In one aspect, a direct coating method is provided for preparing a bifunctional assay surface comprising a capture target hybrid and a thiol-containing secondary reagent. As used herein, "direct coating" means that either a proteinaceous primary reagent or a capture target hybrid and a thiol-containing secondary reagent are immobilized on the assay surface at the same time. In one aspect, either a proteinaceous primary reagent or a capture target hybrid and a thiol-containing secondary reagent are dispensed onto the assay surface in a coating solution. In one aspect, either a proteinaceous primary reagent or a capture target hybrid and a thiol-containing secondary reagent are dispensed onto the assay surface in the same coating solution. In one aspect, the coating solution comprises both a proteinaceous primary reagent and a thiol-containing secondary reagent. In one aspect, the coating solution comprises both a capture target hybrid and a thiol-containing secondary reagent. In one aspect, either the proteinaceous primary reagent or the capture target hybrid and the thiol-containing secondary reagent are printed onto separate binding domains on the assay surface. In one aspect, a liquid handling system is used to print the proteinaceous primary reagent or the capture target hybrid and the thiol-containing secondary reagent onto the assay surface. In one aspect, the reagents are printed onto the assay surface using a non-contact dispenser, including, for example, an inkjet printer or a piezoelectric printer. In one aspect, the reagents are printed onto the assay surface using a contact printer, for example, using pins, capillaries, or ink stamps.

[0085] In one aspect, the primary reagent and the thiol-containing secondary reagent are immobilized on the assay surface via different surface chemistries. In one aspect, the primary reagent is non-covalently immobilized on the assay surface. In one aspect, the primary reagent is non-covalently immobilized on the assay surface by passive adsorption. In one aspect, the primary reagent is covalently immobilized on the carbon-containing assay surface. In one aspect, the primary reagent is immobilized on the carbon-containing assay surface via a binding pair. In one aspect, the primary reagent comprises a first member of a binding pair and the assay surface comprises a second member of the binding pair. In one aspect, the primary reagent comprises biotin and the assay surface is coated with avidin or streptavidin.

[0086] In one embodiment, the thiol-containing secondary reagent is immobilized on the assay surface by the formation of a bond between the thiol group and a reactive functional group on the assay surface. In one embodiment, the thiol-containing secondary reagent is immobilized on the carbon-containing assay surface via a reactive functional group on the assay surface. In one embodiment, the thiol-containing secondary reagent is immobilized on the assay surface via a maleimide group. In one embodiment, the assay surface is treated to introduce one or more maleimide groups prior to immobilizing the thiol-containing secondary reagent on the carbon-containing assay surface.

[0087] In one aspect, the primary reagents are printed onto the assay surface in an array. In one aspect, a plurality of primary reagents are printed into an array. In one aspect, a plurality of primary reagents are printed into separate binding domains. In one aspect, the proteinaceous primary reagents or the capture target hybrids are printed onto the assay surface in an array. In one aspect, a plurality of proteinaceous primary reagents or the capture target hybrids are printed into an array. In one aspect, a plurality of proteinaceous primary reagents or the capture target hybrids are printed into separate binding domains.

[0088] In one aspect, a plurality of secondary reagents are printed in an array. In one aspect, a plurality of secondary reagents are printed on separate binding domains. In one aspect, a plurality of thiol-containing secondary reagents are printed in an array. In one aspect, a plurality of thiol-containing secondary reagents are printed on separate binding domains.

[0089] In one aspect, a unique primary reagent and a unique thiol-containing secondary reagent are printed onto each binding domain. In one aspect, a unique primary reagent and a common thiol-containing secondary reagent are printed onto each binding domain. In one aspect, multiple primary reagents are printed onto multiple primary binding domains. In one aspect, multiple thiol-containing secondary reagents are printed onto multiple secondary binding domains.

[0090] In one aspect, the method comprises dispensing about 10 nl, 15 nl, 20 nl, 25 nl, 30 nl, 35 nl, 40 nl, 45 nl, or 50 up to about 55 nl, 60 nl, 65 nl, 70 nl, 75 nl, 80 nl, 85 nl, 90 nl, 95 nl, or 100 nl of coating solution onto the assay surface. In one aspect, the method comprises dispensing about 25 nl to about 75 nl, about 40 nl to about 60 nl, or about 30 nl to about 50 nl of coating solution onto the assay surface. In one aspect, the method comprises dispensing about 10 nl, 15 nl, 20 nl, 25 nl, 30 nl, 35 nl, 40 nl, 45 nl, or 50 up to about 55 nl, 60 nl, 65 nl, 70 nl, 75 nl, 80 nl, 85 nl, 90 nl, 95 nl, or 100 nl of coating solution onto the carbon-containing assay surface. In one aspect, the method comprises dispensing about 25 nl to about 75 nl, about 40 nl to about 60 nl, or about 30 nl to about 50 nl of coating solution onto the carbon-containing assay surface. In one aspect, the method comprises dispensing about 50 nl or 75 nl of coating solution onto the carbon-containing assay surface.

[0091] In one embodiment, the coating solution comprises from about 50 μg / ml, 75 μg / ml, 100 μg / ml, 125 μg / ml, or 150 μg / ml up to about 200 μg / ml, 250 μg / ml, 300 μg / ml, 350 μg / ml, or 400 μg / ml of the primary reagent, or from about 100 μg / ml to about 400 μg / ml of the primary reagent. In one embodiment, the coating solution comprises from about 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 75 nM, 100 nM, 200 nM, 300 nM, 400 nM, or 500 nM up to about 500 nM, 1000 nM, 1250 nM, or 1500 nM of the secondary reagent, or from about 15 nM to about 1500 nM of the secondary reagent. In one embodiment, the coating solution comprises from about 100 μg / ml to about 400 μg / ml of the primary reagent and from about 15 nM to about 1500 nM of the secondary reagent.

[0092] In one aspect, the coating solution comprises from about 50 μg / ml, 75 μg / ml, 100 μg / ml, 125 μg / ml, or 150 μg / ml up to about 200 μg / ml, 250 μg / ml, 300 μg / ml, 350 μg / ml, or 400 μg / ml of proteinaceous primary reagent or capture target hybrid, or from about 100 μg / ml to 750 μg / ml, from about 500 μg / ml to 750 μg / ml, or from about 100 μg / ml to about 400 μg / ml of proteinaceous primary reagent or capture target hybrid. In one embodiment, the coating solution comprises from about 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 75 nM, 100 nM, 200 nM, 300 nM, 400 nM, or 500 nM up to about 500 nM, 1000 nM, 1250 nM, or 1500 nM of thiol-containing secondary reagent, or from about 15 nM to about 1500 nM of thiol-containing secondary reagent. In one embodiment, the coating solution comprises from about 100 μg / ml to about 400 μg / ml of proteinaceous primary reagent or capture target hybrid and from about 15 nM to about 1500 nM of thiol-containing secondary reagent.

[0093] In one aspect, the coating solution comprises from about 50 μg / ml, 75 μg / ml, 100 μg / ml, 125 μg / ml, or 150 μg / ml up to about 200 μg / ml, 250 μg / ml, 300 μg / ml, 350 μg / ml, or 400 μg / ml of a proteinaceous capture reagent, e.g., an antibody, an antigen-binding antibody fragment, or a receptor, or from about 100 μg / ml to about 400 μg / ml of a proteinaceous capture reagent. In one embodiment, the coating solution comprises from about 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 75 nM, 100 nM, 200 nM, 300 nM, 400 nM, or 500 nM up to about 500 nM, 1000 nM, 1250 nM, or 1500 nM of thiolated oligonucleotide, or from about 15 nM to about 1500 nM of thiolated oligonucleotide. In one embodiment, the coating solution comprises from about 100 μg / ml to about 400 μg / ml of proteinaceous capture reagent and from about 15 nM to about 1500 nM of thiolated oligonucleotide.

[0094] In one embodiment, the coating solution includes a non-ionic detergent such as TRITON X-100. In one embodiment, the coating solution includes about 0.01%, 0.02%, or 0.03% up to about 0.04% or 0.05% TRITON X-100. In one embodiment, the coating solution includes about 0.01%, 0.02%, 0.03%, 0.04%, or 0.05% TRITON X-100. In one embodiment, the coating solution includes a stabilizing agent such as trehalose. In one embodiment, the coating solution includes about 0.01%, 0.02%, 0.03%, 0.04%, or 0.05% up to about 0.1% or 0.5% trehalose. In one embodiment, the coating solution comprises about 0.01% to about 0.1%, about 0.01% to about 0.05%, or about 0.1% to about 0.5% trehalose. In one embodiment, the coating solution comprises a buffer such as phosphate buffered saline. In one embodiment, the coating solution comprises a buffer such as Dulbecco's Phosphate Buffered Saline (DPBS). In one embodiment, DPBS comprises 2.67 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, and 138 mM NaCl. In one embodiment, the buffer comprises about 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM up to about 10 mM, 15 mM, or 20 mM ethylenediaminetetraacetic acid (EDTA). In one aspect, the coating solution comprises a polyethylene glycol derivative, such as polyethylene glycol tert-octylphenyl ether, which is commercially available and referred to herein as TRITON X-100. In one aspect, the coating solution comprises about 100 μg / ml to 750 μg / ml, about 500 μg / ml to 750 μg / ml, or about 100 μg / ml to about 400 μg / ml of a proteinaceous primary reagent or capture-target hybrid, about 15 nM to about 1500 nM of a thiol-containing secondary reagent, about 0.01% to about 0.1% TRITON X-100, about 0.1% to about 0.5% trehalose in DPBS, and about 1 mM EDTA to about 20 mM EDTA.In one embodiment, the coating solution comprises about 100 μg / ml to about 400 μg / ml of a proteinaceous primary reagent or capture-target hybrid, about 15 nM to about 1500 nM of a thiol-containing secondary reagent, about 0.03% TRITON X-100, about 0.4% trehalose in DPBS, and about 10 mM EDTA.

[0095] In one aspect, the bifunctional assay surface is prepared by dispensing a coating solution comprising a primary reagent and a thiol-containing secondary reagent onto an assay surface to form a coated assay surface. In one aspect, the assay surface is a carbon-containing assay surface. In one aspect, the coated assay surface is incubated under conditions in which the proteinaceous primary reagent and the thiol-containing secondary reagent are immobilized on the carbon-containing assay surface. In one aspect, the coated assay surface is incubated under conditions in which the capture target hybrid and the thiol-containing secondary reagent are immobilized on the carbon-containing assay surface. In one aspect, the coated assay surface is incubated overnight. In one aspect, the coated assay surface is incubated at room temperature. In one aspect, the coated assay surface is incubated overnight at room temperature. In various embodiments, the coated assay surface is incubated at temperatures above and below room temperature, for example, between 4° C. and about 37° C. In one aspect, the coated assay surface is incubated for at least about 6, 7, or 8 hours to up to about 10, 11, or 12 hours. In one aspect, the coated assay surface is incubated at a controlled humidity of at least about 30%, 35%, or 40% up to about 40%, 45%, or 50%, or from about 30% to about 50%. In one aspect, the coated assay surface is incubated at a controlled humidity of about 30%, 35%, 40%, 45%, or 50%. In one aspect, the coated assay surface is incubated at a controlled humidity of about 40%.

[0096] Overcoating Method In one aspect, an overcoating method is provided for preparing a bifunctional assay surface comprising a primary reagent and a secondary reagent. In one aspect, an overcoating method is provided for preparing a bifunctional assay surface comprising either a proteinaceous primary reagent or a capture-target hybrid, and a thiol-containing secondary reagent. As used herein, "overcoating" means that the primary and secondary reagents are immobilized sequentially on the assay surface.

[0097] In one aspect, a coating solution comprising a primary reagent is dispensed onto the assay surface. In one aspect, the primary reagent is printed onto distinct binding domains on the assay surface. In one aspect, the primary reagent is printed onto the assay surface in an array. In one aspect, the primary reagent is printed onto the assay surface using a liquid handling system. In one aspect, the primary reagent is printed onto the assay surface using a non-contact dispenser, including, for example, an inkjet printer or a piezoelectric printer. In one aspect, the primary reagent is printed onto the assay surface using a contact printer, for example, using pins, capillaries, or ink stamps. In one aspect, an assay surface is obtained with the primary reagent already immobilized. In one aspect, the primary reagent is a proteinaceous primary reagent or a capture target hybrid. In one aspect, the primary reagent is a proteinaceous capture reagent.

[0098] In one aspect, the assay surface comprises a multi-well plate. In one aspect, the assay surface is a carbon-containing assay surface. In one aspect, the assay surface comprises a carbon-containing electrode.

[0099] In one aspect, the primary reagent is non-covalently immobilized on the assay surface. In one aspect, the primary reagent is non-covalently immobilized on the assay surface by passive adsorption. In one aspect, the primary reagent is covalently immobilized on the carbon-containing assay surface. In one aspect, the capture reagent is immobilized on the carbon-containing assay surface via a binding pair. In one aspect, the primary reagent comprises a first member of a binding pair and the assay surface comprises a second member of the binding pair. In one aspect, the primary reagent comprises biotin and the carbon-containing assay surface is coated with avidin or streptavidin.

[0100] In one aspect, the thiol-containing secondary reagent is dispensed onto an assay surface on which a primary reagent has previously been immobilized by dispensing an overcoating solution comprising the thiol-containing secondary reagent onto the assay surface. In one aspect, an overcoating solution comprising the thiol-containing secondary reagent is dispensed onto the assay surface to form a coated assay surface. In one aspect, the thiol-containing secondary reagent is immobilized onto the assay surface by applying a droplet of the overcoating solution onto the assay surface. In one aspect, about 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, or 40 μL up to about 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, 85 μL, 90 μL, 95 μL, or 100 μL of the overcoating solution comprising the thiol-containing secondary reagent is dispensed onto the assay surface. In one aspect, about 25 μL to about 75 μL of the overcoating solution comprising the thiol-containing secondary reagent is dispensed onto the assay surface. In one embodiment, about 30 μL to about 50 μL of an overcoating solution containing a thiol-containing secondary reagent is dispensed onto the assay surface. In one embodiment, the thiol-containing secondary reagent is immobilized on the assay surface by applying a drop of the overcoating solution onto the assay surface and incubating the assay surface with shaking. In one embodiment, the coated assay surface is incubated on a shaker at about 500 rpm, 600 rpm, or 700 rpm up to about 800 rpm, 900 rpm, or 1000 rpm. In one embodiment, the coated assay surface is incubated on a shaker at about 650 rpm, 675 rpm, 700 rpm up to about 725 rpm, or 750 rpm. In one embodiment, the coated assay surface is incubated on a shaker at about 700 rpm, 705 rpm, 710 rpm, 715 rpm, 720 rpm, or 725 rpm. In one embodiment, the coated assay surface is incubated on a shaker at about 705 rpm.

[0101] In one embodiment, the overcoating solution comprises from about 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, or 5 μM up to about 10 μM, 15 μM, or 20 μM of a thiol-containing secondary reagent. In one embodiment, the overcoating solution comprises from about 0.01 μM to about 20 μM of a thiol-containing secondary reagent. In one embodiment, the overcoating solution comprises from about 1 μM to about 10 μM of a thiol-containing secondary reagent. In one embodiment, the overcoating solution comprises from about 1 μM to about 10 μM of a thiolated oligonucleotide. In one embodiment, the overcoating solution comprises a buffer selected from a deprotection conjugation buffer (Meso Scale Diagnostics, LLC), 1×PBS (phosphate buffered saline) / 10 mM EDTA (ethylenediaminetetraacetic acid), Diluent 100 (Meso Scale Diagnostics, LLC), or a combination thereof. In one embodiment, the deprotection conjugation buffer comprises 10 mM phosphate, pH 7.4, 150 mM NaCl, and 10 mM EDTA.

[0102] In one aspect, the thiol-containing secondary reagent is printed onto a distinct binding domain on the assay surface where the primary reagent was previously immobilized. In one aspect, the thiol-containing secondary reagent is printed onto the assay surface using a liquid handling system. In one aspect, the thiol-containing secondary reagent is printed onto the assay surface using a non-contact dispenser, including, for example, an inkjet printer or a piezoelectric printer. In one aspect, the thiol-containing secondary reagent is printed onto the assay surface using a contact printer, for example, using a pin, capillary, or ink stamp.

[0103] In one embodiment, the coated assay surface is incubated under conditions where the thiol-containing secondary reagent is immobilized on the carbon-containing assay surface via the thiol group. In one embodiment, the coated assay surface is incubated for about 1 hour, 2 hours, 3 hours, or 4 hours up to about 5 hours. In one embodiment, the coated assay surface is incubated for about 1 hour to about 5 hours. In one embodiment, the coated assay surface is incubated for about 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours. In one embodiment, the coated assay surface is incubated at room temperature. In one embodiment, the coated assay surface is incubated at room temperature for 4 hours.

[0104] In one aspect, the thiol-containing secondary reagent is immobilized on the assay surface by the formation of a bond between a thiol group and a reactive functional group on the assay surface. In one aspect, the thiol-containing secondary reagent is immobilized on the assay surface via a maleimide group on the assay surface. In one aspect, the thiol-containing secondary reagent is immobilized on the assay surface by the formation of a covalent bond between a thiol group and a maleimide group on the assay surface. In one aspect, the assay surface is pretreated to introduce one or more maleimide groups before the thiol-containing secondary reagent is dispensed onto the assay surface. In one aspect, the carbon-containing assay surface is pretreated to introduce one or more maleimide groups before the thiol-containing secondary reagent is dispensed onto the carbon-containing assay surface. In one aspect, the ... n (n=2-24). In one embodiment, n=1, n=2, n=3, n=4, or n=5. In one embodiment, n=4.

[0105] In one aspect, the thiol-containing secondary reagent comprises a thiolated polyethylene glycol (PEG) species, eg, to passivate the assay surface and / or reduce non-specific binding.

[0106] In one embodiment, the assay surface is pre-treated with streptavidin, for example, to facilitate immobilization of biotin-containing reagents.

[0107] Incorporation by Reference All references cited herein, including patents, patent applications, articles, textbooks, and the like, and references cited therein, to the extent they have not already been included, are hereby incorporated by reference in their entirety. EXAMPLES

[0108] [Table 1-1] [Table 1-2] [Table 1-3]

[0109] Example 1. Method for preparing a bifunctional assay surface with a proteinaceous capture antibody and a BSA-conjugated anchor oligonucleotide This example describes how to prepare bifunctional assay surfaces on 96-well 7-spot and 10-spot assay plates (Meso Scale Diagnostics, LLC). Briefly, proteinaceous capture antibodies were printed onto the surface and immobilized with BSA-conjugated anchor oligonucleotides (BSA-oligos) or thiol-modified anchor oligonucleotides (thiol-oligos) as shown in Table 1.

[0110] Briefly, maleimide-modified BSA and thiol-modified anchor oligonucleotides were prepared and conjugated at a molar ratio of 1:10. Coating solutions containing 100 μg / ml-400 μg / ml capture antibody and 5 μg / ml-50 μg / ml BSA-oligos or thiol-oligos were prepared in a coating solution containing 0.03% TRITON X-100 and 0.4% trehalose in phosphate-buffered saline (PBS) with or without 750 μg / ml BSA. 50 nl or 75 nl of coating solution was printed onto each binding domain of a 10-spot or 7-spot MSD 96-well plate assay surface using a custom dispenser and allowed to dry overnight at a controlled humidity of 40%.

[0111] Example 2. Method for preparing a bifunctional assay surface with biotinylated capture antibodies and biotinylated anchor oligonucleotides This example describes an alternative method for preparing a bifunctional assay surface on a streptavidin-coated small spot or 10 spot 96-well assay plate (Meso Scale Diagnostics, LLC). In this example, a biotinylated capture antibody was co-immobilized with a biotinylated anchor oligonucleotide on a streptavidin-coated plate (ssSA).

[0112] Briefly, biotinylated capture antibody and biotinylated anchor oligonucleotide were diluted to 0.25 μg / ml and 25 pM, respectively, in Diluent 100 and immobilized on the SA-coated plate surface by incubation for 1 hour with shaking at room temperature.

[0113] Example 3. Overcoating of plates with thiol-modified anchor oligonucleotides This example describes a method for preparing a bifunctional assay surface by overcoating an MSD V-Plex immunoassay plate (Meso Scale Diagnostics, LLC) on which capture antibodies have been immobilized in an array with an overcoating solution containing a thiol-modified anchor oligonucleotide (Anchor-SH).

[0114] Briefly, thiol-modified anchor oligonucleotides (Anchor-SH) ranging in length from 9 nucleotides to 43 nucleotides were prepared. The anchor oligonucleotides were deprotected using either dithiothreitol (DTT) or triscarboxyethylphosphene (TCEP) reducing reagent according to the product insert instructions (ThermoScientific) to generate anchor oligos with reactive thiol groups. Some of the thiol-modified anchor oligonucleotides contained one or more modifications selected from 2,6-diaminopurine (2-amino-dA); 5-methyldeoxycytidine; Super T (5-hydroxybutynyl-2'-deoxyuridine); locked DNA bases; and peptide nucleic acid (PNA) oligonucleotides (see Table 1).

[0115] The thiol-modified anchor oligonucleotide was diluted in one of the following buffers: (i) Deprotection Conjugation Buffer (DCB) (Meso Scale Diagnostics, LLC) containing 10 mM phosphate, pH 7.4, 150 mM NaCl, 10 mM EDTA; (ii) Phosphate Buffered Saline (PBS) / 10 mM EDTA; or (iii) Diluent 100.

[0116] MSD V-Plex immunoassay plates were washed with phosphate-buffered saline (PBS), and 35-50 μL of overcoating solution containing 0.5-20 μM thiol-modified anchor oligonucleotide was added to each well and incubated for 2 h at room temperature with shaking at 705 rpm to immobilize the thiol-modified anchor oligonucleotide to the binding domains onto which the capture antibodies were immobilized.

[0117] Example 4. Direct coating of plates with capture antibodies and thiol-modified anchor oligonucleotides This example describes a method for preparing a bifunctional assay surface by directly coating a 10-spot MSD assay plate (Meso Scale Diagnostics, LLC) with a proteinaceous capture antibody and a thiol-modified anchor oligonucleotide (Anchor-SH). In this example, the proteinaceous capture antibody was immobilized by passive adsorption of the antibody to the plate surface, and the thiol-modified anchor oligonucleotide was immobilized by covalent attachment of a thiol group to the plate surface.

[0118] Thiol-modified anchor oligonucleotides were prepared as described in Example 3.

[0119] A coating solution containing 100 μg / ml-400 μg / ml of capture antibody and 50 nM-1500 nM of thiol-modified anchor oligonucleotide was prepared in a coating solution containing 0.03% TRITON X-100 and 0.4% trehalose (with 10 mM EDTA) in phosphate-buffered saline (PBS). 50 nl of the coating solution was printed onto each binding domain of the assay surface using a custom dispenser and allowed to dry overnight at a controlled humidity of 40%.

[0120] Example 5. Direct coating of plates with streptavidin and thiol-modified anchor oligonucleotides This example describes a method for preparing a bifunctional assay surface by directly coating a 10-spot MSD assay plate (Meso Scale Diagnostics, LLC) with proteinaceous streptavidin and thiol-modified anchor oligonucleotides (Anchor-SH). In this example, the proteinaceous streptavidin was immobilized by passive adsorption of streptavidin to the plate surface, and the thiol-modified anchor oligonucleotides were immobilized by covalent attachment of thiol groups to the plate surface.

[0121] Thiol-modified anchor oligonucleotides were prepared as described in Example 3.

[0122] A coating solution containing 500 μg / ml streptavidin and 50 nM to 1500 nM of thiol-modified anchor oligonucleotide was prepared in a coating solution containing 0.03% TRITON X-100 and 0.4% trehalose (with 10 mM EDTA) in phosphate-buffered saline (PBS). 50 nl of the coating solution was printed onto each binding domain of the assay surface using a custom dispenser and allowed to dry overnight at a controlled humidity of 40%.

[0123] Example 6. IL-4 assay with overcoated and directly coated anchor oligonucleotides A bifunctional assay plate was prepared using a capture antibody that specifically binds IL-4 and a thiolated anchor oligonucleotide (Anchor-SH, #2 in Table 1) by the overcoating and direct coating methods described in Examples 3 and 4. Assay sensitivity was compared to that using an S-Plex plate without an anchor (Anchorless) (Meso Scale Diagnostics, LLC); a streptavidin-coated plate (ssSA) with a biotinylated anchor oligonucleotide prepared as described in Example 2, a V-Plex plate (Meso Scale Diagnostics, LLC), and a BSA anchor oligonucleotide prepared as described in Example 1 (data not shown).

[0124] Plates with bifunctional surfaces were washed with PBS or MSD wash buffer (the same wash buffer was used for all wash steps). Dilutions of analyte (calibration curve) (including a condition without analyte) were prepared and 25-50 μL were added to the plate and incubated for 1 hour at room temperature (RT) with shaking at 700 rpm. Plates were washed and 40-50 μL of Turbo Boost antibody was added to the plate and incubated for 1 hour at room temperature with shaking. Plates were washed and 30-50 μL of Enhance solution was added to the plate and incubated for 0.5 hour at room temperature with shaking. Plates were washed and 30-50 μL of Detect solution was added to the plate and incubated for 1 hour at 27°C with shaking. V-Plex plates were tested according to the MSD product insert.

[0125] The assay sensitivity was determined using a 4PL fit function. The limit of detection (LOD) was calculated as the concentration corresponding to a signal above background of 2.5 standard deviations.

[0126] Assay sensitivity was improved approximately 5-fold in all conditions using anchor oligonucleotides compared to the no-anchor control. Results are shown in Table 2 (below) and Figure 1. Experiments performed with other capture antibodies (IL6, IL10, IL12p70) showed similar improvements in assay performance as seen with the IL4 assay using the overcoating or direct coating approach of the present invention (data not shown). [Table 2]

[0127] Example 7. IL-4, IL10, and GM-CSF assays on streptavidin plates using directly coated and biotinylated anchor oligonucleotides Bifunctional assay plates were prepared using streptavidin-coated plates and procedures described in Example 2 to immobilize biotinylated capture antibodies and biotinylated anchors specific for IL-4, IL-10, and GM-CSF. Bifunctional assay plates were also prepared using streptavidin- and anchor-SH-coated plates described in Example 5 to immobilize biotinylated capture antibodies specific for IL-4, IL-10, and GM-CSF. Assay sensitivity was compared for both types of bifunctional plates, where the capture antibody and anchor were attached onto the surface via a streptavidin-biotin bond (shown in Table 3 as SA), the capture antibody was attached onto the surface via a streptavidin-biotin bond, and the thiol-modified anchor oligonucleotide was immobilized by covalent attachment of the thiol group to the plate surface (shown in Table 3 as SA A-direct).

[0128] The assay was performed and the assay sensitivity was determined as described in Example 6. The assay sensitivity was similar for both types of bifunctional plates. [Table 3]

[0129] Example 8. Assay robustness testing The effect of thiolated anchor oligonucleotides on S-Plex assay performance was evaluated in an assay robustness study using stringent washing to assess the ability of the oligonucleotide anchors to retain the RCA products on the surface. No reduction in sensitivity (signal / NSB) was observed and there was no increase in variability (%CV) compared to the S-Plex assay format (data not shown).

[0130] Example 9. Biotin-PEG-SH Reagent In this example, heterobifunctional thiolation reagents containing biotin and a PEG spacer (Bio-PEG-SH), Nanocs (MW 400, 600, and 1000), were immobilized on the surface of V-Plex MSD plates and 10-spot 96-well assay plates using overcoating and direct coating methods. Bio-PEG-SH was used in the overcoating method at concentrations ranging from 1 μM to 5 μM. Bio-PEG-SH was used in the direct coating method at concentrations ranging from 1 μM to 10 μM. The same solvents were used as described in Examples 3 and 4 above. The concentration ranges were based on thiol group concentrations measured using Ellmann's reagent (ThermoScientific) according to the manufacturer's product insert instructions.

[0131] Both overcoating and direct coating methods provide detectable / functional amounts of biotin on the plate surface, which is then used to attach biotinylated anchor oligonucleotides (Table 1) bound to streptavidin to create bifunctional S-PLEX plates. Addition of anchor oligonucleotides via immobilized biotin allows for the addition of anchor sequences at any other step of the S-PLEX protocol assay (e.g., with Turbo Boost antibody, Enchance or Detect reagent incubation steps) in addition to the bifunctional surface preparation steps described in Examples 1-4, thereby avoiding potential sample-anchor interferences such as, for example, anti-DNA antibodies.

[0132] As shown in Figure 2 and Table 4, plates overcoated with Bio-PEG-SH reagent performed similarly to those overcoated with Anchor-SH oligos, and both performed approximately 6-fold better than plates without anchor oligonucleotides. Plates directly coated with Bio-PEG-SH performed similarly or better than plates directly coated with Anchor-SH oligonucleotides when used in the S-Plex assay described in Example 8 (Figures 3A, 3B, 3C). The overall sensitivity improvement was approximately 6-fold compared to no anchor, similar to Bio-PEG-SH overcoated plates. [Table 4]

[0133] Example 10. FITC-PEG-SH Reagent In this example, a fluorescent reagent was immobilized on an assay plate using the overcoating method described herein. Briefly, a fluorescent (FITC-PEG-SH, Nanocs) reagent was immobilized on the V-Plex assay plate surface using the overcoating method described in Example 3. The presence of the fluorescent reagent was detected using a specific anti-FITC antibody. As shown in Figure 4, signal generation was concentration dependent.

[0134] Example 11. PEG-SH Reagents In some cases, assay and sample components may adhere to surface areas not covered with the capture antibody, increasing background and adversely affecting assay sensitivity. It was hypothesized that surface modification with PEG oligomers could reduce nonspecific binding (NSB) in standard sandwich format assays.

[0135] Five PEG-SH reagents, Nanocs (MW 350, 550, 750, 1000 and 2000), were immobilized onto V-Plex assay plates with capture antibodies immobilized thereon using the overcoating method described in Example 3 and tested in a standard 10-plex sandwich format according to the V-Plex product insert. As shown in Figures 5A and 5B, in both the IFNg and TNFα assays, plates overcoated with PEG-SH reduced NSB in a concentration-dependent manner.

[0136] Coating the same reagents directly onto the plate surface with a-IFNg and a-TNFα antibodies did not produce the same results.

Claims

1. 1. A method for preparing a bifunctional assay surface, the method comprising: a. dispensing a coating solution comprising a proteinaceous primary reagent and a thiol-containing secondary reagent onto a carbon-containing assay surface to form a coated assay surface; b. incubating the coated assay surface under conditions whereby the proteinaceous primary reagent and the thiol-containing secondary reagent are immobilized onto the carbon-containing assay surface, wherein the thiol-containing secondary reagent is immobilized onto the carbon-containing assay surface via a thiol group of the thiol-containing secondary reagent.

2. 2. The method of claim 1, comprising dispensing about 10 nl to about 100 nl, about 25 nl to about 75 nl, about 40 nl to about 60 nl, or about 50 nl of the coating solution onto the carbon-containing assay surface, wherein the coating solution covers the assay surface in a ratio of about 50 nl per binding domain of the assay surface.

3. 10. The method of claim 1, wherein the coating solution comprises about 100 μg / ml to 750 μg / ml, about 500 μg / ml to 750 μg / ml, or about 100 μg / ml to about 400 μg / ml of a proteinaceous primary reagent.

4. The method of claim 1 , wherein the coating solution comprises from about 15 nM to about 1500 nM of a thiol-containing secondary reagent.

5. 1. A method for preparing a bifunctional assay surface, the method comprising: (a) dispensing an overcoating solution comprising a thiol-containing secondary reagent onto a carbon-containing assay surface having one or more proteinaceous primary reagents immobilized thereon to form a coated assay surface; (b) incubating the coated assay surface under conditions in which the thiol-containing secondary reagent is immobilized on the carbon-containing assay surface via the thiol group.

6. 6. The method of claim 5, comprising dispensing about 10 μL to about 100 μL, about 25 μL to about 75 μL, about 30 μL to about 50 μL of the overcoating solution comprising the thiol-containing secondary reagent onto the carbon-containing assay surface.

7. 7. The method of claim 6, wherein the overcoating solution comprises from about 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, or 5 μM up to about 10 μM, 15 μM, or 20 μM of a thiol-containing secondary reagent.

8. The method of any one of claims 1 to 7, wherein the proteinaceous primary reagent is immobilized on the carbon-containing assay surface via a binding pair.

9. The method of claim 8, wherein the proteinaceous primary reagent comprises a first member of a binding pair and the carbon-containing assay surface comprises a second member of the binding pair.

10. The method of any one of claims 1 to 7, wherein the thiol-containing secondary reagent comprises a thiolated oligonucleotide.

11. The method of claim 10, wherein the thiolated oligonucleotide comprises an oligonucleotide sequence having a 5' and a 3' end and a thiol group incorporated at the 5' end (5' terminally thiolated oligonucleotide), the 3' end (3' terminally thiolated oligonucleotide), an internal position of the oligonucleotide, or a combination thereof.

12. 11. The method of claim 10, wherein the thiolated oligonucleotide comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or a combination thereof.

13. The method of claim 10 , wherein the thiolated oligonucleotide comprises one or more non-natural nucleotide bases.

14. The method of any one of claims 1 to 7, wherein the thiol-containing secondary reagent comprises a thiolated member of a binding pair.

15. The method of claims 1-7, wherein the proteinaceous primary reagent comprises a proteinaceous capture reagent that specifically binds to a target analyte.

16. The method of claim 15, wherein the proteinaceous primary reagent comprises a first member of a binding pair and the target analyte comprises a second member of the binding pair.

17. The method of any one of claims 1 to 7, wherein the carbon-containing assay surface comprises a multiwell plate, and one or more wells of the multiwell plate comprise one or more electrodes.

18. A bifunctional assay surface prepared by the method of any one of claims 1 to 7.

19. 1. A method for preparing a bifunctional assay surface, the method comprising: a. dispensing a coating solution comprising a capture target hybrid and a thiol-containing secondary reagent onto a carbon-containing assay surface to form a coated assay surface; b. Incubating the coated assay surface under conditions whereby the capture target hybrid and a thiol-containing secondary reagent are immobilized on the carbon-containing assay surface, wherein the thiol-containing secondary reagent is immobilized on the carbon-containing assay surface via a thiol group.

20. 1. A method for preparing a bifunctional assay surface, the method comprising: a. dispensing an overcoating solution comprising a thiol-containing secondary reagent onto a carbon-containing assay surface having one or more capture target hybrids immobilized thereon to form a coated assay surface; b) incubating the coated assay surface under conditions in which the thiol-containing secondary reagent is immobilized on the carbon-containing assay surface via the thiol group.

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