Improved assay methods

Abstract

The present invention relates to methods of detecting an analyte of interest in a sample, directed to methods for improving assay specificity and performance in binding assays.

Description

[0001] This application is a divisional application of the patent application with application number 202010970676.9.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application Nos. 61 / 993,581, filed May 15, 2014; 62 / 013,823, filed June 18, 2014; 62 / 048,489, filed September 10, 2014; 62 / 049,520, filed September 12, 2014; and 62 / 055,093, filed September 25, 2014, the entire contents of which are incorporated herein by reference. Reference is also made to USSN 14 / 206,284 (Publication No. US-2014 / 0272939); USSN 14 / 208,040 (Publication No. US-2014 / 0274775); and USSN 14 / 203,638 (Publication No. US-2014 / 0256588), the disclosures of which are also incorporated herein by reference. Field of the Invention

[0004] The present invention relates to improved methods for performing immunoassays. The methods are designed to amplify the signal in immunoassays and anchor the immunoassay complexes employed therein. Background of the Invention

[0006] A large literature has been developed on techniques that employ binding reactions (e.g., antigen-antibody reactions, nucleic acid hybridization, and receptor-ligand reactions) for sensitive measurement of analytes of interest in samples. The high specificity of many biochemical binding systems has led to many valuable analytical methods and systems in a variety of markets, including basic research, human and veterinary diagnostics, environmental monitoring, and industrial testing. The presence of an analyte of interest can be measured by directly measuring the participation of the analyte in a binding reaction. In some methods, the participation can be indicated by measuring an observable marker attached to one or more binding materials.

[0007] While the sandwich immunoassay format offers excellent sensitivity and specificity in many applications, some analytes are present in concentrations that are too low to be detected by traditional immunoassay techniques. The performance of sandwich immunoassays may also be limited by nonspecific binding of the detection antibody and the instability of sandwich mixtures containing antibodies with high dissociation rates. However, efforts to modify traditional methods to improve sensitivity and specificity often result in more complex, labor-intensive protocols that may be hampered by inefficiencies at each step, which may greatly affect the sensitivity and specificity of the assay. For example, in a complex assay requiring multiple binding events and / or reactions, if any one of the events or reactions is less than ideal, the sensitivity and specificity of the overall assay may be affected. There is a need for new technologies that improve the performance of sandwich immunoassays by improving sensitivity, reducing nonspecific binding, and improving the stability of the sandwich complex. SUMMARY OF THE INVENTION

[0009] The present invention contemplates the following specific embodiments. Those skilled in the art may make various modifications, additions and changes to the embodiments described herein without departing from the spirit and scope of the present invention. These modifications, additions and changes are intended to fall within the scope of the claims.

[0010] Embodiment (1): A method for detecting an analyte of interest in a sample, comprising: allowing the analyte to bind to: (i) a capture reagent on a surface, the surface comprising a capture reagent for the analyte, and an anchoring reagent; and (ii) a detection reagent for the analyte connected to a nucleic acid probe; thereby forming a complex comprising the capture reagent, the analyte and the detection reagent on the surface; extending the probe to form an extended sequence comprising an anchor region bound to the anchoring reagent; allowing the extended sequence to bind to the anchoring reagent; and measuring the amount of the extended sequence bound to the surface.

[0011] In embodiment (1), the capture reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer. In a specific embodiment, the capture reagent is an antibody. The detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer, and in a specific embodiment, the detection reagent is an antibody. In a specific example of embodiment (1), the capture and detection reagents are antibodies against the analyte. The anchoring reagent can include an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope or a mimotope; and optionally, the anchoring region can include an aptamer, and the anchoring reagent can include an aptamer ligand. The anchoring region can include a nucleic acid sequence, and the anchoring reagent can include a DNA-binding protein. The anchoring reagent can include an oligonucleotide sequence, and the anchoring sequence can include a complementary oligonucleotide sequence. The anchoring region can include a single-stranded oligonucleotide sequence or a double-stranded oligonucleotide sequence.

[0012] The binding step of embodiment (1) may further include forming a triple helix between the anchor region and the anchoring agent. The method may further include denaturing the anchor region to expose the single-stranded sequence before the binding step; exposing the anchor region to helicase activity before the binding step; and / or exposing the anchor region to nuclease treatment before the binding step. In this embodiment, the anchor region may include one or more hapten-modified bases, and the anchoring agent may include one or more antibodies specific for the hapten; and / or the anchor region may include one or more ligand-modified bases, and the anchoring agent may include one or more receptors specific for the ligand. The extension sequence may further include one or more detection sequences, and the measuring step may further include contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences; the extension sequence may include one or more modified bases, and the measuring step may include contacting the extension sequence with a plurality of detection moieties capable of binding to the one or more modified bases; and / or the extension sequence may include one or more labeled bases, and the measuring step may further include detecting the presence of the one or more labeled bases. In this embodiment, one or more modified bases comprise an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope or a mimotope, and each of the plurality of detectable moieties comprises a binding partner of the one or more modified bases and a detectable label. The one or more modified bases may comprise streptavidin, and each of the plurality of detectable moieties comprises biotin and a detectable label; and / or the one or more modified bases may comprise biotin, and each of the plurality of detectable moieties comprises streptavidin and a detectable label; and / or the one or more modified bases may comprise avidin, and each of the plurality of detectable moieties comprises biotin and a detectable label; and / or the one or more modified bases may comprise biotin, and each of the plurality of detectable moieties comprises avidin and a detectable label.

[0013] The first step of embodiment (1) may comprise binding the analyte to the following species in the following order: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte; or the first step of embodiment (1) may comprise binding the analyte to the following species in the following order: (i) a detection reagent for the analyte; and (ii) a capture reagent on the surface; and / or the first step may comprise binding the analyte to the following species simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte.

[0014] The extension step of embodiment (1) may include binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction; and / or binding the probe to a template nucleic acid sequence to form a circular nucleic acid template, and extending the circular template by rolling circle amplification. In this embodiment, the extended probe may still be located on the surface after the probe is extended. Therefore, the complex may still be bound to the surface after the extension step, for example, the extended probe is bound to the anchoring agent at a position within 10-100 μm of the position of the complex on the surface. In a specific embodiment, the extended probe is bound to the anchoring agent at a position less than 100 μm, less than 50 μm, or more particularly less than 10 μm of the position of the complex on the surface.

[0015] The extension step of embodiment (1) may comprise PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustained synthesis reaction), or isothermal amplification method. In one embodiment, the extension step may comprise an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0016] The surface referred to in embodiment (1) may include particles and / or holes of a porous plate. The surface may include multiple different binding domains, and the capture agent and the anchoring agent are positioned on two different binding domains on the surface. If the surface is a hole of a plate, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains in the hole; and / or the surface may include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain on the surface. In one embodiment, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain in the hole. The capture agent and the anchoring agent may be within 10-100nm on the surface. The surface may include electrodes and the measuring step may further include applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal, and optionally, the method includes collecting particles on the electrodes and applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal.

[0017] The measuring step of embodiment (1) may further comprise binding the extended sequence to a detection probe having a detectable label, measuring the detectable label, and correlating the measurement with the amount of analyte in the sample, wherein the detection probe comprises a nucleic acid sequence complementary to a region of the extended sequence. The detectable label may be measured by measuring light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a specific embodiment of embodiment (1), the detectable label is an ECL label and the measuring step may comprise measuring the ECL signal.

[0018] Embodiment (2): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an anchoring reagent; and (b) a detection reagent for the analyte attached to a nucleic acid probe.

[0019] The anchoring agent of embodiment (2) may comprise an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope or a mimotope, and the capture agent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer. In a specific embodiment, the capture agent may comprise an antibody, and / or the detection agent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer. In a specific embodiment of the kit, the detection agent is an antibody.

[0020] The surface of the test kit of embodiment (2) may include particles and / or holes of a porous plate. The surface may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface of the test kit is a hole of a plate, the surface may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains in the hole; and / or the surface may include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain on the surface. In a specific embodiment of the test kit, the surface is a hole, and the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain in the hole. The capture agent and the anchoring agent may be within 10-100nm on the surface. In addition, the surface of the test kit may include an electrode.

[0021] Embodiment (3): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent on a surface, the surface comprising a capture reagent for the analyte, and an anchor reagent comprising an anchor oligonucleotide sequence; and (ii) a detection reagent for the analyte linked to a nucleic acid probe; thereby forming a complex comprising the capture reagent, the analyte and the detection reagent on the surface; (b) extending the probe to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence; (c) hybridizing the anchor sequence with the anchor sequence complement; and (d) measuring the amount of the extended sequence bound to the surface.

[0022] In embodiment (3), the capture reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific embodiment, the capture reagent is an antibody. Similarly, the detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific embodiment of embodiment (3), the detection reagent is an antibody. In an example of embodiment (3), the capture reagent and the detection reagent are antibodies against the analyte. The anchor oligonucleotide sequence can comprise a single-stranded oligonucleotide sequence or a double-stranded oligonucleotide sequence. The extension sequence can further comprise one or more detection sequences, and the measuring step further comprises contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences; or or in addition, the extension sequence can also comprise one or more modified bases, and the measuring step can also comprise contacting the extension sequence with a plurality of detectable portions capable of binding to the one or more modified bases. In a specific embodiment, the extension sequence can also comprise one or more labeled bases, and the measuring step can also comprise detecting the presence of the one or more labeled bases. The one or more modified bases include aptamers, aptamer ligands, antibodies, antigens, ligands, receptors, haptens, epitopes or mimotopes, and the plurality of detectable moieties each include a binding partner of the one or more modified bases and a detectable label. The one or more modified bases may include streptavidin, and the plurality of detectable moieties each include biotin and a detectable label; the one or more modified bases include biotin, and the plurality of detectable moieties each include streptavidin and a detectable label; the one or more modified bases include avidin, and the plurality of detectable moieties each include biotin and a detectable label; and / or the one or more modified bases include biotin, and the plurality of detectable moieties each include avidin and a detectable label.

[0023] Step (a) of embodiment (3) may comprise binding the analyte to the following species in the following order: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte. Alternatively, step (a) may comprise binding the analyte to the following species in the following order: (i) a detection reagent for the analyte; and (ii) a capture reagent on a surface. In another embodiment, step (a) may comprise simultaneously or substantially simultaneously binding the analyte to the following species: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte.

[0024] The extension step of embodiment (3) may include binding the probe to the template nucleic acid sequence and extending the probe by polymerase chain reaction. Alternatively, the extension step may include binding the probe to the template nucleic acid sequence to form a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extended probe may still be positioned on the surface after the probe is extended. For example, after the extension step, the complex is still attached to the surface. In one embodiment, the extended probe is attached to the anchoring agent at a position within 10-100um of the complex position on the surface. In a specific embodiment, the extended probe is attached to the anchoring agent at a position less than 100um, less than 50um, or more particularly less than 10um of the complex position on the surface. In this specific embodiment, the extension step may include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (single-strand displacement amplification), 3SR (automatic maintenance synthesis reaction), or isothermal amplification method. For example, the extension step may include isothermal amplification methods, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0025] The surface of embodiment (3) may comprise a hole of a particle and / or a porous plate. The surface may comprise a plurality of different binding domains, and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole of a plate, it may comprise a plurality of different binding domains, and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may comprise a plurality of different binding domains, and the capture agent and the anchoring agent are located on the same binding domain on the surface. If the surface is a hole, it may comprise a plurality of different binding domains, and the capture agent and the anchoring agent are located on the same binding domain in the hole. The capture agent and the anchoring agent may be within 10-100 nm on the surface. In a specific embodiment, the surface may comprise an electrode, and the measuring step may further comprise applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. The method may further comprise collecting particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. The measuring step may further comprise binding an extension sequence to a detection probe having a detectable label, measuring the detectable label, and correlating the measurement with the amount of analyte in the sample, wherein the detection probe comprises a nucleic acid sequence complementary to the region of the extension sequence. In this embodiment, the detectable label is measured by measurement of light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field or a combination thereof. For example, the detectable label is an ECL label, and the measuring step may include measuring the ECL signal.

[0026] Embodiment (4): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an anchoring reagent comprising an anchoring oligonucleotide sequence; and (b) a detection reagent for the analyte attached to a nucleic acid probe.

[0027] The kit of embodiment (4) comprises a capture reagent comprising an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer. In a specific embodiment, the capture reagent may comprise an antibody. Similarly, the detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer, and specifically, the detection reagent may comprise an antibody.

[0028] The kit of embodiment (4) includes a surface, which may include particles and / or holes of a porous plate. The surface may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain on the surface, for example, if the surface is a hole, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain in the hole. For example, the capture agent and the anchoring agent may be within 10-100nm on the surface. The surface of embodiment (4) may include an electrode.

[0029] Embodiment (5): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent on a surface, the surface comprising a capture reagent for the analyte, and an anchor reagent comprising an anchor oligonucleotide sequence; (ii) a first detection reagent for the analyte linked to a first nucleic acid probe; and (iii) a second detection reagent for the analyte linked to a second nucleic acid probe; thereby forming a complex on the surface, the surface comprising a binding reagent, the analyte, and the first and second detection reagents; (b) extending the second probe to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence using an extension process that requires proximity of the first and second proximal probes; (c) hybridizing the anchor sequence with the anchor sequence complement; and (d) measuring the amount of the extended sequence bound to the surface.

[0030] The capture reagent of embodiment (5) can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer. In a specific embodiment, the capture reagent is an antibody. Similarly, the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer, and in a specific embodiment, the first detection reagent is an antibody. The second detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer, and in a specific embodiment, the second detection reagent is an antibody. More specifically, the capture reagent and the first and second detection reagents are antibodies to the analyte.

[0031] In embodiment (5), the anchor oligonucleotide sequence may include a single-stranded oligonucleotide sequence or a double-stranded oligonucleotide sequence. In this embodiment, the extended sequence may further include one or more detection sequences, and the measuring step may also include contacting the extended sequence with a plurality of labeled probes complementary to the one or more detection sequences. The extended sequence may also include one or more modified bases, and the measuring step may also include contacting the extended sequence with a plurality of detectable portions capable of being combined with the one or more modified bases. The extended sequence may further include one or more labeled bases, and the measuring step may also include detecting the presence of one or more labeled bases. The one or more modified bases may include an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimotope, and each of the multiple detectable portions may include a binding partner of the one or more modified bases and a detectable marker. For example, the one or more modified bases comprise streptavidin and the multiple detectable moieties each comprise biotin and a detectable label; the one or more modified bases comprise biotin and the multiple detectable moieties each comprise streptavidin and a detectable label; the one or more modified bases comprise avidin and the multiple detectable moieties each comprise biotin and a detectable label; and / or the one or more modified bases comprise biotin and the multiple detectable moieties each comprise avidin and a detectable label.

[0032] Step (a) of embodiment (5) may include binding the analyte to the following species in the following order: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte. Alternatively, step (a) may include binding the analyte to the following species in the following order: (i) a detection reagent for the analyte; and (ii) a capture reagent on a surface; or step (a) may include binding the analyte to the following species simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte.

[0033] The extension step of embodiment (5) may include binding the probe to the template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step may also include binding the probe to the template nucleic acid sequence to form a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extended probe may still be positioned on the surface after the probe is extended, for example, the complex is still bound to the surface after the extension step. The extended probe is bound to the anchoring agent at a position within 10-100um of the complex position on the surface. In a specific embodiment, the extended probe is bound to the anchoring agent at a position less than 100um, less than 50um, or more particularly less than 10um of the complex position on the surface. The extension step may include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (single-strand displacement amplification), 3SR (automatic maintenance synthesis reaction), or isothermal amplification method. In a specific embodiment, the extension step may include isothermal amplification method, for example, helicase-dependent amplification or rolling circle amplification (RCA).

[0034] The extension process of embodiment (5) may include contacting the complex formed in step (a) with a connector sequence, wherein the connector sequence comprises (i) an internal sequence complementary to the second probe and (ii) a double-end sequence complementary to the non-overlapping region of the first probe. The method may also include connecting the double-end sequence of the connector oligonucleotide to form a circular target sequence hybridized with both the first and second probes. Alternatively, the extension process may include contacting the complex formed in step (a) of embodiment (5) with a first connector oligonucleotide sequence and a second connector oligonucleotide sequence, wherein the first connector oligonucleotide sequence comprises a first connection probe sequence complementary to a first region of the first probe and a first region on the second probe, and the second connector oligonucleotide comprises a second probe sequence complementary to a second non-overlapping region of the first probe and a second non-overlapping region of the second probe; and optionally, connecting the first and second connector oligonucleotides to form a circular target sequence hybridized with both the first and second probes.

[0035] The surface of embodiment (5) may include particles and / or holes of a porous plate. The surface may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole of a plate, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may also include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain on the surface. If the surface is a hole of a plate, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain in the hole. The capture agent and the anchoring agent are within 10-100nm on the surface. In a specific embodiment, the surface may include electrodes, and the measuring step may also include applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal, and optionally, the method of embodiment (5) also includes collecting particles on the electrodes and applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal.

[0036] The measuring step of embodiment (5) may also include combining the extended sequence with a detection probe having a detectable label, measuring the detectable label, and correlating the measurement with the amount of analyte in the sample, wherein the detection probe comprises a nucleic acid sequence complementary to the region of the extended sequence. The detectable label may be measured by measurement of light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a specific embodiment, the detectable label is an ECL label, and the measuring step may include measuring the ECL signal.

[0037] Embodiment (6): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an anchor reagent comprising an anchor oligonucleotide sequence; (b) a first detection reagent for the analyte linked to a first nucleic acid probe; and (c) a second detection reagent for the analyte linked to a second nucleic acid probe.

[0038] The capture reagent of embodiment (6) may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer, and in a specific embodiment, the capture reagent may include an antibody. Similarly, the first detection reagent may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer, and in a specific embodiment, the first detection reagent may include an antibody. Similarly, the second detection reagent may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope or an aptamer, and in a specific embodiment, the second detection reagent may include an antibody.

[0039] The surface of embodiment (6) may comprise particles and / or holes of a porous plate. The surface may comprise a plurality of different binding domains, and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole, the hole may comprise a plurality of different binding domains, and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may comprise a plurality of different binding domains, and the capture agent and the anchoring agent are located on the same binding domain on the surface; and / or if the surface is a hole, the hole may comprise a plurality of different binding domains, and the capture agent and the anchoring agent are located on the same binding domain in the hole. The capture agent and the anchoring agent may be within 10-100 nm on the surface. In a specific embodiment, the surface may comprise an electrode.

[0040] Embodiment (7): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent for the analyte on a surface, the surface comprising a capture reagent and an anchoring reagent; (ii) a first detection reagent for the analyte comprising a first proximal probe, and (iii) a second detection reagent for the analyte comprising a second proximal probe; thereby forming a detection complex on the surface, the detection complex comprising the capture reagent, the analyte, and the first and second detection reagents; (b) contacting the detection complex formed in (c) with a linker sequence, the linker sequence comprising (i) an internal sequence complementary to the second proximal probe and (ii) a double-end sequence complementary to a non-overlapping region of the first proximal probe; (c) hybridizing the linker sequence to the first and second proximal probes; (d) connecting the double-end sequences of the linker oligonucleotides to form a circular target sequence that hybridizes to both the first and second proximal probes; (e) extending the second proximal probe by rolling circle amplification of the target sequence to produce an amplicon comprising a binding domain that binds to the anchoring reagent; (f) binding the amplicon to the anchoring reagent; and (g) measuring the amount of the amplicon on the surface.

[0041] Embodiment (8): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent for the analyte on a surface, the surface comprising a capture reagent and an anchoring reagent; (ii) a first detection reagent for the analyte comprising a first proximal probe, and (iii) a second detection reagent for the analyte comprising a second proximal probe; thereby forming a detection complex on the surface, the detection complex comprising the capture reagent, the analyte, and the first and second detection reagents; (b) contacting the detection complex formed in (c) with a first linker oligonucleotide and a second linker oligonucleotide, wherein (i) the first linker oligonucleotide The first end and the first end of the second linker are complementary to two non-overlapping regions of the first proximal probe, and (ii) the second end of the first linker and the second end of the second linker are complementary to two non-overlapping regions of the first proximal probe; (c) hybridizing the first and second linker oligonucleotides to the first and second proximal probes; (d) connecting the first and second linker oligonucleotides to form a circular target sequence that hybridizes to both the first and second proximal probes; (e) extending the second proximal probe by rolling circle amplification of the target sequence to produce an amplicon comprising a binding domain that binds an anchoring agent; (f) binding the amplicon to the anchoring agent; and (g) measuring the amount of the amplicon on the surface.

[0042] The capture reagent of embodiments (7) and (8) can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific embodiment, the capture reagent is an antibody. Similarly, the first detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the first detection reagent is an antibody. In addition, the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the second detection reagent is an antibody. In a specific embodiment of embodiments (7) and (8), the capture reagent and the first and second detection reagents are antibodies to the analyte.

[0043] The anchoring agent of embodiments (7) and (8) may include an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimotope. In one embodiment, the binding domain may include an aptamer, and the anchoring agent may include an aptamer ligand. The binding domain may include a nucleic acid sequence, and the anchoring agent may include a DNA binding protein; and / or the anchoring agent may include an oligonucleotide sequence, and the amplicon may include a complementary oligonucleotide sequence.

[0044] The amplicons of embodiments (7) and (8) may further comprise one or more detection sequences, and the measuring step may further comprise contacting the extended sequence with a plurality of labeled probes complementary to the one or more detection sequences. In addition, the amplicons may further comprise one or more modified bases, and the measuring step may further comprise contacting the extended sequence with a plurality of detectable moieties capable of binding to the one or more modified bases. In addition, the amplicons may further comprise one or more labeled bases, and the measuring step may further comprise detecting the presence of the one or more labeled bases. The one or more modified bases may comprise an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimotope and the plurality of detectable moieties each comprise a binding partner for the one or more modified bases and a detectable label. The one or more modified bases may comprise streptavidin, and the plurality of detectable moieties each comprise biotin and a detectable label; the one or more modified bases may comprise biotin, and the plurality of detectable moieties each comprise streptavidin and a detectable label; the one or more modified bases may comprise avidin, and the plurality of detectable moieties each comprise biotin and a detectable label; and / or the one or more modified bases may comprise biotin, and the plurality of detectable moieties each comprise avidin and a detectable label.

[0045] Step (a) of embodiments (7) and (8) may include binding the analyte to the following species in the following order: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte. Alternatively, step (a) may include binding the analyte to the following species in the following order: (i) a first and a second detection reagent for the analyte; and (ii) a capture reagent on a surface. Additionally, step (a) may include simultaneously or substantially simultaneously binding the analyte to the following species: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte.

[0046] The amplicons of embodiments (7) and (8) can still be located on the surface after the probe is extended. The complex can still be bound to the surface after the extension step. For example, the extended probe is bound to the anchoring agent at a position within 10-100um of the position of the complex on the surface. In a specific embodiment, the extended probe is bound to the anchoring agent at a position less than 100um, less than 50um, or more particularly less than 10um from the position of the complex on the surface.

[0047] The surface of embodiments (7) and (8) may comprise the hole of particle and / or porous plate. The surface may comprise multiple different binding domains, and the capture agent and anchoring agent are located on two different binding domains on the surface. If the surface is the hole of a plate, the hole may comprise multiple different binding domains, and the capture agent and anchoring agent are located on two different binding domains in the hole. The surface may comprise multiple different binding domains, and the capture agent and anchoring agent are located on the same binding domain on the surface. If the surface is the hole of a plate, the hole may comprise multiple different binding domains, and the capture agent and anchoring agent are located on the same binding domain in the hole. In a specific embodiment, the capture agent and anchoring agent are within 10-100nm on the surface.

[0048] In addition, the surface may include electrodes, and the measuring step may include applying a voltage waveform to the electrodes to produce an electrochemiluminescent signal. In these embodiments ((7) and (8)), the method may also include collecting particles on the electrodes and applying a voltage waveform to the electrodes to produce an electrochemiluminescent signal. The measuring step may include binding the amplicon to a detection probe with a detectable label, measuring the detectable label, and associating the measurement with the amount of analyte in the sample, wherein the detection probe comprises a nucleic acid sequence complementary to the amplicon region. The detectable label is measured by light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. For example, the detectable label is an ECL label, and the measuring step may include measuring the ECL signal.

[0049] Embodiment (9): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an anchoring reagent; (b) a first detection reagent for the analyte comprising a first proximal probe; (c) a second detection reagent for the analyte comprising a second proximal probe; and (d) a linker sequence comprising (i) an internal sequence complementary to the second proximal probe and (ii) a double-end sequence complementary to a non-overlapping region of the first proximal probe.

[0050] Embodiment (10): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an anchoring reagent; and (b) a first detection reagent for the analyte comprising a first proximal probe; (c) a second detection reagent for the analyte comprising a second proximal probe; and (d) (i) a first linker oligonucleotide and (ii) a second linker oligonucleotide, wherein (x) the first end of the first linker and the first end of the second linker are complementary to two non-overlapping regions of the first proximal probe, and (y) the second end of the first linker and the second end of the second linker are complementary to two non-overlapping regions of the first proximal probe.

[0051] The capture reagents of embodiments (9) and (10) may include antibodies, antigens, ligands, receptors, oligonucleotides, haptens, epitopes, mimotopes, or aptamers. In a specific embodiment, the capture reagent may include an antibody. The first detection reagent may include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in a specific embodiment, the first detection reagent may include an antibody. The second detection reagent may include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in a specific embodiment, the second detection reagent may include an antibody.

[0052] The surface of embodiments (9) and (10) may include a particle and / or a hole of a porous plate. The surface may include a plurality of different binding domains, and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole of a plate, the hole may include a plurality of different binding domains, and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may contain a plurality of different binding domains, and the capture agent and the anchoring agent are located on the same binding domain on the surface. If the surface is a hole, the hole may include a plurality of different binding domains, and the capture agent and the anchoring agent are located on the same binding domain in the hole. In a specific embodiment, the capture agent and the anchoring agent may be within 10-100nm on the surface.

[0053] The surface of embodiments (9) and (10) may include an electrode.

[0054] Embodiment (11): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent for the analyte on a surface, the surface comprising a capture reagent and an anchoring reagent comprising an anchoring oligonucleotide sequence; (ii) a first detection reagent for the analyte comprising a first proximal probe; and (iii) a second detection reagent for the analyte comprising a second proximal probe; thereby forming a detection complex on the surface, the detection complex comprising the capture reagent, the analyte, and the first and second detection reagents; (b) contacting the detection complex formed in (c) with a linker sequence, the linker sequence comprising (i) an internal sequence complementary to the second proximal probe, (ii) a double-end sequence complementary to the non-overlapping region of the first proximal probe, and (iii) a sequence paired with the anchor sequence; (c) hybridizing a linker sequence to the first and second proximal probes; (d) connecting the double-end sequence of the linker oligonucleotide to form a circular target sequence that hybridizes to both the first and second proximal probes; (e) extending the second proximal probe by rolling circle amplification of the target sequence to produce an amplicon comprising multiple anchor sequence complements complementary to the anchor sequence; (f) hybridizing the anchor sequence to one of the anchor sequence complements; and (g) measuring the amount of the amplicon on the surface.

[0055] Embodiment (12): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent for the analyte on a surface, the surface comprising a capture reagent and an anchoring reagent comprising an anchoring oligonucleotide sequence; (ii) a first detection reagent for the analyte comprising a first proximal probe; and (iii) a second detection reagent for the analyte comprising a second proximal probe; thereby forming a detection complex on the surface, the detection complex comprising the capture reagent, the analyte, and the first and second detection reagents; (b) contacting the detection complex formed in (a) with a first linker oligonucleotide and a second linker oligonucleotide, wherein (i) the first end of the first linker and the first end of the second linker are The invention relates to a method for preparing an oligonucleotide comprising: a first proximal probe and a second proximal probe comprising: a first linker oligonucleotide and a second linker oligonucleotide that are complementary to two non-overlapping regions of the first proximal probe, (ii) the second end of the first linker and the second end of the second linker are complementary to the two non-overlapping regions of the first proximal probe, and (iii) the first and / or second linkers further comprise a sequence that pairs with an anchor sequence; (c) hybridizing the first and second linker oligonucleotides with the first and second proximal probes; (d) connecting the first and second linker oligonucleotides to form a circular target sequence that hybridizes with both of the first and second proximal probes; (e) extending the second proximal probe by rolling circle amplification of the target sequence to produce an amplicon comprising a plurality of anchor sequence complements that are complementary to the anchor sequence; (f) hybridizing the anchor sequence with one of the anchor sequence complements; and (g) measuring the amount of the amplicon on the surface.

[0056] The capture reagent of embodiments (11) and (12) is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer. In a specific embodiment, the capture reagent is an antibody. The first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in a specific embodiment, the first detection reagent is an antibody. Similarly, the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in a specific embodiment, the second detection reagent is an antibody. In one embodiment, the first and second detection reagents are antibodies to the analyte.

[0057] The amplicons of embodiments (11) and (12) may further comprise one or more detection sequences, and the measuring step may further comprise contacting the extended sequence with a plurality of labeled probes complementary to the one or more detection sequences. In addition, the amplicons may further comprise one or more modified bases, and the measuring step may comprise contacting the extended sequence with a plurality of detectable moieties capable of binding to the one or more modified bases. The amplicons may further comprise one or more labeled bases, and the measuring step may comprise detecting the presence of the one or more labeled bases. The one or more modified bases comprise an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimotope, and the plurality of detectable moieties each comprise a binding partner for the one or more modified bases and a detectable label. The one or more modified bases may comprise streptavidin and the multiple detectable moieties each comprise biotin and a detectable label; the one or more modified bases may comprise biotin and the multiple detectable moieties each comprise streptavidin and a detectable label; the one or more modified bases may comprise avidin and the multiple detectable moieties each comprise biotin and a detectable label; and / or the one or more modified bases may include biotin and the multiple detectable moieties each comprise avidin and a detectable label.

[0058] Step (a) of embodiments (11) and (12) may include binding the analyte to the following species in the following order: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte. Alternatively, step (a) may include binding the analyte to the following species in the following order: (i) a first and a second detection reagent for the analyte; and (ii) a capture reagent on a surface. Additionally, step (a) may include simultaneously or substantially simultaneously binding the analyte to the following species: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte.

[0059] The amplicons of embodiments (11) and (12) can still be located on the surface after the probe is extended, and optionally, the complex is still bound to the surface after the extension step. For example, the amplicons are bound to the anchoring agent at a position within 10-100 um of the position of the complex on the surface. In a specific embodiment, the extended probe is bound to the anchoring agent at a position less than 100 um, less than 50 um, or more particularly less than 10 um of the position of the complex on the surface.

[0060] The surface of embodiments (11) and (12) may include particles and / or holes of a porous plate. Optionally, the surface may include multiple different binding domains, and the capture agent and anchoring agent are located on two different binding domains on the surface. If the surface is a hole, the hole may include multiple different binding domains, and the capture agent and anchoring agent are located on two different binding domains in the hole. The surface may contain multiple different binding domains, and the capture agent and anchoring agent are located on the same binding domain on the surface. If the surface is a hole, the hole may include multiple different binding domains, and the capture agent and anchoring agent are located on the same binding domain in the hole. The capture agent and anchoring agent are within 10-100nm on the surface.

[0061] The surface of embodiments (11) and (12) may include electrodes, and the measuring step may also include applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal, optionally, embodiments (11) and (12) further include collecting particles on the electrodes and applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The measuring step may also include combining the amplicon with a detection probe having a detectable label, measuring the detectable label, and correlating the measurement with the amount of analyte in the sample, wherein the detection probe comprises a nucleic acid sequence complementary to the region of the amplicon. The detectable label may be measured by light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In one embodiment, the detectable label is an ECL label, and the measuring step may include measuring the ECL signal.

[0062] The sample of embodiments (11) and (12) may contain one or more analyte molecules, and the surface may include multiple capture reagents for one or more analyte molecules, the capture reagents being distributed on multiple distinguishable binding regions located on the surface, and the method may include: (x) binding the one or more analyte molecules to one or more capture reagents on the surface; (y) determining the presence or absence of analyte molecules in each binding region; and (z) identifying the number of binding regions containing analyte molecules and / or the number of analyte binding domains that do not contain analyte molecules. The measuring step may include imaging the optical signal from the surface to generate an image comprising multiple pixels, and each distinguishable binding region is mapped to one or more pixels in the image. The distinguishable binding regions may be elements of an array and / or the distinguishable binding regions are configured to separate individual particles. Each distinguishable binding area can be a single nanopore having a volume of <100 nL, e.g., wherein at least 99% of the binding areas contain zero or one analyte molecule, wherein at least about 95% of the binding areas contain zero or one analyte molecule, wherein at least about 80% of the binding areas contain zero or one analyte molecule, and / or wherein at least about 50% of the binding areas contain zero or one analyte molecule. The concentration of analyte molecules in the sample in embodiments (11) and (12) can be determined at least in part using a calibration curve, a Poisson distribution analysis, and / or a Gaussian distribution analysis of the number of binding areas containing at least one or one analyte molecule.

[0063] In embodiments (11) and (12), the sample may contain one or more analyte molecules, the surface may include multiple particles, each particle includes multiple binding reagents for the analyte molecules, wherein the multiple particles are distributed in multiple distinguishable binding areas, and the method may include: (i) binding one or more analyte molecules to one or more binding reagents on the surface, and (ii) distributing the multiple particles on an array of distinguishable binding areas; and (iii) determining the presence or absence of analyte molecules in each distinguishable binding area so as to identify the number of binding areas containing analyte molecules and / or identify the number of binding areas that do not contain analyte molecules.

[0064] Embodiment (13): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an anchor reagent comprising an anchor oligonucleotide sequence; and (b) a first detection reagent for the analyte comprising a first proximal probe; (c) a second detection reagent for the analyte comprising a second proximal probe; and (d) a linker sequence comprising (i) an internal sequence complementary to the second proximal probe and (ii) a double-end sequence complementary to a non-overlapping region of the first proximal probe.

[0065] Embodiment (14): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an anchor reagent comprising an anchor oligonucleotide sequence; and (b) a first detection reagent for the analyte comprising a first proximal probe; (c) a second detection reagent for the analyte comprising a second proximal probe; and (d) (i) a first linker oligonucleotide and (ii) a second linker oligonucleotide, wherein (x) the first end of the first linker and the first end of the second linker are complementary to two non-overlapping regions of the first proximal probe, and (y) the second end of the first linker and the second end of the second linker are complementary to two non-overlapping regions of the first proximal probe.

[0066] The capture reagent of embodiments (13) and (14) may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the capture reagent may comprise an antibody. Similarly, the first detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the first detection reagent may comprise an antibody. Similarly, the second detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the second detection reagent may comprise an antibody.

[0067] The surface of embodiments (13) and (14) may include particles and / or holes of a porous plate. The surface may include multiple different binding domains, and the capture agent and anchoring agent are located on two different binding domains on the surface. If the surface is a hole, the hole may include multiple different binding domains, and the capture agent and anchoring agent are located on two different binding domains in the hole. The surface may contain multiple different binding domains, and the capture agent and anchoring agent are located on the same binding domain on the surface. If the surface is a hole, the hole may include multiple different binding domains, and the capture agent and anchoring agent are located on the same binding domain in the hole. The capture agent and anchoring agent are within 10-100nm on the surface, and optionally, the surface may include an electrode.

[0068] Embodiment (15): A method for detecting an analyte in a sample, wherein the method may include: (a) binding the analyte to a first and a second detection reagent to form a detection complex, each detection complex comprising the analyte, a first detection reagent and a second detection reagent, wherein the first detection reagent has a first detectable label and the second detection reagent has a second detectable label, (b) separating the analyte between a plurality of reaction vessels such that a majority of the reaction vessels contain one or fewer analytes; and (c) detecting the number of analyte molecules by counting the number of reaction vessels containing the first and second detectable labels. In this embodiment (15), the first detection reagent may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the first detection reagent is an antibody. Similarly, the second detection reagent may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or a ligand, for example, the second detection reagent is an antibody. In a specific embodiment, the first and second detection reagents are antibodies to the analyte.

[0069] Step (a) of embodiment (15) may further include forming a solution comprising the analyte and the detection reagent, and step (b) may include partitioning the solution between multiple reaction vessels, such that the probability of finding unbound first detection reagent and unbound second detection reagent in the same container is less than 1 in 10. Alternatively, step (a) of embodiment (15) may further include forming a solution comprising the analyte and the detection reagent, and step (b) may include partitioning the solution between multiple reaction vessels, such that the probability of finding unbound first detection reagent and unbound second detection reagent in the same container is less than 1 in 100. In addition, step (a) of embodiment (15) may further include forming a solution comprising the analyte and the detection reagent, and step (b) may include partitioning the solution between multiple reaction vessels, such that the probability of finding unbound first detection reagent and unbound second detection reagent in the same container is less than 1 in 1000. In addition, step (a) of embodiment (15) may further include forming a solution comprising the analyte and the detection reagent, and step (b) may include partitioning the solution between multiple reaction containers, such that the probability of finding unbound first detection reagent and unbound second detection reagent in the same container is less than 1 in 10,000.

[0070] Embodiment (16): A method for detecting an analyte in a sample, the method comprising: (a) binding the analyte to a capture reagent and a first and a second detection reagent to form a detection complex, each detection complex comprising a capture reagent, an analyte, a first detection reagent, and a second detection reagent, wherein (i) the first detection reagent has a first detectable label and the second detection reagent has a second detectable label, (ii) the capture reagent is on a surface; (b) separating the analyte between a plurality of reaction vessels such that the plurality of reaction vessels contain one or less analyte; and (c) detecting the number of analyte molecules by counting the number of reaction vessels containing the first and the second detectable labels. In this embodiment, the capture reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the capture reagent is an antibody. Similarly, the first detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the first detection reagent is an antibody. In addition, the second detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the second detection reagent is an antibody. For example, the capture reagent, the first and second detection reagents are antibodies to the analyte.

[0071] Step (b) of embodiment (16) may further include partitioning the solution among multiple reaction containers, so that the probability of finding unbound first detection reagent and unbound second detection reagent in the same container is less than 1 in 10. In addition, step (b) of embodiment (16) may further include partitioning the solution among multiple reaction containers, so that the probability of finding unbound first detection reagent and unbound second detection reagent in the same container is less than 1 in 100. Step (b) of embodiment (16) may further include partitioning the solution among multiple reaction containers, so that the probability of finding unbound first detection reagent and unbound second detection reagent in the same container is less than 1 in 1000. In addition, step (b) of embodiment (16) may further include partitioning the solution among multiple reaction containers, so that the probability of finding unbound first detection reagent and unbound second detection reagent in the same container is less than 1 in 10000.

[0072] The capture agent in the detection complex of embodiment (16) can be on the surface before the capture agent is bound to the analyte; or the capture agent in the detection complex is bound to the analyte before the capture agent is fixed on the surface. In one embodiment, the detection agent can include a targeting portion, and the surface can include a targeting portion complement. The targeting portion and the targeting agent binding partner are selected from the following binding pairs: avidin-biotin, streptavidin-biotin, receptor-ligand, antibody-antigen, nucleic acid-nucleic acid complement.

[0073] The surface of embodiment (16) is a particle, and optionally, the capture reagent is fixed on a plurality of particles, and the distribution of the analyte is achieved by binding the analyte to the capture reagent and distributing the particles into a plurality of reaction vessels. The capture reagent can be fixed on a plurality of particles, and the distribution of the analyte is achieved by distributing the particles into a plurality of reaction vessels and then binding the analyte to the capture reagent.

[0074] Embodiment (16) may further include separating the plurality of particles into a plurality of reaction vessels, wherein the plurality of particles comprise a targeting moiety, the capture reagent comprises a targeting moiety complement, and separation of the analytes is achieved by binding the targeting moiety complement to the targeting moiety. Embodiment (16) may also include washing the particles before and / or after the separation step.

[0075] The surface of embodiment (16) can be a position within one of the reaction vessels. In this embodiment, the capture reagent can be fixed on the surface of multiple reaction vessels, and the separation of the analyte is achieved by binding the analyte to the capture reagent. Optionally, the reaction vessel has a surface fixed with a targeting portion, the capture reagent includes a targeting portion complement, and the distribution of the analyte is achieved by binding the targeting portion complement to the targeting portion. In this specific embodiment, the method can further include washing the reaction vessel before the detection step.

[0076] The plurality of reaction vessels of embodiment (16) may comprise a nanopore array. The plurality of reaction vessels may comprise at least 10,000 reaction vessels. In one embodiment, the reaction vessel has a volume of less than 100 nL. Optionally, less than 50% of the reaction vessels contain analyte at the time of detection, less than 10% of the reaction vessels contain analyte at the time of detection, less than 1% of the reaction vessels contain analyte at the time of detection, and / or less than 0.1% of the reaction vessels contain analyte at the time of detection.

[0077] In one aspect of embodiment (16), the first detectable label is the first enzyme of the coupled enzyme reaction system, and the second detectable label is the second enzyme of the coupled enzyme reaction system, and step (d) may include adding one or more substrates of the reaction system, producing the product of the enzyme reaction system and counting the reaction vessels containing the product. In this embodiment, the product can be produced only when the first enzyme and the second enzyme are extremely close (e.g., the first and second enzymes are within 200nM of each other, or the first and second enzymes are within 50nM of each other). For example, the first enzyme is an oxidase, the second enzyme is a peroxidase, and the substrate comprises an oxidase substrate and a labeled AmplexRed or luminol derivative. In this embodiment, the oxidase can be glucose oxidase, and the oxidase substrate is glucose. In one embodiment, the reaction catalyzed by the first and second enzymes in the detection complex results in the immobilization of labeled AmplexRed or luminol on the surface, and optionally, the method may include measuring the labeled AmplexRed or luminol on the surface. The labeled AmplexRed or Luminol is optionally Biotin-AmplexRed or Luminol, and the method may comprise adding labeled streptavidin and measuring the label on the streptavidin.

[0078] The step (d) of embodiment (16) may include measuring the proximity dependent signal (proximity dependent signal) produced when the first and second detectable markers are combined with the same analyte molecule, and calculating the number of reaction vessels producing proximity dependent signals, for example, producing proximity dependent signals by PLA-RCA. For example, the first detectable marker may be a FRET donor, and the detectable marker is a FRET acceptor, and the proximity dependent signal is measured by exciting the FRET donor and measuring the emission from the FRET acceptor. In an example, the first and second detectable markers may be measured independently. Optionally, the first and second detectable markers are luminescent markers different from each other in terms of spectral characteristics. In an example, the first detectable marker is a first enzyme that reacts with a first substrate to produce a first signal, and the second detectable marker is a second enzyme that reacts with a second substrate to produce a second signal, and the step (d) of embodiment (16) may include adding the first enzyme substrate and the second enzyme substrate, and calculating the number of reaction vessels producing the first and second signals. The first and second signals may be changes in optical absorbance with different spectral characteristics. Optionally, the first and second signals are luminescent signals with different spectral characteristics. The first and second enzymes can be hydrolases, for example selected from phosphatases, sulfatases, beta-galactosidases, glucuronidases, or combinations thereof, and the first and second substrates are selected from phosphate, sulfate, beta-galactosidase and glucuronidase modified stabilized dioxetane, 4-methylumbelliferyl, fluorescein or combinations thereof. In a specific example, the first and second enzymes are selected from horseradish peroxidase, beta-galactosidase, and alkaline phosphatase. The detection step of embodiment (16) can include detection via light scattering, light absorbance, fluorescence, chemiluminescence, electrochemiluminescence, luminescence, radioactivity, magnetic field or a combination thereof.

[0079] Embodiment (17): A kit for detecting an analyte in a sample, the kit comprising in one or more vials, containers or compartments: (a) a first detection reagent comprising a first detectable marker; (b) a second detection reagent comprising a second detectable marker; and (c) a plurality of reaction containers configured to contain one or fewer analyte molecules.

[0080] Embodiment (18): A kit for detecting an analyte in a sample, the kit comprising in one or more vials, containers or compartments: (a) a first detection reagent comprising a first detectable label; (b) a second detection reagent comprising a second detectable label; (c) a surface comprising a capture reagent; and (d) a plurality of reaction vessels configured to contain one or fewer analyte molecules.

[0081] The first and second detection reagents of embodiments (17) and (18) may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, an aptamer, or a combination thereof. In one example, the first and second detection reagents comprise an antibody. The capture antibody may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the capture antibody may comprise an antibody. In one embodiment, the capture reagent may comprise a targeting moiety and the surface may comprise a targeting moiety complement, for example, the targeting moiety and the targeting agent binding partner are selected from the following binding pairs: avidin-biotin, streptavidin-biotin, receptor-ligand, antibody-antigen, nucleic acid-nucleic acid complement.

[0082] The surface of embodiments (17) and (18) can be a particle, and for example, the capture reagent is fixed on a plurality of particles. Alternatively, the surface is a position within one of the reaction vessels, and for example, the capture reagent is fixed on the surface of a plurality of reaction vessels. Alternatively, the surface is a position within one of the reaction vessels, and for example, the capture reagent is fixed on the surface of a plurality of reaction vessels. Optionally, the reaction vessel has a surface on which a targeting portion is fixed, and the capture reagent comprises a targeting portion complement. The plurality of reaction vessels may comprise water droplets dispersed in a nanopore array or an oil-in-water emulsion. The plurality of reaction vessels may comprise at least 10,000 reaction vessels, and optionally, the plurality of reaction vessels have a volume of less than 100 nL.

[0083] In the kit of embodiments (17) and (18), the first detectable label can be the first enzyme of the coupled enzyme reaction system, and the second detectable label is the second enzyme of the coupled enzyme reaction system, and the kit can include one or more substrates of the reaction system in one or more additional vials, containers or compartments. For example, the first enzyme is an oxidase, the second enzyme is a peroxidase, and the substrate includes an oxidase substrate and a labeled Amplex Red or luminol derivative. In a specific embodiment, the oxidase is glucose oxidase, and the oxidase substrate is glucose. The first and second detectable labels can be components of a proximity-dependent system, for example, the first detectable label is a FRET donor, and the detectable label is a FRET acceptor. The first and second detectable labels can be measured independently. Optionally, the first and second detectable labels are luminescent labels that are different from each other in terms of spectral characteristics.

[0084] In the kit of embodiments (17) and (18), the first detectable label is a first enzyme that reacts with the first substrate to produce a first signal, and the second detectable label is a second enzyme that reacts with the second substrate to produce a different second signal, and the kit can include the first enzyme substrate and the second enzyme substrate in one or more vials, containers or compartments. Optionally, the first and second signals are changes in light absorption with different spectral characteristics. In one embodiment, the first and second signals are luminescent signals with different spectral characteristics. The first and second enzymes can be hydrolases. In one embodiment, the first and second enzymes are selected from phosphatases, sulfatases, beta-galactosidases, glucuronidases, or a combination thereof. The first and second substrates can be selected from stable dioxetanes, 4-methylumbelliferone, fluorescein modified with phosphates, sulfates, beta-galactosidase and glucuronidase, or a combination thereof. Optionally, the first and second enzymes are selected from horseradish peroxidase, beta-galactosidase and alkaline phosphatase.

[0085] Embodiment (19): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to a capture reagent, a first detection reagent having a first detectable label, and a second detection reagent having a second detectable label to form a complex, wherein the capture reagent in the complex is immobilized on a surface; (b) cross-linking the first and second detection reagents to form a cross-linked product; (c) releasing the cross-linked product from the surface into an eluate; (d) counting the individual cross-linked products in the eluate containing both the first and second detectable labels. In this embodiment (19), the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific embodiment, the capture reagent is an antibody. Similarly, the first detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific embodiment, the first detection reagent is an antibody. In addition, the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and specifically, the second detection reagent can be an antibody. In a specific embodiment, the capture reagent and the first and second detection reagents are antibodies to the analyte.

[0086] Embodiment (19) may further include adding a cross-linking agent to cross-link the first and second detection reagents, for example, the first and second detection reagents include reactive moieties, and the cross-linking agent is a multifunctional cross-linking agent attached to the reactive moieties. For example, the reactive moieties include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The cross-linking agent may include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The first and second detection reagents may include a binding moiety, and the cross-linking agent is a multivalent binding partner of the binding moiety. In one embodiment, the first and second detection reagents are antibodies of an animal species, and the cross-linking agent is a multivalent anti-species antibody targeting antibodies of the animal species. The first and second detection reagents may include biotin and the cross-linker is streptavidin; the first and second detection reagents include streptavidin and the cross-linker is biotin; the first and second detection reagents are linked to streptavidin and the cross-linker is a polymer comprising multiple biotin molecules; and / or the first and second detection reagents comprise first and second nucleic acid probes, respectively, and the cross-linker is an oligonucleotide, which may comprise a sequence complementary to the first nucleic acid probe and a different sequence complementary to the second nucleic acid probe.

[0087] The surface of embodiment (19) may include particles, reaction vessels, such as tubes or ampoules, and / or the surface may include holes of a multiwell plate. The method of embodiment (19) may further include collecting the particles and washing the particles to remove impurities, and optionally, measuring the first and second detectable labels by measurement of light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a specific embodiment, the first and second detectable labels include ECL labels, and the counting step may include measuring the ECL signal.

[0088] Embodiment (20): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers, or compartments: (a) a surface comprising an immobilized capture reagent; (b) a first detection reagent having a first detectable label; (c) a second detection reagent having a second detectable label; and (d) a cross-linking agent that reacts with the first and second detection reagents.

[0089] The first and second detection reagents of embodiment (20) may include a reactive portion, and the crosslinker is a multifunctional crosslinker connected to the reactive portion. The reactive portion may include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof; and the crosslinker may include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The first and second detection reagents of embodiment (20) may include a binding portion, and the crosslinker is a multivalent binding partner of the binding portion, for example, the first and second detection reagents are antibodies of animal species, and the crosslinker is a multivalent anti-species antibody targeting antibodies of animal species; the first and second detection reagents include biotin, and the crosslinker is streptavidin; the first and second detection reagents include streptavidin, and the crosslinker is biotin; the first and second detection reagents are connected to streptavidin, and the crosslinker is a polymer comprising multiple biotin molecules; and / or the first and second detection reagents respectively include first and second nucleic acid probes, and the crosslinker is an oligonucleotide that can include a sequence complementary to the first nucleic acid probe and a different sequence complementary to the second nucleic acid probe.

[0090] The surface of embodiment (20) may include a particle, a hole of a porous plate, or a reaction vessel, such as a tube or an ampoule. In addition, the surface may include a plurality of different binding domains, and the capture reagents are located on different binding domains on the surface. If the surface is a hole, the hole may include a plurality of different binding domains, and the capture reagents are located on different binding domains in the hole. The surface may also include an electrode.

[0091] Embodiment (21): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to a capture reagent, a first and a second detection reagent to form a detection complex, wherein the first detection reagent may include a first detectable label and a first nucleic acid probe, the second detection reagent may include a second detectable label and a second nucleic acid probe, and the capture reagent in the complex is immobilized on a surface; (b) cross-linking the first and second detection reagents as follows: (i) hybridizing the first probe with the second probe, (ii) hybridizing the first and second probes with a third nucleic acid having a region complementary to the first and second probes, or (iii) connecting the first and second probes; (c) releasing the cross-linked product from the surface into an eluate; and (d) counting individual cross-linked products in the eluate containing the first and second detectable labels.

[0092] The capture reagent of embodiment (21) can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the capture reagent is an antibody. Similarly, the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the first detection reagent is an antibody; the second detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a template or an aptamer, for example, the second detection reagent is an antibody. In a specific embodiment, the capture reagent and the first and second detection reagents are antibodies to the analyte.

[0093] The surface of embodiment (21) may include particles, reaction vessels, such as tubes or ampoules, or wells of a multiwell plate. The method of embodiment (21) may further include collecting the particles and washing the particles to remove impurities. The first and second detectable labels may be measured by light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a specific embodiment, the first and second detectable labels include ECL labels, and the counting step may include measuring the ECL signal

[0094] Embodiment (22): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising an immobilized capture reagent; (b) a first detection reagent having a first detectable label and a first nucleic acid probe; (c) a second detection reagent having a second detectable label and a second nucleic acid probe; and (d) a third nucleic acid having regions complementary to the first and second nucleic acid probes.

[0095] The surface of embodiment (22) may include a particle, a hole of a porous plate, or a reaction vessel, such as a tube or an ampoule. The surface may include a plurality of different binding domains, and the capture reagents are located on different binding domains on the surface. If the surface is a hole, the hole may include a plurality of different binding domains, and the capture reagents are located on different binding domains in the hole. The surface may optionally include an electrode.

[0096] Embodiment (23): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to a capture reagent, a first detection reagent, and a second detection reagent to form a complex, wherein the first detection reagent may include a first nucleic acid probe, the second detection reagent may include a second nucleic acid probe, and the capture reagent in the complex is immobilized on a surface; (b) extending the second nucleic acid probe to form an extended sequence comprising a detectable label, the extension being dependent on the colocalization of the first and second nucleic acid probes in the complex; (c) releasing the extended sequence from the surface into an eluate; and (d) counting individual extended sequences in the eluate. In this embodiment, the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer. In a specific example, the capture reagent is an antibody. Similarly, the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific embodiment, the first detection reagent is an antibody. The second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope or aptamer, and in particular, the second detection reagent is an antibody. In a specific embodiment, the capture reagent and the first and second detection reagents are antibodies to the analyte.

[0097] The surface of embodiment (23) may include particles, reaction vessels, such as tubes or ampoules; or wells of a multiwell plate. The method of embodiment (23) may further comprise collecting and washing the particles to remove impurities.

[0098] The label of embodiment (23) can be measured by light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a specific embodiment, the label can include an ECL label, and the counting step can include measuring the ECL signal.

[0099] The extension step of embodiment (23) may include combining the probe with the template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step may also include combining the first probe with the template nucleic acid sequence to form a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extension step may include combining the first probe with the template nucleic acid sequence, combining the second probe with the template sequence, and connecting the first and second probes. Optionally, the label is a fluorescent label, and the counting of individual extended sequences may include single-molecule fluorescence detection, for example, it may include fluorescence correlation spectroscopy and / or fluorescence cross-correlation spectroscopy. Single-molecule fluorescence detection may include flowing an eluent through a capillary, focusing a light source on a volume within the capillary to produce an interrogation zone, and observing the interrogation zone with a light detector to detect the passage of fluorescent molecules passing through the interrogation zone. Single-molecule fluorescence detection may also include flowing an eluent through a capillary, focusing a light source on a volume within the capillary to produce an interrogation zone, and observing the interrogation zone with a light detector to detect the passage of fluorescent molecules passing through the interrogation zone.

[0100] Embodiment (24): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to a capture reagent, a first detection reagent having a first detectable label, and a second detection reagent having a second detectable label to form a complex, wherein the capture reagent in the complex is immobilized on a surface; (b) releasing the formed complex from the surface by dissociating the immobilized capture reagent from the surface into an eluent; and (c) counting individual products in the eluent that contain both the first and second detectable labels. In this embodiment, the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the capture reagent is an antibody; the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the first detection reagent is an antibody; the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the second detection reagent is an antibody; and in a specific embodiment, the capture reagent and the first and second detection reagents are antibodies to the analyte.

[0101] The surface of embodiment (24) may include particles, reaction vessels, such as tubes or ampoules, and / or wells of a multiwell plate. The method of embodiment (24) may include collecting the particles and washing the particles to remove impurities. The first and second detectable labels may be measured by light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof, and in a specific embodiment, the first and second detectable labels include ECL labels, and the counting step may include measuring the ECL signal.

[0102] Embodiment (25): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent on a surface comprising a capture reagent for the analyte; (ii) a first detection reagent for the analyte linked to a first nucleic acid probe; and (iii) a second detection reagent for the analyte linked to a second nucleic acid probe; thereby forming a complex on the surface, the complex comprising a binding reagent, the analyte, and the first and second detection reagents; (b) extending the second probe to form an extended sequence using an extension process that requires the first and second probes to be in proximity; and (c) measuring the amount of the extended sequence bound to the surface. In this embodiment, the capture reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, for example, the capture reagent is an antibody; the first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, for example, the first detection reagent is an antibody; the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, for example, the second detection reagent is an antibody; and in a specific embodiment, the capture reagent and the first and second detection reagents are antibodies to the analyte.

[0103] The extension sequence of embodiment (25) may comprise one or more detection sequences, and the measuring step may comprise contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences; the extension sequence may comprise one or more modified bases, and the measuring step may comprise contacting the extension sequence with a plurality of detectable moieties capable of binding to the one or more modified bases; and / or the extension sequence may comprise one or more labeled bases, and the measuring step may comprise detecting the presence of the one or more labeled bases. The one or more modified bases comprise an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimotope, and the plurality of detectable moieties each comprise a binding partner for the one or more modified bases and a detectable label. The one or more modified bases may comprise streptavidin and the multiple detectable moieties each comprise biotin and a detectable label; the one or more modified bases comprise biotin and the multiple detectable moieties each comprise streptavidin and a detectable label; the one or more modified bases comprise avidin and the multiple detectable moieties each comprise biotin and a detectable label; and / or the one or more modified bases comprise biotin and the multiple detectable moieties each comprise avidin and a detectable label.

[0104] Step (a) of embodiment (25) may include binding the analyte to the following species in the following order: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte; binding the analyte to the following species in the following order: (i) a detection reagent for the analyte; and (ii) a capture reagent on a surface; or binding the analyte to the following species simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte. The extension step may include binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction; or binding the probe to a template nucleic acid sequence to form a circular nucleic acid template, and extending the circular template by rolling circle amplification. In this embodiment, the extended probe may still be located on the surface after the probe is extended, for example, the complex is still bound to the surface after the extension step. The extension step may include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (single strand displacement amplification), 3SR (self-sustained synthesis reaction), or an isothermal amplification method. In a specific embodiment, the extension step may include an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0105] The extension process of embodiment (25) may include contacting the complex formed in step (a) with a linker sequence, wherein the linker sequence comprises (i) an internal sequence complementary to the second probe and (ii) a double-end sequence complementary to the non-overlapping region of the first probe. The process may further include connecting the double-end sequence of the linker oligonucleotide to form a circular target sequence that hybridizes with the first and second probes. The extension process of embodiment (25) may also include contacting the complex formed in step (a) with a first linker oligonucleotide sequence and a second linker oligonucleotide sequence, wherein the first linker oligonucleotide sequence comprises a first linker probe sequence that is complementary to the first region of the first probe and the first region of the second probe, and the second linker oligonucleotide comprises a second linker probe sequence that is complementary to the second non-overlapping region of the first probe and the second non-overlapping region of the second probe. The process may also include connecting the first and second linker oligonucleotides to form a circular target sequence that hybridizes with both the first and second probes.

[0106] The surface of embodiment (25) may include a particle or a hole of a porous plate. The surface may include a plurality of different binding domains, and the capture reagent is located on two different binding domains on the surface. If the surface is a hole, the hole may include a plurality of different binding domains, and the capture reagent is located on two different binding domains in the hole. The surface may contain a plurality of different binding domains, and the capture reagent is located on the same binding domain on the surface, and if the surface is a hole, the hole may include a plurality of different binding domains, and the capture reagent is located on the same binding domain in the hole. The surface may include electrodes, and the measuring step may include applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. The method optionally includes collecting particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. The measuring step may further include binding the extended sequence to a detection probe having a detectable label, measuring the detectable label, and correlating the measurement with the amount of analyte in the sample, wherein the detection probe comprises a nucleic acid sequence complementary to the region of the extended sequence. The detectable label can be measured by light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a specific embodiment, the detectable label is an ECL label, and the measuring step may include measuring the ECL signal.

[0107] Embodiment (26): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers, or compartments: (a) a surface comprising a capture reagent for the analyte; (b) a first detection reagent for the analyte linked to a first nucleic acid probe; and (c) a second detection reagent for the analyte linked to a second nucleic acid probe.

[0108] The capture reagent of embodiment (26) may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the capture reagent may include an antibody; the first detection reagent may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the first detection reagent may include an antibody; the second detection reagent may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the second detection reagent may include an antibody; and the surface may include particles and / or wells of a multi-well plate. The surface may include a plurality of different binding domains, and the capture reagent is located on two different binding domains on the surface; and if the surface is a well, the well may include a plurality of different binding domains, and the capture reagent is located on two different binding domains within the well. Optionally, the surface may include a plurality of different binding domains, and the capture reagent is located on the same binding domain on the surface, and if the surface is a well, the well may include a plurality of different binding domains, and the capture reagent is located on the same binding domain within the well. The surface may include an electrode.

[0109] The surface of embodiments 1-26 may include the inner surface of an assay container (e.g., a test tube, a cuvette, a flow cell, a FACS cell sorter, a cartridge, or a well of a multi-well plate). The surface may also include a slide, an assay chip, or an assay array; a needle, a probe, a bead, or a filter medium; a lateral flow medium, such as a filter membrane.

[0110] Embodiment (27): A method for detecting an analyte of interest in a sample comprising one or more analyte molecules, the method comprising: (a) contacting the sample with a surface comprising a plurality of distinguishable binding regions located on the surface, each distinguishable binding region comprising a plurality of capture reagents for one or more analyte molecules in the sample; (b) binding the one or more analyte molecules to (i) one or more capture reagents on the surface; (ii) a first detection reagent for the analyte comprising a first detectable label, and (iii) a second detection reagent for the analyte comprising a second detectable label; thereby forming a detection complex on the distinguishable binding domains on the surface, the detection complex comprising the capture reagent, the analyte, and a first and a second detection reagent, wherein the first and the second detectable labels are different labeled compounds; (c) determining the presence or absence of the analyte molecule in each binding region; and (d) identifying the number of binding regions containing the analyte molecules and / or identifying the number of binding regions that do not contain the analyte molecules. The identification step may include imaging the optical signal from the surface to generate an image comprising a plurality of pixels, and each distinguishable binding region is mapped to one or more pixels in the image. The distinguishable binding regions may be elements of an array and / or configured to separate individual particles. Each resolvable binding area can be a single nanopore with a volume <100 nL and / or at least 99% of the binding areas contain zero or one analyte molecule; at least about 95% of the binding areas contain zero or one analyte molecule; wherein at least about 80% of the binding areas contain zero or one analyte molecule; or at least about 50% of the binding areas contain zero or one analyte molecule. The concentration of analyte molecules in the sample can be determined, at least in part, using a calibration curve of the number of binding areas containing at least one or one analyte molecule, a Poisson distribution analysis, and / or a Gaussian distribution analysis.

[0111] The surface of embodiment (27) may include multiple particles, each particle including multiple capture reagents for analyte molecules, wherein the multiple particles are distributed in multiple distinguishable binding areas, and the method may include: (i) binding one or more analyte molecules to one or more binding reagents on the surface, and first and second detection reagents for each of the one or more analyte molecules, wherein the first and second detection reagents include first and second detectable labels, respectively; (ii) distributing multiple particles between the array of fecal binding areas; and (iii) determining the presence or absence of analyte molecules in each distinguishable binding area to identify the number of binding areas containing analyte molecules and / or identify the number of binding areas without analyte molecules, wherein optionally, each distinguishable binding area can be a single nanopore with a volume of <100nL, and / or at least 99% of the binding areas contain zero or one analyte molecule; at least about 95% of the binding areas contain zero or one analyte molecule; wherein at least about 80% of the binding areas contain zero or one analyte molecule; or at least about 50% of the binding areas contain zero or one analyte molecule.

[0112] In embodiment (27), the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the capture reagent is an antibody; the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the first detection reagent is an antibody; the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the second detection reagent is an antibody. In a specific embodiment, the capture reagent and the first and second detection reagents are antibodies to the analyte.

[0113] Step (a) of embodiment (27) may include binding the analyte to the following species in the following order: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte; binding the analyte to the following species in the following order: (i) a first and a second detection reagent for the analyte; and (ii) a capture reagent on a surface; or binding the analyte to the following species simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte.

[0114] The surface of embodiment (27) may include particles or holes of a porous plate. In a specific embodiment, the surface may include electrodes, and the identification step may include applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The method of embodiment (27) may further include collecting particles on the electrodes and applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The first detectable marker is measured by light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof; and / or the second detectable marker is measured by light scattering, optical absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. The first and second detectable markers can be measured independently, and in one example, the first and second detectable markers are luminescent markers that are different from each other with respect to spectral characteristics.

[0115] The surface of embodiment (27) may include the inner surface of an assay container (e.g., a test tube, a cuvette, a flow cell, a FACS cell sorter, a cartridge, or a well of a multiwell plate). The surface may also include a slide, an assay chip, or an assay array; a needle, a probe, a bead, or a filter medium; a lateral flow medium, such as a filter membrane.

[0116] Embodiment (28): A kit for detecting an analyte of interest in a sample containing one or more analyte molecules, the kit comprising: (a) a surface comprising a plurality of distinguishable binding areas located on the surface, each distinguishable binding area comprising a plurality of capture reagents for one or more analyte molecules in the sample; (b) a first detection reagent for the analyte comprising a first detectable label, and (c) a second detection reagent for the analyte comprising a second detectable label; wherein the first and second detectable labels are different labeling compounds.

[0117] The distinguishable binding areas of embodiment (28) can be elements of an array and / or configured as separate individual particles. Optionally, each distinguishable binding area is optionally an individual nanopore with a volume of <100 nL. The surface can include a plurality of particles, each particle including a plurality of capture reagents for an analyte molecule, wherein the plurality of particles are distributed over a plurality of distinguishable binding areas, and the kit can include: a first and a second detection reagent for each of the one or more analyte molecules, wherein the first and the second detection reagents include a first and a second detectable label, respectively.

[0118] In embodiment (28), the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the capture reagent is an antibody; the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the first detection reagent is an antibody; the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the second detection reagent is an antibody. In a specific embodiment, the capture reagent and the first and second detection reagents are antibodies to the analyte.

[0119] The surface of embodiment (28) may include holes of a particle or a porous plate. In a specific embodiment, the surface may include electrodes, and the identification step may include applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The first detectable label is measured by light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof; and / or the second detectable label is measured by light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. The first and second detectable labels can be measured independently, and in one embodiment, the first and second detectable labels are luminescent labels that are different from each other with respect to spectral characteristics.

[0120] The surface of embodiment (28) may include the inner surface of an assay container (e.g., a test tube, a cuvette, a flow cell, a FACS cell sorter, a cartridge, or a well of a multiwell plate). The surface may also include a slide, an assay chip, or an assay array; a needle, a probe, a bead, or a filter medium; a lateral flow medium, such as a filter membrane.

[0121] Embodiment (29): A method for detecting HIV p24 in a sample, comprising: (a) binding HIV p24 to: (i) a capture reagent on a surface, the surface comprising a capture reagent for HIV p24, and an anchor reagent comprising an anchor oligonucleotide; (ii) a first detection reagent for HIV p24 linked to a first nucleic acid probe; and (iii) a second detection reagent for HIV p24 linked to a second nucleic acid probe; thereby forming a complex on the surface, the complex comprising the binding reagent, HIV p24, and the first and second detection reagents; (b) extending the second probe to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence using an extension process that requires the first and second probes to be in proximity; (c) hybridizing the anchor sequence with the anchor sequence complement; and (d) measuring the amount of the extended sequence bound to the surface.

[0122] The capture reagent of embodiment (29) can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer. In a specific embodiment, the capture reagent is an antibody. Similarly, the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific embodiment, the first detection reagent is an antibody. The second detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific example, the second detection reagent is an antibody. More specifically, the capture reagent and the first and second detection reagents are antibodies to HIV p24.

[0123] In embodiment (29), the anchor oligonucleotide sequence may include a single-stranded oligonucleotide sequence or a double-stranded oligonucleotide sequence. In this embodiment, the extension sequence may include one or more detection sequences, and the measuring step may include contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences. The extension sequence may also include one or more modified bases, and the measuring step may include contacting the extension sequence with a plurality of detectable portions capable of binding to the one or more modified bases. The extension sequence may further include one or more labeled bases, and the measuring step may include detecting the presence of the one or more labeled bases. The one or more modified bases may include an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimotope, and the plurality of detectable portions each include a binding partner of the one or more modified bases and a detectable label. For example, one or more modified bases comprise streptavidin and the plurality of detectable moieties each comprise biotin and a detectable label; one or more modified bases comprise biotin and the plurality of detectable moieties each comprise streptavidin and a detectable label; or one or more modified bases comprise avidin and the plurality of detectable moieties each comprise biotin and a detectable label; and / or one or more modified bases comprise biotin and the plurality of detectable moieties each comprise avidin and a detectable label.

[0124] Step (a) of embodiment (29) may comprise binding HIV p24 to the following species in the following order: (i) a capture reagent on a surface; and (ii) a detection reagent for HIV p24. Alternatively, step (a) may comprise binding HIV p24 to the following species in the following order: (i) a detection reagent for HIV p24; and (ii) a capture reagent on a surface; or step (a) may comprise binding HIV p24 to the following species simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a detection reagent for HIV p24.

[0125] The extension step of embodiment (29) may include binding the probe to the template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step may further include binding the probe to the template nucleic acid sequence to form a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extended probe may still be located on the surface after the probe is extended, for example, the complex is still bound to the surface after the extension step. The extended probe is bound to the anchoring agent at a position within 10-100um of the complex position on the surface. In a specific embodiment, the extended probe is bound to the anchoring agent at a position less than 100um, less than 50um, or more particularly less than 10um of the complex position on the surface. The extension step may include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (single-strand displacement amplification), 3SR (automatic maintenance synthesis reaction), or isothermal amplification method. In a specific embodiment, the extension step may include an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0126] The extension process of embodiment (29) may include contacting the complex formed in step (a) with a linker sequence, wherein the linker sequence comprises (i) an internal sequence complementary to the second probe and (ii) a double-end sequence complementary to the non-overlapping region of the first probe. The method may further include connecting the double-end sequence of the oligonucleotide to form a circular target sequence that hybridizes with both the first and second probes. Alternatively, the extension process may include contacting the complex formed in step (a) of embodiment (29) with a first linker oligonucleotide sequence and a second linker oligonucleotide sequence, wherein the first linker oligonucleotide sequence comprises a first linker probe sequence complementary to a first region on the first probe and a first region on the second probe, and the second linker oligonucleotide sequence comprises a second probe sequence complementary to a second non-overlapping region of the first probe and a second non-overlapping region of the second probe; and optionally, connecting the first and second linker oligonucleotides to form a circular target sequence that hybridizes with both the first and second probes.

[0127] The surface of embodiment (29) may include particles and / or holes of a porous plate. The surface may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole of a plate, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may also include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain on the surface. If the surface is a hole of a plate, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain in the hole. The capture agent and the anchoring agent are within 10-100nm on the surface. In a specific embodiment, the surface may include electrodes, and the measuring step may also include applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal, and optionally, the method of embodiment (29) further includes collecting particles on the electrodes and applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal.

[0128] The measuring step of embodiment (29) may include combining the extended sequence with a detection probe having a detectable label, measuring the detectable label, and correlating the measurement with the amount of p24 in the sample, wherein the detection probe comprises a nucleic acid sequence complementary to a region of the extended sequence. The detectable label may be measured by measurement of light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, a magnetic field, or a combination thereof. In a specific embodiment, the detectable label is an ECL label, and the measuring step may include measuring an ECL signal.

[0129] Embodiment (30): A kit for detecting HIV p24 in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising (i) a capture reagent for HIV p24, and (ii) an anchoring reagent comprising an anchoring oligonucleotide sequence; (b) a first detection reagent for HIV p24 linked to a first nucleic acid probe; and (c) a second detection reagent for HIV p24 linked to a second nucleic acid probe.

[0130] The capture reagent of embodiment (30) may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific embodiment, the capture reagent may include an antibody. Similarly, the first detection reagent may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific embodiment, the first detection reagent may include an antibody. Similarly, the second detection reagent may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific embodiment, the second detection reagent may include an antibody.

[0131] The surface of embodiment (30) may include particles and / or holes of a porous plate. The surface may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may also include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain on the surface; and / or if the surface is a hole, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain in the hole. The capture agent and the anchoring agent are within 10-100nm on the surface. In a specific embodiment, the surface may include an electrode.

[0132] Embodiment (31): A method for detecting HIV p24 in a sample, comprising: (a) binding HIV p24 to: (i) a capture reagent for HIV p24 on a surface, the surface comprising a capture reagent and an anchoring reagent; (ii) a first detection reagent for HIV p24 comprising a first proximal probe, and (iii) a second detection reagent for HIV p24 comprising a second proximal probe; thereby forming a detection complex on the surface, the detection complex comprising the capture reagent, HIV p24, and an anchoring reagent; p24 and first and second detection reagents; (b) contacting the detection complex formed in (c) with a linker sequence, wherein the linker sequence comprises (i) an internal sequence complementary to the second proximal probe and (ii) a double-end sequence complementary to a non-overlapping region of the first proximal probe; (c) hybridizing the linker sequence to the first and second proximal probes; (d) connecting the double-end sequences of the linker oligonucleotides to form a circular target sequence that hybridizes to both the first and second proximal probes; (e) extending the second proximal probe by rolling circle amplification of the target sequence to produce an amplicon comprising a binding domain that binds to an anchoring reagent; (f) binding the amplicon to the anchoring reagent; and (g) measuring the amount of the amplicon on the surface.

[0133] Embodiment (32): A method for detecting HIV p24 in a sample, comprising: (a) binding HIV p24 to: (i) a capture reagent for HIV p24 on a surface, the surface comprising a capture reagent and an anchoring reagent; (ii) a first detection reagent for HIV p24 comprising a first proximal probe, and (iii) a second detection reagent for HIV p24 comprising a second proximal probe; thereby forming a detection complex on the surface, the detection complex comprising the capture reagent, HIV p24, and an anchoring reagent; p24 and a first and a second detection reagent; (b) contacting the detection complex formed in (c) with a first linker oligonucleotide and a second linker oligonucleotide, wherein (i) the first end of the first linker and the first end of the second linker are complementary to two non-overlapping regions of the first proximal probe, and (ii) the second end of the first linker and the second end of the second linker are complementary to two non-overlapping regions of the first proximal probe; (c) hybridizing the first and the second linker oligonucleotides with the first and the second proximal probes; (d) connecting the first and the second linker oligonucleotides to form a circular target sequence that hybridizes to both of the first and the second proximal probes; (e) extending the second proximal probe by rolling circle amplification of the target sequence to produce an amplicon comprising a binding domain that binds to an anchoring reagent; (f) binding the amplicon to the anchoring reagent; and (g) measuring the amount of the amplicon on the surface.

[0134] The capture reagent of embodiments (31) and (32) can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific embodiment, the capture reagent is an antibody. Similarly, the first detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the first detection reagent is an antibody. In addition, the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, for example, the second detection reagent is an antibody. In a specific embodiment of embodiments (31) and (32), the capture reagent and the first and second detection reagents are antibodies to HIV p24.

[0135] The anchoring agent of embodiments (31) and (32) may include an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimotope. In one embodiment, the binding epitope may include an aptamer, and the anchoring agent may include an aptamer ligand. The binding domain may include a nucleic acid sequence, and the anchoring agent may include a DNA binding protein; and / or the anchoring agent may include an oligonucleotide sequence, and the amplicon may include a complementary oligonucleotide sequence.

[0136] The amplicons of embodiments (31) and (32) may comprise one or more detection sequences, and the measuring step may comprise contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences. In addition, the amplicons may further comprise one or more modified bases, and the measuring step may comprise contacting the extension sequence with a plurality of detectable moieties capable of binding to the one or more modified bases. In addition, the amplicons may further comprise one or more labeled bases, and the measuring step may comprise detecting the presence of the one or more labeled bases. The one or more modified bases may comprise an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimotope, and the plurality of detectable moieties each comprise a binding partner for the one or more modified bases and a detectable label. The one or more modified bases may comprise streptavidin and the plurality of detectable moieties each comprise biotin and a detectable label; the one or more modified bases may comprise biotin and the plurality of detectable moieties each comprise streptavidin and a detectable label; the one or more modified bases may comprise avidin and the plurality of detectable moieties each comprise biotin and a detectable label; and / or the one or more modified bases may comprise biotin and the plurality of detectable moieties each comprise avidin and a detectable label.

[0137] Step (a) of embodiments (31) and (32) may include binding HIV p24 to the following species in the following order: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for HIV p24. Alternatively, step (a) may include binding HIV p24 to the following species in the following order: (i) a first and a second detection reagent for HIV p24; and (ii) a capture reagent on a surface. Additionally, step (a) may include simultaneously or substantially simultaneously binding HIV p24 to the following species: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for HIV p24.

[0138] The amplifier of embodiments (31) and (32) may still be located on the surface after the probe is extended. The complex may still be bound to the surface after the extension step. For example, the amplifier is bound to the anchoring agent at a position within 10-100um of the position of the complex on the surface. In a specific embodiment, the extended probe is bound to the anchoring agent at a position less than 100um, less than 50um, or more particularly less than 10um of the position of the complex on the surface.

[0139] The surface of embodiments (31) and (32) may comprise particles and / or holes of a porous plate. The surface may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole in a plate, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain on the surface. If the surface is a hole in a plate, the hole may include multiple different binding domains, and the capture agent and the anchoring agent are located on the same binding domain in the hole. In a specific embodiment, the capture agent and the anchoring agent are within 10-100nm on the surface.

[0140] In addition, the surface may include electrodes, and the measuring step may include applying a voltage waveform to the electrodes to produce an electrochemiluminescent signal. In these embodiments ((31) and (32)), the method may also include collecting particles on the electrodes and applying a voltage waveform to the electrodes to produce an electrochemiluminescent signal. The measuring step may include binding the amplicon to a detection probe with a detectable label, measuring the detectable label, and associating the measurement with the amount of analyte in the sample, wherein the detection probe comprises a nucleic acid sequence complementary to the amplicon region. The detectable label is measured by light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. For example, the detectable label is an ECL label, and the measuring step may include measuring the ECL signal.

[0141] Embodiment (33): A method for detecting an analyte of interest in a sample, comprising: (a) concentrating the sample under conditions sufficient to form an analyte complex comprising the analyte bound to a first detection reagent, wherein the first detection reagent is linked to a first nucleic acid probe; (b) binding the analyte complex formed in step (a) to: (i) a capture reagent on a surface, the surface comprising a capture reagent for the analyte, and an anchor reagent comprising an anchor oligonucleotide sequence; and (ii) a second detection reagent for the analyte linked to a second nucleic acid probe; thereby forming a complex on the surface comprising the capture reagent, the analyte, and the first and second detection reagents; (c) extending the second probe using an extension process that requires proximity of the first and second probes to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence; (d) hybridizing the anchor sequence to the anchor sequence complement; and (e) measuring the amount of the extended sequence bound to the surface. The concentration step (a) may further include (i) contacting a sample containing the analyte with a solid phase, the solid phase being linked to a targeting agent complementary to at least a portion of the first nucleic acid probe, thereby forming a concentrated complex containing the analyte bound to the solid phase through a binding reaction between the first nucleic acid probe and the targeting agent; (ii) collecting the concentrated complex; (iii) separating unbound components of the sample from the concentrated complex; and (iv) releasing the concentrated complex to separate the solid phase from the analyte to form an analyte complex.

[0142] Embodiment (34): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an anchoring reagent comprising an anchoring oligonucleotide sequence; (b) a first detection reagent for the analyte linked to a first nucleic acid probe; (c) a second detection reagent for the analyte linked to a second nucleic acid probe; and (d) a solid phase comprising a targeting agent complementary to at least a portion of the first nucleic acid probe.

[0143] Embodiment (35): A method for detecting exosomes in a sample, comprising: (a) binding the exosomes to: (i) a capture reagent on a surface, the surface comprising a capture reagent for the exosomes, and an anchor reagent comprising an anchor oligonucleotide sequence; (ii) a first detection reagent for the exosomes linked to a first nucleic acid probe; and (iii) a second detection reagent for the exosomes linked to a second nucleic acid probe; thereby forming a complex on the surface, the complex comprising the binding reagent, the exosomes, and the first and second detection reagents; (b) extending the second probe to form an extended sequence comprising a complement to the anchor sequence, the complement being complementary to the anchor sequence, using an extension process that requires proximity of the first and second probes; (c) hybridizing the anchor sequence to the complement to the anchor sequence; and (d) measuring the amount of the extended sequence bound to the surface.

[0144] Embodiment (36): A kit for detecting exosomes of interest in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising (i) a capture reagent for exosomes, and (ii) an anchoring reagent comprising an anchoring oligonucleotide sequence; (b) a first detection reagent for exosomes linked to a first nucleic acid probe; and (c) a second detection reagent for exosomes linked to a second nucleic acid probe.

[0145] The present invention includes the following embodiments:

[0146] Embodiment 1. A method for detecting an analyte of interest in a sample, comprising:

[0147] a. binding an analyte to: (i) a capture reagent on a surface, the surface comprising a capture reagent for the analyte, and an anchoring reagent comprising an anchoring oligonucleotide sequence; (ii) a first detection reagent for the analyte attached to a first nucleic acid probe; and (iii) a second detection reagent for the analyte attached to a second nucleic acid probe; thereby forming a complex comprising the binding reagent, the analyte, and the first and second detection reagents on the surface;

[0148] b. extending the second probe to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence using an extension process that requires the first and second probes to be in proximity;

[0149] c. hybridizing the anchor sequence to the complement of the anchor sequence; and

[0150] d. measuring the amount of extended sequence bound to the surface,

[0151] The analytes are G-CDF, GM-CSF, IFNgamma, IL-lbeta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 / 23p40, IL12p70, IL-17A, IL21, IL-22, IL-23, IL-31, IL-33, TNFalpha, TSLP, VEGF, complexed PSA, free PSA, Abeta42, Abeta40, Abeta38, tau, cardiac troponin I, cardiac troponin T, HIV p24, C-peptide and / or FGF21.

[0152] Embodiment 2. The method of embodiment 1, wherein the capture agent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0153] Embodiment 3. The method of embodiment 1, wherein the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0154] Embodiment 4. The method of embodiment 1, wherein the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0155] Embodiment 5. The method of embodiment 1, wherein the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0156] Embodiment 6. The method of embodiment 1, wherein the anchor oligonucleotide sequence comprises a single-stranded oligonucleotide sequence.

[0157] Embodiment 7. The method of embodiment 1, wherein the extension sequence further comprises one or more detection sequences, and the measuring step further comprises contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences.

[0158] Embodiment 8. The method of embodiment 1, wherein the extending step comprises binding the probe to a template nucleic acid sequence to form a circular nucleic acid template, and extending the circular template by rolling circle amplification.

[0159] Embodiment 9. The method of embodiment 1, wherein the extended probe remains positioned on the surface after the probe is extended.

[0160] Embodiment 10. The method of embodiment 9, wherein the complex remains bound to the surface after the extension step.

[0161] Embodiment 11. The method of embodiment 10, wherein the extended probe is bound to the anchoring agent at a position less than 100 um of the position of the complex on the surface.

[0162] Embodiment 12. The method of embodiment 1, wherein the extension step comprises PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (single-strand displacement amplification), 3SR (self-sustained synthesis reaction), or an isothermal amplification method.

[0163] Embodiment 13. The method of embodiment 12, wherein the extension step comprises an isothermal amplification method.

[0164] Embodiment 14. The method of embodiment 13, wherein the isothermal amplification method is helicase-dependent amplification or rolling circle amplification (RCA).

[0165] Embodiment 15. The method of embodiment 1, wherein the extension process comprises contacting the complex formed in step (a) with a connector sequence, wherein the connector sequence comprises (i) an internal sequence complementary to the second probe and (ii) two end sequences complementary to the non-overlapping region of the first probe.

[0166] Embodiment 16. The method of embodiment 15, further comprising ligating the double-terminal sequences of the linker oligonucleotide to form a circular target sequence that hybridizes to both the first and second probes.

[0167] Embodiment 17. The method of embodiment 1, wherein the extension process includes contacting the complex formed in step (a) with a first linker oligonucleotide sequence and a second linker oligonucleotide sequence, wherein the first linker oligonucleotide sequence comprises a first linker probe sequence complementary to a first region on the first probe and a first region on the second probe, and the second linker oligonucleotide comprises a second probe sequence complementary to a second non-overlapping region of the first probe and a second non-overlapping region of the second probe.

[0168] Embodiment 18. The method of embodiment 17, further comprising ligating the first and second linker oligonucleotides to form a circular target sequence that hybridizes to both the first and second probes.

[0169] Embodiment 19. The method of Embodiment 1, wherein the surface comprises particles.

[0170] Embodiment 20. The method of Embodiment 1, wherein the surface comprises wells of a multiwell plate.

[0171] Embodiment 21. The method of embodiment 1, wherein the surface comprises a plurality of different binding domains, and the capture reagent and the anchoring reagent are located on two different binding domains on the surface.

[0172] Embodiment 22. The method of embodiment 20, wherein the pore comprises a plurality of different binding domains, and the capture reagent and the anchoring reagent are located on two different binding domains within the pore.

[0173] Embodiment 23. The method of embodiment 1, wherein the surface comprises a plurality of different binding domains, and the capture reagent and the anchor reagent are located on the same binding domain on the surface.

[0174] Embodiment 24. The method of embodiment 20, wherein the pore comprises a plurality of different binding domains, and the capture reagent and the anchoring reagent are located on the same binding domain within the pore.

[0175] Embodiment 25. The method of embodiment 1, wherein the capture reagent and the anchoring reagent are less than 10 nm on the surface.

[0176] Embodiment 26. The method of embodiment 1, wherein the surface comprises an electrode and the measuring step further comprises applying a voltage waveform to the electrode to generate an electrochemiluminescent signal.

[0177] Embodiment 27. The method of Embodiment 19 further comprises collecting particles on an electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescent signal.

[0178] Embodiment 28. The method of embodiment 1, wherein the measuring step further comprises binding the extension sequence to a detection probe having a detectable label, measuring the detectable label and correlating the measurement with the amount of analyte in the sample, wherein the detection probe comprises a nucleic acid sequence complementary to a region of the extension sequence.

[0179] Embodiment 29. A method according to embodiment 28, wherein the detectable label is measured by measurement of light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field or a combination thereof.

[0180] Embodiment 30. A method according to embodiment 29, wherein the detectable label is an ECL label and the measuring step comprises measuring the ECL signal.

[0181] Embodiment 31. A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers or compartments:

[0182] a. a surface comprising (i) a capture reagent for the analyte, and (ii) an anchoring reagent comprising an anchoring oligonucleotide sequence;

[0183] b. a first detection reagent for said analyte attached to a first nucleic acid probe; and

[0184] c. A second detection reagent for the analyte attached to a second nucleic acid probe

[0185] The analytes are G-CDF, GM-CSF, IFNgamma, IL-lbeta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 / 23p40, IL12p70, IL-17A, IL21, IL-22, IL-23, IL-31, IL-33, TNFalpha, TSLP, VEGF, composite PSA, free PSA, Abeta42, Abeta40, Abeta38, tau, cardiac troponin I, cardiac troponin T, HIV p24, C-peptide and / or FGF21.

[0186] Embodiment 32. The kit of embodiment 31, wherein the capture reagent comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0187] Embodiment 33. The kit of embodiment 31, wherein the first detection reagent comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0188] Embodiment 34. The kit of embodiment 31, wherein the second detection reagent comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0189] Embodiment 35. The kit of Embodiment 31, wherein the surface comprises particles.

[0190] Embodiment 36. The kit of Embodiment 31, wherein the surface comprises wells of a multi-well plate.

[0191] Embodiment 37. The kit of embodiment 31, wherein the surface comprises a plurality of different binding domains, and the capture reagent and the anchoring reagent are located on two different binding domains on the surface.

[0192] Embodiment 38. The kit of embodiment 36, wherein the well comprises a plurality of different binding domains, and the capture reagent and the anchor reagent are located on two different binding domains within the well.

[0193] Embodiment 39. The kit of embodiment 31, wherein the surface comprises a plurality of different binding domains, and the capture reagent and the anchor reagent are located on the same binding domain on the surface.

[0194] Embodiment 40. The kit of embodiment 36, wherein the well comprises a plurality of different binding domains, and the capture reagent and the anchor reagent are located on the same binding domain within the well.

[0195] Embodiment 41. The kit of embodiment 31, wherein the capture reagent and the anchoring reagent are within 10-100 nm of the surface.

[0196] Embodiment 42. The kit of Embodiment 31, wherein the surface comprises an electrode.

[0197] Embodiment 43. The method of embodiment 1, wherein the analyte is a health care associated infection selected from Klebsiella pneumonia, Acinetobacter baumannii, Pseudomon asaeruginosa, Enterobacter species, and extraintestinal pathogenic Escherichia coli.

[0198] Embodiment 44. The kit of embodiment 31, wherein the marker is a healthcare-associated infection selected from Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and extraintestinal pathogenic Escherichia coli.

[0199] Embodiment 45. The method of embodiment 1, wherein the analyte is 50S ribosomal protein L20, 30S ribosomal protein S7, 30S ribosomal protein S2, 50S ribosomal protein L21, 50S ribosomal protein L17, 30S ribosomal protein S4, 50S ribosomal protein L15, 30S ribosomal protein S5, 50S ribosomal protein L16, 30S ribosomal protein S3, 50S ribosomal protein L22, 50S ribosomal protein L4, ribosomal protein L25, 50S ribosomal protein L5, 30S ribosomal protein S2, ribosomal protein L30, L31 and L32 and combinations thereof.

[0200] Embodiment 46. The method of embodiment 1, wherein the analytes are elongation factors EF-TU, ACP, acyl carrier protein, RplL, ribosomal protein GroS (MopB, 65,000), components of the molecular chaperone systems Gro-EL-Gro-ES and GapA, enzymes in glycolysis, and combinations thereof.

[0201] Embodiment 47. A kit according to embodiment 31, wherein the analyte is 50S ribosomal protein L20, 30S ribosomal protein S7, 30S ribosomal protein S2, 50S ribosomal protein L21, 50S ribosomal protein L17, 30S ribosomal protein S4, 50S ribosomal protein L15, 30S ribosomal protein S5, 50S ribosomal protein L16, 30S ribosomal protein S3, 50S ribosomal protein L22, 50S ribosomal protein L4, ribosomal protein L25, 50S ribosomal protein L5, 30S ribosomal protein S2, ribosomal protein L30, L31 and L32, and combinations thereof.

[0202] Embodiment 48. The kit of embodiment 31, wherein the analytes are elongation factors EF-TU, ACP, acyl carrier protein, RplL, ribosomal protein GroS (MopB, 65,000), components of the molecular chaperone systems Gro-EL-Gro-ES and GapA, enzymes in glycolysis, and combinations thereof.

[0203] Embodiment 49. A method for detecting an analyte of interest in a sample, comprising:

[0204] a. concentrating the sample under conditions sufficient to form an analyte complex comprising an analyte bound to a first detection reagent, wherein the first detection reagent is linked to a first nucleic acid probe;

[0205] b. binding the analyte complex formed in step (a) to: (i) a capture reagent on a surface, the surface comprising a capture reagent for the analyte, and an anchoring reagent comprising an anchoring oligonucleotide sequence; and (ii) a second detection reagent for the analyte linked to a second nucleic acid probe; thereby forming a complex on the surface, the complex comprising the capture reagent, the analyte, and the first and second detection reagents;

[0206] c. extending the second probe to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence using an extension process that requires the first and second probes to be in proximity;

[0207] d. hybridizing the anchor sequence to the complement of the anchor sequence; and

[0208] e. Measuring the amount of extended sequence bound to the surface.

[0209] Embodiment 50. The method of Embodiment 49, wherein the concentration step (a) further comprises

[0210] (i) contacting a sample including the analyte with a solid phase, the solid phase being linked to a targeting agent complementary to at least a portion of the first nucleic acid probe, thereby forming a concentrated complex, the concentrated complex comprising the analyte bound to the solid phase by a binding reaction between the first nucleic acid probe and the targeting agent;

[0211] (ii) collecting the concentrated complex;

[0212] (iii) separating unbound components of the sample from the concentrated complex; and

[0213] (iv) releasing the concentrated complex to separate the solid phase from the analyte to form the analyte complex.

[0214] Embodiment 51. A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers or compartments:

[0215] a. a surface comprising (i) a capture reagent for the analyte, and (ii) an anchoring reagent comprising an anchoring oligonucleotide sequence;

[0216] b. a first detection reagent for the analyte attached to a first nucleic acid probe;

[0217] c. a second detection reagent for the analyte attached to a second nucleic acid probe; and

[0218] d. a solid phase comprising a targeting agent complementary to at least a portion of the first nucleic acid probe.

[0219] Embodiment 52. The kit of embodiment 51, wherein the solid phase is a particle.

[0220] Embodiment 53. The kit of embodiment 51, wherein the capture reagent comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0221] Embodiment 54. The kit of embodiment 51, wherein the first detection reagent comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0222] Embodiment 55. The kit of embodiment 51, wherein the second detection reagent comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0223] Embodiment 56. The kit of Embodiment 51, wherein the surface comprises wells of a multi-well plate.

[0224] Embodiment 57. The kit of embodiment 56, wherein the surface comprises a plurality of different binding domains, and the capture reagent and the anchoring reagent are located on two different binding domains on the surface.

[0225] Embodiment 58. The kit of embodiment 56, wherein the well comprises a plurality of different binding domains, and the capture reagent and the anchor reagent are located on two different binding domains within the well.

[0226] Embodiment 59. The kit of embodiment 56, wherein the surface comprises a plurality of different binding domains, and the capture reagent and the anchor reagent are located on the same binding domain on the surface.

[0227] Embodiment 60. The kit of embodiment 59, wherein the well comprises a plurality of different binding domains, and the capture reagent and the anchor reagent are located on the same binding domain within the well.

[0228] Embodiment 61. The kit of embodiment 51, wherein the capture reagent and the anchoring reagent are within 10-100 nm on the surface.

[0229] Embodiment 62. The kit of Embodiment 51, wherein the surface comprises an electrode.

[0230] Embodiment 63. A method for detecting an analyte of interest in a sample, comprising:

[0231] a. binding the analyte to: (i) a capture reagent on a particle, the particle comprising the capture reagent for the analyte, and an anchoring reagent comprising an anchoring oligonucleotide sequence; (ii) a first detection reagent for the analyte attached to a first nucleic acid probe; and (iii) a second detection reagent for the analyte attached to a second nucleic acid probe; thereby forming a complex on the surface, the complex comprising the binding reagent, the analyte, and the first and second detection reagents;

[0232] b. extending the second probe to form an extended sequence comprising a complement of an anchor sequence that is complementary to the anchor sequence using an extension process that requires the first and second probes to be in proximity, wherein the extension process is performed during or after the particles settle by gravity onto the surface of the wells of the multiwell plate;

[0233] c. hybridizing the anchor sequence to the complement of the anchor sequence; and

[0234] d. Measuring the amount of extended sequence bound to the particle.

[0235] Embodiment 64. The method of embodiment 63, wherein the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0236] Embodiment 65. The method of embodiment 64, wherein the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0237] Embodiment 66. The method of Embodiment 65, wherein the first detection reagent is an antibody.

[0238] Embodiment 67. The method of embodiment 63, wherein the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0239] Embodiment 68. The method of Embodiment 63, wherein the capture reagent and the first and second detection reagents are antibodies to the analyte.

[0240] Embodiment 69. The method of embodiment 63, wherein the anchor oligonucleotide sequence comprises a single-stranded oligonucleotide sequence.

[0241] Embodiment 70. The method of embodiment 63, wherein the extension sequence further comprises one or more detection sequences, and the measuring step further comprises contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences.

[0242] Embodiment 71. The method of embodiment 63, wherein the extension step comprises PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (single-strand displacement amplification), 3SR (self-sustained synthesis reaction), or an isothermal amplification method.

[0243] Embodiment 72. The method of Embodiment 71, wherein the extension step comprises an isothermal amplification method.

[0244] Embodiment 73. The method of embodiment 72, wherein the isothermal amplification method is helicase-dependent amplification or rolling circle amplification (RCA).

[0245] Embodiment 74. A method for detecting an analyte of interest in a sample, comprising:

[0246] a. binding the analyte to: (i) a capture reagent on a particle, the particle comprising a capture reagent for the analyte; (ii) a first detection reagent for the analyte attached to a first nucleic acid probe; and (iii) a second detection reagent for the analyte attached to a second nucleic acid probe; thereby forming a complex on the surface, the complex comprising the binding reagent, the analyte, and the first and second detection reagents;

[0247] b. extending the second probe to form an extended sequence comprising a complement of an anchor sequence that is complementary to the anchor sequence using an extension process that requires the first and second probes to be in proximity, wherein the extension process is performed during or after the particles settle by gravity onto the surface of the wells of a multiwell plate;

[0248] c. Measuring the amount of extended sequence bound to the particle.

[0249] Embodiment 75. The method of embodiment 74, wherein the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0250] Embodiment 76. The method of embodiment 74, wherein the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0251] Embodiment 77. The method of embodiment 74, wherein the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0252] Embodiment 78. The method of Embodiment 74, wherein the capture reagent and the first and the second detection reagents are antibodies to the analyte.

[0253] Embodiment 79. The method of embodiment 74, wherein the extension sequence further comprises one or more detection sequences, and the measuring step further comprises contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences.

[0254] Embodiment 80. The method of embodiment 74, wherein the extension step comprises PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (single-strand displacement amplification), 3SR (self-sustained synthesis reaction), or an isothermal amplification method.

[0255] Embodiment 81. The method of Embodiment 80, wherein the extension step comprises an isothermal amplification method.

[0256] Embodiment 82. The method of embodiment 81, wherein the isothermal amplification method is helicase-dependent amplification or rolling circle amplification (RCA).

[0257] Embodiment 83. A method for detecting exosomes in a sample, comprising:

[0258] a. binding the exosome to: (i) a capture reagent on a surface, the surface comprising a capture reagent for the exosome, and an anchoring reagent comprising an anchoring oligonucleotide sequence; (ii) a first detection reagent for the exosome linked to a first nucleic acid probe; and (iii) a second detection reagent for the exosome linked to a second nucleic acid probe; thereby forming a complex on the surface, the complex comprising the binding reagent, the exosome, and the first and second detection reagents;

[0259] b. extending the second probe to form an extended sequence comprising a complement of an anchor sequence, the complement being complementary to the anchor sequence, using an extension process that requires the first and second probes to be in proximity;

[0260] c. hybridizing the anchor sequence to the complement of the anchor sequence; and

[0261] d. Measuring the amount of extended sequence bound to the surface.

[0262] Embodiment 84. The method of embodiment 83, wherein the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0263] Embodiment 85. The method of embodiment 83, wherein the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0264] Embodiment 86. The method of embodiment 83, wherein the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0265] Embodiment 87. The method of embodiment 83, wherein the capture reagent and the first and second detection reagents are antibodies to target molecules in or on the surface of the exosome.

[0266] Embodiment 88. The method of embodiment 87, wherein the capture reagent binds a common exosomal target protein selected from CD9, CD63, CD81, Hsp70, PDCD6IP or Tsg101.

[0267] Embodiment 89. The method of embodiment 87, wherein at least one of the first or second detection reagents is an antibody to a disease-specific target molecule in or on the surface of an exosome.

[0268] Embodiment 90. The method of embodiment 83, wherein said first and said second detection reagents each bind to an interacting exosomal target molecule.

[0269] Embodiment 91. The method of embodiment 90, wherein the interacting target molecules include ligand-receptor pairs and / or mRNA molecules and RNA binding proteins.

[0270] Embodiment 92. The method of embodiment 83, wherein the extension sequence further comprises one or more detection sequences, and the measuring step further comprises contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences.

[0271] Embodiment 93. The method of embodiment 83, wherein the extending step comprises binding the probe to a template nucleic acid sequence to form a circular nucleic acid template, and extending the circular template by rolling circle amplification.

[0272] Embodiment 94. A method according to Embodiment 83, wherein the extended probe remains positioned on the surface after the probe is extended.

[0273] Embodiment 95. The method of Embodiment 94, wherein the complex remains bound to the surface after the extension step.

[0274] Embodiment 96. The method of Embodiment 94, wherein the extended probe is bound to the anchoring agent at a position less than 100 um of the location of the complex on the surface.

[0275] Embodiment 97. The method of embodiment 83, wherein the extension step comprises PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (single-strand displacement amplification), 3SR (self-sustained synthesis reaction), or an isothermal amplification method.

[0276] Embodiment 98. The method of Embodiment 97, wherein the extension step comprises an isothermal amplification method.

[0277] Embodiment 99. The method of embodiment 98, wherein the isothermal amplification method is helicase-dependent amplification or rolling circle amplification (RCA).

[0278] Embodiment 100. A method according to embodiment 83, wherein the extension process comprises contacting the complex formed in step (a) with a linker sequence comprising (i) an internal sequence complementary to the second probe and (ii) a double-end sequence complementary to a non-overlapping region of the first probe.

[0279] Embodiment 101 The method of embodiment 100 further comprises connecting the double-terminal sequences of the linker oligonucleotide to form a circular target sequence that hybridizes to both the first and second probes.

[0280] Embodiment 102. A method according to embodiment 83, wherein the extension process comprises contacting the complex formed in step (a) with a first linker oligonucleotide sequence and a second linker oligonucleotide sequence, wherein the first linker oligonucleotide sequence comprises a first linker probe sequence complementary to a first region on the first probe and a first region on the second probe, and the second linker oligonucleotide comprises a second probe sequence complementary to a second non-overlapping region of the first probe and a second non-overlapping region of the second probe.

[0281] Embodiment 103. The method of embodiment 102, further comprising ligating the first and second linker oligonucleotides to form a circular target sequence that hybridizes to both the first and second probes.

[0282] Embodiment 104. The method of Embodiment 83, wherein the sample comprises purified exosomes.

[0283] Embodiment 105. The method of Embodiment 83, wherein the measuring step further comprises imaging the amplicons present on the surface.

[0284] Embodiment 106. The method of embodiment 105, wherein the amplicons are labeled with a plurality of fluorescent probes, and the subsequent measuring step comprises fluorescent staining of the exosomes, imaging the fluorescently labeled amplicons and the stained exosomes, and correlating the imaging step with the results.

[0285] Embodiment 107. A kit for detecting exosomes of interest in a sample, comprising in one or more vials, containers, or compartments:

[0286] a. a surface comprising (i) a capture reagent for exosomes, and (ii) an anchoring reagent comprising an anchoring oligonucleotide sequence;

[0287] b. a first detection reagent for said exosomes linked to a first nucleic acid probe; and

[0288] c. a second detection reagent for the exosome linked to a second nucleic acid probe.

[0289] Embodiment 108. The kit of embodiment 107, wherein the capture reagent comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0290] Embodiment 109. The kit of embodiment 107, wherein the first detection reagent comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0291] Embodiment 110. The kit of embodiment 107, wherein the second detection reagent comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

[0292] Embodiment 111. The kit of Embodiment 107, wherein the surface comprises particles.

[0293] Embodiment 112. The kit of Embodiment 107, wherein the surface comprises wells of a multi-well plate.

[0294] Embodiment 113. The kit of embodiment 107, wherein the surface comprises a plurality of different binding domains, and the capture reagent and the anchoring reagent are located on two different binding domains on the surface.

[0295] Embodiment 114. The kit of embodiment 112, wherein the well comprises a plurality of different binding domains, and the capture reagent and the anchoring reagent are located on two different binding domains within the well.

[0296] Embodiment 115. The kit of embodiment 113, wherein the surface comprises a plurality of different binding domains, and the capture reagent and the anchoring reagent are located on the same binding domain on the surface.

[0297] Embodiment 116. The kit of embodiment 112, wherein the well comprises a plurality of different binding domains, and the capture reagent and the anchor reagent are located on the same binding domain within the well.

[0298] Embodiment 117. The kit of Embodiment 107, wherein the capture reagent and the anchoring reagent are within 10-100 nm of the surface.

[0299] Embodiment 118. The kit of Embodiment 107, wherein the surface comprises an electrode.

[0300] Embodiment 119. The kit of embodiment 107, wherein the capture reagent binds a common exosomal target protein selected from CD9, CD63, CD81, Hsp70, PDCD6IP or Tsg101.

[0301] Embodiment 120. The kit of embodiment 107, wherein the first and second detection reagents each bind to an interacting exosome target protein.

[0302] Embodiment 121. The kit of embodiment 102, wherein the interacting target molecules include ligand-receptor pairs and / or mRNA molecules and RNA binding proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0304] Figures 1(a)-(c) illustrate the use of anchoring agents in immunoassays. Figure 1(a) shows the use of an anchoring agent to bind and stabilize a detection complex comprising a capture reagent, an analyte of interest, and a detection reagent comprising a nucleic acid probe. The nucleic acid probe is extended to bind the anchoring agent. In Figure 1(b), the anchoring agent comprises an oligonucleotide sequence comprising a region complementary to a portion of an extended sequence formed on the detection reagent. Figure 1(c) shows that in a specific embodiment, two detection reagents are used to bind the analyte, each detection reagent comprising a nucleic acid probe. The probes on the detection reagent are subjected to an amplification process that causes an extended probe to hybridize with the anchoring oligonucleotide sequence.

[0305] Figure 2(a) shows a specific embodiment in which immune complexes formed on a surface carrying an anchoring agent are subjected to a PLA-RCA process to incorporate multiple detectable species in the extended sequence attached to the immune complex. Figure 2(b) and 2(c) There are two alternative configurations of linker oligonucleotides which may be employed in the methods of the invention.

[0306] Figure 3 Methods for attaching oligonucleotides to proteins are shown.

[0307] Figure 4(a) illustrates a preferred embodiment of the present invention, wherein a surface-bound complex is formed between a capture reagent, an analyte, and two detection reagents, wherein the two detection reagents are each attached to a first and a second proximal probe, respectively, which are ligated to a linker probe to form a circular DNA template, which is amplified by rolling circle amplification. Figure 4(a) also includes an amplification reagent, which includes an anchor oligonucleotide sequence complementary to the sequence of the amplicon formed as the assay progresses. Figure 4(b) shows an exemplary sequence of the first circular DNA template Circ-1, the detection oligonucleotide sequence, the inert region of the amplicon, and a portion PP2 designed to hybridize with the second proximal probe. An alternative embodiment is depicted in Figure 4(c).

[0308] Figure 5 and Figures 6(a)-(b) illustrate an alternative method for generating amplicons that can be amplified by rolling circle amplification.

[0309] Figure 7 An alternative embodiment is illustrated in which a portion of each proximal probe in the sandwich complex is temporarily protected by a short strand of RNA that hybridizes to each segment. Those strands are enzymatically removed to allow the proximal probes to hybridize to each other and to the strand to be extended.

[0310] Figure 8 A further embodiment is shown in which the proximal probes are attached to the capture reagent and the detection reagent, and a portion of each proximal probe is temporarily protected by a short strand of RNA to which it hybridizes, as described above with respect to Figure 8 Descriptive.

[0311] Fig. 9 The calibration curve for the IL-10 assay performed using the method described in Example 1 is shown.

[0312] Figure 10 (a)-(b) shows fluorescence microscopy images with (a) and without (b) an anchoring agent.

[0313] Figure 11 (a) shows the configuration of a single linear linker oligonucleotide sequence including ligation site 1 or 2, and the use of these linkers in the assays of the present invention. Figure 11 (b) shows comparative performance data for an assay using a combination of Circ-1 and Circ-2 vs. a single linear linker oligonucleotide sequence including ligation site 1 or a single linear linker oligonucleotide sequence including ligation site 2.

[0314] Fig.12 Calibration curves for the HIV p24 assay performed using the methods described in Examples 1 and 6 are shown.

[0315] Fig.13 Results of analysis of the seroconversion panel using the methods described in Examples 1 and 6 are shown.

[0316] Figures 14(a)-(c) show the results of an assay for HIV p24 including an analyte concentration step.

[0317] Fig.15 Shown is the calibration curve for the HIV p24 assay including the analyte concentration step.

[0318] Fig.16 is a schematic diagram of an assay method as described herein including an analyte concentration step.

[0319] Fig.17 is a schematic diagram of an assay method as described herein, wherein amplicons are formed in solution prior to binding to a surface via a capture reagent and / or an anchoring reagent.

[0320] Fig.18 Schematic diagram of an assay method that incorporates the use of multiple U-shaped staple sequences to attach to amplicons to form a more compact structure on a surface.

[0321] Fig.19 is a schematic diagram of a bridging immunoassay format combining 3AB PLA-RCA technology and using a targeting moiety and its complement to bind a capture reagent to a surface.

[0322] 20(a)-(b) are schematic diagrams of the biosynthetic method for template formation.

[0323] Fig.21 It is a schematic diagram of sample multiplexing.

[0324] Fig. 22 is a schematic diagram of detecting lipoprotein complexes using the method described herein. DETAILED DESCRIPTION OF THE INVENTION

[0326] Unless otherwise defined herein, the technical and scientific terms used in the present invention should have the same meaning as generally understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms should include plural terms and plural terms should include singular terms. The articles "a" and "a kind" are used herein to refer to the grammatical objects of one or more than one (i.e., at least one) of the articles. For example, "a / kind of element" means one / kind of element or more than one / kind of element.

[0327] The present invention includes improved immunoassay methods, which include (i) anchoring a detection complex formed between a target analyte and one or more analyte binding reagents used in the assay; and / or (ii) amplifying a signal from a labeled detection complex. Anchoring can be used to stabilize complexes involving low binding affinity interactions and / or one or more high molecular weight markers or marker sites. Signal amplification can be achieved by attaching an extended probe to a binding complex containing multiple markers or detection marker sites, thereby amplifying a detectable signal for each detection complex. In a preferred embodiment, the method includes attaching an extended probe containing multiple markers or detection marker sites to a detection complex, and anchoring the complex to a surface to ensure that the complex remains on the surface. This improved assay method can be used to detect extremely low numbers of binding events, even single analyte-binding reagent complexes. This basic approach is not limited to immunoassays and can be used to implement binding assays using other binding reagent categories.

[0328] One method that can be used to improve binding assays is to use a surface-bound anchoring agent to adhere a detection complex containing an analyte of interest to a surface and stabilize the detection complex. This approach can be used to overcome low binding affinity between reagents that form the detection complex and / or prevent the complex from dissociating from the surface prior to subsequent processing. The use of an anchoring agent in a binding assay is illustrated in FIG. 1( a). A surface (101) comprises a capture reagent (102) that binds analyte A and an anchoring agent (103). In one or more steps, the analyte binds to the capture reagent and the detection reagent (104) also binds to the analyte, wherein the detection reagent is connected to a nucleic acid probe (105). The analyte may bind to the capture and detection reagents simultaneously or substantially simultaneously, or the analyte may bind to each of the capture and detection reagents sequentially (in either order). Thus, a complex (106) is formed on the surface, which comprises a capture reagent, an analyte, and a detection reagent. The probe is extended to form an extension sequence (107), which comprises an anchor region that binds to the anchoring agent. The extension sequence binds to the anchoring agent, and the amount of the extension sequence bound to the surface is measured.

[0329] Those skilled in the art of binding assays will readily appreciate the range of capture reagents and companion binding partners that can be used in the methods. Non-limiting examples of such pairs include (in any order) receptor / ligand pairs, antibody / antigen, natural or synthetic receptor / ligand pairs, hapten / antibody pairs, antigen / antibody pairs, epitope / antibody pairs, mimotope / antibody pairs, aptamer / target molecule pairs, hybridization partners, and intercalator / target molecule pairs. In one embodiment, the binding assay employs antibodies or other receptor proteins as capture and / or detection reagents for the analyte of interest. The term "antibody" includes intact antibody molecules (including hybrid antibodies assembled by in vitro reassociation of antibody subunits), antibody fragments and recombinant protein constructs comprising the antigen-binding domain of the antibody (as described, for example, in Porter, RR and Weir, RC J Cell Physiol., 67 (Suppl); 51-64 (1966) and Hochman, I. Inbar, D. and Givol, D. Biochemistry 12: 1130 (1973)), as well as antibody constructs that have been chemically modified (for example, by the introduction of a detectable label).

[0330] Similarly, anchoring agent and corresponding anchoring member or region can comprise any suitable binding pair, for example receptor / ligand pair, antibody / antigen, natural or synthetic receptor / ligand pair, hapten / antibody pair, antigen / antibody pair, epitope / antibody pair, analog position / antibody pair, aptamer / target molecule pair, hybridization partner and inserter / target molecule pair, and use the surface and anchoring agent that are combined by electrostatic charge.For example, anchoring agent can be oligonucleotide sequence, aptamer, aptamer ligand, antibody, antigen, ligand, receptor, hapten, epitope or analog position, and corresponding anchoring region comprises complementary oligonucleotide sequence, aptamer ligand, aptamer, antigen, antibody, receptor, ligand or antibody respectively.In a specific embodiment, anchoring region is oligonucleotide sequence and anchoring agent comprises DNA binding protein.Or, if anchoring region is double-stranded oligonucleotide sequence, anchoring agent can comprise intercalator.In another embodiment, anchoring region can comprise one or more modified oligonucleotide bases and corresponding anchoring agent comprises one or more modules of modified base in conjunction with anchoring region. For example, the modified base may comprise a hapten or a ligand, and the corresponding anchoring reagent comprises one or more antibodies or ligands specific for the hapten or ligand, respectively. Moreover, the anchoring region may comprise a plurality of labeled nucleotide bases, which can be used to detect the detection complex.

[0331] In a specific embodiment depicted in FIG. 1 (b), the anchoring reagent of the binding surface comprises an oligonucleotide for anchoring the detection complex to the surface. The anchoring oligonucleotide sequence binds to a complementary oligonucleotide sequence attached to the detection complex. In this embodiment, the surface (108) comprises a capture reagent (109) that binds analyte A, and an anchoring reagent (110) that comprises an anchoring oligonucleotide sequence (111). In one or more steps, the analyte is bound to a capture reagent and a detection reagent (112) that also binds to the analyte, wherein the detection reagent is connected to a nucleic acid probe (113). As described above for FIG. 1 (a), the analyte can be bound to the capture and detection reagents simultaneously or substantially simultaneously, or the analyte can be sequentially bound to each of the capture and detection reagents (in any order). Therefore, a complex (114) is formed on the surface, which comprises a binding reagent, an analyte, and a detection reagent. The probe is extended to form an extension sequence (115), which comprises an anchor sequence complement complementary to the anchor sequence. The anchor sequence hybridizes to the anchor sequence complement and the amount of extended sequence bound to the surface is measured.

[0332] The detection complex may include one or more detection reagents, for example, to enhance the specificity of the assay for the analyte. The use of multiple detection reagents can enhance the specificity of the assay, if, for example, the assay is designed to emit a detectable signal when each detection reagent is near the analyte, or the signal from a single detection reagent bound to the analyte can be distinguished from the signal emitted from multiple detection reagents bound to the analyte. One embodiment of this type of assay is shown in Figure 1 (c). The surface (116) includes a capture reagent (117) that binds to analyte A and an anchoring reagent (118) that includes an anchoring oligonucleotide sequence (119). In one or more steps, the analyte is bound to each of the two (or more) detection reagents (120 and 121, respectively) that bind the capture reagent and the analyte, wherein each of the first and second detection reagents is connected to a nucleic acid probe (122 and 123, respectively, the first and second nucleic acid probes). The analyte can be combined with the capture and detection reagents simultaneously or substantially simultaneously, or in a sequential, stepwise manner. Therefore, a complex (124) is formed on the surface, which includes a capture reagent, an analyte, and one and a second detection reagent. Using an extension process that requires the first and second probes to be in proximity to each other, the first probe is extended to form an extended sequence (125) comprising an anchor sequence complement complementary to the anchor sequence. In the penultimate step, the anchor sequence hybridizes to the anchor sequence complement and the amount of extended sequence bound to the surface is measured.

[0333] A specific embodiment of the method depicted in Figure 1 (c) is shown in Figure 2 (a), wherein an anchoring agent is used to adhere the detection complex to the surface and the probe attached to the detection complex is extended to generate an extended area that binds the anchoring agent. In this embodiment, the complex is detected using two detection agents that are bound to the proximal probe. The method also includes combining the detection agent with a connector sequence, which is then connected to form a circular target sequence, and performing rolling circle amplification to generate an amplicon that binds the anchoring agent. The surface (201) contains a capture agent (202) and an anchoring agent (203). In one or more steps, the analyte is bound to a capture agent, a first detection agent (204) containing a first proximal probe (205), and a second detection agent (206) containing a second proximal probe (207), thereby forming a detection complex (208) on the surface. The detection complex is contacted with two connector sequences (209a and 209b), each of which contains an end sequence complementary to the non-overlapping region of the first proximal probe and an end sequence complementary to the non-overlapping region of the second proximal probe. The connector sequence is hybridized with the first and second proximal probes, and the end sequences of the connector oligonucleotides are connected to form a circular target sequence (210), which hybridizes with both the first and second proximal probes. The second proximal probe is extended by rolling circle hybridization to generate an amplicon comprising a binding reagent that binds to an anchoring reagent, and the amount of the amplicon bound to the surface is measured. The first proximal probe can be capped or otherwise modified to prevent extension of the first probe. (In an alternative embodiment, the first proximal probe is extended and the second proximal probe can be capped or otherwise modified to prevent extension). In the embodiment depicted in Figure 2 (a), the amplicon also comprises two or more detection sequences complementary to a labeled detection probe that hybridizes with the amplicon for measuring the amount of the amplicon bound to the surface. In an alternative embodiment (not depicted in Figure 2 (a)), the extension process incorporates labeled nucleotide bases into the amplicon, which is used to directly detect the amplicon on the surface without adding one or more labeled probes that are complementary to the amplicon. FIG. 2( b ) is a schematic diagram of the sequence composition of the linker, which shows the first and second linker oligonucleotides (209 a and 209 b , respectively), wherein the first end of the first linker (C 1(E1) ) and the first end (C 2(E1) ) are complementary to two non-overlapping regions of the first proximal probe, and the second end of the first connector (C 1(E2) ) and the second end of the second connector (C 2(E2) ) are complementary to two non-overlapping regions of the second proximal probe. The first and second linkers hybridize to the first and second proximal probes and the first and second linkers are connected to form a circular target sequence, which hybridizes to both the first and second proximal probes.

[0334] Figure 2(c) shows an alternative embodiment of the linker. The linker sequence 211 comprises an internal sequence complementary to the second proximal probe (C IS ) and two terminal sequences complementary to the non-overlapping regions of the first proximal probe (C E1 and C E2 In this embodiment, only one ligation event is required to form a circular target sequence for rolling circle amplification (i.e., the C terminal that hybridizes to the first proximal probe). E1 and C E2 However, since the initiation / extension comes from the second proximal probe, the two proximal probes still need to be in proximity. Preferably, the first proximal probe is capped or otherwise modified to prevent extension of the first probe.

[0335] Thereafter, the second proximal probe is extended by rolling circle amplification of the circular target sequence to generate an amplicon comprising a binding region that binds the anchoring agent, and the amount of amplicon bound to the surface is measured.

[0336] The sequence of the first and second proximal probes can be designed by methods known to those skilled in the art. For example, every kind of probe length is about 20-50 bases, preferably 25-40 bases, most preferably about 30-35 bases. The first and second proximal probes also include one or more connector sequences used in the process as described herein or sequences complementary to their parts. In one embodiment, the detection complex is contacted with two kinds of connector sequences (209a and 209b), each of which includes an end sequence complementary to the non-overlapping region of the first proximal probe and an end sequence complementary to the non-overlapping region of the second proximal probe. Therefore, in this embodiment, the first and second proximal probes each include a non-overlapping region complementary to the end sequence of the connector. Alternatively, only a kind of connector and the connector sequence (211) can be used to include an internal sequence (CIS) complementary to the second proximal probe and two end sequences (CE1 and CE2, respectively) complementary to the non-overlapping region of the first proximal probe. Thus, in this embodiment, the first proximal probe comprises non-overlapping regions complementary to the two terminal sequences CE1 and CE2 of the connector, respectively, and the second proximal probe comprises a sequence complementary to the internal sequence (CIS) of the connector. The first proximal probe may be capped or otherwise modified to prevent extension of the first probe. (In an alternative embodiment, the first proximal probe is extended and the second proximal probe may be capped or otherwise modified to prevent extension.)

[0337] Therefore, the embodiments illustrated in Figures 1(a)-(c) and 2(a)-(c) show that the binding assay can be modified to include an anchoring agent and / or the signal from the detection complex can be amplified. In a preferred embodiment, an anchoring agent and a signal amplification method are used in the binding assay. In an embodiment including the use of an anchoring agent, the concentration of the anchoring agent present on the surface is 0.2-200ug / mL, in particular, 1.0-50ug / mL, and more particularly 3.0-10ug / mL. Alternatively, only one or another method can be used to achieve enhanced binding assays. The present invention therefore includes an assay having a signal amplification method as described in Figures 1(a)-(c) and 2(a)-(c), omitting the anchoring agent.

[0338] In those embodiments in which the anchoring agent comprises an anchor sequence that is directly or indirectly bound to a surface (e.g., via a binding reaction), methods established in the art for immobilizing oligonucleotides can be used to generate the anchoring agent, including covalent and non-covalent attachment methods. In one embodiment, the anchoring agent comprises a protein that is connected or otherwise bound to an anchor sequence. In this embodiment, any protein that can be immobilized on a surface (covalently or non-covalently) and modified by an anchoring oligonucleotide can be used. Non-limiting examples include streptavidin, avidin, or bovine serum albumin (BSA). In a preferred embodiment, the anchoring agent comprises BSA. Proteins can be modified by anchoring oligonucleotides and attached to a surface using known methods, such as Figure 3 As exemplified in , sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC), an established heterobifunctional crosslinker, is used. The reaction of the N-hydroxysuccinimide (NHS) group of SMCC with bovine serum albumin (BSA) labels BSA with a thiol-reactive maleimide group. The maleimide group reacts accordingly with a thiol-modified oligonucleotide to form a BSA-oligonucleotide conjugate, which is linked via a stable thioether bond. In a specific example, an array is formed by printing a series of BSA-oligonucleotide conjugates onto a graphitic carbon surface (preferably a screen printed carbon ink electrode). Alternatively, if the protein is avidin or streptavidin, the anchor sequence can be linked to biotin and associated to the immobilized avidin or streptavidin via a biotin-avidin or biotin-streptavidin interaction.

[0339] The anchoring oligonucleotide attached to the anchoring agent can be any sequence that will hybridize with the extension sequence (or amplicon) formed during the extension process. The anchoring oligonucleotide can also include a non-complementary region (e.g., poly (A) sequence), which is used as a joint sequence between the surface and the complementary (hybridization) region to extend the complementary region to leave the surface. In one embodiment, the hybridization sequence is selected as an amplicon region ("inactive" region) that is unrelated to the binding proximal end or detection probe. In a more specific embodiment, a hybridization sequence (preferably about 25 nucleotides in length) complementary to the full-length inactive region of the amplicon is included alone or in combination with a poly (A) arm (e.g., up to 30 nucleotides in length). Preferably, the anchoring oligonucleotide is selected from: (i) (full-length complement of the inactive region of the amplicon, 25 nucleotides in length)-(20 nucleotide poly (A) arms); or (ii) (complement of a part of the inactive region of the amplicon, 15 nucleotides in length)-(30 nucleotide poly (A) arms).

[0340] In one embodiment, the second proximal probe is extended by proximal connection amplification (PLA). As described above to Fig. 2 (a)-(c), a complex comprising two proximal probes is contacted with one or more linker oligonucleotides (209a-209b or 211), and the connection of the linker sequence of the hybridization forms a circular oligonucleotide, which is then used to extend the second proximal probe by the rolling circle amplification (RCA) of the ring. Suitable probe design and amplification conditions for proximal connection amplification are established in the art. A unique aspect of the present invention is to include the same sequence used in the anchoring agent in one of the linkers. Thus during the extension of the second proximal probe, the extended region includes the complement of the anchoring sequence, which hybridizes with the anchoring agent, thereby stabilizing the sandwich complex and preventing the dissociation of the second proximal probe. The second proximal probe extended may contain a detectable label (e.g., by including labeled nucleotides during the RCA extension reaction), which can be measured to measure the amount of the analyte on the surface. Alternatively, a plurality of labeled probes comprising a detectable label are added and hybridized to the extended second proximal probe, and the amount of analyte bound to the surface is measured.

[0341] Any suitable amplification technique may be used to generate the extended sequence (or amplicon), including, but not limited to, PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustaining synthesis reaction), and isothermal amplification methods such as helicase-dependent amplification and rolling circle amplification (RCA). In a preferred embodiment, RCA is used because it has significant advantages in terms of sensitivity, multiplexity, dynamic range, and scalability. The technique of RCA is known in the art (see, e.g., Baner et al., Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al., Nature Genetics 19:226, 1998; Schweitzer et al., Proc. Natl. Acad. Sci. USA 97:10113-119, 2000; Faruqi et al., BMC Genomics 2:4, 2000; Nallur et al., Nucl. Acids Res. 29:e118, 2001; Dean et al., Genome Res. 11:1095-1099, 2001; Schweitzer et al., Nature Biotech. 20:359-361, 2003). 365, 2002; US Pat. Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329 and 6,368,801). Several different variations of RCA are known, including linear RCA (LRCA) and exponential RCA (ERCA). RCA generates thousands of copies of a circular template, the strands of which are attached to the original target DNA, allowing spatial resolution of the target and rapid amplification of the signal. RCA facilitates (i) detection of a single target molecule; (ii) amplification of signals from proteins as well as DNA and RNA; (iii) identification of the location of the amplified molecule on a solid surface; (iv) simultaneous measurement of many different targets; and (v) analysis of one or more targets in solution or in a solid phase. Spatial localization of the RCA product and the detection complex is particularly advantageous when performing multiplex binding assays in an array- or particle-based format.

[0342] A specific embodiment of the present invention is described in Figure 4 (a), in which both anchoring agents and signal amplification processes are used. A complex between a capture agent (402), an analyte (403) and two detection agents (304 and 305) is formed on a surface (401), each detection agent comprising a first and a second proximal probe (406 and 407), respectively. The first and second linker oligonucleotides Circ-1 (408) and Circ-2 (409) in Figure 4 (a) are added respectively, and when both proximal probes are present in the complex, each hybridizes and bridges the two proximal probes. The bound linker probes are connected at connection sites 1 and 2 (410 and 411), respectively, to form a circular DNA template (412). The circular DNA template is amplified by rolling circle amplification to extend the second proximal probe, thereby generating an amplicon (including a partial anchor sequence complement (415)) comprising one or more detection sequences (413) and an anchor oligonucleotide sequence complement (414). The anchor oligonucleotide sequence (416) (attached to the capture portion (417)) hybridizes with its complement, multiple detection probes hybridize with multiple detection probe sequences, and the amount of analyte bound to the surface is measured (not shown, but illustrated in Figure 1(a)). Figure 4(b) shows a first circular DNA template Circ-1 (408) (which is designed to hybridize with a first proximal probe (PP1)), a detection oligonucleotide sequence, an inactive region of the amplicon (which can be used in whole or in part to bind to the anchor oligonucleotide sequence) and a portion PP2 (which is designed to hybridize with a second proximal probe). Another embodiment is shown in Figure 4(c), in which the circular DNA template is amplified by rolling circle amplification to produce an amplicon comprising multiple detection sequences (418 and 419, respectively). In a further embodiment, the anchor oligonucleotide sequence (416) attached to the capture portion 417 can serve as a primer with a free 3' end. In this embodiment, the second proximal probe includes a sequence complementary to the detection sequence (413).

[0343] In one embodiment, the assay format described herein utilizes a detection reagent coupled to the 5' end of the detection sequence, wherein the 3' end is exposed to facilitate ligation with a linker probe to form a circular DNA template, which is then amplified by rolling circle to extend a second proximal probe (PP2). In this embodiment, PP1 is potentially independently available for extension by a polymerase, but this problem can be solved by adding modified bases to prevent polymerase priming. Alternatively, the ligation template PP1 can be directly coupled to the detection reagent via its 3' end, preventing the oligonucleotide from participating as a primer for a DNA polymerase, even if it is degraded.

[0344] Another approach to generating target sequences for amplification by RCA or any suitable amplification method is exemplified in Figure 5In this embodiment, each proximal probe can be folded into a circular hairpin structure. The formation of these hairpin structures generates a single-stranded loop and a double-stranded portion, which contains a recombination signal. A recombinase is added to drive the recombination of the two hairpin structures to form a circular DNA template, which is then subjected to RCA as described above. The amplicon is labeled and optionally anchored to an anchoring agent, and the analyte is detected. The key element of this embodiment is the ability of the recombinase to catalyze the site-specific recombination of DNA containing sequence-specific recombination sites. For example, the Cre recombinase from bacteriophage P1 catalyzes recombination at a site containing a loxP site, and other non-limiting examples include but are not limited to an engineered (evolved) version of flippase (flp, from Yeast), Hin (Salmonella) and Tre, Cre. This alternative approach does not require the addition of additional components such as oligonucleotide templates, ATP and dNTP. In this embodiment, the loxP (recombination) site is preferably modified to be asymmetric, thereby producing a migration in the normal equilibrium toward the formation of the desired recombination product. This is in Figure 5 As illustrated in FIG, recombination sites have light / dark shading.

[0345] Moreover, Fig. 6 (a) illustrates another method for generating a target sequence, which is amplified by RCA or any suitable amplification method. Each proximal probe attached to the detection reagent includes a loxP site, which enables site-specific recombination between two oligonucleotides by Cre recombinase, resulting in the formation of a new oligonucleotide sequence, which includes a 5' part of a proximal probe flanking the lox P site and a 3' part of another proximal probe. The newly created target sequence can then be amplified, labeled, optionally anchored and detected as described above by any suitable method. Fig. 6 (a) illustrates this embodiment, using T7 RNA polymerase promoter as an operable element for amplification. It should also be understood that other RNA polymerase sites such as T3 and SP6 connected at any one of the 3 or 5' parts of the proximal probe are also applicable to the method. In this embodiment, the loxP (recombination) site is preferably modified to asymmetric, thereby producing a migration in the normal balance formed towards the desired recombination product. As shown in Fig. 6 (b), the method can also be used to generate a circular DNA template that can be used in RCA.

[0346] The present invention includes a method for detecting an analyte, including making the capture reagent and two detection reagents on the analyte binding surface to form a detection complex. The method includes measuring the detection complex, wherein relative to the complex containing only one of the two detection reagents, the measurement method preferentially measures the complex containing the two detection reagents. In one embodiment, the method includes forming a complex, then cross-linking the detection reagent and detecting the cross-linked reagent. Any suitable cross-linking chemistry can be used to connect the components of the detection complex. For example, the first and second detection reagents can include a reaction module, which reacts and connects by adding a multifunctional cross-linking agent that connects the reaction module. In this embodiment, the reaction module and the cross-linking agent can include amines, thiols, hydrazides, aldehydes, esters, iodoacetamide (iodoacetamide), maleimide, click chemistry reagents and combinations thereof. In another embodiment, the first and second detection reagents can include a binding module and the cross-linking agent is a multivalent binding partner of the binding module. Several non-limiting examples of this embodiment include: (a) the first and second detection reagents are antibodies to an animal species and the cross-linker is a multivalent anti-species antibody targeting the antibodies to the animal species; (b) the first and second detection reagents comprise biotin and the cross-linker is streptavidin (or vice versa); (c) the first and second detection reagents are linked to streptavidin and the cross-linker is a polymer comprising multiple biotin molecules (or vice versa); or (d) the first and second detection reagents comprise first and second nucleic acid probes, respectively, and the cross-linker is an oligonucleotide comprising a sequence complementary to the first nucleic acid probe and another sequence complementary to the second nucleic acid probe.

[0347] In a specific embodiment, the target analyte in the sample can be detected by binding the analyte to an immobilized capture reagent, a first detection reagent, and a second detection reagent to form a complex, wherein the first detection reagent comprises a first detectable label and a first nucleic acid probe, and the second detection reagent comprises a second detectable label and a second nucleic acid probe. In this embodiment, the first and second detection reagents are cross-linked by: (i) hybridizing the first probe to the second probe, (ii) hybridizing the first and second probes to a third nucleic acid having a region complementary to the first and second probes, or (iii) connecting the first and second probes.

[0348] Once the cross-linked product binding surface can detect the product, or optionally, the cross-linked product can be released from the surface into the eluate and detected. In this regard, only those individual cross-linked products containing both the first and second detectable labels in the eluate are calculated. Any suitable detection method can be used to detect the presence of markers in the eluate. In a preferred embodiment, the marker is a fluorescent molecule and is detected by single molecule fluorescence, such as fluorescence correlation spectroscopy and / or fluorescence cross-correlation spectroscopy, to calculate the labeled cross-linked products present in the eluate. In this embodiment, single molecule fluorescence detection includes passing the eluate through a capillary, focusing a light source on a certain volume in the capillary to create an interrogation zone, and observing the interrogation zone with a light detector to detect the passage of the fluorescent molecule via the interrogation zone. The detection method may further include detecting a first fluorescent signal related to the first marker and a second fluorescent signal related to the second marker, and counting detection events when two signals are detected from the interrogation zone. Alternatively, one label is a fluorescence resonance energy transfer (FRET) donor and the other label is a FRET acceptor, and the detection method may further comprise exciting the FRET donor in the interrogation zone and detecting a fluorescent signal from the FRET acceptor.

[0349] In a specific embodiment, an analyte in a sample can be detected by binding the analyte to an immobilized capture reagent, a first detection reagent, and a second detection reagent to form a complex, wherein the first detection reagent comprises a first nucleic acid probe and the second detection reagent comprises a second nucleic acid probe; extending the second nucleic acid probe to form an extended sequence comprising a detectable label, wherein the extension depends on the co-localization of the first and second nucleic acid probes in the complex; releasing the extended sequence from the surface into the eluate; and counting each extended sequence in the eluate. The extension step can include binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction. Alternatively, the extension step includes binding the first probe to a template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extension step can also include binding the first probe to a template nucleic acid sequence, binding the second probe to the template sequence, and connecting the first and second probes.

[0350] In the methods of the present invention employing capture agents, the capture agents may be directly immobilized on the solid phase or they may be indirectly immobilized via a secondary binding agent such as a targeting agent described below. For example, the capture agent may be attached to or comprise a targeting agent that binds to a targeting agent complement immobilized on the solid phase. The binding of the targeting agent to its complement may be direct (e.g., the targeting agent may be streptavidin and the complement may be biotin), or may be indirect via a bridging agent (e.g., the targeting agent and the complement may be biotin, and the bridging agent may be a multivalent biotin binding receptor such as streptavidin). In one embodiment, the targeting agent and its complement include a first oligonucleotide and a complementary oligonucleotide, a receptor-ligand pair, an antigen-antibody pair, a hapten-antibody pair, an epitope-antibody pair, a mimetic position-antibody pair, an aptamer-target molecule pair, a hybridization partner, or an intercalator-target molecule pair. The targeting agent and complement used in the assay are selected so that the targeting agent and complement associated with the capture or detection agent of one analyte measured by the assay are substantially non-cross-reactive with the targeting agent and complement associated with the capture or detection agent of other analytes measured by the assay. For example, the binding of the binding agent to its associated binding domain (via its associated targeting agent and targeting agent complement) should be substantially greater than its binding to the binding domain associated with other analytes (and presenting different targeting agent complements). Preferably, the cross-reactivity of the binding of the capture or detection agent of one analyte to the binding domain associated with other analytes relative to the binding to the correct binding domain is <1%, more preferably <0.1% and more preferably <0.01%. In a preferred embodiment, the targeting agent / targeting agent complement comprises a pair of oligonucleotides comprising complementary sequences and the targeting agent and its complement are contacted under conditions sufficient to hybridize the targeting agent to its complement.

[0351] When using a targeting agent, there is some flexibility as to when to immobilize the capture reagent used in the assay on a solid phase. In one embodiment, the capture reagent is provided to the user pre-immobilized on a solid phase via a targeting agent-targeting agent complement interaction. In another embodiment, the capture reagent attached to the targeting agent and the solid phase supporting the immobilized targeting agent complement are provided as separate components. Thus, the assay method further comprises the step of immobilizing the capture reagent on a solid phase by binding the targeting agent to its complement (directly or via the use of a bridging agent). This step may be performed before, simultaneously with, or after the step associated with forming the detection complex.

[0352] In a specific embodiment, the multifunctional targeting agent can be used in the assay methods and components described herein. The multifunctional targeting agent can include (a) a first segment designed to bind to a capture agent through a first segment complement (i.e., the capture agent includes a targeting agent complement that is complementary to the first segment of the multifunctional targeting agent), and (b) a second segment designed to bind to an amplicon (i.e., a second segment of a multifunctional targeting agent that is used as an anchoring agent on the surface). Therefore, in this embodiment, the surface includes a multifunctional targeting agent that is contacted with a capture agent that is bound to the targeting agent through a connection between the first segment and the first segment complement. The 3-AB RCA / PLA assay is performed as described herein, and the amplicon binds to the anchoring segment of the multifunctional targeting agent before the measurement step. This method can be used to ensure that a 1:1 ratio of capture agent to anchoring agent is used in the assay method.

[0353] A wide variety of surfaces are suitable for use in the methods of the present invention, including conventional surfaces in the field of binding assays. Surfaces can be prepared from a variety of different materials, including polymers (e.g., polystyrene and polypropylene), ceramics, glass, composite materials (e.g., carbon polymer composites such as carbon-based inks). Suitable surfaces include surfaces of macroscopic objects such as assay containers (e.g., test tubes, cuvettes, flow cells, FACS cell sorters, cartridges, holes in multiwell plates, etc.), slides, assay chips (such as those used in gene or protein chip measurements), pins or probes, beads, filter media, lateral flow media (e.g., filter membranes used in lateral flow test strips), etc.

[0354] Suitable surfaces also include particles (including but not limited to colloids or beads) commonly used in other types of particle-based assays, such as magnetic, polypropylene and latex particles, materials commonly used in solid phase synthesis such as polystyrene and polyacrylamide particles, and materials commonly used in chromatography applications such as silica, aluminum, polyacrylamide, polystyrene. The material can also be a fiber such as carbon fiber. Microparticles can be inanimate or can contain living biological entities such as cells, viruses, bacteria, etc. The particles used for the method can be made of any material suitable for attaching one or more capture or detection reagents, and they can be collected via, for example, centrifugation, gravity, filtration or magnetic collection. Many different types of particles that can be attached to capture or detection reagents are commercially available for use in binding assays. These include non-magnetic particles and particles comprising magnetizable materials, which allow particles to be collected with a magnetic field. In one embodiment, particles are made of electrical conductivity and / or semiconductor materials, such as colloidal gold particles. Microparticles can have a variety of sizes and shapes. For example and without limitation, microparticles can be 5 nanometers to 100 nanometers. Preferably, microparticles have materials from 20nm to 10 microns. The particles may be spherical, oblong, rod-shaped, etc., or they may be irregularly shaped.

[0355] The particles used in the method can be coded to allow identification of specific particles or particle sub-populations in a particle mixture. The use of such coded particles has been used to realize the multiplexing of an assay method that uses particles as a solid support for binding assays. In one approach, particles are manufactured to include one or more fluorescent dyes and identify specific particle populations based on the intensity and / or relative intensity of fluorescence emission at one or more wavelengths. This approach has been used in Luminex xMAP systems (see, e.g., U.S. Patent No. 6,939,720) and Becton Dickinson Cytometric Bead Array systems. Alternatively, particles can be encoded via differences in other physical properties such as size, shape, embedded optical modes, etc. One or more particles provided in a mixture or particle set can be encoded to distinguish from other particles in the mixture by particle optical properties, size, shape, embedded optical modes, etc.

[0356] In a specific embodiment, the method of the invention can be used in a multiplex format by binding a plurality of different analytes to a plurality of capture reagents for those analytes, the capture analytes being immobilized on coded beads, such that the code identifies the capture reagent (and analyte target) of a particular bead. The method further comprises counting the number of beads with bound analytes (using the detection method described herein).

[0357] Alternatively or additionally, the detection complex and / or capture reagent can directly or indirectly bind to different discrete binding domains on one or more solid phases, such as in a binding array, where the binding domains are individual array elements, or in a set of beads, where the binding domains are individual beads, so that discrete assay signals are generated and measured from each binding domain. If capture reagents for different analytes are immobilized in different binding domains, different analytes bound to those domains can be measured independently. In one example of such an embodiment, the binding domains are prepared by immobilizing discrete domains of capture reagents that bind to the analyte of interest on one or more surfaces. Optionally, the one or more surfaces can partially define one or more boundaries of a container (e.g., a flow cell, a well, a cuvette, etc.) that holds the sample or through which the sample passes. In a preferred embodiment, each binding domain is formed on an electrode for use in an electrochemical or electrochemiluminescent assay. Multiplexed measurements of analytes on a surface comprising multiple binding domains using electrochemiluminescence have been used in Meso Scale Diagnostics, LLC, and Imager product line (see, e.g., U.S. Patent Nos. 7,842,246 and 6,977,722, the disclosures of which are incorporated herein by reference in their entireties).

[0358] Further, the detection complex and / or capture reagent can be directly or indirectly bound to the electrode surface, which optionally comprises different discrete binding domains as described above. The electrode surface can be a component of a porous wall and / or a flow cell. The electrode can comprise an electrically conductive material, such as a metal such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, conductive allow, and the like. They can also comprise a metal coated with an oxide, such as aluminum coated with aluminum oxide. The electrode can comprise a working electrode and a corresponding electrode, which can be prepared from the same or different materials, such as a metal corresponding electrode and a carbon working electrode. In a specific embodiment, the electrode comprises a carbon-based material such as carbon, carbon black, graphite carbon, carbon nanotubes, carbon filaments, graphite, graphene, carbon fiber, and a mixture thereof. In one embodiment, the electrode comprises elemental carbon, such as graphite, carbon black, carbon nanotubes, etc. Advantageously, they can comprise conductive carbon polymer composites, conductive particles (such as carbon ink, carbon glue, metal ink, graphene ink) dispersed in a matrix, and / or conductive polymers. A specific embodiment of the present invention is an assay module, preferably a multiwell plate, having an electrode (eg a working and / or counter electrode) comprising carbon, such as a carbon layer and / or a screen-printed carbon ink layer.

[0359] The present invention includes methods for detecting and calculating each detection complex. In a specific embodiment, the surface may include a plurality of capture reagents for one or more analytes present in the sample, and the plurality of capture reagents are distributed across a plurality of resolvable binding regions located on the surface. Under conditions for implementation and analysis of measurements, a "resolvable binding region" is the minimum surface region associated with a single binding event that can be resolved and distinguished from another region where another single binding event occurs. Therefore, the method includes combining one or more analyte molecules with one or more capture reagents on the surface, determining the presence or absence of analyte molecules in a plurality of resolvable binding regions on the surface, and identifying the number of resolvable binding regions containing analyte molecules and / or the number of analyte domains that do not contain analyte molecules.

[0360] The resolvable binding region can be optically interrogated in whole or in part, i.e., each resolvable binding region can be optically interrogated separately and / or the entire surface containing multiple resolvable binding regions can be imaged and one or more pixels or pixel grouping maps in the image can be located (mapped) to each resolvable binding region. The resolvable binding region can also be a microparticle within a plurality of microparticles. The resolvable binding region showing changes in its optical signature can be identified by a conventional optical detection system. Depending on the type of detection (e.g., the type of fluorescent entity, etc.) and the operating wavelength, an optical fiber designed for a specific wavelength can be used to perform optical interrogation of the resolvable binding region. In an embodiment in which optical interrogation is used, the system may include more than one light source and / or multiple optical fibers to adjust the wavelength and / or the intensity of the light source. In some embodiments, a CCD camera is used to capture light signals from multiple resolvable binding regions. Other non-limiting examples of camera imaging systems that can be used to capture images include charge injection devices (CIDs), complementary metal oxide semiconductor (CMOS) devices, scientific CMOS (sCMOS) devices, and time delay interrogation (time delay integration) (TDI) devices, as known to those of ordinary skill in the art. In some embodiments, a scanning mirror system coupled to a photodiode or photomultiplier tube (PMT) can be used for imaging.

[0361] The measuring step of the method may include imaging the light signal from the surface (or part thereof) to generate an image consisting of a plurality of pixels, wherein each resolvable binding region map is located to one or more pixels or pixel groups in the image. The method recognized in the art can be used to complete image analysis to identify the pixel or pixel set with the signal indicating the binding event (detection complex), such as a large number of image analysis algorithms and software present in the method to identify and calculate the biological structure labeled in the fluorescence microscope image. In one embodiment, after filtering the image to remove the large-scale signal gradient, the image is converted into a binary image using a segmentation threshold. The resolvable binding region is found by identifying the continuous region with a higher than threshold intensity. If the binding domain meets the size and intensity requirements, it is classified as a binding event.

[0362] In one embodiment, the resolvable binding region is an element of an array. In a preferred embodiment, the array is an array of micropores or nanopores, such as individual depressions or wells of a unitary substrate. Preferably, the volume of the pore is less than 100 nL, preferably less than 50 nL. In one embodiment, the pore volume ranges from about 10 aL-100 pL. Optionally, the pore can be configured to store microparticles.

[0363] In one embodiment, at least 50% of the resolvable binding areas located on the substrate and addressed during the assay contain 0 or 1 analyte molecules. Preferably, at least 80%, more preferably at least 95%, and most preferably at least 99% of the resolvable binding areas contain 0 or more analyte molecules. The concentration of the analyte molecules in the sample is determined at least in part using a calibration curve, a Poisson distribution analysis, and / or a Gaussian distribution analysis of the number of binding areas containing at least 0 or 1 analyte molecules. In a specific embodiment, the surface comprises a plurality of particles, each of which comprises a plurality of capture reagents for the analyte molecules, and the plurality of particles are distributed across a plurality of resolvable binding areas (e.g., an array of micropores or nanopores). Therefore, the method comprises: (i) binding one or more analyte molecules to one or more capture reagents on the surface, (ii) distributing a plurality of particles across an array of resolvable binding areas; and (iii) determining the presence or absence of analyte molecules in each resolvable binding area, thereby identifying the number of binding domains containing analyte molecules and / or the number of binding domains that do not contain analyte molecules.

[0364] It can also be advantageous to detect analytes in a confined volume using one or more methods of the present invention. In these embodiments, the analyte molecules in the sample are combined with a pair of detection reagents, each of which carries a distinguishable marker, and the analyte is divided in multiple positions on a substrate (e.g., plate, dish, chip, optical fiber, grid, etc.), such as a hole or a reaction vessel (referred to herein as "reaction vessel"), so that most reaction vessels contain one or less analytes. The method allows the user to detect analyte molecules by counting the number of reaction vessels containing each distinguishable marker attached to the analyte. In some cases, the multiple reaction vessels of addressing are part or substantially all of the total amount of reaction vessels that may contain at least one analyte molecule (such as combined with at least one analyte molecule or not combined with any analyte molecule). Reference is made to the following published U.S. patent applications: U.S. Patent Application No. 20070259448; U.S. Patent Application No. 20070259385; U.S. Patent Application No. 20070259381; and International Patent Application No. PCT / US07 / 019184; and International Patent Application No. PCT / US09 / 005428. Each of these publications is incorporated herein by reference. At least a portion of the reaction vessels can be addressed and a measurement indicating the number / percentage of reaction vessels containing at least one analyte molecule or particle can be made. In some cases, based on the number / percentage, a measure of the concentration of analyte molecules in the fluid sample can be determined.

[0365] In a specific embodiment of realizing the detection of analyte molecules in a confined volume, the analyte in the sample can be detected by making the analyte bind to the first and second detection reagents to form a detection complex. Each detection complex comprises an analyte, a first detection reagent and a second detection reagent, and the first detection reagent and the second detection reagent have a first and a second detectable label, respectively. The detection complex can be formed simultaneously, substantially simultaneously or sequentially. The detection complex is divided across multiple reaction vessels so that most reaction vessels contain one or less detection complexes, and the number of analyte molecules is detected by calculating the number of each reaction vessel containing the first and second detectable labels. Preferably, the detection complex is divided across multiple reaction vessels so that the probability of detecting the unbound first detection reagent and the unbound second detection reagent in the same container is less than about one tenth, preferably less than about one hundredth, more preferably less than about one thousandth, and most preferably less than about one ten-thousandth. The detection complex is divided across multiple reaction vessels, i.e., divided or divided into parts, for example, by manually separating the parts of the detection complex equally in multiple reaction vessels, and / or by flowing a solution containing the detection complex through multiple reaction vessels so that the detection complex is divided into each reaction vessel on the support.

[0366] In another embodiment, the analyte in the sample can be detected by (a) binding the analyte to a surface-bound capture reagent and a first and second detection reagent to form a detection complex, wherein (i) each detection complex comprises a capture reagent, an analyte, a first detection reagent and a second detection reagent, and (ii) the first detection reagent has a first detectable label and the second detection reagent has a second detectable label. The detection complex can be formed by adding components in any order, for example, by bringing the components close together simultaneously or substantially simultaneously, or by sequentially adding each component to establish the detection complex in a stepwise manner. The detection complex is divided across multiple reaction vessels so that most reaction vessels contain one or less analyte, and the number of analyte molecules is detected by counting the number of reaction vessels containing the first and second detectable labels. The method can be performed with or without cleaning after each step and before the detection step.

[0367] The surface can be a particle and optionally, a plurality of capture reagents are immobilized on the particle or a plurality of particles. In this embodiment, the partitioning step can be performed in a variety of ways: (i) the capture reagent is immobilized on a plurality of particles and the partitioning of the analyte is achieved by allowing the analyte to bind to the capture reagent and partitioning the particles into a plurality of reaction vessels; or (ii) the capture reagent is immobilized on a plurality of particles and the partitioning of the analyte is achieved by partitioning the particles into a plurality of reaction vessels and then allowing the analyte to bind to the capture reagent.

[0368] Multiple reaction vessels can also contain water droplets dispersed in a water-in-oil emulsion. The emulsion can be prepared as droplets with a diameter of up to 100um and a volume of nearly 1nL. High loading, i.e., more than 1010 droplets in the emulsion, the ease of preparing the emulsion and its high stability under a wide range of conditions make it an ideal means for compartmentalized biochemical assays. Each droplet acts as an independent reaction vessel and the detection complex optionally attached to the particle can be divided across multiple droplets.

[0369] Alternatively, for example, if the reaction vessels are wells of a plate, the surface is located in one of the reaction vessels, then the surface can be a domain or region within one of the wells of the plate. In this embodiment, the capture reagent can be immobilized on the domain or region of multiple reaction vessels, and the partitioning step is achieved by binding the analyte molecules to the capture reagent. In another embodiment, multiple reaction vessels include regions to which the targeting module is immobilized, the capture reagent includes a targeting module complement, and the partitioning step is achieved by binding the targeting module complement to the targeting module located in multiple reaction vessels.

[0370] In an additional or alternative embodiment, the binding assays described herein may also include a pre-concentration step to improve assay performance, for example by increasing the concentration of the analyte in the sample and / or by reducing the concentration of extraneous materials that may be present in the sample that may hinder assay performance. This may be accomplished by (a) contacting a sample comprising the analyte of interest with particles of a first binding reagent attached to the binding analyte, thereby forming a complex comprising the analyte bound to the first binding reagent; (b) collecting the complex; (c) separating the sample unbound components from the complex; (d) releasing the complex. Such a pre-concentration method may be performed prior to the binding assays described herein to remove impurities that may hinder assay performance. In this regard, reference is made to U.S. Application Publication No. US2010 / 0261292, the disclosure of which is incorporated herein by reference.

[0371] Specifically, the concentration step includes subjecting the sample containing the analyte to conditions sufficient to form an analyte complex, the analyte complex including the analyte bound to the first detection reagent, wherein the first detection reagent is connected to the first nucleic acid probe. Then, the analyte complex formed at the end of the concentration step is bound to (i) a capture reagent on the surface, the surface comprising a capture reagent for the analyte, and an anchoring reagent comprising an anchoring oligonucleotide sequence; and (ii) a second detection reagent for the analyte connected to the second nucleic acid probe; thereby forming a complex comprising the capture reagent, the analyte, and the first and second detection reagents on the surface. The surface-bound complex is subjected to an extension process requiring the proximity of the first and second probes, extending the second probe to form an extended sequence comprising an anchor sequence complement complementary to the anchor sequence. In a specific embodiment, the concentration step further includes: (i) contacting the sample containing the analyte with a solid phase connected to a targeting agent, the targeting agent being complementary to at least a portion of the first nucleic acid probe, thereby forming a concentrated complex, the concentrated complex comprising the analyte bound to the solid phase through a binding reaction between the first nucleic acid probe and the targeting agent. (ii) collecting the concentrated complex; (iii) separating unbound components of the sample from the concentrated complex; and (iv) releasing the concentrated complex to separate the solid phase from the analyte to form an analyte complex.

[0372] As used herein, collection refers to the physical positioning of materials in a mixture. Collection includes positioning of materials by binding reactions or adsorption. For example, materials in a mixture can be collected on a solid phase by adsorbing the material on a solid phase or by binding the material to a binding agent on a solid phase. However, collection is not limited to positioning on a solid phase, and can also include techniques in the art for positioning materials at a position / volume within a larger fluid volume, such as by using optical tweezers (which use light to manipulate microscopic objects as small as a single atom, where radiation pressure from a focused laser beam can capture small particles), electric or magnetic fields, focused flow, density gradient centrifugation, etc. to position materials.

[0373] Certain embodiments of the present invention include collecting microparticles or materials bound to microparticles. Suitable collection methods include many methods known in the art of microparticle-based assays that achieve localization of microparticles from suspensions. These methods include filtering on filters or porous membranes under gravity or by centrifugal precipitation, localization by applying a magnetic field (magnetizable particles), binding or adsorption of particles to a macroscopic solid phase, using optical tweezers, etc.

[0374] As used herein, release refers to the delocalization of previously collected materials. Materials that are held in a localized position by chemical bonds or by specific or nonspecific binding interactions can be allowed to be delocalized by breaking bonds or interactions, so that the material can diffuse or mix into the surrounding medium. There are many mature cleavable chemical linkers that can be used to provide covalent bonds that can be cleaved without harsh conditions. For example, linkers containing disulfide bonds can be cleaved using thiol or other reducing agents, linkers containing cis-diols can be cleaved using periodate, and metal-ligand interactions (such as nickel-histidine) can be cleaved by changing pH or introducing competitive ligands. Similarly, there are many mature reversible binding pairs that can be used (including those identified in the field of affinity chromatography). For example, the binding of many antibody-ligand pairs can be reversed by changes in pH, adding protein denaturants or chaotropes, adding competitive ligands, etc. Other suitable reversible binding pairs include complementary nucleic acid sequences, and their hybridization can be reversed under a variety of conditions, including changing pH, reducing salt concentration, increasing temperature to a melting temperature higher than the pair and / or adding nucleic acid denaturants (such as formamide). Such reversible binding pairs can be used as targeting agents (as described above), for example, a first targeting agent can be attached to a first binding reagent that binds an analyte, a second targeting agent can be attached to a solid phase, and the binding interaction of the first and second targeting agents can be used to reversibly immobilize the first binding reagent on the solid phase.

[0375] Release also includes the physical removal of the material by, for example, mixing, oscillation, vortex, convection fluid flow, by applying magnetic force, electric power or optical force mixing, etc. In the case of having collected particulates or materials that are bound to particulates, such physical methods can be used to resuspend the particles in the surrounding matrix. Release can be simply opposite to the previous collection step (for example, by any of the above-mentioned mechanisms), or collection and release can be carried out by two different mechanisms. In one such example, the collection of the material (for example, analyte or the complex comprising analyte) that is bound to the particle can be realized by the physical collection of the particle. Then the material is released by cutting a bond or reversing the binding reaction that keeps the material on the particle. In a second such example, by interacting with the binding of the binding reagent connected to the surface, the material (for example, the analyte of the complex comprising analyte) is collected on the surface. Then the material is released by destroying a bond or connecting the binding reagent to the second binding interaction on the surface.

[0376] Collect and then release the analyte that can be used to concentrate and / or purify the sample.By collecting in the first volume and being released in the second smaller volume, the analyte in the concentrated sample can be.By concentrating, the sensitivity of subsequent measurement steps can be significantly improved usually.By collecting and removing some or all of the samples that are not collected from the sample, the potential determination interfering substance in the sample can be reduced or eliminated.Optionally, the removal of unbound sample can include washing with a liquid reagent and releasing the material to a liquid reagent species (for example, assay or washing buffer) that is limited, so that uniform matrix is ​​provided for subsequent determination steps.

[0377] As in US2010 / 0261292 Figure 3 As shown in (a), which is incorporated herein by reference, the method includes contacting a sample containing a target analyte with a particle connected to a first binding reagent, the first binding reagent binds to the target analyte, wherein the first binding reagent is connected to a first targeting agent, and the particle is connected to a second targeting agent, and the first binding reagent and the particle are connected by a binding reaction between the first and second targeting agents to form a complex containing the target analyte bound to the first binding reagent. The complex is then collected, and unbound components in the sample are separated from the complex. The complex is released, and the released complex is contacted with a second binding reagent bound to a solid phase, wherein the second binding reagent binds to the complex. This specific embodiment is as shown in Fig.16As shown. The modified particle (1601) is to include a capture oligonucleotide sequence, 1602, which is at least partially complementary to the sequence of the proximal probe, 1603, which is bound to the detection antibody 1604. The particle is mixed with the proximal probe to hybridize the capture sequence with the probe sequence to form a complex, 1605. The complex is then mixed with a sample containing an analyte, 1606, and optionally, one or more contaminants, 1607-1608. The analyte is bound to the detection antibody (1609) and the contaminants are removed (1610). The particle is removed from the complex including the bound analyte under suitable conditions to form a concentrated analyte solution bound to the proximal probe (1611), which can be used in an immunoassay as described herein, wherein an additional proximal probe is bound to the analyte, and the 3-antibody complex is subjected to RCA-PLA to detect the presence of the analyte in the sample (1612), for example, as described in Figure 2 (a) and the accompanying instructions.

[0378] In another embodiment, the immunoassay complex between the detection antibody and the analyte is formed in solution, then amplified, and the amplified product is then attached to the particle via a capture reagent and / or anchor. Optionally, the amplified product can be filtered and then captured on the particle via a capture reagent and / or anchor. This method is shown in Fig.17 In. The analyte, A (1701) is bound to detection antibodies (1702 and 1703, respectively), each of which is bound to a proximal probe (1704 and 1705, respectively) and a detection complex (1706) is formed comprising the analyte bound to each detection antibody. The detection complex is contacted with two linker sequences (1707a and 1707b), each of which includes a terminal sequence complementary to a non-overlapping region of the first proximal probe and a terminal sequence complementary to a non-overlapping region of the second proximal probe. The linker sequence is hybridized with the first and second proximal probes, and the terminal sequence of the linker oligonucleotide is ligated to form a circular target sequence (1708) that hybridizes to both the first and second proximal probes. The second proximal probe is extended by rolling circle hybridization to produce an amplicon comprising a binding reagent complementary to the anchoring reagent. The amplicons are contacted with a surface (1709) comprising a capture agent (1710) and an anchoring agent (1711), and the amount of amplicons bound to the surface is measured by labeling with a plurality of labeled probes (1712).

[0379] In addition, the method described herein can be used for the detection and quantification of oligomeric analytes. If the target antigen is a homo-oligomer, i.e., an antigen with two or more identical subunits, the detection antibody needs to be independently labeled with both a ligation templating oligonucleotide and a primer oligonucleotide. This may lead to inefficiency, because only two antibodies with template oligonucleotides or only two antibodies with priming oligonucleotides can be combined, and this non-productive complex can reduce the sensitivity of the method. When the method is applied to the detection of specific oligomeric structures, this problem may be aggravated, wherein additional specific template oligonucleotides are used to allow specific detection of oligomers. Therefore, the method described herein may include the pre-formation of a group of detection antibodies, which are configured to effectively bind to the desired oligomeric analyte and a template for forming a rolling circle template. This can be achieved using additional oligonucleotide sequences, such as scaffold oligonucleotides, which can be specifically hybridized with the oligonucleotides coupled to the antibody, so that the antibody is formulated in the form of an oligomer in the detection antibody mixture, and is paired with the target antigen oligomer. This pre-organization step allows for optimal binding of the detection antibody to the target oligomer. After the detection antibody binds to the oligomer antigen, the scaffold oligonucleotide can be removed by exonuclease degradation or another suitable chemical method or endonuclease treatment. Once the scaffold oligonucleotide is removed, a proximity ligation rolling circle amplification assay can be performed as described herein.

[0380] In other embodiments, the assay format described herein also includes one or more control assays. A negative control may be included on the binding domain, which includes a capture antibody without a corresponding detection antibody, thereby providing a consistent background signal for all samples. The measurement of a signal above a preset threshold may indicate an inappropriate assay treatment or the presence of a sample-dependent matrix effect that causes non-specific binding of a labeled detection probe. In addition, a sample sample may also be included in the assay of a human target antigen (e.g., secreted or intracellular protein) for multiple control functions. A positive signal will indicate the presence of human material, so the test sample is added and the quality. Human antigens may also be used as a process control for bacterial antigen extraction. For example, intracellular human analytes may be selected as samples, which also require lysis and extraction for detection, such as Akt. The measurement of a signal below a predetermined threshold will indicate that no sample has been added, indicating that a reagent or treatment failure has occurred, or indicating that there is a substance that interferes with amplification or detection. In addition to internal controls, external positive and negative controls may also be used with methods and / or kits. Positive controls may contain a mixture of non-infectious bacterial extracts representing all specific target antigens detected by the multiplexed panel, providing positive signals for all assays tested. Negative controls include a representative matrix without any target protein.

[0381] Examples of samples that can be analyzed by the methods of the present invention include, but are not limited to, food samples (including food extracts, food homogenates, beverages, etc.), environmental samples (e.g., oil samples, environmental sludges, collected environmental aerosols, environmental wipes, water filtrates, etc.), industrial samples (e.g., starting materials, products or intermediates from industrial production processes), human clinical samples, veterinary samples, and other samples of biological origin. Biological samples that can be analyzed include, but are not limited to, feces, mucosal swabs, physiological samples, and / or samples containing cell suspensions. Specific examples of biological samples include blood, serum, plasma, feces, mucosal swabs, tissue aspirates, tissue homogenates, cell cultures and cell culture supernatants (including cultures of eukaryotic and prokaryotic cells), urine, saliva, sputum, and cerebrospinal cord samples.

[0382] Analytes that can be measured using the methods of the present invention include, but are not limited to, proteins, toxins, nucleic acids, microorganisms, viruses, cells, fungi, spores, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, drugs, hormones, steroids, nutrients, metabolites, and any modified derivatives of the above molecules, or any complex comprising one or more of the above molecules or a combination thereof. The level of the analyte of interest in a sample may indicate a disease or disease condition, or it may simply indicate whether a patient has been exposed to the analyte.

[0383] In a specific embodiment, the analyte of interest is an exosome, i.e., a small membrane vesicle released by most cell types. The release and subsequent uptake of exosomes is a method of cell-to-cell communication and plays a role in the regulation of many physiological and pathological processes. Exosomes have been shown to contain a variety of signaling molecules, including but not limited to surface-bound and cytoplasmic proteins, lipids, mRNAs, and miRNAs, and it has been shown that the properties and concentrations of these substances in each exosome can be used to infer their cellular origin and function. Therefore, the genomic or proteomic profile of the patient's total exosome population can provide valuable prognostic information for various pathological conditions, including cancer, infectious diseases, kidney and liver diseases, and traumatic brain injury, etc.

[0384] Exosomes are usually measured in aggregates, which are measured by separating a large number of aggregates from biological fluids, breaking their membranes, and measuring the contents by conventional methods, such as western blotting, PCR or sequencing. However, the method and kit of the present invention can be used to characterize exosomes by capturing exosomes on the surface and subsequently detecting target molecules expressed by exosomes. Several proteins common to most exosomes have been identified, such as CD9, CD63, CD81, Hsp70, PDCD6IP, Tsg101, and in one embodiment, these targets can be used individually or in combination to capture exosomes on the surface by binding interactions between one or more capture agents and one or more shared exosome target proteins. In alternative or additional embodiments, disease-related markers present on or in exosomes are used to capture exosomes on the surface by binding interactions between one or more capture agents and disease-related exosome markers. The captured exosomes can then be probed using one or more detection reagents directed against one or more common exosomal proteins, or one or more additional molecular targets present in or on the surface of some portion of the exosome. When different targets are chosen for the capture and detection reagents, only those exosomes containing both targets will be detected, thereby having additional specificity in identifying or quantifying exosome subpopulations. In a specific embodiment, the sample used is a purified exosome preparation.

[0385] In a specific embodiment, a pair of detection reagents can be used to achieve the detection of specific phenotypically different exosome subpopulations, and the detection reagent only generates a signal when it is close to the target (proximal targets) by binding interaction as described herein. In this embodiment, the close targets can be different epitopes on the same molecule, which can be different copies of the same molecular species close to each other inside a single exosome or bound to the same exosome membrane, or they can be different interaction species in or on a single exosome, such as two interacting proteins (such as tetraserine-integrin complexes), protein receptors and ligands (such as EFG and EGFR), or mRNA molecules and RNA binding proteins (such as Argonaute 1-4 or GW182). In addition, the use of the methods described herein allows the analysis of up to three different target molecules, which may not be functionally connected, but connected by being present in a single exosome. The proximal probe used in the PLA / RCA method described herein can be extended to allow connection and subsequent amplification of target molecules that are farther away on or in the exosome. In one embodiment, a PLA / RCA assay is performed using a specific fluorescent probe for the amplicon. Thereafter, a universal fluorescent reagent (e.g., acridine orange for RNA in exosomes) is used to label the captured exosomes, and the captured exosomes are imaged to determine the correlation between the two fluorescent signatures. The use of a universal fluorescent reagent allows selection of signals from the desired exosomes or a subset thereof based on size and staining intensity.

[0386] To detect internal target molecules (carrier proteins, lipids or RNA molecules), exosomes can be fixed and permeablized before or after capture but before adding detection reagents. If permeabilized exosomes are interrogated using the methods described herein, one or both of the detection reagents can contain oligonucleotide probes capable of binding to specific RNAs, enabling detection of mRNA, miRNA and / or interactions between proteins and RNA.

[0387] Exosomes can be measured using the methods described herein with or without anchoring agents. As previously described, the use of anchoring agents in the binding assay is shown in FIG. 1 (a), and if the analyte is an exosome, the surface (101) includes a capture agent, such as a capture agent for a target protein of a common exosome. In a specific embodiment, the surface also includes an anchoring agent (103). In one or more steps, the exosome is bound to a capture agent and a detection agent (104), which also binds to the exosome through the same or different exosome target protein, wherein the detection agent is connected to a nucleic acid probe (105). Thus, a complex including a capture agent, an exosome, and a detection agent is formed on the surface. The probe is extended to form an extended sequence (107) including an anchor region that binds to the anchoring agent. The extended sequence is bound to the anchoring agent, and the amount of the extended sequence bound to the surface is measured.

[0388] Similarly, the method shown in FIG. 1 (b) can also be used for the detection of exosomes. In a specific embodiment, the detection complex may include one or more detection reagents to enhance the specificity of the exosome assay, for example, as shown in FIG. 1 (c). In one or more steps, the exosome is combined with each of a capture reagent and two (or more) detection reagents (120 and 121, respectively), wherein the detection reagent binds to a target protein expressed by the exosome, wherein each of the first and second detection reagents is connected to a nucleic acid probe (122 and 123, respectively, the first and second nucleic acid probes). The exosome can be combined with the capture and detection reagents simultaneously or substantially simultaneously or in a sequential, stepwise manner. Thus, a complex (124) including a capture reagent, an exosome, and the first and second detection reagents is formed on the surface. The first probe is extended using an extension process that requires the first and second probes to be close to each other to form an extension sequence (125), wherein the extension sequence comprises an anchor sequence complement complementary to the anchor sequence. In the penultimate step, the anchor sequence is hybridized with the anchor sequence complement and the amount of the extension sequence bound to the surface is measured. Similarly, exosomes can be detected using the methods shown in each of Figures 2(a)-(c).

[0389] The assays of the present invention can be used to determine the concentration of one or more, e.g., two or more, analytes in a sample. Thus, two or more analytes can be measured in the same sample. Groups of analytes that can be measured in the same sample include, for example, groups of assays of analytes or activities associated with a disease state or physiological condition.Certain such groups include cytokines and / or their receptors (e.g., one or more of TNF-alpha, TNF-beta, IL1-alpha, IL1-beta, IL2, IL4, IL6, IL-10, IL-12, IFN-y, etc.), growth factors and / or their receptors (e.g., one or more of EGF, VGF, TGF, VEGF, etc.), drugs of abuse, therapeutic drugs, vitamins, pathogen-specific antibodies, autoantibodies (e.g., antibodies to one or more of Sm, RNP, SS-A, SS-alpha, J0-1, and Scl-70 antigens), allergen-specific antibodies, tumor markers (e.g., CEA, PSA, CA-125II, CA 15-3, CA 19-9, CA 72-4, CYFRA, etc.), 21-1, NSE, AFP, etc.), markers of heart disease including congestive heart disease and / or acute myocardial infarction (e.g., one or more of troponin T, troponin I, troponin C, myoglobin, CKMB, myeloperoxidase, glutathione peroxidase, beta-natriuretic protein (BNP), alpha-natriuretic protein (ANP), endothelin, aldosterone, C-reactive protein (CRP), etc.), markers related to hemostasis (e.g., fibrin monomer, D-dimer, thrombin-anticoagulant enzyme complex, prothrombin fragments 1 & 2, anti-factor Xa, etc.), markers of acute viral hepatitis infection (such as IgM antibodies to hepatitis A virus, IgM antibodies to hepatitis B core antigen, hepatitis B surface antigen, antibodies to hepatitis C virus, etc.), markers of Alzheimer's disease (alpha-amyloid protein, beta-amyloid protein, Aβ42, Aβ40, Aβ38, Aβ39, Aβ37, Aβ34, tau-protein, etc.), markers of osteoporosis (such as cross-linked Nor One or more of C-terminal telopeptide, total deoxypyridinoline, free deoxypyridinoline, osteocalcin, alkaline phosphatase, C-terminal propeptide of type I collagen, bone-specific alkaline phosphatase, etc.), markers of fertility status or fertility-related disorders (e.g., one or more of estradiol, progesterone, follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, hCG, testosterone, etc.), markers of thyroid disorders (e.g., one or more of thyroid stimulating hormone (TSH), total T3, free T3, total T4, free T4 and reverse T3), and markers of prostate cancer (e.g., one or more of total PSA, free PSA, composite PSA, prostatic acid phosphatase, creatine kinase, etc.).Certain embodiments of the invention include measuring one or more, two or more, four or more, or ten or more analytes (e.g., analytes grouped together in a panel, such as those listed above; for example, a panel useful for diagnosing a thyroid disorder may include, for example, one or more of thyroid stimulating hormone (TSH), total T3, free T3, total T4, free T4, and reverse T3) associated with, for example, a particular disease state or physiological condition.

[0390] In a preferred embodiment, the panel includes one or more low abundance analytes in a traditional sample matrix, such as analytes at a concentration of less than about 100 fg / mL, and preferably less than about 10 fg / mL. A non-limiting list of analytes that may be included in the panel includes, for example, IL-17, IL-21, IL-31, Ab-38, Ab-40, Ab-42, Ab-39, Ab-43, Ab-15, Ab-16, Ab-17, Abeta oligomers, C-peptide, IL-13, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-8, IL-12 / 23p40, IL-12p70, IN F-g, PSA, PSAc, Tau, phospho-Tau, TNFa, troponin I, troponin T, troponin C, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, EPO, LC3B, albumin, CHO-P, E. coli HCP, IgA, IgE, IgG, IgG1, IgG4, IgM, NSO-P, Per-C6, residual protein protein)A, IgG2, IgG3, IgG4, AFP, CA125, caspase-3 activity, CXCL11 / I-TAC, ErbB2 / HER2, HGFR / o-MET, IFN-beta, MMP1, MMP2, MMP3, MMP9, beta-NGF, TFF3, TIMP1, Kim-1, alpha-2 macroglobulin, D-dimer, ICAM-1, myeloperoxidase, myoglobin, PAI-1, PCSK9, plasminogen, renin / prorenin, tPA, CXCL1 / GRO-alpha, CCL2 / MCP1, CCL3 / MIP-1alpha, CCL4 / MIP-1beta, CCL5 / Rantes, CRP, CXCL9 / MIG, CXCL10 / IL-10, G-CSF, GM-CSF, IFN-alpha, IFN-gamma, IL1alpha, IL-1beta, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL12(p70), IL13, IL15, IL18, IL-22, IL-23, IL-33, c-MET, adiponectin, FGF21, TSLP, GLP-1, growth hormone, IGF1, IGF2, insulin, leptin, prolactin, HIV p24, HB-EGF, AKT, phospho-AKT, and combinations thereof.

[0391] In a specific embodiment, the panel comprises one or more low abundance analytes in a traditional sample matrix, for example, analytes at a concentration of less than about 100 fg / mL, and preferably less than about 10 fg / mL, and the panel includes one or more of the following human targets: G-CDF, GM-CSF, IFNgamma, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 / 23p40, IL12p70, IL-17A, IL21, IL-22, IL-23, IL-31, IL-33, TNFalpha, TSLP, VEGF, composite PSA, free PSA, Abeta42, Abeta40, Abeta38, tau, cardiac troponin I, cardiac troponin T, HIV p24, C-peptide, and / or FGF21.

[0392] In addition, the methods and kits described herein can be used in immunoassays to detect single organism susceptibility to antimicrobial resistance markers (PBP2a / mecA (Staphylococcus aureus, Gram-positive), TEM1 (Escherichia coli, Gram-negative). These assays can be used for such analytes in both Gram-positive and Gram-negative bacteria. One or more of the following target proteins involved in antimicrobial resistance to vancomycin, beta-lactams, carbapenems, aminoglycosides, and macrolide antibiotics can be included in the assay; erm family, vanA, vanB, aac(6')-aph(2"), KPC, NDM, OXA-48, VIM, OXA-23-like, OXA-40-like, OXA-58-like, ESBL genes of the CTX-M-1 and CTX-M-9 families, and combinations thereof. In addition, one or more of the following proteins can be included in the assay: White: 50S ribosomal protein L20, 30S ribosomal protein S7, 30S ribosomal protein S2, 50S ribosomal protein L21, 50S ribosomal protein L17, 30S ribosomal protein S4, 50S ribosomal protein L15, 30S ribosomal protein S5, 50S ribosomal protein L16, 30S ribosomal protein S3, 50S ribosomal protein L22, 50S ribosomal protein L4, ribosomal protein L25, 50S ribosomal protein L 5, 30S ribosomal protein S2, ribosomal proteins L30, L31 and L32, and combinations thereof. In addition, one or more of the following targets, such as the elongation molecule EF-TU, ACP, acyl carrier protein, RolL, ribosomal protein GroS (MoB, 65,000), components of the molecular chaperone systems Gro-EL-Gro-ES and GapA, enzymes in glycolysis, and combinations thereof, can be evaluated alone or together with one or more markers identified herein.

[0393] In addition, the methods and kits described herein can also be used for immunoassays to detect health care-associated infections (HAI organisms), including but not limited to Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomon asaeruginosa, Enterobacter species. The resistance markers for each HAI organism are provided in the table below:

[0394]

[0395]

[0396] Specific outer membrane proteins can be assessed in the immunoassays described herein:

[0397]

[0398] In addition, the methods and kits described herein can be used in immunoassays to detect one or more of the following categories of biomarkers: cytokines, circulating tumor-specific proteins, proteins associated with one or more infectious diseases, intracellular markers, etc., and combinations thereof.

[0399] Still further, the methods and kits described herein can be used to identify the following autoimmune diseases by evaluating the presence or absence of one or more of the relevant antigens listed below:

[0400]

[0401]

[0402] In a specific embodiment, the group includes one or more low-abundance analytes in a traditional sample matrix, such as an analyte having a concentration of less than about 100 fg / mL, and preferably less than about 10 fg / mL. The group preferably includes one or more of the following analytes: IL-17, IL-21, IL-31, IL-22, IL-23, IL-33, cardiac troponin T, and combinations thereof. In a specific embodiment, the concentration of the analyte detected in the sample is in the range of 0.01 fM to 100 fM, 0.03 fM–50 fM, or 0.03 fM–10 fM. In some embodiments, the concentration of the analyte molecule in the sample that can be substantially accurately determined is less than about 100 fM, less than about 10 fM, less than about 3 fM, less than about 1 fM, less than about 0.3 fM, less than about 0.1 fM, less than about 0.03 fM, or less. The concentration of analyte molecules in a sample can be considered to be substantially accurately determined if the measured concentration of analyte molecules in the sample is within about 20% of the actual concentration of analyte molecules in the sample. In certain embodiments, the measured concentration of analyte molecules in the sample can be within about 10%, about 3%, or about 1% of the actual concentration of analyte molecules in the sample. The detection limit of the assay is the concentration that produces a signal that is at least 2.5 standard deviations above the background signal, and preferably the assay can detect about 10-10,000 molecules in a sample, or 100-5,000 molecules in a sample, or 100-1000 molecules in a sample.

[0403] In another embodiment, the methods described herein can be used to detect analytes that are in low abundance due to recent exposure and / or infection. Early diagnosis of a variety of diseases or conditions, such as cancer, bacterial infections, such as Bacillus anthracis (anthrax), viral infections, such as HIV, hepatitis, HPV, etc., toxin exposure, such as ricin, botulinum toxin A, B or E, etc., is limited due to the fact that the limits of detection (LOD) of existing technologies such as ELISA are higher than the circulating concentration of low-abundance proteins that can indicate the onset of disease. The panel can include one or more low-abundance analytes in a traditional sample matrix, such as analytes at a concentration of less than about 100 fg / mL, and preferably less than about 10 fg / mL. A non-limiting list of analytes that can be included in the panel includes, for example, HIVgp41, HIVgp120, HIVgp160, HIVp24, HIVp66, HIVp51, HIVp17, HIVp31, Tat, Nef, Viv, hepatitis A, B, C, D, or E antigens, HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and / or 82, HPV-E6 and E7 proteins, IL-17, IL-21, IL-31, IL-22, IL-23, IL-33, cardiac troponin T, and combinations thereof.Still further, the panel may also include one or more of the following analytes, which may be in low abundance due to recent disease onset, exposure and / or infection: Ab-38, Ab-40, Ab-42, Ab-39, Ab-43, Ab-15, Ab-16, Ab-17, Abeta oligomers, C-peptide, IL-13, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-8, INF-g, PSA, Tau, Phospho-Tau, TNFa, Troponin I, Troponin T, Troponin C, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, EPO, LC3B, albumin, CHO-P, E. coli HCP, IgA, IgE, IgG, IgG1, IgG4, IgM, NSO-P, Per-C6, remaining protein A, IgG2, IgG3, IgG4, AFP, CA125, caspase-3 activity, CXCL11 / I-TAC, ErbB2 / HER2, HGFR / o-MET, IFN-be ta, MMP1, MMP2, MMP3, MMP9, beta-NGF, TFF3, TIMP1, Kim-1, alpha-2 macroglobulin, D-dimer, ICAM-1, myeloperoxidase, myoglobin, PAI-1, PCSK9, plasminogen, renin / prorennin, tPA, CXCL1 / GRO-alpha, CCL2 / MCP1, CCL3 / MIP-1alpha, CCL4 / MIP-1beta, CCL5 / Rantes, C RP, CXCL9 / MIG, CXCL10 / IL-10, G-CSF, GM-CSF, IFN-alpha, IFN-gamma, IL1alpha, IL-1beta, IL-3, IL-7, IL-12(p70), IL-13, IL-15, IL-18, c-MET, adiponectin, FGF21, GLP-1, growth hormone, IGF1, IGF2, insulin, leptin, prolactin, HB-EGF, AKT, phospho-AKT, and combinations thereof.

[0404] The methods of the present invention are designed to allow detection of a wide variety of biological and biochemical agents, such as those described above. In one embodiment, the methods can be used to detect pathogenic and / or potentially pathogenic viruses, bacteria and toxins, including biological warfare agents ("BWA") in a variety of relevant clinical and environmental matrices, including, but not limited to, blood, sputum, stool, filtrate, swabs, etc. A non-limiting list of pathogens and toxins that can be analyzed (alone or in combination) using the methods of the present invention are Bacillus anthracis (anthrax), Yersinia pestis (plague), Vibrio cholerae (cholera), Francisella tularensis (tularemia), Brucella species (brucellosis), Coxiella burnetii (Q fever), listeria, salmonella, shigella, V. cholera, Chlamydia trachomatis, Burkholderia pseudomallei, orthopox viruses including smallpox, viral encephalitis, Venezuelan equine encephalitis virus (VEE), western equine encephalitis virus (WEE), encephalitis virus (WEE), eastern equine encephalitis virus (EEE), Alpha viruses, viral hemorrhagic fevers, Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae, Ebola virus, Staphylococcal enterotoxins, Ricin, Botulinum toxins (A, B, E), Clostridium botulinum, Mycotoxins, Fusarium, Myrotecium, Cephalosporium, Trichoderma, Verticimonosporium, Stachybotrys, glanders, wheat fungi, Bacillus globigii, Serratia marcescens, marcescens, yellow rain, trichothecene mycotoxins,Salmonella typhimurium, aflatoxins, Xenopsylla cheopis, Diamanus montanus, alastrim, monkeypox, arenaviruses, Hantaviruses, Lassa fever, Argentine hemorrhagic fevers, Bolivian hemorrhagic fevers, Rift Valley fever virus, Crimean-Congo virus, Hantaviruses, Marburg hemorrhagic fevers, yellow fever virus, dengue virus, influenza (including human and animal strains, including avian influenza H5N1, influenza A, influenza A, H1-specific, influenza A, H3-specific, influenza A, H5-specific, influenza A, 2009-H1N1-specific, influenza B), RSV, human immunodeficiency virus I and II (HIV I and II), hepatitis A, hepatitis B, hepatitis C, hepatitis (non-A, B or C), enterovirus, Epstein-Barr virus, cytomegalovirus, herpes simplex virus, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, human papillomavirus, Treponema pallidum, Streptococcus pneumonia, Borellia burgdorferi, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Staphylococcus aureus, Staphylococcal enterotoxin B (SEB), abrin, Shiga toxin 1, Shiga toxin 2, Moraxella catarrhalis catarrhalis, Streptococcus pyogenes, Clostridium difficile, Neisseria meningitidis, Klebsiella pneumoniae, Mycobacterium tuberculosis, Group A Streptococcus, Escherichia coli O157, Coronavirus,Coxsackie A virus, rhinovirus, parainfluenza virus, respiratory syncytial virus (RSV), metapneumovirus, vaccinia, and adenovirus.

[0405] The improvements to the binding assay described herein can be used to expand the dynamic range of the binding assay, i.e., the concentration range of analyte molecules in a fluid sample that can be quantified by a system or method without diluting or concentrating the sample or changing the assay conditions (e.g., the concentration of the reagents used, etc.) that produce similar results, and wherein the measured concentration of the analyte molecules can be determined substantially accurately. The concentration of the analyte molecules in the fluid sample can be considered to be substantially accurately determined if the concentration of the analyte molecules in the measured fluid sample is within about 10% of the actual (e.g., true) concentration of the analyte molecules in the fluid sample. In certain embodiments, the concentration of the analyte molecules in the measured fluid sample is substantially accurately determined in an embodiment, wherein the measured concentration is within about 5% of the actual concentration of the analyte molecules in the fluid sample, within about 4%, within about 3%, within about 2%, within about 1%, within about 0.5%, within about 0.4%, within about 0.3%, within about 0.2%, or within about 0.1%. In some cases, the measured concentration brightness differs from the true (e.g., actual) concentration by no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, or no more than about 0.5%. In some embodiments, the accuracy of the assay method can be determined by determining the concentration of analyte molecules in a fluid sample of known concentration using a selected assay method and comparing the measured concentration to the actual concentration.

[0406] In some embodiments, the system or method may be capable of measuring the concentration of analyte molecules in a fluid sample over a dynamic range of greater than about 1000 (3log), about 10,000 (4log), about 100,000 (5log), about 350,000 (5.5log), 1,000,000 (6log), about 3,500,000 (6.5log), about 10,000,000 (7log), about 35,000,000 (7.5log), about 100,000,000 (8log), or more.

[0407] In some embodiments, the concentration of analyte molecules in a fluid sample (e.g., an unknown concentration) that can be substantially accurately determined is less than about 5000 fM (femtomolar), less than about 3000 fM, less than about 2000 fM, less than about 1000 fM, less than about 500 fM, less than about 300 fM, less than about 200 fM, less than about 100 fM, less than about 50 fM, less than about 25 fM, less than about 10 fM, less than about 5 fM, less than about 2 fM, less than about 1 fM, less than about 500 aM (attomolar), less than about 100 aM, less than about 10 aM, less than about 5 aM, less than about 1 aM, less than about 0.1 aM, less than about 500 zM (zeptomolar), less than about 100 zM, less than about 10 zM, less than about 5 zM, less than about 1 zM, less than about 0.1 zM, or less. In some cases, the limit of detection (e.g., the lowest concentration of analyte molecules that can be measured in a solution) is about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, about 1 fM, about 500 aM (attomolar), about 100 aM, about 50 aM, about 10 aM, about 5 aM, about 1 aM, about 0.1 aM, about 500 zM (zeptomolar), about 100 zM, about 50 zM, about 10 zM, about 5 zM, about 1 zM, about 0.1 zM, or lower. In some embodiments, the concentration of analyte molecules or particles in the fluid sample that can be basically accurately determined is about 5000fM to about 0.1fM, about 3000fM to about 0.1fM, about 1000fM to about 0.1fM, about 1000fM to about 0.1zM, about 100fM to about 1zM, about 100aM to about 0.1zM, or lower. The upper detection limit (e.g., the upper limit concentration of the analyte molecules that can be determined in the solution) is at least about 100fM, at least about 1000fM, at least about 10pM (picomolar), at least about 100pM, at least about 100pM, at least about 10nM (nanomolar), at least about 100nM, at least about 1000nM, at least about 10uM, at least about 100uM, at least about 1000uM, at least about 10mM, at least about 100mM, at least about 1000mM, or greater. In some embodiments, the concentration of analyte molecules or particles in the fluid sample determined is less than about 50 x 10 -15 M, or less than about 40x 10 -15 M, or less than about 30X10 -15 M, or less than about 20x 10 -15 M, or less than about 10x 10 -15 M, or less than about, or less than about 1x10 -15 M.

[0408] In some embodiments, the concentration of the analyte molecule in the sample that can be substantially accurately determined is less than about 100 fM, less than about 10 fM, less than about 3 fM, less than about 1 fM, less than about 0.3 fM, less than about 0.1 fM, less than about 0.03 fM, or less. In some embodiments, the concentration of the analyte molecule in the sample that can be substantially accurately determined is about 5000 fM to about 0.1 fM, about 3000 fM to about 0.1 fM, about 1000 fM to about 0.1 fM, about 1000 fM to about 1 fM, about 100 fM to about 1 fM, about 100 fM to about 0.1 fM. The concentration of the analyte molecule in the sample can be considered to be substantially accurately determined if the measured concentration of the analyte molecule in the sample is within about 20% of the actual concentration of the analyte molecule in the sample. In certain embodiments, the concentration of the analyte molecules in the sample measured is within about 10%, within about 3%, or within about 1% of the actual concentration of the analyte molecules in the sample. In some embodiments, the accuracy of the assay can be determined by determining the concentration of the analyte molecules in a sample of known concentration using a selected assay. Preferably, the assay can detect 10-10,000 molecules in the sample, preferably 100-5,000 molecules in the sample, and more preferably 100-1000 molecules in the sample.

[0409] Relative to conventional sandwich immunoassay techniques, as measured, for example, using the same capture antibody and either of two detection antibodies and the same labeling and detection technology, the use of the assay formats described herein can improve the detection signal and assay sensitivity by up to 10-fold, preferably up to 50-fold, 100-fold, or up to 1000-fold. Preferably, the use of the assay formats described herein can improve the detection signal and assay sensitivity by up to 100-fold relative to standard sandwich immunoassays.

[0410] An advantageous aspect of the methods of the invention, especially when coupled with sensitive optical detection techniques, is that signal amplification allows detection of single binding events as bright spots of light. Quantification of the signal can then be performed by counting individual events (which can provide better sensitivity for low analyte concentrations, by providing improved discrimination of binding events from background noise) or by signal integration for all binding events (which can provide better dynamic range for measuring high analyte concentrations).

[0411] The method of the present invention can be used in a variety of assay devices and / or forms. The assay device may include, for example, an assay module, such as an assay plate, a cartridge, a porous assay plate, a reaction vessel, a test tube, a cuvette, a flow cell, an assay chip, a flow measuring device, etc., with an assay reagent (which may include a targeting reagent or other binding reagent) added as the assay progresses or preloaded in the hole, chamber or assay region of the assay module. These devices can be used in a variety of assay forms for specific binding assays, such as immunoassays or immunochromatographic assays. Exemplary assay devices and forms are described below. In certain embodiments, the method of the present invention may use an assay reagent stored in a dry state and the assay device / kit may further include or be supplied with a desiccant material to maintain the assay reagent in a dry state. The assay device preloaded with an assay reagent can greatly improve speed and reduce the complexity of the assay measurement, while maintaining excellent stability during storage. The dried assay reagent can be any assay reagent that can be dried, and then reconstructed before use in the assay. These include, but are not limited to, binding reagents, enzymes, enzyme substrates, indicator dyes and other reactive compounds that can be used to detect the analyte of interest that can be used in conjunction with the assay. Assay reagents may also include substances that are not directly involved in the detection mechanism but play an auxiliary role in the assay, including, but not limited to, blocking agents, stabilizers, detergents, salts, pH buffers, preservatives, etc. The reagents may be present in free form or supported on a solid phase, including the surface of a compartment (e.g., chamber, channel, flow cell, well, etc.) in an assay module or on the surface of a colloid, bead or other particulate support.

[0412] The method of the present invention can be used together with a variety of methods for measuring the amount of analyte, particularly the amount of analyte bound to a solid phase. Available techniques include, but are not limited to, techniques known in the art such as cell culture-based assays, binding assays (including agglutination tests, immunoassays, nucleic acid hybridization assays, etc.), enzyme assays, colorimetric assays, etc. Other suitable techniques will be readily apparent to those of ordinary skill in the art. Some measurement techniques allow for measurement by visual inspection, and other techniques may require or benefit from the use of instruments for measurement.

[0413] Methods for measuring the amount of an analyte include label-free techniques, including but not limited to i) techniques that measure changes in the mass or refractive index of a surface after the analyte binds to the surface (e.g., surface acoustic wave techniques, surface plasmon resonance sensors, ellipsometric techniques, etc.), ii) mass spectrometry techniques (including techniques that can measure analytes on a surface such as MALDI, SELDI, etc.), iii) chromatography or electrophoresis techniques, iv) fluorescence techniques (which may be based on the intrinsic fluorescence of the analyte), and the like.

[0414] The method for measuring the amount of analyte also includes the technology of measuring analyte via the detection of the marker that may be attached to analyte directly or indirectly (for example, via the labeled binding partner using analyte). Suitable markers include markers that can be directly visualized (for example, particles that can be seen by the naked eye and markers that can generate measurable signals such as light scattering, light absorbance, fluorescence, chemiluminescence, electrochemiluminescence, radioactivity, magnetic field, etc.). Useable markers also include enzymes or other chemically reactive species, which have the chemical activity that produces measurable signals such as light scattering, absorbance, fluorescence, etc. Enzyme is used as markers in enzyme-linked immunosorbent assay, also referred to as ELISA, enzyme immunoassay or EIA to be determined. In the ELISA form, the antigen of unknown quantity is attached to the surface, and then the specific antibody is cleaned from the surface, so that it can bind antigen. The antibody is connected to the enzyme, and in the final step, material is added so that the enzyme converts it into a product that can be detected and changes in the signal. The formation of the product can be detectable, for example, due to the difference relative to the substrate in measurable characteristics such as absorbance, fluorescence, chemiluminescence, light scattering, etc. Some (but not all) measurement methods that can be used with the solid phase binding method according to the present invention may benefit from or require a washing step to remove unbound components (e.g., labels) from the solid phase. Thus, the method of the present invention may comprise such a washing step.

[0415] In those embodiments using a pair of detectable markers, based on when a pair of markers are close to each other, i.e., each directly or indirectly combined with the target analyte in the detection complex, its ability to be independently detected and / or the ability of those substances to work together to generate detectable signals select those labeled substances. In one embodiment, the first detectable marker is the first enzyme of the coupled enzyme reaction system and the second detectable label is the second enzyme of the coupled enzyme reaction system, and the method further includes the step of adding one or more substrates of the reaction system, thereby producing a detectable product of the enzyme reaction system. Those reaction vessels comprising detectable products can be distinguished from those reaction vessels not comprising. In a preferred embodiment, detectable products are produced only when the first enzyme and the second enzyme are close (e.g., less than 200nm, ideally less than 50nm). In one embodiment, the first enzyme is an oxidase, such as glucose oxidase, the second enzyme is a peroxidase, and the substrate comprises an oxidase substrate, such as glucose, and a labeled tyramide, Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine), or luminol derivative (collectively referred to herein as labeled reactive derivatives and in a preferred embodiment, the labeled reactive derivative comprises Amplex Red or luminol). In this embodiment, the first enzyme reacts with the substrate to generate a product, which reacts with the second enzyme to generate a second product, which reacts with the labeled reactive derivative to generate a detectable species. Preferably, the reaction catalyzed by the first and second enzymes in the detection complex results in the immobilization of the labeled reactive derivative on the surface, which can be measured to determine the number of analyte molecules present on the surface. In one embodiment, the labeled reactive derivative is biotin-tyramide, which method further comprises adding labeled streptavidin and measuring the label on the streptavidin.

[0416] Another proximal dependent labeling system that can be used for the method is a FRET pair, for example, the first detectable label is a FRET donor and the detectable label is a FRET acceptor. Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules, wherein the excitation is transferred from the donor molecule to the acceptor molecule without emitting photons. The efficiency of FRET depends on the inverse sixth power of the intermolecular spacing, making it available in a distance comparable to the size of biomacromolecules. In this labeling system, the proximal dependent signal is measured by exciting the FRET donor and measuring the emission from the FRET acceptor. The donor and acceptor molecules are preferably in close proximity, for example, about 10-100 angstroms, and the absorption spectrum of the acceptor is preferably overlapped with the fluorescence emission spectrum of the donor, and the orientation of the donor and acceptor transition dipoles (transition dipole) should be roughly parallel. The non-limiting table of FRET pairs is provided in Table 1 below.

[0417] Table 1. Examples of FRET pairs

[0418]

[0419]

[0420] There are various FRET detection methods for light microscopy, such as acceptor photobleaching, donor photobleaching, ratiometric imaging, sensitized emission, and fluorescence lifetime measurement.

[0421] Another suitable labeling system that can be used in the embodiment that adopts a pair of detection labels is a system in which the first and second detectable labels can be measured independently. For example, the first and second detectable labels can be luminescent labels, and their spectral characteristics are different from each other. Or, the first detectable label is a first enzyme that reacts with the first substrate to produce a first signal, and the second detectable label is a second enzyme that reacts with the second substrate to produce different second signals, and the method also includes adding the first enzyme substrate and the second enzyme substrate, and calculating the number of reaction vessels that wherein generate the first and second signals. The first and second signals can be changes in light absorbance and / or luminescent signals with different spectral characteristics.

[0422] If the first and second detectable labels comprise a first and second enzyme, each of them can be a hydrolase, such as a phosphatase, a sulfatase, a β-galactosidase, a glucuronidase, or a combination thereof, and thus, the first and second substrates are selected from phosphate, sulfate, β-galactoside and glucuronide modified stabilized dioxetanes, 4-methylumbelliferyl, fluorescein, or a combination thereof. Alternatively, the first and second enzymes are selected from horseradish peroxidase, beta-galactosidase and alkaline phosphatase.

[0423] Alternatively, the label used to detect the analyte molecule can be a fluorescent species that can be used in single molecule fluorescence detection, such as fluorescence correlation spectroscopy and / or fluorescence cross-correlation spectroscopy. Single molecule fluorescence detection includes passing an eluent containing the detectable species through a capillary, focusing a light source on a volume within the capillary to create an interrogation zone, and observing the interrogation zone with a light detector to detect the passage of fluorescent molecules through the interrogation zone.

[0424] In one embodiment, an electrochemiluminescence-based assay format can be used to measure one or more target analytes in a sample, such as an immunoassay based on electrochemiluminescence (ECL). The high sensitivity, wide dynamic range and selectivity of ECL are important factors for medical diagnosis. Commercialized ECL instruments have shown superior performance, and due to their excellent sensitivity, dynamic range, precision and tolerance to complex sample matrices, they have been widely used. Species that can be induced to emit ECL (ECL active species) have been used as ECL markers, such as i) organometallic compounds, wherein the metal is from, for example, a noble metal of group VIII, including Ru-containing and Os-containing organometallic compounds such as tris-bipyridine-ruthenium (RuBpy) modules and ii) luminol and related compounds. The species participating in the ECL marker in the ECL process are referred to herein as ECL co-reactants. Commonly used co-reactants include tertiary amines for ECL from RuBpy (see, e.g., U.S. Pat. No. 5,846,485), oxalates, and persulfates, and hydrogen peroxide for ECL from luminol (see, e.g., U.S. Pat. No. 5,240,863). The light generated by the ECL marker can be used as a reporter signal in diagnostic procedures (Bard et al., U.S. Pat. No. 5,238,808, incorporated herein by reference). For example, the ECL marker can be covalently coupled to a binding agent such as an antibody, a nucleic acid probe, a receptor, or a ligand; the participation of the binding agent in the binding interaction can be monitored by measuring the ECL emitted from the ECL marker. Alternatively, the ECL signal from the ECL-active compound can indicate the chemical environment (see, e.g., U.S. Pat. No. 5,641,623, which describes an ECL assay that monitors the formation or destruction of an ECL co-reactant). For more background on ECL, ECL labels, ECL assays, and apparatus for performing ECL assays, see U.S. Pat. Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623; 5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434; 5,786,141; 5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754; 5,240,863; 6,207,369; 6,214,552 and 5,589,136 and the published PCT Nos. WO99 / 63347; WO00 / 03233; WO99 / 58962; WO99 / 32662; WO99 / 14599; WO98 / 12539; WO97 / 36931 and WO98 / 57154, all of which are incorporated herein by reference.

[0425] The methods of the present invention can be applied in a single channel or multiplexed format where multiple assay measurements are performed in a single sample. Multiplexed measurements that can be used in the present invention include, but are not limited to, multiplexed measurements i) involving the use of multiple sensors; ii) using discrete assay domains on a surface (e.g., an array) that are distinguishable based on position on the surface; iii) involving the use of reagents coated on particles that are distinguishable based on particle properties such as size, shape, and color; iv) generating assay signals that are distinguishable based on optical properties (e.g., absorbance or emission spectra) or v) based on temporal properties of the assay signal (e.g., the time, frequency, or phase of the signal).

[0426] In some embodiments, the measurement of the concentration of analyte molecules in the sample can be determined at least in part by comparing the measured parameters and calibration standards. For example, the score of the binding surface comprising the analyte molecule can be compared with the calibration curve to measure the measurement of the concentration of analyte molecules in the sample. The calibration curve can be produced by completing the determination of standardized samples of multiple known concentrations under the conditions for analyzing the test sample. For each standardized sample, the signal reading related to the detection / quantification of the analyte molecule can be taken, so that the calibration curve related to the detection of the analyte molecule and the analyte molecule of known concentration is allowed to be formed. Then, the determination can be completed on the sample comprising the analyte molecule of unknown concentration, and the detection of the analyte molecule from the determination is plotted on the calibration curve, so the measurement of the concentration of analyte molecules in the sample is determined.

[0427] In one specific case where imaging techniques are used to measure optical signals (such as fluorescence, chemiluminescence, or electrochemiluminescence), binding events can be detected as bright point sources of light. When the surface density of point sources is low (e.g., when the probability of finding a point source in an area RxR (where R is the spatial resolution of the detection system) is less than 10%), it is likely that any observed point source is due to a single binding event. Under these conditions, counting events can provide the most sensitive measurement. As the surface density increases, it becomes increasingly difficult to resolve and count individual binding events. Under these conditions, integrating the optical signals on the binding surface provides a more accurate measurement.

[0428] It is obvious to the skilled person that the method described herein can be applied to a large number of immunoassay platforms known to those skilled in the art. Multiple features of the immunoassay platform can be adjusted to fit a specific platform, but those adjustments are completely within the capabilities of ordinary technicians. For example, the method described herein can be applied to a bead-based format using coded particles. In such systems, the beads used can be magnetic or non-magnetic, and the surface of the beads is modified to contain one or more copies of a capture reagent. The detection reagent used in the system is a pair of detection reagents. In one embodiment, the two detection reagents include distinguishable fluorescent markers. Alternatively, the two detection reagents are modified with nucleic acid probes, as described herein, in which case the immunoassay method includes an extension process such as RCA-PLA to generate amplified products, which indicate the presence of each detectable detection reagent. If the detection reagent includes two distinguishable fluorescent markers, the measurement step includes introducing the beads into a flow cell, and if the beads are magnetic, the beads are captured in the flow cell. If the detection reagent is modified with nucleic acid probes, the measurement step includes forming a sandwich complex on the beads, implementing RCA-PLA and marking the amplicon with a fluorescently labeled detection probe. Then, the labeled beads are introduced into the flow cell and if the beads are magnetic, the beads are captured in the flow cell. In each embodiment, the spectrum can be multiplexed based on the identification of fluorescently labeled coded beads. The binding events in each embodiment can be detected using an excitation light source and an emission light detector for multicolor detection, and quantitative analysis is performed by counting beads with two detectable labels or beads containing detectable labeled extension products, and quantitative analysis is also performed by integrated intensity, such as by integrating signal detection for all binding events. Therefore, a kit for the method described above can be provided, which includes one or more of the following in one or more vials, containers or compartments: (a) magnetic or non-magnetic beads with capture reagents; (b) two detection reagents with distinguishable fluorescent markers; and (c) optional buffers and / or diluents for determination schemes. Another kit useful for the methods described above may include one or more of the following in one or more vials, containers or compartments: (a) magnetic or non-magnetic beads with capture reagents; (b) two detection reagents modified with nucleic acid probes (optionally, the detection reagents are provided separately and the proximal probes (1 and 2) are additionally provided, with instructions for modifying the detection reagents with the probes); and (c) fluorescently labeled probes; optional reagents required for modifying the detection reagents with the proximal probes; assay diluents, calibrators, circularized oligonucleotides, ligation mixtures or components thereof, such as ligation buffer, ATP, BSA, Tween 20, T4 DNA ligase; RCA mixtures or components thereof, such as BSA, buffer, dNTPs, Tween 20, Phi29 DNA polymerase.

[0429] In another embodiment, the method described herein can be used for the bead-based malignancy of flow cell analysis. In such systems, the beads used can be magnetic and the bead surface is modified to include one or more copies of the capture reagent. The detection reagent used in the system is a pair of detection reagents modified with nucleic acid probes, as described herein, in which case, the immunoassay method includes an extension process such as RCA-PLA to generate amplified products, which indicates the presence of each detectable detection reagent. The measurement step includes forming a sandwich complex on the beads, implementing RCA-PLA and labeling amplicon with an ECL-labeled detection probe. Then, the labeled beads are introduced into the flow cell and the beads are captured in the flow cell. Specifically, a magnetic field is applied to attract magnetic particles such as beads to the electrode surface, which may include multiple metals such as platinum. A voltage source is used to apply voltage to the electrode and an emission light detector can be used to detect binding events; Quantification is achieved by counting beads with detectable labeled extension products, and quantification is also achieved by the intensity of integration, such as by integrating signal detection for all binding events. The kits useful for the methods described above may comprise one or more of the following in one or more vials, containers or compartments: (a) magnetic beads with capture reagents; (b) two detection reagents modified with nucleic acid probes (optionally, the detection reagents are provided separately and the proximal probes (1 and 2) are additionally provided, with instructions for modifying the detection reagents with the probes); and (c) ECL-labeled probes; optional reagents required for modifying the detection reagents with the proximal probes; assay diluents, calibrators, circularized oligonucleotides, ligation mixtures or components thereof, such as ligation buffer, ATP, BSA, Tween 20, T4 DNA ligase; RCA mixtures or components thereof, such as BSA, buffer, dNTPs, Tween 20, Phi29 DNA polymerase.

[0430] In a specific embodiment of the bead-based format of flow cell analysis, the sample is incubated with a mixture of analyte-specific biotinylated monoclonal capture antibodies and analyte-specific monoclonal antibodies, each of which is conjugated to an oligonucleotide, which reacts to form a sandwich complex. After adding microparticles coated with streptavidin, the complex becomes bound to the solid phase via the interaction between biotin and streptavidin. A connection mixture is added to the mixture, and the mixture is incubated with the connection mixture, washed to remove excess cyclic oligonucleotides, and incubated with an RCA mixture. The mixture is washed and a mixture of biotin-labeled detection probes is added. In order to incorporate suitable labels such as luminescent, chemiluminescent or electrochemiluminescent labels, such as SULFO-TAG, a detection probe with a terminal biotin label is synthesized and pre-bound to streptavidin labeled with SULFO-TAG. The reaction mixture is aspirated into a measuring well, where the microparticles are magnetically captured on the surface of an electrode such as a metal electrode (such as a platinum electrode). Unbound substances are then removed with a suitable washing buffer such as ProCell (buffer containing TPA). Application of a voltage to the electrodes then induces chemiluminescent emission, which is measured by a photomultiplier tube.Application of voltage and measurement of the resulting emission can be accomplished in any suitable flow cell, such as a Cobas and / or Elecsys instrument (available from Hoffmann-La Roche LTD.).

[0431] In yet another embodiment, the method described herein can be applied to a bead-based format with capillary flow to count individual molecular numbers. In such systems, the beads used can be magnetic and the surface of the beads can be modified to contain one or more copies of a capture reagent. The detection reagent used in the system is a pair of detection reagents containing distinguishable fluorescent markers. The measurement step includes forming a sandwich complex comprising a capture reagent, an analyte, and a detection reagent, cross-linking the detection reagent, eluting the detection reagent, and introducing the beads into a flow cell. An excitation light source and an emission light detector for multicolor detection can be used to detect binding events, and quantification is achieved by associating the detection of two fluorophores in the flow cell, and quantification is also achieved by integrated intensity, such as by integrating signal detection for all binding events. The kit that can be used for the method described above can include one or more of the following in one or more vials, containers, or compartments: (a) magnetic beads with a capture reagent; (b) two cross-linkable detection reagents with distinguishable fluorescent markers; and (c) optional buffers and / or diluents for the assay protocol.

[0432] Moreover, the method described herein can be applied to the form based on beads, which comprises dividing beads into each nanopore. In this type of system, the beads used can be magnetic and the surface modification of beads is to comprise one or more copies of capture reagents. The detection reagent adopted in this system is a pair of detection reagents comprising distinguishable enzyme labels. The measuring step comprises forming a sandwich complex comprising capture reagent, analyte and detection reagent, and adding the substrate of two enzyme labels. Then capture beads in each nanopore. Based on the identification of enzyme products with different spectral characteristics, determination can be spectral multiplexing. Excitation light source and the emission light detector for multicolor detection can be used to detect binding events, and quantitative is realized by counting the nanopores with two enzyme products, and quantitative is also realized by integrated intensity, such as by integrating signal detection to all nanopores. Kits useful for the methods described above may include one or more of the following in one or more vials, containers or compartments: (a) magnetic beads with capture reagents; (b) two detection reagents each modified with a distinguishable enzyme label, such as a biotinylated detection reagent and a detection reagent conjugated with a hapten; (c) streptavidin-beta galactosidase, an enzyme conjugated with an anti-hapten, resorufin-beta-d-galactopyranoside; (d) an array, such as a Quanterix DVD format array; (e) fluorocarbon oil; and (f) op...

Claims

1. A method for detecting an analyte of interest in a sample, comprising: a. binding an analyte to: (i) a capture reagent on a surface, the surface comprising a capture reagent for the analyte, and an anchoring reagent comprising an anchoring oligonucleotide sequence; (ii) a first detection reagent for the analyte attached to a first nucleic acid probe; and (iii) a second detection reagent for the analyte attached to a second nucleic acid probe; thereby forming a complex comprising the binding reagent, the analyte, and the first and second detection reagents on the surface; b. extending the second probe to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence using an extension process that requires the first and second probes to be in proximity; c. hybridizing the anchor sequence to the complement of the anchor sequence; and d. measuring the amount of extended sequence bound to the surface, The analytes are G-CDF, GM-CSF, IFNgamma, IL-lbeta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 / 23p40, IL12p70, IL-17A, IL21, IL-22, IL-23, IL-31, IL-33, TNFalpha, TSLP, VEGF, complexed PSA, free PSA, Abeta42, Abeta40, Abeta38, tau, cardiac troponin I, cardiac troponin T, HIV p24, C-peptide and / or FGF21.

2. The method of claim 1, wherein the capture agent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

3. The method of claim 1, wherein the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

4. The method of claim 1, wherein the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer.

5. The method of claim 1, wherein the capture reagent and the first and second detection reagents are antibodies directed against the analyte.

6. The method of claim 1, wherein the anchor oligonucleotide sequence comprises a single-stranded oligonucleotide sequence.

7. The method of claim 1, wherein the extension sequence further comprises one or more detection sequences, and the measuring step further comprises contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences.

8. The method of claim 1, wherein the extending step comprises binding the probe to a template nucleic acid sequence to form a circular nucleic acid template, and extending the circular template by rolling circle amplification.

9. The method of claim 1, wherein the extended probe remains positioned on the surface after the probe is extended.

10. The method of claim 9, wherein the complex remains bound to the surface after the extension step.

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