Improved assay method

Abstract

To cope with the necessity for a new technology for improving sandwich immunoassay performance by improving sensitivity, reducing nonspecific bond, and improving stability of sandwich complex.SOLUTION: A method includes an improved immunoassay which includes (i) anchoring a detection complex formed between a target analyte used in the assay and one or more analyte-binding reagents; and / or (ii) amplifying a signal from the labeled detection complex.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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.

[0002] The present invention is directed to improved methods for performing immunoassays, which are designed to amplify signals in and anchor immunoassay complexes used therein. [Background technology]

[0003] A significant body of literature describes techniques that use binding reactions, such as antigen-antibody reactions, nucleic acid hybridization, and receptor-ligand reactions, for the sensitive measurement of analytes of interest in a sample. The high degree of specificity in many biochemical binding systems has led to many assay methods and systems that are valuable in a variety of markets, including basic research, human and veterinary diagnostics, environmental monitoring, and industrial testing. The presence of the analyte of interest can be measured by directly measuring the participation of the analyte in the binding reaction. In some approaches, this participation can be indicated through the measurement of an observable label attached to one or more binding substances.

[0004] Although sandwich immunoassay formats offer good sensitivity and specificity in many applications, some analytes are present at concentrations that are too low for detection by conventional immunoassay techniques. Sandwich immunoassay performance can also be limited by non-specific binding of detection antibodies and the instability of sandwich complexes that contain antibodies with high off-rates. However, efforts to modify conventional immunoassay techniques to improve sensitivity and specificity often result in more complex and labor-intensive protocols that can be hampered by inefficiencies at each step, which can affect the sensitivity and specificity of the assay. For example, in a complex assay that requires multiple binding events and / or reactions, if one event or reaction is less than optimal, the sensitivity and specificity of the assay as a whole can be affected. There is a need for new techniques to improve sandwich immunoassay performance by improving sensitivity, reducing non-specific binding, and improving the stability of the sandwich complex. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention contemplates the following specific embodiments. Various modifications, additions, and modifications may be made to the embodiments described herein by those skilled in the art without departing from the spirit and scope of the present invention. Such modifications, additions, and modifications are intended to fall within the scope of the following claims. [Means for solving the problem]

[0006] Embodiment (1): A method for detecting an analyte of interest in a sample, comprising binding the analyte to (i) a capture reagent on a surface comprising the capture reagent for the analyte, and an anchor reagent; and (ii) a detection reagent for the analyte linked to a nucleic acid probe; thereby forming a complex on the surface comprising the capture reagent, the analyte and the detection reagent; extending the probe to form an extended sequence comprising an anchor region that binds to the anchor reagent; binding the extended sequence to the anchor reagent; and measuring the amount of extended sequence bound to the surface.

[0007] In embodiment (1), the capture reagent may be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer. In a particular embodiment, the capture reagent is an antibody. The detection reagent may be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, and in a particular embodiment, the detection reagent is an antibody. In one particular example of embodiment (1), the capture and detection reagent is an antibody to the analyte. The anchor reagent may comprise an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, ligand, receptor, hapten, epitope, or mimetope; optionally, the anchor region may comprise an aptamer and the anchor reagent may comprise an aptamer ligand. The anchor region may comprise a nucleic acid sequence and the anchor reagent may comprise a DNA binding protein. The anchor reagent may comprise an oligonucleotide sequence and the anchor reagent may comprise a complementary oligonucleotide sequence. The anchor region can comprise a single-stranded oligonucleotide sequence or a double-stranded oligonucleotide sequence.

[0008] The binding step of embodiment (1) may further comprise forming a triple helix between the anchor region and the anchor reagent. The method may further comprise denaturing the anchor region to expose a single-stranded sequence prior to the binding step; exposing the anchor region to helicase activity prior to the binding step; and / or exposing the anchor region to nuclease treatment prior to the binding step. In this embodiment, the anchor region may comprise one or more hapten-modified bases and the anchor reagent may comprise one or more antibodies specific for the haptens; and / or the anchor region may comprise one or more ligand-modified bases and the anchor reagent may comprise one or more receptors specific for the ligands. The extended sequence may further comprise one or more detection sequences, and the measuring step may comprise contacting the extended sequence with a plurality of labeled probes complementary to the one or more detection sequences; the extended sequence may 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; and / or the extended sequence may comprise one or more labeled bases, and the measuring step may further comprise detecting the presence of the one or more labeled bases. In this embodiment, the one or more modified bases comprise an aptamer, aptamer ligand, antibody, antigen, ligand, receptor, hapten, epitope, or mimetope, and the plurality of detectable moieties each comprise a binding partner of the one or more modified bases and a detectable label. The one or more modified bases can include streptavidin, and the plurality of detectable moieties each include biotin and a detectable label; and / or the one or more modified bases can include biotin, and the plurality of detectable moieties each include streptavidin and a detectable label; and / or the one or more modified bases can include avidin, and the plurality of detectable moieties each include biotin and a detectable label; and / or the one or more modified bases can include biotin, and the plurality of detectable moieties each include avidin and a detectable label.

[0009] The first step of embodiment (1) may comprise binding the analyte to the following species in the following order: (i) a capture reagent on the 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 the surface; and (ii) a detection reagent for the analyte.

[0010] The extension step of embodiment (1) can include binding a probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction; and / or binding a probe to a template nucleic acid sequence, forming a circular nucleic acid template; and extending the circular template by rolling circle amplification. In this embodiment, the probe to be extended can remain localized on the surface after the probe extension. Thus, the complex can remain bound to the surface after the extension step, for example, the probe to be extended is bound to an anchoring reagent at a position within 10-100 um of the location of the complex on the surface. In one particular embodiment, the extension probe is bound to an anchoring reagent at a position less than 100 um, less than 50 um, or more particularly less than 10 um from the location of the complex on the surface.

[0011] The extension step of embodiment (1) can include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustained synthesis reaction), or an isothermal amplification method. In one embodiment, the extension step can include an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0012] The surface referred to in embodiment (1) may include particles and / or wells of a multi-well plate. The surface may include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at two distinct binding domains on the surface. If the surface is a well of a plate, the well may include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at two distinct binding domains in the well; and / or the surface may include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at the same binding domain on the surface. In one embodiment, the well may include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at the same binding domain in the well. The capture reagent and anchor reagent may be within 10-100 nm on the surface. The surface may include an electrode, and the measuring step may further include applying a voltage waveform to generate an electrochemiluminescence signal, and optionally the method includes collecting the particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescence signal.

[0013] The measuring step of embodiment (1) can further include 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, where the detection probe comprises a nucleic acid sequence that is complementary to a region of the extension sequence. The detectable label can be measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a particular example of embodiment (1), the detectable label is an ECL label, and the measuring step can include measuring an ECL signal.

[0014] Embodiment (2): A kit for detection of 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; and (b) a detection reagent for the analyte linked to a nucleic acid probe.

[0015] The anchor reagent of embodiment (2) can comprise an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimetope, and the capture reagent can comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer. In certain embodiments, the capture reagent can comprise an antibody, and / or the detection reagent can comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer. In certain embodiments of the kit, the detection reagent is an antibody.

[0016] The surface of the kit of embodiment (2) may comprise a particle and / or a well of a multi-well plate. The surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains on the surface. When the surface of the kit is a well of a plate, the surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains in the well; and / or the surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain on the surface. In a particular example of the kit, the surface is a well, and the well may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain in the well. The capture reagent and the anchor reagent may be within 10-100 nm on the surface. Furthermore, the surface of the kit may comprise an electrode.

[0017] Embodiment (3): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to (i) a 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 on the surface comprising the capture reagent, the analyte and the detection reagent; (b) extending the probe to form an extension sequence comprising an anchor sequence complement that is complementary to the anchor sequence; (c) hybridizing the anchor sequence to the anchor sequence complement; and (d) measuring the amount of extension sequence bound to the surface.

[0018] In embodiment (3), the capture reagent may be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, and in a particular example, the capture reagent is an antibody. Similarly, the detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, and in a particular example of embodiment (3), the detection reagent is an antibody. In one example of embodiment (3), the capture and detection reagent is an antibody against the analyte. The anchor oligonucleotide sequence may comprise a single-stranded oligonucleotide sequence or a double-stranded oligonucleotide sequence. The extension sequence may further comprise one or more detection sequences, and the measuring step may further comprise contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences; alternatively or additionally, the extension sequence may further comprise one or more modified bases, and the measuring step may further comprise contacting the extension sequence with a plurality of detectable moieties capable of binding to the one or more modified bases. In certain examples, the extended sequence can further comprise one or more labeled bases, and the measuring step can further comprise detecting the presence of the one or more labeled bases. The one or more modified bases can comprise an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimetope, and the plurality of detectable moieties each comprise a binding partner of the one or more modified bases and a detectable label. The one or more modified bases can comprise streptavidin, and the plurality of detectable moieties each comprise biotin and a detectable label; the one or more modified bases can comprise biotin, and the plurality of detectable moieties each comprise streptavidin and a detectable label; the one or more modified bases can comprise avidin, and the plurality of detectable moieties each comprise biotin and a detectable label; and / or the one or more modified bases can comprise biotin, and the plurality of detectable moieties each comprise avidin and a detectable label.

[0019] Step (a) of embodiment (3) can include binding the analyte to the following species in the following order: (i) a capture reagent on the surface; and (ii) a detection reagent for the analyte. Alternatively, step (a) can 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 the surface. In yet another example, step (a) can include simultaneously or substantially simultaneously binding the analyte to the following species: (i) a capture reagent on the surface; and (ii) a detection reagent for the analyte.

[0020] The extension step of embodiment (3) can include binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction. Alternatively, the extension step can include binding the probe to a template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extended probe can remain localized on the surface after probe extension, e.g., the complex remains attached to the surface after the extension step. In one example, the extended probe is attached to an anchoring reagent at a position within 10-100 um of the location of the complex on the surface. In one particular embodiment, the extension probe is attached to an anchoring reagent at a position less than 100 um, less than 50 um, or more particularly less than 10 um from the location of the complex on the surface. In this particular embodiment, the extension step can include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustained synthesis reaction), or an isothermal amplification method. For example, the extension step can include an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0021] The surface of embodiment (3) may include particles and / or wells of a multi-well plate. The surface may include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at two distinct binding domains on the surface. If the surface is a well of a plate, it may include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at two distinct binding domains in the well; the surface may include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at the same binding domain on the surface. If the surface is a well, it may include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at the same binding domain in the well; the capture reagent and anchor reagent may be within 10-100 nm on the surface. In a particular example, the surface may include an electrode, and the measuring step may further include applying a potential waveform to the electrode to generate an electrochemiluminescence signal. The method may further include collecting the particles on the electrode and applying a potential waveform to the electrode to generate an electrochemiluminescence signal. The measuring step can further include 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, where the detection probe comprises a nucleic acid sequence that is complementary to a region of the extension sequence. In this embodiment, the detectable label is measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. For example, the detectable label can be an ECL label, and the measuring step can include measuring an ECL signal.

[0022] Embodiment (4): A kit for detection of 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 detection reagent for the analyte linked to a nucleic acid probe.

[0023] The kit of embodiment (4) includes a capture reagent that includes an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer. In a particular example, the capture reagent can include an antibody. Similarly, the detection reagent can include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, and in particular, the detection reagent can include an antibody.

[0024] The kit of embodiment (4) includes a surface that can include particles and / or wells of a multi-well plate. The surface can include a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains on the surface. When the surface is a well, the well can include a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains in the well. The surface can include a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain on the surface, for example, when the surface is a well, the well can include a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain in the well. For example, the capture reagent and the anchor reagent are within 10-100 nm on the surface. The surface of embodiment (4) can include an electrode.

[0025] 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 comprising the 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 comprising a binding reagent, the analyte, and the first and second detection reagents; (b) extending the second probe using an extension process that requires the first and second probes to be brought into close proximity to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence; (c) hybridizing the anchor sequence to the anchor sequence complement; and (d) measuring the amount of extended sequence bound to the surface.

[0026] The capture reagent of embodiment (5) may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer. In a particular 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 mimitope, or an aptamer, and in a particular example, the first detection reagent is an antibody. The second detection reagent may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, and in a particular example, the second detection reagent is an antibody. More specifically, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0027] In embodiment (5), the anchor oligonucleotide sequence can comprise a single stranded oligonucleotide sequence or a double stranded oligonucleotide sequence. In this embodiment, the extension sequence can further comprise one or more detection sequences, and the measuring step can further comprise contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences. The extension sequence can also comprise one or more modified bases, and the measuring step can further comprise contacting the extension sequence with a plurality of detectable moieties capable of binding to the one or more modified bases. The extension sequence can further comprise one or more labeled bases, and the measuring step can further comprise detecting the presence of the one or more labeled bases. The one or more modified bases can comprise an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimetope, and the plurality of detectable moieties each comprise a binding partner of the one or more modified bases and a detectable label. For example, the one or more modified bases comprise streptavidin and the plurality of detectable moieties each comprise biotin and a detectable label; the one or more modified bases comprise biotin and the plurality of detectable moieties each comprise streptavidin and a detectable label; the one or more modified bases comprise avidin and the plurality of detectable moieties each comprise biotin and a detectable label; and / or the one or more modified bases comprise biotin and the plurality of detectable moieties each comprise avidin and a detectable label.

[0028] Step (a) of embodiment (5) can comprise binding the analyte to the following species in the following order: (i) a capture reagent on the surface; and (ii) a detection reagent for the analyte. Alternatively, step (a) can 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; or step (a) can comprise simultaneously or substantially simultaneously binding the analyte to the following species: (i) a capture reagent on the surface; and (ii) a detection reagent for the analyte.

[0029] The extension step of embodiment (5) can include binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step can further include binding the probe to the template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extended probe can remain localized on the surface after probe extension, e.g., the complex remains bound to the surface after the extension step. The extended probe can be bound to an anchoring reagent at a position within 10-100 um of the location of the complex on the surface. In one particular embodiment, the extension probe is bound to an anchoring reagent at a position less than 100 um, less than 50 um, or more particularly less than 10 um from the location of the complex on the surface. The extension step can include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustained synthesis reaction), or an isothermal amplification method. In certain instances, the extension step can include an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0030] The extension process of embodiment (5) may include contacting the complex formed in step (a) with a connector sequence comprising (i) an internal sequence complementary to the second probe and (ii) two terminal sequences complementary to the non-overlapping regions of the first probe. The method may further include ligating the two terminal sequences of the connector oligonucleotide to form a circular target sequence that hybridizes to 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 comprising a first connector probe sequence complementary to a first region of the first probe and a first region in the second probe, and a second connector oligonucleotide comprising 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, ligating the first and second connector oligonucleotides to form a circular target sequence that hybridizes to both the first and second probes.

[0031] The surface of embodiment (5) can include particles and / or wells of a multi-well plate. The surface can include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at two distinct binding domains on the surface. If the surface is a well of a plate, the well can include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at two distinct binding domains in the well. The surface can also include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at the same binding domain on the surface. If the surface is a well of a plate, the well can include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at the same binding domain in the well. The capture reagent and anchor reagent can be within 10-100 nm on the surface. In a particular example, the surface can include an electrode, and the measuring step can further include applying a potential waveform to generate an electrochemiluminescence signal, and optionally the method of embodiment (5) further includes collecting the particles on the electrode and applying a potential waveform to the electrode to generate an electrochemiluminescence signal.

[0032] The measuring step of embodiment (5) can further include 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, where the detection probe comprises a nucleic acid sequence that is complementary to a region of the extension sequence. The detectable label can be measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a particular example, the detectable label is an ECL label, and the measuring step can include measuring an ECL signal.

[0033] Embodiment (6): A kit for detection of 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.

[0034] The capture reagent of embodiment (6) can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, and in certain examples, the capture reagent can include an antibody. Similarly, the first detection reagent can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, and in certain examples, the first detection reagent can include an antibody. Similarly, the second detection reagent can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, and in certain examples, the second detection reagent can include an antibody.

[0035] The surface of embodiment (6) may comprise a particle and / or a well of a multi-well plate. The surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains on the surface. If the surface is a well, the well may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains within the well. The surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain on the surface; and / or if the surface is a well, the well may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain within the well. The capture reagent and the anchor reagent may be within 10-100 nm on the surface. In a particular example, the surface may comprise an electrode.

[0036] Embodiment (7): A method of detecting an analyte of interest in a sample, comprising: (a) binding the analyte to (i) a capture reagent for the analyte on a surface comprising a capture reagent, and an anchor reagent; (ii) a first detection reagent for the analyte comprising a first proximity probe, and (iii) a second detection reagent for the analyte comprising a second proximity probe; thereby forming a detection complex on the surface comprising the capture reagent, the analyte and the first and second detection reagents; (b) detecting the detection complex formed in (c) by binding the capture reagent to an internal sequence complementary to (i) the second proximity probe and (ii) a non-overlapping region of the first proximity probe. (c) contacting the surface with a connector sequence comprising two complementary end sequences; (d) hybridizing the connector sequence to the first and second proximity probes; (e) ligating the two end sequences of a connector oligonucleotide to form a circular target sequence that hybridizes to both the first and second proximity probes; (f) extending the second proximity probe by rolling circle amplification of the target sequence to generate an amplicon comprising a binding domain that binds to the anchor reagent; (g) binding the amplicon to the anchor reagent; and (h) measuring the amount of amplicon on the surface.

[0037] Embodiment (8): A method of detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent for the analyte on a surface comprising a capture reagent, and an anchor reagent; (ii) a first detection reagent for the analyte comprising a first proximity probe, and (iii) a second detection reagent for the analyte comprising a second proximity probe; thereby forming a detection complex on the surface comprising the capture reagent, the analyte and the first and second detection reagents; (b) contacting the detection complex formed in (c) with a first connector oligonucleotide and a second connector oligonucleotide, wherein (i) a first end of the first connector and a first end of the second connector are complementary to two non-overlapping regions of the first proximity probe. and (ii) a second end of the first connector and a second end of the second connector are complementary to two non-overlapping regions of the first proximity probe; (c) hybridizing the first and second connector oligonucleotides to the first and second proximity probes; (d) ligating the first and second connector oligonucleotides to form a circular target sequence that hybridizes to both the first and second proximity probes; (e) extending the second proximity probe by rolling circle amplification of the target sequence to generate an amplicon comprising a binding domain that binds to the anchor reagent; (f) binding the amplicon to the anchor reagent; and (g) measuring the amount of amplicon on the surface.

[0038] The capture reagent of embodiment (7) and (8) may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, in a particular example, the capture reagent is an antibody. Similarly, the first detection reagent may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, for example, the first detection reagent is an antibody. Furthermore, the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, for example, the second detection reagent is an antibody. In a particular example of embodiment (7) and (8), the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0039] The anchor reagent of embodiments (7) and (8) can comprise an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimetope. In one example, the binding domain can comprise an aptamer and the anchor reagent can comprise an aptamer ligand. The binding domain can comprise a nucleic acid sequence and the anchor reagent can comprise a DNA binding protein; and / or the anchor reagent can comprise an oligonucleotide sequence and the amplicon can comprise a complementary oligonucleotide sequence.

[0040] The amplicon 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. Additionally, the amplicon 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. Additionally, the amplicon 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 mimetope, and the plurality of detectable moieties each comprise a binding partner of the one or more modified bases and a detectable label. The one or more modified bases can include streptavidin, and the plurality of detectable moieties each include biotin and a detectable label; the one or more modified bases can include biotin, and the plurality of detectable moieties each include streptavidin and a detectable label; the one or more modified bases can include avidin, and the plurality of detectable moieties each include biotin and a detectable label; and / or the one or more modified bases can include biotin, and the plurality of detectable moieties each include avidin and a detectable label.

[0041] Step (a) of embodiments (7) and (8) can include binding the analyte to the following species in the following order: (i) a capture reagent on the surface; and (ii) a first and second detection reagent for the analyte. Alternatively, step (a) can include binding the analyte to the following species in the following order: (i) a first and second detection reagent for the analyte; and (ii) a capture reagent on the surface. Furthermore, step (a) can include simultaneously or substantially simultaneously binding the analyte to the following species: (i) a capture reagent on the surface; and (ii) a first and second detection reagent for the analyte.

[0042] The amplicons of embodiments (7) and (8) can remain localized on the surface after probe extension. The complexes can remain bound to the surface after the extension step. For example, the amplicons bind to the anchoring reagent at a position within 10-100 um of the location of the complexes on the surface. In one particular embodiment, the extension probes are bound to the anchoring reagents at a position less than 100 um, less than 50 um, or more particularly less than 10 um from the location of the complexes on the surface.

[0043] The surface of embodiments (7) and (8) may comprise a particle and / or a well of a multi-well plate. The surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains on the surface. If the surface is a well of a plate, the well may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains in the well. The surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain on the surface. If the surface is a well of a plate, the well may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain in the well. In a particular example, the capture reagent and the anchor reagent are within 10-100 nm on the surface.

[0044] Furthermore, the surface can include an electrode, and the measuring step can include applying a potential waveform to the electrode to generate an electrochemiluminescence signal. In these embodiments ((7) and (8)), the method can further include harvesting the particles on the electrode and applying a potential waveform to the electrode to generate an electrochemiluminescence signal. The measuring step can include binding the amplicon to a detection probe having a detectable label, measuring the detectable label, and correlating the measurement with the amount of analyte in the sample, where the detection probe includes a nucleic acid sequence that is complementary to a region of the amplicon. The detectable label is measured by measuring light scattering, optical absorbance, 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 can include measuring an ECL signal.

[0045] Embodiment (9): A kit for detection of 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; (b) a first detection reagent for the analyte comprising a first proximity probe; (c) a second detection reagent for the analyte comprising a second proximity probe; and (d) a connector sequence comprising (i) an internal sequence complementary to the second proximity probe and (ii) two terminal sequences complementary to the non-overlapping regions of the first proximity probe.

[0046] Embodiment (10): A kit for detection of 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; (b) a first detection reagent for the analyte comprising a first proximity probe; (c) a second detection reagent for the analyte comprising a second proximity probe; and (d) (i) a first connector oligonucleotide and (ii) a second connector oligonucleotide, wherein (x) a first end of the first connector and a first end of the second connector are complementary to two non-overlapping regions of the first proximity probe, and; (y) a second end of the first connector and a second end of the second connector are complementary to two non-overlapping regions of the first proximity probe.

[0047] The capture reagent of embodiments (9) and (10) can include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer. In certain examples, the capture reagent can include an antibody. The first detection reagent can include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, and in certain examples, the first detection reagent can include an antibody. The second detection reagent can include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, and in certain examples, the second detection reagent can include an antibody.

[0048] The surface of embodiments (9) and (10) may comprise a particle and / or a well of a multi-well plate. The surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains on the surface. If the surface is a well of a plate, the well may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains in the well. The surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain on the surface. If the surface is a well, the well may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain in the well. In a particular example, the capture reagent and the anchor reagent are within 10-100 nm on the surface.

[0049] The surfaces of embodiments (9) and (10) may include electrodes.

[0050] 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 comprising a capture reagent, and an anchor reagent comprising an anchor oligonucleotide sequence; (ii) a first detection reagent for the analyte comprising a first proximity probe, and (iii) a second detection reagent for the analyte comprising a second proximity probe; thereby forming a detection complex on the surface comprising the capture reagent, the analyte and the first and second detection reagents; (b) detecting the detection complex formed in (c) as a complex comprising (i) an internal sequence complementary to the second proximity probe, (ii) two terminal sequences complementary to non-overlapping regions of the first proximity probe, and (iii) contacting the anchor sequence with a connector sequence comprising a sequence matching the anchor sequence; (c) hybridizing the connector sequence to the first and second proximity probes; (d) ligating the two end sequences of a connector oligonucleotide to form a circular target sequence that hybridizes to both the first and second proximity probes; (e) extending the second proximity probe by rolling circle amplification of the target sequence to generate an amplicon comprising a plurality of anchor sequence complements that are complementary to the anchor reagent; (f) hybridizing the anchor sequence to one of the anchor sequence complements; and (g) measuring the amount of amplicon on the surface.

[0051] Embodiment (12): A method of detecting an analyte of interest in a sample, comprising: (a) binding the analyte to (i) a capture reagent for the analyte on a surface comprising a capture reagent, and an anchor reagent comprising an anchor oligonucleotide sequence; (ii) a first detection reagent for the analyte comprising a first proximity probe, and (iii) a second detection reagent for the analyte comprising a second proximity probe; thereby forming a detection complex on the surface comprising the capture reagent, the analyte and the first and second detection reagents; (b) contacting the detection complex formed in (a) with a first connector oligonucleotide and a second connector oligonucleotide, wherein (i) a first end of the first connector and a first end of the second connector are complementary to two non-overlapping regions of the first proximity probe, (ii) a second end of the first connector and a second connector oligonucleotide are complementary to two non-overlapping regions of the first proximity probe. a second end of a second connector is complementary to two non-overlapping regions of the first proximity probe; and (iii) the first and / or second connector also comprises a sequence matching the anchor sequence; (c) hybridizing the first and second connector oligonucleotides to the first and second proximity probes; (d) ligating the first and second connector oligonucleotides to form a circular target sequence that hybridizes to both the first and second proximity probes; (e) extending the second proximity probe by rolling circle amplification of the target sequence to generate an amplicon comprising a plurality of anchor sequence complements that are complementary to the anchor reagent; (f) hybridizing the anchor sequence to one of the anchor sequence complements; and (g) measuring the amount of amplicon on the surface.

[0052] The capture reagent of the embodiment (11) and (12) is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer. In a particular 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 mimitope, or an aptamer, and in a particular example, the first detection reagent is an antibody. Similarly, the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, and in a particular example, the second detection reagent is an antibody. In one example, the first and second detection reagents are antibodies against the analyte.

[0053] The amplicon of embodiments (11) and (12) may further comprise one or more detection sequences, and the measuring step may comprise contacting the extended sequence with a plurality of labeled probes complementary to the one or more detection sequences. Additionally, the amplicon may also 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 amplicon may additionally 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 mimetope, and the plurality of detectable moieties may each comprise a binding partner of the one or more modified bases and the detectable label. The one or more modified bases can include streptavidin, and the plurality of detectable moieties each include biotin and a detectable label; the one or more modified bases can include biotin, and the plurality of detectable moieties each include streptavidin and a detectable label; the one or more modified bases can include avidin, and the plurality of detectable moieties each include biotin and a detectable label; and / or the one or more modified bases can include biotin, and the plurality of detectable moieties each include avidin and a detectable label.

[0054] Step (a) of embodiments (11) and (12) can include binding the analyte to the following species in the following order: (i) a capture reagent on the surface; and (ii) a first and second detection reagent for the analyte. Alternatively, step (a) can include binding the analyte to the following species in the following order: (i) a first and second detection reagent for the analyte; and (ii) a capture reagent on the surface. Furthermore, step (a) can include simultaneously or substantially simultaneously binding the analyte to the following species: (i) a capture reagent on the surface; and (ii) a first and second detection reagent for the analyte.

[0055] The amplicons in embodiments (11) and (12) can remain localized on the surface after probe extension, and optionally the complexes remain bound to the surface after the extension step. For example, the amplicons bind to the anchor reagent at a position within 10-100 um of the location of the complexes on the surface. In one particular embodiment, the extension probes are bound to the anchor reagent at a position less than 100 um, less than 50 um, or more particularly less than 10 um from the location of the complexes on the surface.

[0056] The surface of embodiments (11) and (12) may comprise particles and / or wells of a multi-well plate. Optionally, the surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains on the surface. If the surface is a well, the well may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains within the well. The surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain on the surface. If the surface is a well, the well may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain within the well. The capture reagent and the anchor reagent may be within 10-100 nm on the surface.

[0057] The surface of embodiments (11) and (12) can include an electrode, and the measuring step can include applying a potential waveform to the electrode to generate an electrochemiluminescence signal. Optionally, embodiments (11) and (12) further include harvesting the particles on the electrode and applying a potential waveform to the electrode to generate an electrochemiluminescence signal. The measuring step can include binding the amplicon to a detection probe having a detectable label, measuring the detectable label, and correlating the measurement with the amount of analyte in the sample, where the detection probe also includes a nucleic acid sequence that is complementary to a region of the amplicon. The detectable label can be measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In one example, the detectable label is an ECL label, and the measuring step can include measuring an ECL signal.

[0058] The sample of embodiments (11) and (12) can include one or more analyte molecules, and the surface can include a plurality of capture reagents for the one or more analyte molecules distributed across a plurality of resolvable binding regions located on the surface, and the method can include (x) binding the one or more analyte molecules to the one or more capture reagents on the surface; (y) determining the presence or absence of the analyte molecule in each binding region; and (z) identifying the number of binding regions containing the analyte molecule and / or the number of analyte domains not containing the analyte molecule. The measuring step can include imaging the optical signal from the surface to generate an image comprising a plurality of pixels, and each resolvable binding region is mapped to one or more pixels in the image. The resolvable binding regions can be elements of an array and / or the resolvable binding regions are configured to be isolated to individual particles. Each resolvable binding region may be an individual nano-well having a volume of <100 nL, for example, where at least 99% of the binding regions contain zero or one analyte molecule, at least about 95% of the binding regions contain zero or one analyte molecule, at least about 80% of the binding regions contain zero or one analyte molecule, and / or at least about 50% of the binding regions contain zero or one analyte molecule. In embodiments (11) and (12), the concentration of analyte molecules in the sample may be determined, at least in part, using a calibration curve of the number of binding regions containing at least one or one analyte molecule, Poisson distribution analysis, and / or Gaussian distribution analysis.

[0059] In embodiments (11) and (12), the sample can include one or more analyte molecules and the surface can include a plurality of particles each including a plurality of binding reagents for the analyte molecules, where the plurality of particles are distributed across a plurality of resolvable binding regions, and the method can include (i) binding one or more analyte molecules to one or more binding reagents on the surface; and (ii) distributing the plurality of particles across the array of resolvable binding regions; and (iii) determining the presence or absence of an analyte molecule in each resolvable binding region to identify the number of binding regions containing the analyte molecule and / or the number of binding regions not containing the analyte molecule.

[0060] Embodiment (13): A kit for detection of 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 comprising a first proximity probe; (c) a second detection reagent for the analyte comprising a second proximity probe; and (d) a connector sequence comprising (i) an internal sequence complementary to the second proximity probe and (ii) two terminal sequences complementary to non-overlapping regions of the first proximity probe.

[0061] Embodiment (14): A kit for detection of 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 comprising a first proximity probe; (c) a second detection reagent for the analyte comprising a second proximity probe; and (d) (i) a first connector oligonucleotide and (ii) a second connector oligonucleotide; wherein (x) a first end of the first connector and a first end of the second connector are complementary to two non-overlapping regions of the first proximity probe, and; (y) a second end of the first connector and a second end of the second connector are complementary to two non-overlapping regions of the first proximity probe.

[0062] The capture reagent of embodiments (13) and (14) can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, e.g., the capture reagent can include an antibody. Similarly, the first detection reagent can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, e.g., the first detection reagent can include an antibody. Similarly, the second detection reagent can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, e.g., the second detection reagent can include an antibody.

[0063] The surface of embodiments (13) and (14) may comprise a particle and / or a well of a multi-well plate. The surface may comprise a plurality of distinct binding domains, with the capture reagent and the anchor reagent located at two distinct binding domains on the surface. When the surface is a well, the well may comprise a plurality of distinct binding domains, with the capture reagent and the anchor reagent located at two distinct binding domains within the well. The surface may comprise a plurality of distinct binding domains, with the capture reagent and the anchor reagent located at the same binding domain on the surface. When the surface is a well, the well may comprise a plurality of distinct binding domains, with the capture reagent and the anchor reagent located at the same binding domain within the well. The capture reagent and the anchor reagent may be within 10-100 nm on the surface, and optionally the surface may comprise an electrode.

[0064] Embodiment (15): A method for detecting an analyte in a sample, the method can include: (a) binding the analyte to a first and a second detection reagent to form a detection complex, each detection complex including the analyte, a first detection reagent and a second detection reagent, where the first detection reagent has a first detectable label and the second detection reagent has a second detectable label; (b) distributing the analyte across a plurality of reaction vessels, such that a majority of the reaction vessels contain one or less analytes; and (c) detecting the number of analyte molecules by counting the number of reaction vessels containing the first and second detectable labels. In embodiment (15), the first detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, for example, the first detection reagent is an antibody. Similarly, the second detection reagent may be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, for example, the second detection reagent is an antibody. In a particular example, the first and second detection reagents are antibodies against the analyte.

[0065] Step (a) of embodiment (15) can further include forming a solution comprising the analyte and the detection reagent, and step (b) can include distributing the solution across a plurality of reaction vessels such that the likelihood of finding an unbound first detection reagent and an unbound second detection reagent in the same vessel is less than 1 in 10. Alternatively, step (a) of embodiment (15) can further include forming a solution comprising the analyte and the detection reagent, and step (b) can include distributing the solution across a plurality of reaction vessels such that the likelihood of finding an unbound first detection reagent and an unbound second detection reagent in the same vessel is less than 1 in 100. Still further, step (a) of embodiment (15) can further include forming a solution comprising the analyte and the detection reagent, and step (b) can include distributing the solution across a plurality of reaction vessels such that the likelihood of finding an unbound first detection reagent and an unbound second detection reagent in the same vessel is less than 1 in 1000. Furthermore, step (a) of embodiment (15) can further include forming a solution comprising the analyte and the detection reagent, and step (b) can include distributing the solution across multiple reaction vessels such that the probability of finding an unbound first detection reagent and an unbound second detection reagent in the same vessel is less than 1 in 10,000.

[0066] Embodiment (16): A method for detecting an analyte in a sample, comprising: (a) binding the analyte to a capture reagent and a first and a second detection reagent to form detection complexes, each detection complex comprising a capture reagent, an analyte, a first detection reagent and a second detection reagent, where (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) distributing the analyte across a plurality of reaction vessels, such that the majority of reaction vessels contain one or less analytes; and (c) detecting the number of analyte molecules by counting the number of reaction vessels that contain the first and 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 mimitope, 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 mimitope, or an aptamer, for example, the first detection reagent is an antibody.Furthermore, the second detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, for example, the second detection reagent is an antibody.For example, the capture reagent, the first and the second detection reagent are antibodies against the analyte.

[0067] Step (b) of embodiment (16) may further include distributing the solution across multiple reaction vessels such that the likelihood of finding an unbound first detection reagent and an unbound second detection reagent in the same vessel is less than 1 in 10. Furthermore, step (b) of embodiment (16) may further include distributing the solution across multiple reaction vessels such that the likelihood of finding an unbound first detection reagent and an unbound second detection reagent in the same vessel is less than 1 in 100. Step (b) of embodiment (16) may further include distributing the solution across multiple reaction vessels such that the likelihood of finding an unbound first detection reagent and an unbound second detection reagent in the same vessel is less than 1 in 1000. Furthermore, step (b) of embodiment (16) may further include distributing the solution across multiple reaction vessels such that the likelihood of finding an unbound first detection reagent and an unbound second detection reagent in the same vessel is less than 1 in 10000.

[0068] The capture reagent in the detection complex of embodiment (16) may be on the surface prior to binding of the capture reagent to the analyte; or the capture reagent in the detection complex binds to the analyte prior to immobilization of the capture reagent on the surface. In one example, the capture reagent may include a targeting moiety and the surface may include a targeting moiety complement. 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.

[0069] The surface of embodiment (16) is a particle, and optionally, the capture reagent is immobilized on a plurality of particles, and distribution of the analyte is achieved by binding the analyte to the capture reagent and distributing the particles to a plurality of reaction vessels. The capture reagent can be immobilized on a plurality of particles, and distribution of the analyte is achieved by distributing the particles to a plurality of reaction vessels and then binding the analyte to the capture reagent.

[0070] Embodiment (16) can further include distributing a plurality of particles into a plurality of reaction vessels, where the plurality of particles include a targeting moiety, the capture reagent includes a targeting moiety complement, and distribution of the analyte is achieved by binding the targeting moiety complement to the targeting moiety.Embodiment (16) can also include washing the particles before and / or after the distributing step.

[0071] The surface of embodiment (16) may be at a location within one of the reaction vessels. In this embodiment, the capture reagent may be immobilized on a surface of the plurality of reaction vessels, and distribution of the analyte is achieved by binding the analyte to the capture reagent. Optionally, the reaction vessel has a surface with a targeting moiety immobilized thereon, the capture reagent includes a targeting moiety complement, and distribution of the analyte is achieved by binding the targeting moiety complement to the targeting moiety. In this particular example, the method may further include washing the reaction vessel prior to the detection step.

[0072] The plurality of reaction vessels of embodiment (16) can include an array of nanowells. The plurality of reaction vessels can include at least 10,000 reaction vessels. In one embodiment, the reaction vessels have a volume of less than 100 nL. Optionally, less than 50% of the reaction vessels contain the analyte upon detection, less than 10% of the reaction vessels contain the analyte upon detection, less than 1% of the reaction vessels contain the analyte upon detection, and / or less than 0.1% of the reaction vessels contain the analyte upon detection.

[0073] In one aspect of embodiment (16), the first detectable label is a first enzyme of a pair of enzyme reactions, and the second detectable label is a second enzyme of a coupled enzyme reaction, and step (d) can include adding one or more reaction substrates, producing a product of the enzyme reaction, and counting the reaction vessels containing the product. In this embodiment, the product can only be produced when the first enzyme and the second enzyme are in close proximity, such as when the first and second enzymes are within 200 nM of each other, or when the first and second enzymes are within 50 nM 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 Amplex Red or a 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 immobilization of labeled Amplex Red or luminol on the surface, and optionally the method can include measuring the labeled Amplex Red or luminol on the surface. The labeled Amplex Red or luminol is optionally biotin-Amplex Red or luminol, and the method can include adding labeled streptavidin and measuring the label on the streptavidin.

[0074] Step (d) of embodiment (16) may include measuring the proximity-dependent signal generated when the first and second detectable labels bind to the same analyte molecule, and counting the number of reaction vessels that produce the proximity-dependent signal, for example, the proximity-dependent signal is generated by PLA-RCA. For example, the first detectable label may be a FRET donor, and the detectable label 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 one example, 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 properties. In one example, the first detectable label is a first enzyme that reacts with a first substrate to produce a first signal, and the second detectable label is a second enzyme that reacts with a second substrate to produce a different second signal, and step (d) of embodiment (16) can include adding a first enzyme substrate and a second enzyme substrate and counting the number of reaction vessels in which the first and second signals are produced. The first and second signals can be changes in optical absorbance with different spectral characteristics. Optionally, the first and second signals are luminescence signals with different spectral characteristics. The first and second enzymes can be hydrolases selected from, for example, phosphatases, sulfatases, galactosidases, glucuronidases, or combinations thereof, and the first and second substrates are selected from phosphate, sulfate, galactoside and glucuronide modified and stabilized dioxetanes, 4-methylumbelliferyl, fluorescein, or combinations thereof. In a particular 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, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, luminescence, radioactivity, a magnetic field, or a combination thereof.

[0075] Embodiment (17): A kit for detection of an analyte in a sample, 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; and (c) a plurality of reaction vessels configured to contain one or less analyte molecules.

[0076] Embodiment (18): A kit for detection of an analyte in a sample, 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 less analyte molecules.

[0077] The first and second detection reagents of embodiments (17) and (18) can include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, an aptamer, or a combination thereof. In one example, the first and second detection reagents include an antibody. The capture antibody can include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, for example, the capture antibody can include an antibody. In one example, the capture reagent can include a targeting moiety, and the surface can include a targeting moiety complement, for example, a targeting moiety, and the targeting agent binding partner is selected from the following binding pairs: avidin-biotin, streptavidin-biotin, receptor-ligand, antibody-antigen, nucleic acid-nucleic acid complement.

[0078] The surface of embodiments (17) and (18) may be a particle, and for example, the capture reagent is immobilized on a plurality of particles. Alternatively, the surface is a location within one of the reaction vessels, and for example, the capture reagent is immobilized on a surface of the plurality of reaction vessels. Optionally, the reaction vessel has a surface with a targeting moiety immobilized thereon, and the capture reagent comprises a targeting moiety complement. The plurality of reaction vessels may comprise an array of nanowells or water droplets dispersed in a water-in-oil emulsion. The plurality of reaction vessels may comprise at least 10,000 reaction vessels, and optionally, one reaction vessel of the plurality of reaction vessels has a volume of less than 100 nL.

[0079] In the kits of embodiments (17) and (18), the first detectable label may be a first enzyme of a pair of enzyme reactions, and the second detectable label is a second enzyme of a coupled enzyme reaction, and the kit may include one or more substrates of the reaction 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 label Amplex Red or a luminol derivative. In certain embodiments, the oxidase is glucose oxidase, and the oxidase substrate is glucose. The first and second detectable labels may 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 may be measured independently. Optionally, the first and second detectable labels are luminescent labels that differ from each other in terms of spectral properties.

[0080] In the kit of embodiments (17) and (18), the first detectable label is a first enzyme that reacts with a first substrate to produce a first signal, and the second detectable label is a second enzyme that reacts with a second substrate to produce a different second signal, and the kit can include a first enzyme substrate and a second enzyme substrate in one or more vials, containers, or compartments. Optionally, the first and second signals are changes in optical absorbance with different spectral characteristics. In one example, the first and second signals are luminescence signals with different spectral characteristics. The first and second enzymes can be hydrolases. In one example, the first and second enzymes are selected from phosphatases, sulfatases, galactosidases, glucuronidases, or combinations thereof. The first and second substrates can be selected from phosphate, sulfate, galactoside and glucuronide modified stabilized dioxetanes, 4-methylumbelliferyl, fluorescein, or combinations thereof. Optionally, the first and second enzymes are selected from horseradish peroxidase, beta-galactosidase, and alkaline phosphatase.

[0081] 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, where the capture reagent in the complex is immobilized on a surface; (b) crosslinking the first and second detection reagents to form a crosslinked product; (c) releasing the crosslinked product from the surface into an eluent; (d) counting the individual crosslinked products in the eluent that contain both the first and second detectable labels. In this example (19), the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, in a particular example, the capture reagent is an antibody. Similarly, the first detection reagent may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, in a particular example, the first detection reagent is an antibody. Furthermore, the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, in particular, the second detection reagent may be an antibody. In one particular example, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0082] The embodiment (19) can further include adding a crosslinker to crosslink the first and second detection reagents, for example, the first and second detection reagents include reactive moieties, and the crosslinker is a multifunctional crosslinker that links to the reactive moieties. For example, the reactive moieties include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The crosslinker can include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The first and second detection reagents can include binding moieties, and the crosslinker is a multivalent binding partner of the binding moiety. In one example, the first and second detection reagents are antibodies of an animal species, and the crosslinker is a multivalent anti-species antibody targeting antibody of the animal species. The first and second detection reagents can comprise biotin and the crosslinker is streptavidin; the first and second detection reagents comprise streptavidin and the crosslinker is biotin; the first and second detection reagents are linked to streptavidin and the crosslinker 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 crosslinker is an oligonucleotide that can comprise a sequence complementary to the first nucleic acid probe and a separate sequence complementary to the second nucleic acid probe.

[0083] The surface of embodiment (19) can include particles, reaction vessels, such as tubes or ampoules, and / or the surface can include wells of a multi-well plate. The method of embodiment (19) can further include harvesting the particles and washing the particles to remove impurities, and optionally the first and second detectable labels are measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a particular example, the first and second detectable labels include ECL labels, and the counting step can include measuring an ECL signal.

[0084] Embodiment (20): A kit for detection of 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-linker reactive with said first and second detection reagents.

[0085] The first and second detection reagents of embodiment (20) can include reactive moieties, and the crosslinker is a multifunctional crosslinker linked to the reactive moieties. The reactive moieties can include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof; and the crosslinkers can include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The first and second detection reagents of embodiment (20) can comprise a binding moiety and the crosslinker is a multivalent binding partner of the binding moiety, e.g., the first and second detection reagents are antibodies of the animal species and the crosslinker is a multivalent anti-species antibody targeting antibody of the animal species; the first and second detection reagents comprise biotin and the crosslinker is streptavidin; the first and second detection reagents comprise streptavidin and the crosslinker is biotin; the first and second detection reagents are linked to streptavidin and the crosslinker 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 crosslinker is an oligonucleotide that can comprise a sequence complementary to the first nucleic acid probe and a separate sequence complementary to the second nucleic acid probe.

[0086] The surface of embodiment (20) can include a particle, a well of a multi-well plate, or a reaction vessel, such as a tube or an ampoule. Furthermore, the surface can include a plurality of distinct binding domains, and the capture reagent is located at the distinct binding domains on the surface. When the surface is a well, the well can include a plurality of distinct binding domains, and the capture reagent is located at the distinct binding domains within the well. The surface can also include an electrode.

[0087] Embodiment (21): 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, where the first detection reagent can include a first detectable label and a first nucleic acid probe, and the second detection reagent can include a second detectable label and a second nucleic acid probe, and the capture reagent in the complex is immobilized on a surface; (b) crosslinking the first and second detection reagents 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) ligating the first and second probes; (c) releasing the crosslinked products from the surface into an eluent; and (d) counting individual crosslinked products in the eluent that include both the first and second detectable labels.

[0088] The capture reagent of embodiment (21) may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, for example, the capture reagent is an antibody.Similarly, the first detection reagent may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, for example, the first detection reagent is an antibody; the second detection reagent may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, for example, the second detection reagent is an antibody.In a particular example, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0089] The surface of embodiment (21) can include particles, reaction vessels, such as tubes or ampoules, or wells of a multi-well plate. The method of embodiment (21) can further include harvesting the particles and washing the particles to remove impurities. The first and second detectable labels can be measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a particular example, the first and second detectable labels include ECL labels, and the counting step can include measuring an ECL signal.

[0090] Embodiment (22): A kit for detection of 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 and a first nucleic acid probe having a first detectable label; (c) a second detection reagent and a second nucleic acid probe having a second detectable label; and (d) a third nucleic acid having a region complementary to the first and second nucleic acid probes.

[0091] The surface of embodiment (22) can include a particle, a well of a multi-well plate, or a reaction vessel, such as a tube or an ampoule. The surface can include a plurality of distinct binding domains, with the capture reagent located at the distinct binding domains on the surface, and when the surface is a well, the well can include a plurality of distinct binding domains, with the capture reagent located at the distinct binding domains within the well. The surface can optionally include an electrode.

[0092] 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, where the first detection reagent can include a first nucleic acid probe, and the second detection reagent can 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 that includes a detectable label, the extension being dependent on the co-localization of the first and second nucleic acid probes in the complex; (c) releasing the extended sequence from the surface into an eluent; and (d) counting the individual extended sequences in the eluent. In this embodiment, the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer. In a particular 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 mimitope, or an aptamer, and in a particular 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 mimitope, or an aptamer, and in particular, the second detection reagent is an antibody.In a particular example, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0093] The surface of embodiment (23) can include particles, reaction vessels, such as tubes or ampoules; or wells of a multi-well plate. The method of embodiment (23) can further include harvesting the particles and washing the particles to remove impurities.

[0094] The label of embodiment (23) can be measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a particular example, the label can include an ECL label, and the counting step can include measuring an ECL signal.

[0095] The extension step of embodiment (23) can include binding the probe to the template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step can also include binding the first probe to the template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extension step can include binding the first probe to the template nucleic acid sequence, binding the second probe to the template sequence, and ligating the first and second probes. Optionally, the label is a fluorescent label, and counting of individual extension sequences can include single molecule fluorescence detection, for example, fluorescence correlation spectroscopy and / or fluorescence cross-correlation spectroscopy. Single molecule fluorescence detection can include flowing an eluent through the capillary, focusing a light source onto a volume within the capillary to create an interrogation zone, and observing the interrogation zone with a photodetector to detect passage of fluorescent molecules through the interrogation zone. Single molecule fluorescence detection can also include flowing an eluent through the capillary, focusing a light source onto a volume within the capillary to create an interrogation zone, and observing the interrogation zone with a photodetector to detect passage of fluorescent molecules through the interrogation zone.

[0096] Embodiment (24): A method of 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, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, e.g., the capture reagent is an antibody; the first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, e.g., the first detection reagent is an antibody; the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, e.g., the second detection reagent is an antibody; and, in certain examples, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0097] The surface of embodiment (24) can include particles, reaction vessels, such as tubes and / or ampoules, or wells of a multi-well plate. The method of embodiment (24) can include harvesting the particles and washing the particles to remove impurities. The first and second detectable labels can be measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof, and in certain embodiments, the first and second detectable labels include ECL labels and the counting step can include measuring an ECL signal.

[0098] 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 the 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 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 brought into proximity; and (c) measuring the amount of extended sequence bound to the surface. In this embodiment, the capture reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, e.g., the capture reagent is an antibody; the first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, e.g., the first detection reagent is an antibody; the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, e.g., the second detection reagent is an antibody; and, in certain examples, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0099] The extended sequence of embodiment (25) can include one or more detection sequences and the measuring step can include contacting the extended sequence with a plurality of labeled probes complementary to the one or more detection sequences; the extended sequence can include one or more modified bases and the measuring step can include contacting the extended sequence with a plurality of detectable moieties capable of binding to the one or more modified bases; and / or the extended sequence can include one or more labeled bases and the measuring step can include detecting the presence of the one or more labeled bases. The one or more modified bases include an aptamer, aptamer ligand, antibody, antigen, ligand, receptor, hapten, epitope, or mimetope 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 can 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.

[0100] Step (a) of embodiment (25) can include binding the analyte to the following species in the following order: (i) a capture reagent on the 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 the surface; or simultaneously or substantially simultaneously binding the analyte to the following species: (i) a capture reagent on the surface; and (ii) a detection reagent for the analyte. The extension step can include binding the probe to the template nucleic acid sequence and extending the probe by polymerase chain reaction; or binding the probe to the template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. In this embodiment, the extended probe can remain localized on the surface after the probe extension, e.g., the complex remains bound to the surface after the extension step. The extension step can include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustained synthesis reaction), or an isothermal amplification method. In certain examples, the extension step can include an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0101] The extension process of embodiment (25) may include contacting the complex formed in step (a) with a connector sequence comprising (i) an internal sequence complementary to the second probe and (ii) two terminal sequences complementary to the non-overlapping region of the first probe. The process may further comprise ligating the two terminal sequences of the connector oligonucleotide to form a circular target sequence hybridizing to both the first and second probes. The extension process of embodiment (25) may also comprise contacting the complex formed in step (a) with a first connector oligonucleotide sequence comprising a first connector probe sequence complementary to a first region of the first probe and a first region in the second probe, and a second connector oligonucleotide comprising 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. The process can also include ligating a first and a second connector oligonucleotide to form a circular target sequence that hybridizes to both the first and second probes.

[0102] The surface of embodiment (25) can include particles or wells of a multi-well plate. The surface can include a plurality of distinct binding domains, and the capture reagent is located at two distinct binding domains on the surface. When the surface is a well, the well can include a plurality of distinct binding domains, and the capture reagent is located at two distinct binding domains in the well. The surface can include a plurality of distinct binding domains, and the capture reagent is located at the same binding domain on the surface, and when the surface is a well, the well can include a plurality of distinct binding domains, and the capture reagent is located at the same binding domain in the well. The surface can include an electrode, and the measuring step can include applying a potential waveform to the electrode to generate an electrochemiluminescence signal. The method optionally includes collecting the particles on the electrode and applying a potential waveform to the electrode to generate an electrochemiluminescence signal. The measuring step can further include 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, where the detection probe includes a nucleic acid sequence that is complementary to a region of the extension sequence. The detectable label can be measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a particular example, the detectable label is an ECL label, and the measuring step can include measuring an ECL signal.

[0103] Embodiment (26): A kit for detection of 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.

[0104] The capture reagent of embodiment (26) can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, for example, the capture reagent can include an antibody; the first detection reagent can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, for example, the first detection reagent can include an antibody; the second detection reagent can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, for example, the second detection reagent can include an antibody; and the surface can include a particle and / or a well of a multi-well plate. The surface can include a plurality of distinct binding domains, and the capture reagent is located at two distinct binding domains on the surface; and when the surface is a well, the well can include a plurality of distinct binding domains, and the capture reagent is located at two distinct binding domains within the well. Optionally, the surface can comprise multiple distinct binding domains and the capture reagent is located at the same binding domain on the surface, if the surface is a well, the well can comprise multiple distinct binding domains and the capture reagent is located at the same binding domain within the well. The surface can comprise an electrode.

[0105] The surfaces of embodiments 1-26 can include the interior surfaces of assay vessels, such as test tubes, cuvettes, flow cells, FACS cell sorters, cartridges, or wells of multi-well plates. Surfaces can also include slides, assay chips, or assay arrays; pins, probes, beads, or filtration media; lateral flow media, such as filtration membranes.

[0106] Embodiment (27): A method of detecting an analyte of interest in a sample containing one or more analyte molecules, comprising: (a) contacting the sample with a surface comprising a plurality of resolvable binding regions located thereon, each resolvable binding region comprising a plurality of capture reagents for one or more analyte molecules in the sample; (b) binding 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 at the resolvable binding domains on the surface comprising the capture reagent, the analyte, and the first and second detection reagents, wherein the first and second detectable labels are different labeling compounds; (c) determining the presence or absence of the analyte molecule at each binding region; and (d) identifying the number of binding regions containing the analyte molecule and / or the number of binding regions not containing the analyte molecule. The identifying step can include imaging the optical signal from the surface to generate an image comprising a plurality of pixels, with each resolvable binding region being mapped to one or more pixels in the image. The resolvable binding regions can be elements of an array and / or can be configured to be isolated to individual particles. Each resolvable binding region can be an individual nano-well having a volume of <100 nL and / or at least 99% of the binding regions contain zero or one analyte molecule; at least about 95% of the binding regions contain zero or one analyte molecule; at least about 80% of the binding regions contain zero or one analyte molecule; or at least about 50% of the binding regions contain zero or one analyte molecule. The concentration of the analyte molecule in the sample can be determined at least in part using a calibration curve of the number of binding regions containing at least one or one analyte molecule, Poisson distribution analysis and / or Gaussian distribution analysis.

[0107] The surface of embodiment (27) can include a plurality of particles each comprising a plurality of binding reagents for an analyte molecule, where the plurality of particles are distributed across a plurality of resolvable binding regions, the method can include (i) binding one or more analyte molecules to one or more capture reagents on the surface and first and second detection reagents for each of the one or more analyte molecules, where the first and second detection reagents comprise first and second detectable labels, respectively; (ii) distributing the plurality of particles across the array of resolvable binding regions; and (iii) determining the number and amount of binding regions containing analyte molecules. and / or determining the presence or absence of an analyte molecule in each resolvable binding region to identify the number of binding regions that do not contain an analyte molecule, where, optionally, each resolvable binding region is an individual nano-well having a volume of <100 nL and / or at least 99% of the binding regions contain zero or one analyte molecule; at least about 95% of the binding regions contain zero or one analyte molecule; at least about 80% of the binding regions contain zero or one analyte molecule; and / or at least about 50% of the binding regions contain zero or one analyte molecule.

[0108] The capture reagent in embodiment (27) is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, 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 mimitope, 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 mimitope, or an aptamer, for example, the second detection reagent is an antibody.In a particular example, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0109] Step (a) of embodiment (27) can include binding the analyte to the following species in the following order: (i) a capture reagent on the surface; and (ii) a first and second detection reagent for the analyte; binding the analyte to the following species in the following order: (i) a first and second detection reagent for the analyte; and (ii) a capture reagent on the surface; or simultaneously or substantially simultaneously binding the analyte to the following species: (i) a capture reagent on the surface; and (ii) a first and second detection reagent for the analyte.

[0110] The surface of embodiment (27) can include particles or wells of a multi-well plate. In a particular example, the surface can include an electrode, and the identifying step can include applying a potential waveform to the electrode to generate an electrochemiluminescence signal. The method of embodiment (27) can further include harvesting the particles on the electrode and applying a potential waveform to the electrode to generate an electrochemiluminescence signal. The first detectable label is measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof; and / or the second detectable label is measured by measuring light scattering, optical absorbance, 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 example, the first and second detectable labels are luminescent labels that differ from each other in terms of spectral characteristics.

[0111] The surface of embodiment (27) can include the interior surface of an assay vessel, such as a test tube, cuvette, flow cell, FACS cell sorter, cartridge, or well of a multi-well plate. The surface can also include a slide, assay chip, or assay array; a pin, probe, bead, or filtration medium; a lateral flow media, such as a filtration membrane.

[0112] Embodiment (28): A kit for detecting an analyte of interest in a sample containing one or more analyte molecules, comprising: (a) a surface comprising a plurality of resolvable binding regions located on the surface, each resolvable binding region 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.

[0113] The resolvable binding regions of embodiment (28) may be elements of an array and / or may be configured to be isolated to individual particles. Each resolvable binding region is optionally an individual nano-well having a volume of <100 nL. The surface may include a plurality of particles each including a plurality of binding reagents for the analyte molecule, where the plurality of particles are distributed across the plurality of resolvable binding regions. The kit may include a first and a second detection reagent for each of the one or more analyte molecules, where the first and second detection reagents include a first and a second detectable label, respectively.

[0114] The capture reagent in embodiment (28) is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, 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 mimitope, 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 mimitope, or an aptamer, for example, the second detection reagent is an antibody.In a particular example, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0115] The surface of embodiment (28) can include a particle or a well of a multi-well plate. In a particular example, the surface can include an electrode, and the identifying step can include applying a potential waveform to the electrode to generate an electrochemiluminescence signal. The first detectable label is measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof; and / or the second detectable label is measured by measuring light scattering, optical absorbance, 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 example, the first and second detectable labels are luminescent labels that differ from each other in terms of spectral characteristics.

[0116] The surface of embodiment (28) can include the interior surface of an assay vessel, e.g., a test tube, cuvette, flow cell, FACS cell sorter, cartridge, or well of a multi-well plate. The surface can also include a slide, assay chip, or assay array; a pin, probe, bead, or filtration medium; a lateral flow media, e.g., a filtration membrane.

[0117] Embodiment (29): A method for detecting HIV p24 in a sample, comprising: (a) binding HIV p24 to (i) a capture reagent on a surface comprising the capture reagent for HIV p24, and an anchor reagent comprising an anchor oligonucleotide sequence; (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 comprising a binding reagent, HIV p24, and the first and second detection reagents; (b) extending the second probe using an extension process that requires the first and second probes to be brought into close proximity to form an extension sequence comprising an anchor sequence complement that is complementary to the anchor sequence; (c) hybridizing the anchor sequence to the anchor sequence complement; and (d) measuring the amount of extension sequence bound to the surface.

[0118] The capture reagent of embodiment (29) may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer. In a particular 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 mimitope, or an aptamer, and in a particular example, the first detection reagent is an antibody. The second detection reagent may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, and in a particular example, the second detection reagent is an antibody. More specifically, the capture reagent and the first and second detection reagents are antibodies against HIV p24.

[0119] In embodiment (29), the anchor oligonucleotide sequence can comprise a single stranded oligonucleotide sequence or a double stranded oligonucleotide sequence. In this embodiment, the extension sequence can comprise one or more detection sequences, and the measuring step can comprise contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences. The extension sequence can also comprise one or more modified bases, and the measuring step can comprise contacting the extension sequence with a plurality of detectable moieties capable of binding to the one or more modified bases. The extension sequence can further comprise one or more labeled bases, and the measuring step can comprise detecting the presence of the one or more labeled bases. The one or more modified bases can comprise an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimetope, and the plurality of detectable moieties each comprise a binding partner of the one or more modified bases and the detectable label. For example, the one or more modified bases comprise streptavidin and the plurality of detectable moieties each comprise biotin and a detectable label; the one or more modified bases comprise biotin and the plurality of detectable moieties each comprise streptavidin and a detectable label; the one or more modified bases comprise avidin and the plurality of detectable moieties each comprise biotin and a detectable label; and / or the one or more modified bases comprise biotin and the plurality of detectable moieties each comprise avidin and a detectable label.

[0120] Step (a) of embodiment (29) can comprise binding HIV p24 to the following species in the following order: (i) a capture reagent on the surface; and (ii) a detection reagent for HIV p24. Alternatively, step (a) can 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 the surface; or step (a) can comprise simultaneously or substantially simultaneously binding HIV p24 to the following species: (i) a capture reagent on the surface; and (ii) a detection reagent for HIV p24.

[0121] The extension step of embodiment (29) can include binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step can further include binding the probe to the template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extended probe can remain localized on the surface after probe extension, e.g., the complex remains bound to the surface after the extension step. The extended probe can be bound to an anchoring reagent at a position within 10-100 um of the location of the complex on the surface. In one particular embodiment, the extension probe is bound to an anchoring reagent at a position less than 100 um, less than 50 um, or more particularly less than 10 um from the location of the complex on the surface. The extension step can include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustained synthesis reaction), or an isothermal amplification method. In certain instances, the extension step can include an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0122] The extension process of embodiment (29) can include contacting the complex formed in step (a) with a connector sequence that includes (i) an internal sequence complementary to the second probe and (ii) two terminal sequences complementary to the non-overlapping regions of the first probe. The method can further include ligating the two terminal sequences of the connector oligonucleotide to form a circular target sequence that hybridizes to both the first and second probes. Alternatively, the extension process can include contacting the complex formed in step (a) of embodiment (29) with a first connector oligonucleotide sequence comprising a first connector probe sequence complementary to a first region of the first probe and a first region in the second probe, and a second connector oligonucleotide comprising 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, ligating the first and second connector oligonucleotides to form a circular target sequence that hybridizes to both the first and second probes.

[0123] The surface of embodiment (29) can include particles and / or wells of a multi-well plate. The surface can include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at two distinct binding domains on the surface. If the surface is a well of a plate, the well can include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at two distinct binding domains in the well. The surface can also include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at the same binding domain on the surface. If the surface is a well of a plate, the well can include a plurality of distinct binding domains, and the capture reagent and anchor reagent are located at the same binding domain in the well. The capture reagent and anchor reagent can be within 10-100 nm on the surface. In a particular example, the surface can include an electrode, and the measuring step can include applying a potential waveform to generate an electrochemiluminescence signal, and optionally the method of embodiment (29) further includes collecting the particles on the electrode and applying a potential waveform to the electrode to generate an electrochemiluminescence signal.

[0124] The measuring step of embodiment (29) can include binding the extension sequence to a detection probe having a detectable label, measuring the detectable label, and correlating the measurement with the amount of p24 in the sample, where the detection probe comprises a nucleic acid sequence that is complementary to a region of the extension sequence. The detectable label can be measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. In a particular example, the detectable label is an ECL label, and the measuring step can include measuring an ECL signal.

[0125] Embodiment (30): A kit for the detection of 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 anchor reagent comprising an anchor 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.

[0126] The capture reagent of embodiment (30) can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, and in certain examples, the capture reagent can include an antibody. Similarly, the first detection reagent can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, and in certain examples, the first detection reagent can include an antibody. Similarly, the second detection reagent can include an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer, and in certain examples, the second detection reagent can include an antibody.

[0127] The surface of embodiment (30) may comprise a particle and / or a well of a multi-well plate. The surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains on the surface. If the surface is a well, the well may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains within the well. The surface may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain on the surface; and / or if the surface is a well, the well may comprise a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain within the well. The capture reagent and the anchor reagent may be within 10-100 nm on the surface. In certain examples, the surface may comprise an electrode.

[0128] 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 comprising the capture reagent, and an anchor reagent; (ii) a first detection reagent for HIV p24 comprising a first proximity probe, and (iii) a second detection reagent for HIV p24 comprising a second proximity probe; thereby binding the capture reagent, the HIV (b) forming a detection complex on the surface comprising p24 and the first and second detection reagents; (b) contacting the detection complex formed in (c) with a connector sequence comprising (i) an internal sequence complementary to the second proximity probe and (ii) two terminal sequences complementary to non-overlapping regions of the first proximity probe; (c) hybridizing the connector sequence to the first and second proximity probes; (d) ligating the two terminal sequences of a connector oligonucleotide to form a circular target sequence that hybridizes to both the first and second proximity probes; (e) extending the second proximity probe by rolling circle amplification of the target sequence to produce an amplicon comprising a binding domain that binds to the anchor reagent; (f) binding the amplicon to the anchor reagent; and (g) measuring the amount of amplicon on the surface.

[0129] 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 comprising the capture reagent, and an anchor reagent; (ii) a first detection reagent for HIV p24 comprising a first proximity probe, and (iii) a second detection reagent for HIV p24 comprising a second proximity probe; thereby forming a detection complex on the surface comprising the capture reagent, HIV p24 and the first and second detection reagents; (b) contacting the detection complex formed in (c) with a first connector oligonucleotide and a second connector oligonucleotide, wherein (i) a first end of the first connector and a first end of the second connector are complementary to two non-overlapping regions of the first proximity probe and (ii) a second end of the first connector and a second end of the second connector are complementary to two non-overlapping regions of the first proximity probe; (c) contacting the detection complex formed in (c) with a first connector oligonucleotide and a second connector oligonucleotide, wherein (i) a first end of the first connector and a first end of the second connector are complementary to two non-overlapping regions of the first proximity probe; (d) hybridizing first and second connector oligonucleotides to the first and second proximity probes; (e) ligating the first and second connector oligonucleotides to form a circular target sequence that hybridizes to both the first and second proximity probes; (f) extending the second proximity probe by rolling circle amplification of the target sequence to generate an amplicon comprising a binding domain that binds to the anchor reagent; (f) binding the amplicon to the anchor reagent; and (g) measuring the amount of amplicon on the surface.

[0130] The capture reagent of the embodiment (31) and (32) is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, in a particular 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 mimitope, or an aptamer, for example, the first detection reagent is an antibody. Furthermore, the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer, for example, the second detection reagent is an antibody. In a particular example of the embodiment (31) and (32), the capture reagent and the first and second detection reagents are antibodies against HIV p24.

[0131] The anchor reagent of embodiments (31) and (32) can comprise an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimetope. In one example, the binding domain can comprise an aptamer and the anchor reagent can comprise an aptamer ligand. The binding domain can comprise a nucleic acid sequence and the anchor reagent can comprise a DNA binding protein; and / or the anchor reagent can comprise an oligonucleotide sequence and the amplicon can comprise a complementary oligonucleotide sequence.

[0132] The amplicon of embodiments (31) and (32) can include one or more detection sequences, and the measuring step can include contacting the extended sequence with a plurality of labeled probes complementary to the one or more detection sequences. Additionally, the amplicon can further include one or more modified bases, and the measuring step can include contacting the extended sequence with a plurality of detectable moieties capable of binding to the one or more modified bases. Additionally, the amplicon can further include one or more labeled bases, and the measuring step can include detecting the presence of the one or more labeled bases. The one or more modified bases can include an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimetope, 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 can include streptavidin, and the plurality of detectable moieties each include biotin and a detectable label; the one or more modified bases can include biotin, and the plurality of detectable moieties each include streptavidin and a detectable label; the one or more modified bases can include avidin, and the plurality of detectable moieties each include biotin and a detectable label; and / or the one or more modified bases can include biotin, and the plurality of detectable moieties each include avidin and a detectable label.

[0133] Step (a) of embodiments (31) and (32) can include binding HIV p24 to the following species, in the following order: (i) a capture reagent on the surface; and (ii) a first and second detection reagent for HIV p24. Alternatively, step (a) can include binding HIV p24 to the following species, in the following order: (i) a first and second detection reagent for HIV p24; and (ii) a capture reagent on the surface. Furthermore, step (a) can include simultaneously or substantially simultaneously binding HIV p24 to the following species: (i) a capture reagent on the surface; and (ii) a first and second detection reagent for HIV p24.

[0134] The amplicons of embodiments (31) and (32) remain localized on the surface after probe extension. The complexes can remain bound to the surface after the extension step. For example, the amplicons bind to the anchoring reagent at a position within 10-100 um of the location of the complexes on the surface. In one particular embodiment, the extension probes are bound to the anchoring reagents at a position less than 100 um, less than 50 um, or more particularly less than 10 um from the location of the complexes on the surface.

[0135] The surface of embodiments (31) and (32) can include particles and / or wells of a multi-well plate. The surface can include a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains on the surface. If the surface is a well of a plate, the well can include a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains in the well. The surface can include a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain on the surface. If the surface is a well of a plate, the well can include a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain in the well. In a particular example, the capture reagent and the anchor reagent are within 10-100 nm on the surface.

[0136] Furthermore, the surface can include an electrode, and the measuring step can include applying a potential waveform to the electrode to generate an electrochemiluminescence signal. In these embodiments ((31) and (32)), the method can further include harvesting the particles on the electrode and applying a potential waveform to the electrode to generate an electrochemiluminescence signal. The measuring step can include binding the amplicon to a detection probe having a detectable label, measuring the detectable label, and correlating the measurement with the amount of analyte in the sample, where the detection probe includes a nucleic acid sequence that is complementary to a region of the amplicon. The detectable label is measured by measuring light scattering, optical absorbance, 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 can include measuring an ECL signal.

[0137] 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 linked to a first nucleic acid probe; (b) binding the analyte complex formed in step (a) to: (i) a capture reagent for the analyte on a surface comprising a capture reagent, 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 the first and second probes to be brought into close proximity to form an extension 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 extension sequence bound to the surface. The enrichment step (a) may further comprise: (i) contacting the sample containing the analyte with a solid phase linked to a targeting agent complementary to at least a portion of the first nucleic acid probe, thereby forming an enriched complex containing the analyte bound to the solid phase via a binding reaction between the first nucleic acid probe and the targeting agent; (ii) collecting the enriched complex; (iii) separating unbound components of the sample from the enriched complex; and releasing the enriched complex to separate the solid phase from the analyte to form an analyte complex.

[0138] Embodiment (34): A kit for detection of 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; (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.

[0139] Embodiment (35): A method for detecting exosomes in a sample, comprising: (a) binding the exosomes to (i) a capture reagent on a surface comprising the 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 comprising a binding reagent, the exosomes, and the first and second detection reagents; (b) extending the second probe using an extension process that requires the first and second probes to be brought into close proximity to form an extension sequence comprising an anchor sequence complement that is complementary to the anchor sequence; (c) hybridizing the anchor sequence to the anchor sequence complement; and (d) measuring the amount of extension sequence bound to the surface.

[0140] Embodiment (36): A kit for detection of exosomes of interest in a sample, comprising in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the exosomes, and (ii) an anchor reagent comprising an anchor oligonucleotide sequence; (b) a first detection reagent for the exosomes linked to a first nucleic acid probe; and (c) a second detection reagent for the exosomes linked to a second nucleic acid probe. [Brief description of the drawings]

[0141] [Figure 1-1] Figures 1(a)-(c) are diagrams illustrating the use of immobilized reagents in immunoassays. Figure 1(a) shows the use of immobilized reagents 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 elongated and bound to the immobilized reagent. [Figure 1-2] In FIG. 1(b), the immobilized reagent contains an oligonucleotide sequence that includes a region that is complementary to a portion of the extended sequence formed on the detection reagent. [Figure 1-3] Figure 1(c) illustrates a particular embodiment in which two detection reagents, each containing a nucleic acid probe, are used to bind to the analyte, where the probes on the detection reagent are subjected to an amplification process such that one of the extended probes becomes available to hybridize to the anchor oligonucleotide sequence. [Figure 2-1] FIG. 2(a) illustrates a specific embodiment in which the PLA-RCA process is applied to immune complexes formed on a surface with immobilized reagents, incorporating multiple detectable species into the extended sequences bound to the immune complexes. [Figure 2-2] Figures 2(b) and 2(c) are diagrams of two alternative configurations of linking oligonucleotides that can be used in the methods of the invention. [Diagram 3] FIG. 1 shows one method for attaching oligonucleotides to proteins. [Figure 4-1] Figure 4(a) illustrates a preferred embodiment of the invention in which a surface boundary complex is formed between a capture reagent, an analyte, and two detection reagents, each bound to a first and second proximity probe, respectively, which ligate to a connector probe to form a circular DNA template that is amplified by rolling circle amplification. Figure 4(a) also includes an amplification reagent that includes an anchor oligonucleotide sequence that is complementary to a sequence of an amplicon that is formed as the assay method progresses. [Figure 4-2] FIG. 4(b) shows the first circular DNA template Circ-1, the detection oligonucleotide sequence, the inactive region of the amplicon, and an exemplary sequence of the PP2 portion designed to hybridize to the second proximity probe. [Figure 4-3] An alternative embodiment is shown in FIG. [Diagram 5] FIG. 1 illustrates an alternative method for generating amplicons that can be amplified by rolling circle amplification. [Figure 6-1]FIG. 1 illustrates an alternative method for generating amplicons that can be amplified by rolling circle amplification. [Figure 6-2] FIG. 1 illustrates an alternative method for generating amplicons that can be amplified by rolling circle amplification. [Figure 7] FIG. 1 illustrates an alternative embodiment in which a portion of each of the proximity probes in a sandwich complex is temporarily protected by short strands of RNA hybridized to each segment. These strands are enzymatically removed to allow the proximity probes to hybridize to each other and for the strands to be extended. [Figure 8] FIG. 8 shows a further embodiment in which proximity probes are attached to the capture and detection reagents and a portion of each proximity probe is temporarily protected by a short strand of RNA hybridised to it as described in FIG. [Figure 9] FIG. 1 shows a standard curve for the IL-10 assay derived using the method described in Example 1. [Figure 10] 10(a) and (b) are photographs showing fluorescent microscope images when a fixing reagent was used (a) and when a fixing reagent was not used (b). [Figure 11-1] FIG. 11(a) shows the construction of single linear connector oligonucleotide sequences containing ligation sites 1 or 2 and the use of these connectors in assays of the invention. [Figure 11-2] FIG. 11(b) shows comparative performance data for an assay using a combination of Circ-1 and Circ-2 and an assay using either a single linear connector oligonucleotide sequence containing ligation site 1 or a single linear connector oligonucleotide sequence with ligation site 2. [Figure 12] 1 is a calibration curve for the HIV p24 assay derived using the methods described in Examples 1 and 6. [Figure 13] 1 shows the results of analysis of a seroconversion panel using the methods described in Examples 1 and 6. [Figure 14]14(a)-(c) show the results of an assay for HIVp24 that includes an analyte concentration step. [Figure 15] FIG. 1 shows a calibration curve for an HIVp24 assay that includes an analyte concentration step. [Figure 16] FIG. 1 is a schematic illustration of an assay method described herein that includes an analyte enrichment step. [Figure 17] FIG. 1 is a schematic illustration of the assay method described herein, in which amplicons are formed in solution before being bound to a surface via a capture and / or anchor reagent. [Figure 18] FIG. 1 is a schematic illustration of an assay method that incorporates the use of multiple clasp sequences to attach amplicons, thereby forming more compact structures on the surface. [Figure 19] FIG. 1 is a schematic illustration of a bridging immunoassay format incorporating 3AB PLA-RCA technology and the use of a targeting moiety and its complement to attach a capture reagent to a surface. [Figure 20-1] FIG. 20(a) is a schematic illustration of the biosynthetic method for template formation. [Figure 20-2] FIG. 20(b) is a schematic illustration of the biosynthetic method for template formation. [Figure 21] FIG. 1 is a schematic illustration of sample multiplexing. [Figure 22] FIG. 1 is a schematic illustration of detection of lipoprotein complexes using the methods described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0142] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are generally understood by those skilled in the art. Furthermore, unless otherwise required by context, the singular shall include the plural, and the plural shall include the singular. As used herein, the articles "a" and "an" refer to one or more (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0143] The present invention includes an improved immunoassay comprising (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. An anchor may be used to stabilize complexes that contain low binding affinity interactions and / or high molecular weight labels or label moieties. Signal amplification can be achieved by attaching an extender probe to a binding complex that contains multiple labels or detection label moieties, thereby amplifying the detectable signal for each individual detection complex. In a preferred embodiment, the method includes attaching an extender probe that contains multiple labels or detection label moieties to the detection complex, and anchoring the complex to a surface to ensure that the complex is retained on the surface. This modified assay can be used to detect very low numbers of binding events, even individual analyte-binding reagent complexes. The basic approach is not limited to immunoassays, but can also be used to perform binding assays with other classes of binding reagents.

[0144] One method that can be used to improve binding assays is to use a surface-bound anchoring reagent to attach a detection complex containing the analyte of interest to the surface and to stabilize the detection complex. This approach may be used to overcome low binding affinity between the reagents forming the detection complex and / or to prevent the complex from dissociating from the surface prior to further processing. The use of an anchoring reagent in a binding assay is illustrated in FIG. 1(a). A surface (101) includes a capture reagent (102), which binds analyte A, and an anchoring reagent (103). In one or more steps, the analyte binds to the capture reagent and a detection reagent (104), which also binds to the analyte, where the detection reagent is linked to a nucleic acid probe (105). The analyte can bind to the capture reagent and the detection reagent simultaneously or substantially simultaneously, or the analyte can bind to the capture reagent and the detection reagent sequentially (in either order). Thus, a complex (106) is formed on the surface that includes the capture reagent, the analyte, and the detection reagent. The probe is extended to form an extension sequence (107) that includes an anchor region that binds to the anchoring reagent. The extension sequence binds to the anchoring reagent and the amount of extension sequence bound to the surface is measured.

[0145] Those skilled in the art of binding assays will easily understand the scope of capture reagents and corresponding binding partners that may be used in the present method.A non-limiting list of such pairs includes (in any order) receptor / ligand pairs, antibody / antigen, natural or synthetic receptor / ligand pairs, hapten / antibody pairs, antigen / antibody pairs, epitope / antibody pairs, mimitope / antibody pairs, aptamer / target molecule pairs, hybridization partners and intercalator / target molecule pairs.In one embodiment, binding assays use antibodies or other receptor proteins as capture and / or detection reagents for analytes of interest. The term "antibody" includes intact antibody molecules (including hybrid antibodies constructed in vitro by the reassociation of antibody subunits), antibody fragments and recombinant protein constructs comprising the antigen-binding domains of antibodies (e.g., as described in Porter, RR and Weir, RCJ Cell Physiol., 67(Suppl); 51-64 (1966) and Hochman, 1. Inbar, D. and Givol, D. Biochemistry 12:1130 (1973)), as well as antibody constructs which have been chemically modified, for example by the introduction of a detectable label.

[0146] Similarly, the anchor reagent and the corresponding anchor member or region may include any suitable binding pair, such as receptor / ligand pair, antibody / antigen, natural or synthetic receptor / ligand pair, hapten / antibody pair, antigen / antibody pair, epitope / antibody pair, mimitope / antibody pair, aptamer / target molecule pair, hybridization partner and intercalator / target molecule pair, and the use of electrostatically charged binding surfaces and anchor reagents.For example, the anchor reagent may be an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimetope, and the corresponding anchor region includes a complementary oligonucleotide sequence, an aptamer ligand, an aptamer, an antigen, an antibody, a receptor, a ligand, or an antibody, respectively.In one particular embodiment, the anchor region is an oligonucleotide sequence and the anchor reagent includes a DNA binding protein.Alternatively, if the anchor region is a double-stranded oligonucleotide sequence, the anchor reagent may include an intercalator. In a further embodiment, the anchor region comprises one or more modified oligonucleotide bases, and the corresponding anchor reagent comprises one or more moieties that bind to the modified bases on the anchor region. For example, the modified bases may comprise a hapten or a ligand, and the corresponding anchor reagent may comprise one or more antibodies or ligands specific to the hapten or the ligand, respectively. Furthermore, the anchor region may comprise a number of labeled nucleotide bases that can be used to detect the detection complex.

[0147] In a particular embodiment shown in FIG. 1(b), the anchoring reagent bound to the surface may include an oligonucleotide used to anchor 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) includes a capture reagent (109) that binds to the analyte, A, and an anchoring reagent (110) that includes an anchoring oligonucleotide sequence (111). In one or more steps, the analyte binds to the capture reagent and a detection reagent (112) that also binds to the analyte, where the detection reagent is linked to a nucleic acid probe (113). As previously described in FIG. 1(a), the analyte can bind to the capture reagent and the detection reagent simultaneously or substantially simultaneously, or the analyte can bind to each capture reagent and detection reagent sequentially (in either order). Thus, a complex (114) is formed on the surface that includes the binding reagent, the analyte, and the detection reagent. The probe is extended to form an extension sequence (115) that includes an anchor sequence complement that is complementary to the anchor sequence. The anchor sequence hybridizes to the anchor sequence complement and the amount of extension sequence bound to the surface is measured.

[0148] The detection complex may include one or more detection reagents, for example to enhance the specificity of the assay for the analyte. For example, the assay may be designed such that each detection reagent produces a detectable signal when in proximity to the analyte, or the signal from a single detection reagent bound to the analyte can be distinguished from the signal from multiple detection reagents bound to the analyte by using multiple detection reagents. One embodiment of such an assay is shown in FIG. 1(c). The surface (116) includes a capture reagent (117) that binds to analyte A, and an anchor reagent (118) that includes an anchor oligonucleotide sequence (119). In one or more steps, the analyte binds to the capture reagent and each of two (or more) detection reagents (120 and 121, respectively) that bind to the analyte, and each first and second detection reagent is linked to a nucleic acid probe (122 and 123, first and second nucleic acid probes, respectively). The analyte may bind to the capture reagent and the detection reagent simultaneously or substantially simultaneously, or sequentially, in a stepwise manner. Thus, a complex (124) is formed on the surface containing the capture reagent, the analyte, and the first and second detection reagents. Using an extension process that requires the first and second probes to be in close proximity to one another, the first probe is extended to form an extended sequence (125) that includes an anchor sequence complement that is complementary to the anchor sequence. In a penultimate step, the anchor sequence is hybridized to the anchor sequence complement and the amount of extended sequence bound on the surface is measured.

[0149] A particular embodiment of the method depicted in FIG. 1(c) is depicted in FIG. 2(a) where an anchor reagent is used to attach a detection complex to a surface and the probe attached to the detection complex is extended to generate an extension region that binds to the anchor reagent. In this embodiment, the complex is detected using two detection reagents that bind to the proximity probes. The method includes joining the detection reagent to a connector sequence, which is then ligated to form a circular target sequence and generate an amplicon bound to the anchor reagent by rolling circle amplification. The surface (201) includes a capture reagent (202) and an anchor reagent (203). In one or more steps, the analyte binds to the capture reagent, a first detection reagent (204) that includes a first proximity probe (205), and a second detection reagent (206) that includes a second proximity probe (207), thereby forming a detection complex (208) on the surface. The detection complex is contacted with two connector sequences (209a and 209b), each comprising a terminal sequence complementary to a non-overlapping region of a first proximity probe and a terminal sequence complementary to a non-overlapping region of a second proximity probe. The connector sequences are hybridized to the first and second proximity probes, and the terminal sequences of the connector oligonucleotides are ligated to form a circular target sequence (210) hybridized to both the first and second proximity probes. The second proximity probe is extended by rolling circle hybridization to produce an amplicon that includes a binding reagent that binds to the anchor reagent, and the amount of amplicon bound to the surface is measured. The first proximity probe may be capped or otherwise modified to prevent extension of the first probe. (In alternative embodiments, the first proximity probe is extended and the second proximity probe is capped or otherwise modified to prevent extension.) In the embodiment shown in Figure 2(a), the amplicon also contains two or more detection sequences that are complementary to labeled detection probes that hybridize to the amplicon and are used to measure the amount of amplicon bound to the surface.In an alternative embodiment (not shown in FIG. 2(a)), the extension process incorporates labeled nucleotide bases into the amplicon that are used to detect the amplicon directly on the surface without adding one or more labeled probes complementary to the amplicon. FIG. 2(b) is a schematic illustration of the components of a connector sequence showing a first and a second connector oligonucleotide (209a and 209b, respectively), where the first end of the first connector (C1(E1)) and the first end of the second connector (C2(E1)) are complementary to the two non-overlapping regions of the first proximity probe, and the second end of the first connector (C1(E2)) and the second end of the second connector (C2(E2)) are complementary to the two non-overlapping regions of the second proximity probe. The first and second connectors hybridize to the first and second proximity probes, and the first and second connectors are ligated to form a circular target sequence that hybridizes to both the first and second proximity probes.

[0150] Figure 2(c) shows an alternative embodiment of the connector. The connector sequence 211 comprises an internal sequence (CIS) complementary to the second proximity probe and two end sequences (CE1 and CE2, respectively) complementary to the non-overlapping regions of the first proximity probe. In this embodiment, only one ligation event is required to form a circular target sequence for rolling circle amplification (i.e., ligation of the ends CE1 and CE2 hybridized to the first proximity probe), but the need for proximity of the two proximity probes is preserved since priming / extension is from the second proximity probe. Preferably, the first proximity probe is capped or otherwise modified to prevent extension of the first probe.

[0151] The second proximity probe is then extended by rolling circle amplification of the circular target sequence to generate amplicons that contain a binding region that binds to the anchor reagent, and the amount of amplicon bound to the surface is measured.

[0152] The sequences of the first and second proximity probes can be designed by methods known to those skilled in the art. For example, each probe is about 20-50 bases in length, preferably between 25-40 bases in length, and most preferably between about 30-35 bases in length. The first and second proximity probes also contain sequences complementary to one or more connector sequences or portions thereof used in the processes described herein. In one embodiment, the detection complex is contacted with two connector sequences (209a and 209b), each of which contains a terminal sequence complementary to a non-overlapping region of the first proximity probe and a terminal sequence complementary to a non-overlapping region of the second proximity probe. Thus, in this embodiment, the first and second proximity probes each contain a non-overlapping region complementary to the terminal sequence of the connector. Alternatively, only one connector may be used, with the connector sequence (211) containing an internal sequence (CIS) complementary to the second proximity probe and two terminal sequences (CE1 and CE2, respectively) complementary to the non-overlapping region of the first proximity probe. Thus, in this embodiment, the first proximity probe comprises non-overlapping regions complementary to the two end sequences of the connector, CE1 and CE2, respectively, and the second proximity probe comprises a sequence complementary to the internal sequence (CIS) of the connector. The first proximity probe may be capped or otherwise modified to prevent extension of the first probe. (In an alternative embodiment, the first proximity probe may be extended and the second proximity probe may be capped or otherwise modified to prevent extension.)

[0153] Thus, the embodiments illustrated in Figures 1-2 show that binding assays can be modified to incorporate an anchoring reagent and / or the signal from the detection complex can be amplified. In a preferred embodiment, an anchoring reagent and a signal amplification method are used in the binding assay. In embodiments involving the use of an anchoring reagent, the concentration of anchoring reagent present on the surface is 0.2-200ug / mL, specifically 1.0-50ug / mL, more specifically 3.0-10ug / mL. Alternatively, only one or the other method may be used to achieve an enhanced binding assay. Thus, the present invention includes assays having a signal amplification method as described in Figures 1-2, omitting the anchoring reagent.

[0154] In those embodiments in which the anchor reagent comprises an anchor sequence directly or indirectly bound to a surface (e.g., by a coupling reaction), methods established in the art for immobilizing oligonucleotides, including covalent and non-covalent attachment methods, can be used to generate the anchor reagent. In one embodiment, the anchor reagent comprises a protein linked or otherwise bound to an anchor sequence. In this embodiment, any protein that can be immobilized to a surface (covalently or non-covalently) and modified with an anchor oligonucleotide can be used. Non-limiting examples include streptavidin, avidin, or bovine serum albumin (BSA). In a preferred embodiment, the anchor reagent comprises BSA. Proteins can be modified with anchor oligonucleotides and attached to a surface using known methods, for example, as illustrated in FIG. 3, using the well-established heterobifunctional crosslinker sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid (Sulfo-SMCC). The N-hydroxysuccinimide (NHS) group of SMCC reacts with BSA to label the BSA with a thiol-reactive maleimide group. Alternatively, the maleimide group reacts with a thiol-modified oligonucleotide to form a BSA-oligonucleotide conjugate linked via a stable thioether bond. As one specific example, a series of BSA-oligonucleotide conjugates are printed onto a graphitic carbon surface, preferably a screen-printed carbon ink electrode, to form an array. Alternatively, if the protein is avidin or streptavidin, the anchor sequence can be linked to biotin and can be bound to the immobilized avidin or streptavidin via biotin-avidin or biotin-streptavidin interaction.

[0155] The anchor oligonucleotide attached to the anchor reagent can be any sequence that hybridizes to the extension sequence (or amplicon) that unfolds during the extension process. The anchor oligonucleotide can also contain a non-complementary region (e.g., a poly(A) sequence) that is used as a linker sequence between the surface and the complementary (hybridized) region to extend the complementary region away from the surface. In one embodiment, the hybridization sequence is selected as a region of the amplicon that is not involved in binding to the proximity or detection probe (the "internal" region). In a more specific embodiment, the hybridization sequence is complementary to the entire internal region of the amplicon (preferably about 25 nucleotides in length), either alone or in combination with a poly(A) arm, e.g., up to 30 nucleotides in length. Preferably, the anchor oligonucleotide is selected from: (i) (full complement to an internal region of the amplicon, 25 nucleotides in length) - (20 nucleotide poly(A) arm); or (ii) (complement to a portion of an internal region of the amplicon, 15 nucleotides in length) - (30 nucleotide poly(A) arm).

[0156] In one embodiment, proximity ligation amplification (PLA) is performed to extend the second proximity probe. As described above with reference to Figures 2(a)-(c), the complex containing the two proximity probes is contacted with one or more connector oligonucleotides (209a-209b or 211) and ligation of the hybridized connector sequences forms a circular oligonucleotide which is then used to extend the second proximity probe by rolling circle amplification (RCA) of the circle. Suitable probe designs and amplification conditions for proximity ligation amplification are well established in the art. A unique aspect of the present invention is that it is encompassed by one of the connectors of the same sequence as that used in the anchor reagent. During extension of the second proximity probe, the extended region thereby contains the complement of the anchor sequence hybridized to the anchor reagent, thereby stabilizing the sandwich complex and preventing dissociation of the second proximity probe. The extended second proximity probe may contain a detectable label that can be measured to determine the amount of analyte on the surface (e.g. by incorporation of a labeled nucleotide during the RCA extension reaction). Alternatively, a labeled probe containing multiple detectable labels is added and hybridized to the extended second proximity probe, and the amount of analyte bound to the surface is measured.

[0157] Any suitable amplification technique can be used for generating the extended sequence (or amplicon), including, but not limited to, PCR (Polymerase Chain Reaction), LCR (Ligase Chain Reaction), SDA (Strand Displacement Amplification), 3SR (Self-Sustained Synthetic Reaction) and isothermal amplification, 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, multiplexing, dynamic range and scalability. Techniques for RCA are known in the art (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 97:10113-10119, 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. 20:359-365, 2002; see U.S. Patent Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329 and 6,368,801). Several different variants of RCA are known, including linear RCA (LRCA) and exponential RCA (ERCA). RCA generates thousands of copies of a circular template with a strand of the copy attached to the original target DNA, resulting in spatial resolution of the target and rapid amplification of the signal.RCA facilitates (i) detection of single target molecules; (ii) amplification of signals from proteins as well as DNA and RNA; (iii) identification of the location of amplified molecules on a solid surface; (iv) simultaneous measurement of many different targets; and (v) analysis of one or more targets in solution or on a solid phase. The spatial localization of RCA products with detection complexes is particularly advantageous in performing multiplex binding assays in array or particle-based formats.

[0158] A particular embodiment of the invention using both anchor reagents and signal amplification processes is shown in Figure 4(a). A complex forms on a surface (401) between a capture reagent (402), an analyte (403) and two detection reagents (304 and 305), each containing a first and second proximity probe (406 and 407), respectively. A first and a second connector oligonucleotide (Circ-1 (408) and Circ-2 (409) in Figure 4(a), respectively) are added, each of which hybridizes and bridges the two proximity probes if both proximity probes are present in the complex. A first and a second connector oligonucleotide Circ-1 (408) (SEQ ID NO: 4) and Circ-2 (409) (SEQ ID NO: 5) are added, each of which hybridizes to Circ-1 and Circ-2 if both proximity probes are present in the complex, creating a bridge between the two proximity probes. The bound connector probes are ligated at ligation 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 proximity probe, thereby generating an amplicon that includes one or more detection sequences (413) and anchor oligonucleotide sequence complements (414) (which include partial anchor sequence complements (415)). The anchor oligonucleotide sequence (416) (attached to a capture moiety (417)) and its complement hybridize to multiple detection probes hybridizing to multiple detection probe sequences, and the amount of analyte bound to the surface is measured (not shown, but illustrated in FIG. 1(a)). FIG. 4(b) shows an exemplary sequence of a first circular DNA template Circ-1 (408) (designed to hybridize to a first proximity probe (PP1)), a detection oligonucleotide sequence, an internal region of the amplicon (which can be used in whole or in part to bind to an anchor oligonucleotide sequence), and a portion of PP2 (designed to hybridize to a second proximity probe).A further embodiment is shown in Figure 4(c) showing a circular DNA template being amplified by rolling circle amplification to generate an amplicon containing multiple detection sequences (418 and 419, respectively). In a further embodiment, the anchor oligonucleotide sequence (416) attached to the capture moiety 417 can act as a primer with its free 3' end. In this embodiment, the second proximity probe comprises a sequence complementary to the detection sequence (413).

[0159] In one embodiment, the assay format described herein uses a detection reagent coupled with an exposed 3' end to facilitate ligation with a detection sequence connector probe at the 5' end to form a circular DNA template, which is then amplified by rolling circle amplification to extend the second proximity probe (PR2). In this embodiment, PP1 is potentially and independently available for extension by polymerase, but this issue can be addressed by adding modified bases to prevent priming by polymerase. Alternatively, the ligation template, PP1, can be directly coupled at its 3' end to the detection reagent, preventing this oligonucleotide from participating as a primer for DNA polymerase even if it is degraded.

[0160] Another approach to generate target sequences to be amplified by RCA or any suitable amplification method is illustrated in FIG. 5. In this embodiment, each proximity probe may be folded into a looped hairpin structure. The formation of these hairpin structures generates a double-stranded portion that contains a single-stranded loop and a recombination signal. A recombinase is added to drive 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, optionally anchored to an anchoring reagent, and the analyte is detected. A key element of this embodiment is the ability of the recombinase to catalyze site-specific recombination of DNA that contains sequence-specific recombination sites. For example, Cre recombinase from bacteriophage P1 catalyzes recombination at sites that contain loxP sites, and other non-limiting examples include, but are not limited to, Flippase (flp, from yeast), Hin (Salmonella), and Tre, a genetically engineered (evolved) version of Cre. This alternative approach does not require the addition of additional components such as oligonucleotide templates, ATP, and dNTPs. In this embodiment, the loxP (recombination) sites are preferably modified to be asymmetric, resulting in a shift in the normal equilibrium toward the formation of the desired recombination product, as illustrated in Figure 5 by the light and dark shading of the recombination sites.

[0161] Yet another method for generating amplified target sequences by RCA or any suitable amplification method is illustrated in FIG. 6(a). Each proximity probe attached to a detection reagent contains a loxP site that allows for site-specific recombination between the two oligonucleotides by Cre recombinase, resulting in the formation of a new oligonucleotide sequence that includes the 5' portion of one proximity probe and the 3' portion of the other proximity probe, flanked by the loxP sites. The newly created target sequence can then be amplified, labeled, optionally anchored, and detected by any suitable method, as described above. This embodiment, which uses a T7 RNA polymerase promoter as an operable element for amplification, is illustrated in FIG. 6(a). It will also be appreciated that other RNA polymerase sites such as T3 and SP6 linked to either the 3 or 5' portion of the proximity probe are equally suitable for use in the method. In this embodiment, the loxP (recombination) sites are preferably modified to be asymmetric, resulting in a shift in the normal equilibrium toward the formation of the desired recombination product. As shown in Figure 6(b), this method can also be used to generate circular DNA templates that can be used in RCA.

[0162] The present invention includes a method for detecting an analyte, comprising binding the analyte to a capture reagent on a surface and two detection reagents to form a detection complex. The method includes measuring a detection complex, which preferentially measures a complex that includes both detection reagents compared to a complex that includes only one of the two detection reagents. In one embodiment, the method includes forming a complex, then crosslinking the detection reagents, and detecting the crosslinked reagent. Any suitable crosslinking chemistry can be used to link the components of the detection complex. For example, the first and second detection reagents can include reactive moieties that react and link by the addition of a multifunctional crosslinker that links the reactive moieties. In this embodiment, the reactive moieties and crosslinkers can include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. In another embodiment, the first and second detection reagents can include a binding moiety, and the crosslinker is a multivalent binding partner of the binding moiety. Some non-limiting examples of this embodiment include: (a) the first and second detection reagents are antibodies of the animal species and the crosslinker is a multivalent anti-species antibody that targets the antibodies of the animal species; (b) the first and second detection reagents comprise biotin and the crosslinker is streptavidin (or vice versa); (c) the first and second detection reagents are linked to streptavidin and the crosslinker 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 crosslinker is an oligonucleotide comprising a sequence complementary to the first nucleic acid probe and another sequence complementary to the second nucleic acid probe.

[0163] In certain embodiments, the analyte of interest in a sample can be detected by binding the analyte to the immobilized capture reagent, the first detection reagent, and the second detection reagent to form a complex, where 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 crosslinked 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) ligating the first and second probes.

[0164] The crosslinked products can be detected by being bound to the surface, or in some cases, the crosslinked products can be released from the surface into an eluent and detected. At this point, only those individual crosslinked products in the eluent that contain both the first and second detectable labels are counted. Any suitable detection method can be used to detect the presence of the label in the eluent. In a preferred embodiment, the label is a fluorescent molecule, and the labeled crosslinked products present in the eluent are counted by single molecule fluorescence detection, such as fluorescence correlation spectroscopy, and / or fluorescence cross-correlation spectroscopy. In this embodiment, single molecule fluorescence detection includes flowing the eluent through the capillary, focusing a light source into a volume within the capillary to create an interrogation zone, and observing the interrogation zone with a photodetector to detect the passage of fluorescent molecules through the interrogation zone. The detection method may further include detecting a first fluorescent signal associated with the first label and a second fluorescent signal associated with the second label, and counting detection events in which both 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 include exciting the FRET donor in the interrogation zone and detecting a fluorescent signal from the FRET acceptor.

[0165] In certain embodiments, the analyte in the sample can be detected by binding the analyte to the immobilized capture reagent, the first detection reagent comprising a first nucleic acid probe, and the second detection reagent comprising a second nucleic acid probe to form a complex; extending the second nucleic acid probe to form an extended sequence comprising a detectable label, the extension being dependent on the co-localization of the first and second nucleic acid probes in the complex; releasing the extended sequence from the surface into an eluent; and counting the individual extended sequences in the eluent. The extension step may include binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction. Alternatively, the extension step may include binding the first probe to the template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extension step may include binding the first probe to the template nucleic acid sequence, binding the second probe to the template sequence, and ligating the first and second probes.

[0166] In the methods of the invention using capture reagents, the capture reagents can be directly immobilized on a solid phase or indirectly immobilized by a second binding reagent, such as a targeting reagent described below. For example, the capture reagent can be linked to or include a targeting reagent that binds to the targeting reagent complement immobilized on the solid phase. The binding of the targeting reagent to its complement can be direct (e.g., the targeting reagent can be streptavidin and the complement can be biotin) or indirect through a cross-linking agent (e.g., the targeting reagent and the complement can be biotin and the cross-linking agent can 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 mimetope-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 reagent for the analyte measured by the assay are substantially non-cross-reactive with the targeting agent and complement associated with the capture or detection reagent for the other analyte measured by the assay.For example, the binding of a binding reagent to its associated binding domain (by its associated targeting agent and targeting agent complement) should be substantially stronger than its binding to the binding domain associated with the other analyte (and the different targeting agent complements present).Preferably, the cross-reactivity of the binding of the capture or detection reagent for an analyte to the binding domain associated with the other analyte 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 that contain complementary sequences, and the targeting agent and its complement are contacted under conditions sufficient for the targeting agent to hybridize to its complement.

[0167] When a targeting agent is used, there is some flexibility as to when the capture agent used in the assay method is immobilized on a solid phase. In one embodiment, the capture agent is provided to the user pre-immobilized on a solid phase by targeting agent-targeting agent complement interaction. In another embodiment, the targeting agent and the capture agent linked to a solid phase that supports the immobilized targeting agent complement are provided as separate components. The assay method thus further comprises a step of immobilizing the capture agent on a solid phase by binding the targeting agent to its complement (directly or by using a crosslinking agent). This step may be performed before, simultaneously with, or after the step related to the formation of the detection complex.

[0168] In certain embodiments, multifunctional targeting agents can be used in the assay methods and components described herein. The multifunctional targeting agent comprises (a) a first segment designed to bind to a capture reagent via a first segment complement (i.e., the capture reagent comprises 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., the second segment of the multifunctional targeting agent serves as an anchoring agent on the surface). Thus, in this embodiment, the surface comprises a multifunctional targeting agent that is contacted with a capture agent that binds to the targeting agent via a link between the first segment and the first segment complement. The 3-ABRCA / PLA assay is carried out as described herein, and the amplicon binds to the anchoring segment of the multifunctional targeting agent prior to the measuring step. This method can be used to confirm that a 1:1 ratio of capture agent to anchoring agent is used in the assay method.

[0169] In the method of the present invention, a wide variety of surfaces are suitable for use, including conventional surfaces from the technical field of binding assays. Surfaces may be made from a variety of different materials, including polymers (e.g., polystyrene and polypropylene), ceramics, glass, composites (e.g., carbon-polymer composites, such as carbon-based inks). Suitable surfaces include the surfaces of macroscopic objects, such as the interior surfaces of assay vessels (e.g., test tubes, cuvettes, flow cells, FACS cell sorters, cartridges, wells of multi-well plates, etc.), slides, assay chips (as used in gene or protein chip assays), pins or probes, beads, filtration media, lateral flow media (e.g., filtration membranes used in lateral flow test strips), etc.

[0170] Suitable surfaces also include particles (including but not limited to colloids or beads) typically used in other types of particle-based assays, such as magnetic, polypropylene, and latex particles, materials typically used in solid-phase synthesis, such as polystyrene and polyacrylamide particles, and materials typically used in chromatographic applications, such as silica, alumina, polyacrylamide, polystyrene. The materials may also be fibers, such as carbon fibrils. The microparticles may be inanimate or alternatively may contain living biological entities, such as cells, viruses, bacteria, etc. The particles used in the present method may include any material suitable for attachment to one or more capture or detection reagents, which may be collected, for example, via centrifugation, gravity, filtration, or magnetic collection. A wide variety of different types of particles that may be attached to capture or detection reagents are commercially available for use in binding assays. These include non-magnetic particles, as well as particles that include magnetizable materials so that the particles can be collected by a magnetic field. In one embodiment, the particles include conductive and / or semiconducting materials, such as colloidal gold particles. Microparticles may have a wide variety of sizes and shapes. As a non-limiting example, microparticles may be between 5 nanometers and 100 micrometers. Preferably, microparticles have a size between 20 nm and 10 micrometers. Particles may be spherical, oblong, rod-shaped, etc., or irregular in shape.

[0171] The particles used in the method may be coded to identify a particular particle or subpopulation of particles among a mixture of particles. The use of such coded particles has been used to enable multiplexing of assays utilizing particles as solid supports for binding assays. In one approach, particles are manufactured to include one or more fluorescent dyes, and specific populations of particles are identified based on the intensity and / or relative intensity of fluorescent emission at one or more wavelengths. This approach has been used in the Luminex xMAP systems (see, e.g., U.S. Pat. No. 6,939,720) and Becton Dickinson Cytometric Bead Array systems. Alternatively, particles may be coded for differences in other physical properties, such as size, shape, embedded optical patterns, etc. One or more particles provided in a mixture or set of particles may be coded to be distinguished from other particles in the mixture based on the optical properties, size, shape, embedded optical patterns, etc. of the particles.

[0172] In certain embodiments, the methods of the invention can be used in a multiplexed format by binding multiple different analytes to multiple capture reagents for those analytes. The capture analytes are immobilized on coded beads such that the coding identifies the capture reagent (and analyte target) for a particular bead. The method may further include counting the number of beads with bound analytes (using detection approaches described herein).

[0173] Alternatively or additionally, the detection complexes and / or capture reagents can be directly or indirectly bound to different separate binding domains on one or more solid phases such that separate assay signals are generated and measured on each binding domain, e.g., 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. If capture reagents for different analytes are immobilized on different binding domains, the different analytes bound to those domains can be measured independently. In one example of such an embodiment, the binding domains are prepared to immobilize separate domains of the capture reagent that bind the analytes of interest on one or more surfaces. In some cases, the surfaces may, in part, define one or more boundaries of a vessel (e.g., a flow cell, a well, a cuvette, etc.) that holds the sample or through which the sample passes. For a preferred embodiment, the individual binding domains are formed on electrodes used in electrochemical or electrochemiluminescent assays. Multiplexed measurement of analytes on surfaces containing multiple binding domains using electrochemiluminescence has been used by Meso Scale Diagnostics, LLC in its MULTI-ARRAY® and SECTOR® Imager product lines (e.g., U.S. Pat. Nos. 7,842,246 and 6,977,722, the disclosures of which are incorporated herein by reference in their entireties).

[0174] Still further, the detection complex and / or capture reagent can be directly or indirectly bound to an electrode surface that optionally includes different discrete binding domains as described above. The electrode surface can be a component of a multi-well plate and / or a flow cell. The electrodes can include conductive materials, such as metals, for example, gold, silver, platinum, nickel, steel, iridium, copper, aluminum, a conductive allow, and the like. They can also include oxide-coated metals, for example, aluminum oxide-coated aluminum. The electrodes can include working and counter electrodes that can be made of the same or different materials, for example, a metal counter electrode and a carbon working electrode. In one particular embodiment, the electrodes include carbon-based materials, such as carbon, carbon black, graphitic carbon, carbon nanotubes, carbon fibrils, graphite, graphene, carbon fibers, and mixtures thereof. In one embodiment, the electrodes include elemental carbon, for example, graphite, carbon black, carbon nanotubes, and the like. They may advantageously include conductive carbon polymer composites, conductive particles dispersed in a matrix (e.g., carbon ink, carbon paste, metal ink, graphene ink), and / or conductive polymers.One particular embodiment of the invention is an assay module, preferably a multi-well plate, having electrodes (e.g., working and / or counter electrodes) that comprise carbon, e.g., a carbon layer, and / or a screen-printed layer of carbon ink.

[0175] The present invention includes a method for detecting and counting individual detection complexes. In certain embodiments, a surface comprises a number of capture reagents for one or more analyte molecules present in a sample, the multiple capture reagents being distributed across a number of separable binding regions located on the surface. Under the conditions used to perform and analyze the measurement, a "separable binding region" is the smallest surface area associated with an individual binding event that can be separated and distinguished from another area in which a further individual binding event occurs. Thus, the method includes binding one or more analyte molecules to one or more capture reagents on a surface, determining the presence or absence of the analyte molecules in the multiple separable binding regions on the surface, and identifying the number of separable binding regions that contain the analyte molecules and / or the number of analyte domains that do not contain the analyte molecules.

[0176] The separable binding regions can be optically interrogated, in whole or in part. That is, each individual separable binding region can be optically interrogated individually, and / or the entire surface containing multiple separable binding regions can be imaged and one or more pixels or groupings of pixels of the image can be mapped to individual separable binding regions. The separable binding regions can be microparticles in multiple microparticles. The separable binding regions that show a change in optical signature can be identified by conventional optical detection systems. Depending on the species detected (e.g., the type of fluorescent entity) and the operational wavelength, optical filters designed for specific wavelengths can be used for optical interrogation of the separable binding regions. In embodiments where optical interrogation is used, the system can include multiple light sources and / or multiple filters to adjust the wavelength and / or intensity of the light source. In some embodiments, the optical signals from multiple separable binding regions are captured using a CCD camera. Other non-limiting examples of camera imaging systems that can be used to capture images include charge injection devices (CIDs), complementary metal oxide semiconductors (CMOSs) devices, scientific CMOS (sCMOS) devices, and time delay integration (TDI) devices, as known to those skilled in the art. In some embodiments, a scanning mirror system coupled with a photodiode or photomultiplier tube (PMT) may be used for imaging.

[0177] The measuring step of the method can include imaging the optical signal from the surface (or a portion thereof) to generate an image consisting of a number of pixels, where each separable binding region maps to one or more pixels or groups of pixels in the image. Image analysis to identify pixels or sets of pixels having signals indicative of binding events (detection complexes) can be accomplished using art-recognized methods, such as the wealth of image analysis algorithms and software available for identifying and counting labeled biological structures in fluorescent microscopy images. In one embodiment, after filtering the image to remove large-scale signal gradients, the image is converted to a binary image using a segmentation threshold. Separable binding regions are found by identifying adjacent regions of the above-mentioned threshold intensity. A binding domain is classified as a binding event if it meets the size and intensity requirements.

[0178] In one embodiment, the separable binding regions are elements of an array. In a preferred embodiment, the array is an array of micro- or nanowells, such as individual depressions or wells in a unitary substrate. Preferably, the volume of the wells is less than 100 nL, preferably less than 50 nL. In one embodiment, the volume of the wells is in the range of about 10 aL to 100 pL. In some cases, the wells may be shaped to hold microparticles.

[0179] In one embodiment, at least 50% of the separable binding regions located on the substrate and subject to the assay contain zero or one analyte molecule. Preferably, at least 80%, more preferably at least 95%, and most preferably at least 99% of the separable binding regions contain zero or more analyte molecules. The concentration of the analyte molecule in the sample is determined at least in part using a calibration curve, Poisson distribution analysis, and / or Gaussian distribution analysis of the number of binding regions containing at least one or one analyte molecule. In a particular embodiment, the surface comprises a multiplicity of particles, each of which contains a multiplicity of capture reagents for the analyte molecule, and the multiplicity of particles is distributed across a multiplicity of separable binding regions (e.g., an array of micro- or nano-wells). Thus, the method includes: (i) binding one or more analyte molecules to one or more capture reagents on the surface, (ii) distributing the multiplicity of particles across the array of separable binding regions, and (iii) determining the presence or absence of an analyte molecule in each separable binding region to identify the number of binding domains containing the analyte molecule and / or the number of binding domains not containing the analyte molecule.

[0180] It is also 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 bound to a pair of detection reagents, each of which has a distinguishable label, and the analyte is partitioned across a number of locations, such as wells or reaction vessels (herein referred to as "reaction vessels"), on a substrate, such as a plate, dish, chip, optical fiber, grid, etc., such that the majority of the reaction vessels contain one or fewer analytes. This method allows a user to detect analyte molecules by counting the number of reaction vessels that contain each distinguishable label attached to an analyte. In some cases, the number of reaction vessels of interest is a portion or essentially all of the total amount of reaction vessels that may contain at least one analyte molecule (e.g., associated with at least one analyte molecule or associated with no analyte molecule). The following published US patent applications are cited: US Patent Application No. 20070259448; US Patent Application No. 20070259385; US Patent Application No. 20070259381; International Patent Application No. PCT / US07 / 019184; and International Patent Application No. PCT / US09 / 005428. The disclosures of each of these publications are incorporated herein by reference. At least a portion of the reaction vessels may be subject to the test, and a measurement may be made indicating the number / percentage of reaction vessels that contain at least one analyte molecule or particle. In some cases, based on the number / percentage, a measurement of the concentration of the analyte molecule in the fluid sample may be determined.

[0181] In certain embodiments that allow for the detection of analyte molecules in a confined volume, an analyte in a sample can be detected by binding the analyte to a first and a second detection reagent to form a detection complex. Each detection complex includes 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 complexes can be formed simultaneously, substantially simultaneously, or sequentially. The detection complexes are partitioned across multiple reaction vessels such that the majority of the reaction vessels contain one or fewer detection complexes, and the number of analyte molecules is detected by counting the number of reaction vessels that contain each of the first and the second detectable labels. Preferably, the detection complexes are partitioned across multiple reaction vessels such that the chance of detecting unbound first detection reagent and unbound second detection reagent in the same vessel is less than about 1 in 10, preferably less than about 1 in 100, more preferably less than about 1 in 1000, and most preferably less than about 1 in 10,000. The detection complex is compartmentalized, i.e., divided or separated into portions or parts, across multiple reaction vessels, for example, by manually aliquoting portions of the detection complex across multiple reaction vessels and / or by flowing a solution containing the detection complex across multiple reaction vessels such that the detection complex separates into individual reaction vessels on a support.

[0182] In a further embodiment, an analyte in a sample can be detected by (a) binding the analyte to a capture reagent bound to a surface and a first and a second detection reagent to form a detection complex, where (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 the components in any order, for example by combining the components simultaneously or substantially simultaneously, or by adding each component sequentially to form the detection complex stepwise. The detection complex is partitioned across multiple reaction vessels, with most reaction vessels containing one or fewer analytes, 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 washing after each step and before the detection step.

[0183] The surface can be particles, and optionally a multiplicity of capture reagents are immobilized on the particles or on the multiplicity of particles. In this embodiment, the compartmentalization step can be carried out in a number of ways: (i) the capture reagents are immobilized on a multiplicity of particles, and compartmentalization of the analytes is achieved by binding the analytes to the capture reagents and compartmentalizing the particles into a multiplicity of reaction vessels; (ii) the capture reagents are immobilized on a multiplicity of particles, and compartmentalization of the analytes is achieved by compartmentalizing the particles into a multiplicity of reaction vessels, and then binding the analytes to the capture reagents.

[0184] The multiple reaction vessels also contain water droplets dispersed in a water-in-oil emulsion. The emulsions can be made into droplets with diameters up to 100 um and volumes of approximately 1 nL. The high volume, i.e., more than 10 droplets in 1 mL of emulsion, the ease of emulsion preparation, and their high stability over a wide range of conditions provide an ideal means for compartmentalized biochemical assays. Each water droplet acts as an independent reaction vessel, and detection complexes, optionally attached to particles, can be compartmentalized across multiple water droplets.

[0185] Alternatively, the surface is a location within one of the reaction vessels. For example, if the reaction vessels are wells of a plate, the surface can be a domain or region within the well of the plate. In this embodiment, the capture reagent can be immobilized in a domain or region of the multiple reaction vessels, and the partitioning step is achieved by binding the analyte molecule to the capture reagent. In another embodiment, the multiple reaction vessels include regions with targeting moieties immobilized thereon, the capture reagent includes a targeting moiety complement, and the partitioning step is achieved by binding the targeting moiety complement to a target moiety located in the multiple reaction vessels.

[0186] In further or alternative embodiments, the binding assays described herein may also include a preconcentration step to improve assay performance, for example by increasing the concentration of the analyte in the sample and / or decreasing the concentration of contaminants that may be present in the sample that may interfere with assay performance. This may be done by (a) contacting a sample containing the analyte of interest with a solid phase, e.g., a particle, linked to a first binding reagent that binds to the analyte, thereby forming a complex containing the analyte bound to said first binding reagent; (b) collecting the complex; (c) separating unbound components of the sample from the complex; (d) and releasing the complex. This method of concentrating the analyte is performed before the binding assays described herein are performed in order to remove impurities that may interfere with the performance of the assay. In this regard, reference is made to US Patent Application Publication No. US2010 / 0261292, the disclosure of which is incorporated herein by reference.

[0187] In particular, the enrichment step involves subjecting a sample containing an analyte to conditions sufficient to form an analyte complex comprising the analyte bound to a first detection reagent linked to a first nucleic acid probe. The analyte complex formed as a result of the enrichment step is then bound to (i) a capture reagent on 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. The surface-bound complex is subjected to an extension process that requires the first and second probes to be in close proximity, and extends the second probe to form an extension sequence comprising an anchor sequence complement that is complementary to the anchor sequence. The anchor sequence then hybridizes to the anchor sequence complement; and the amount of extension sequence bound to the surface is measured. In certain embodiments, the enrichment step further comprises: (i) contacting the sample containing the analyte with a solid phase linked to a targeting agent complementary to at least a portion of the first nucleic acid probe, thereby forming an enriched complex containing the analyte bound to the solid phase via a binding reaction between the first nucleic acid probe and the targeting agent; (ii) collecting the enriched complex; (iii) separating unbound components of the sample from the enriched complex; and (iv) releasing the enriched complex and separating the solid phase from the analyte to form an analyte complex.

[0188] As used herein, collection refers to the physical localization of a material in a mixture. Collection includes localization of a material by binding reaction or adsorption. For example, a material in a mixture is collected on a solid phase by adsorption of the material on the solid phase or by binding of the material to a binding reagent on the solid phase. However, collection is not limited to localization on a solid phase, and may include techniques in the art for localizing a material to a location / volume within a larger volume of liquid, such as localization of a material by optical tweezers (using light to manipulate microscopic objects as small as a single atom, where the pressure of irradiation from a focused laser beam allows small particles to be captured), electric or magnetic fields, focusing flow, density gradient centrifugation, etc.

[0189] Certain embodiments of the present invention include collection of 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 suspension. These include settling by gravity or centrifugation, filtration onto a filter or porous membrane, localization (for magnetizable particles) by application of a magnetic field, binding or adsorption of particles to a macroscopic solid phase, use of optical tweezers, etc.

[0190] Release, as used herein, refers to the delocalization of previously collected material. Materials held in a localized location by chemical bonds or specific or nonspecific binding interactions may be delocalized by breaking the bonds or interactions, allowing the material to diffuse or mix into the surrounding media. There are many well-established cleavable chemical linkers that can be used to provide covalent bonds that can be cleaved without the need for harsh conditions. For example, disulfide-containing linkers can be cleaved using thiols or other reducing agents, cis-diol-containing linkers can be cleaved using periodic acid, and metal-ligand interactions (such as nickel-histidine) can be cleaved by changing the pH or introducing a competing ligand. Similarly, there are many well-established reversible binding pairs that can be used (including those that have been identified in the art of affinity chromatography). By way of example, the binding of many antibody-ligand pairs can be reversed by changing the pH, adding protein denaturants or chaotropic agents, adding a competing ligand, and the like. Other suitable reversible binding pairs include complementary nucleic acid sequences, where hybridization can be reversed under various conditions, such as changing the pH, decreasing the salt concentration, raising the temperature above the melting temperature of the pairing, and / or adding a nucleic acid denaturant (such as formamide), etc. Such reversible binding pairs can be used as targeting agents (as described above), e.g., a first targeting agent can be linked to a first binding reagent that binds the analyte, and a second targeting agent can be linked 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.

[0191] Releasing may also include physically delocalizing the material, for example by mixing, shaking, vortex mixing, convective liquid flow, magnetic field, electrical or optical forces, etc. Upon collection of the microparticles or materials bound to the microparticles, such physical methods may be used to resuspend the particles in the surrounding matrix. Releasing may simply be the reversal of a previous collection step (e.g., by any of the mechanisms described above), or collection and release may proceed by two different mechanisms. In one such example, collection of the material (such as an analyte or a complex containing the analyte) bound to the particle can be achieved by physical collection of the particle. The material is then released by breaking the bond holding the material to the particle or reversing the binding reaction. In a second such example, the material (such as an analyte or a complex containing the analyte) is collected on the surface by a binding interaction with a binding reagent linked to the surface. The material is then released by breaking the bond or second binding interaction that links the binding reagent to the surface.

[0192] Collection followed by release may be used to concentrate and / or purify the analyte in the sample. Collection in a first volume and release into a second, smaller volume can concentrate the analyte in the sample. Concentration often allows the sensitivity of the next measurement step to be significantly improved. Removal of some or all of the collected and uncollected sample from the sample can reduce or eliminate potential assay interferences in the sample. Optionally, removal of unbound sample may include washing the collected material with a defined liquid reagent (such as, for example, an assay or wash buffer) and releasing the collected material into a defined liquid reagent to provide a uniform matrix for the next assay step.

[0193] As illustrated in FIG. 3(a) of US2010 / 0261292, which is incorporated herein by reference, the method includes contacting a sample containing a target analyte with particles linked to a first binding reagent that binds to the target analyte, where the first binding reagent is linked to a first targeting agent and the particles are linked to a second targeting agent, and the first binding reagent and the particles are linked via a binding reaction between the first targeting agent and the second targeting agent 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, where the second binding reagent binds to the complex. This particular embodiment is illustrated in FIG. The particles (1601) are modified to include a capture oligonucleotide sequence, 1602, that is at least partially complementary to the sequence of a proximity probe, 1603, bound to a detection antibody, 1604. The particles are mixed with the proximity probe, allowing the capture sequence to hybridize to the probe sequence to form a complex, 1605. The complex is then mixed with a sample containing the analyte, 1606, and optionally one or more contaminants, 1607-1608. The analyte is allowed to bind to the detection antibody (1609) and the contaminants are removed (1610). The particles are removed under appropriate conditions from the complex containing the bound analyte to form an enriched solution of analyte bound to the proximity probe (1611), which may be used in an immunoassay as described herein, where an additional proximity probe binds to the analyte and the three-antibody complex is subjected to RCA-PLA to detect the presence of the analyte in the sample (1612), for example as described in FIG. 2(a) and the accompanying description.

[0194] In another embodiment, an immunoassay complex between a detection antibody and an analyte is formed in solution and subsequently amplified, after which the amplified product is attached to a particle via a capture reagent and / or anchor. Optionally, the amplified product is filtered and then captured on a particle via a capture reagent and / or anchor. This method is illustrated in FIG. 17. An analyte, A (1701), is bound to a detection antibody (1702 and 1703, respectively), each bound to a proximity probe (1704 and 1705, respectively), forming a detection complex that includes the analyte bound to each of the detection antibodies (1706). The detection complex is contacted with two connector sequences (1707a and 1707b), each including a terminal sequence complementary to a non-overlapping region of a first proximity probe and a terminal sequence complementary to a non-overlapping region of a second proximity probe. The connector sequence hybridizes to the first and second proximity probes, and the end sequences of the connector oligonucleotides are ligated to form a circular target sequence (1708) hybridized to both the first and second proximity probes. The second proximity probe is extended by rolling circle hybridization to generate an amplicon that contains a binding reagent complementary to the anchor reagent. The amplicon is contacted with a surface (1709) that contains a capture reagent (1710) and an anchor reagent (1711), and the amount of amplicon bound to the surface is measured using a plurality of labeled probes (1712).

[0195] Furthermore, the methods described herein can be used for the detection and quantification of oligomeric analytes. When 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 to both the ligation template oligonucleotide and the primer oligonucleotide. This can lead to inefficiencies, since two antibodies with only the template oligonucleotide or two antibodies with only the primer oligonucleotide can bind, and such non-productive complexes can reduce the sensitivity of the approach. This problem can be exacerbated when the approach is applied to the detection of specific oligomeric structures, such that an additional specific template oligonucleotide is used to enable specific detection of the oligomer. Thus, the methods described herein can include the preformation of a set of detection antibodies configured to efficiently bind to the desired oligomeric analyte, together with a template for the formation of a rolling circle template. This is achieved by using additional oligonucleotide sequences, for example, scaffolding oligonucleotides that can specifically hybridize with the oligonucleotides coupled to the antibodies, so that the antibodies are configured as a detection antibody mixture of oligomeric types that match the target antigen oligomer. This pre-assembly step allows optimal binding of the detection antibody to the target oligomer. Following binding of the detection antibody to the oligomer antigen, the scaffolding oligonucleotides can be removed via exonuclease degradation or other suitable chemical or endonuclease treatment. Once the scaffolding oligonucleotides are removed, the proximity ligation rolling circle amplification assay proceeds as described herein.

[0196] In further embodiments, the assay formats described herein further include one or more control assays. A negative control can be included on the binding domain that includes a capture antibody without a corresponding detection antibody, thereby providing a consistent background signal for all samples. A measured signal above a pre-set threshold indicates that improper assay processing has occurred or that there is a sample-dependent matrix effect that causes non-specific binding of the labeled detection probe. In addition, a specimen control can also be included in the assay for a human target antigen (such as a secreted or intracellular protein) that performs a number of control functions. A positive signal would indicate the presence of human material, and thus a test of sample addition and quality. Human antigens can also serve as processing controls for bacterial antigen extraction. For example, intracellular human analytes can also be selected as specimen controls that require lysis and extraction to be detected, such as Akt. A measured signal below a pre-defined threshold would indicate that no sample was added, that a reagent or processing failure has occurred, or that there is a substance that interferes with amplification or detection. In addition to internal controls, external positive and negative controls can also be used with the present methods and / or kits. Positive controls can also include a mixture of non-infected bacterial extracts representative of all specific target antigens detected by the multiplexed panel that, when tested, provide a positive signal for all assays. Negative controls include a representative matrix that does not contain any target proteins.

[0197] Examples of samples that may be analyzed by the methods of the present invention include, but are not limited to, food samples (including food extracts, food shreds, beverages, etc.), environmental samples (e.g., soil samples, environmental sludge, collected environmental aerosols, environmental wipes, filtered water, etc.), industrial samples (e.g., starting materials, products or intermediates from industrial production processes), human medical samples, veterinary samples, and samples of other biological origin. Biological samples that may be analyzed include, but are not limited to, samples containing stool, mucosal swabs, physiological specimens, and / or cell suspensions. Specific examples of biological samples include blood, serum, plasma, stool, mucosal swabs, tissue aspirates, tissue homogenates, cell cultures and cell culture supernatants (including eukaryotic and prokaryotic cell cultures), urine, saliva, sputum, and cerebrospinal samples.

[0198] Analytes that may be measured using the methods of the 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 complexes containing one or more of the above molecules, or combinations thereof. The level of an analyte of interest in a sample may be indicative of a disease or condition, or it may simply be indicative of whether a patient has been exposed to the analyte.

[0199] In certain embodiments, the analyte of interest is an exosome, i.e., a small membrane vesicle released from most cell types. The release and subsequent uptake of exosomes is a method of cell-to-cell communication and plays a role in regulating many physiological and pathological processes. It has been shown that exosomes contain a wide variety of signaling molecules, including but not limited to surface-bound and cytoplasmic proteins, lipids, mRNA, and miRNA, and it has been suggested that the identification and concentration of these specimens in each exosome can be used to infer its cellular origin and function. Thus, genomic or proteomic profiling of a patient's total exosome population could provide valuable predictive information for a variety of pathological conditions, including cancer, infectious diseases, kidney and liver diseases, and traumatic brain injury, among others.

[0200] Exosomes are typically measured in aggregates by isolating exosomes in bulk from biological fluids, disrupting their membranes, and assaying their contents by conventional methods such as Western blot, PCR, or sequencing. However, the methods and kits of the present invention can be used to characterize exosomes through capturing them on a surface and subsequently detecting target molecules expressed by the exosomes. Several proteins have been identified that are common among most exosomes (e.g., CD9, CD63, CD81, Hsp70, PDCD6IP, Tsg101), and in one embodiment, these targets can be used independently or in combination to capture exosomes on a surface via binding interactions between one or more capture reagents and one or more common exosome target proteins. In alternative or further embodiments, disease-associated markers present on or within exosomes are used to capture exosomes on a surface via binding interactions between one or more capture reagents and disease-associated exosome markers. The captured exosome(s) are then probed with one or more detection reagents directed against either one or more common exosomal proteins, or one or more additional molecular targets present in or on the surface of some fraction of exosomes. If separate targets are selected for the capture and detection reagents, only exosomes containing both targets will be detected, allowing additional specificity in identifying or quantifying exosome subpopulations. In certain embodiments, the sample used is a purified exosome preparation.

[0201] In certain embodiments, detection of specific phenotypically distinct exosome subpopulations is achieved using a pair of detection reagents that only generate a signal when brought into proximity by binding interactions at the proximity targets described herein. In this embodiment, the proximity targets may be distinct epitopes on the same molecule, different copies of the same molecular species that are in close proximity inside a single exosome or bound to the membrane of the same exosome, or different interacting species within or on a single exosome, such as two interacting proteins (e.g., tetraspanin-integrin complex), a protein receptor and a ligand (e.g., EFG and EGFR), or an mRNA molecule and an RNA binding protein (e.g., Argonaute 1-4 or GW182). Furthermore, the use of the methods described herein allows the analysis of up to three separate target molecules that may not be functionally linked, but are linked by their presence within a single exosome. The proximity probes used in the PLA / RCA method described herein can be extended to allow ligation and subsequent amplification of target molecules located further away on or within exosomes. In one embodiment, the PLA / RCA assay is performed using a specific fluorescent probe for the amplicon. The captured exosomes are then labeled with a general fluorescent reagent, e.g., acridine orange for RNA in exosomes, and the captured exosomes are imaged to determine the correlation between the two fluorescent signatures. The use of a general fluorescent reagent allows the selection of the signal from the desired exosome.

[0202] To probe for internal target molecules (cargo proteins, lipids or RNA molecules), exosomes can be fixed and permeabilized before or after capture, but before adding detection reagents. If permeabilized exosomes are being interrogated using the methods described herein, one or both of the detection reagents can include oligonucleotide probes capable of binding to specific RNA sequences, allowing detection of mRNA, miRNA and / or interactions between proteins and RNA.

[0203] Exosomes are measured with or without the use of anchoring reagents using the methods described herein. As described above, the use of anchoring reagents in a binding assay is illustrated in FIG. 1(a), where if the analyte is an exosome, the surface (101) includes a capture reagent, for example, directed to a common exosome target protein. In certain embodiments, the surface also includes an anchoring reagent (103). In one or more steps, the exosomes are bound to the capture reagent and a detection reagent (104) that also binds to the exosomes via the same or a different exosome target protein. Here, the detection reagent is linked to a nucleic acid probe (105). Thus, a complex is formed on the surface that includes the capture reagent, the exosomes, and the detection reagent. The probe extends to form an extension sequence (107) that includes an anchor region that binds the anchoring reagent. The extension sequence binds to the anchoring reagent, and the amount of extension sequence bound to the surface is measured.

[0204] Similarly, the method illustrated in FIG. 1(b) can also be applied to the detection of exosomes. In certain embodiments, the detection complex can include one or more detection reagents to enhance the specificity of the assay for exosomes, for example, as illustrated in FIG. 1(c). In one or more steps, the exosomes bind to each of the capture reagent and two (or more) detection reagents (120 and 121, respectively) that bind the target protein expressed by the exosomes, where each of the first and second detection reagents is linked to a nucleic acid probe (122 and 123, respectively, the first and second nucleic acid probes). The exosomes can bind to the capture reagent and the detection reagent simultaneously or substantially simultaneously, or sequentially, in a stepwise manner. Thus, a complex (124) is formed on the surface that includes the capture reagent, the exosome, and the first and second detection reagents. Using an extension process that requires the first and second probes to be in close proximity to each other, the first probe is extended to form an extension sequence (125) that includes an anchor sequence complement that is complementary to the anchor sequence. In the penultimate step, the anchor sequence is hybridized to the anchor sequence complement and the amount of extension sequence bound to the surface is measured. Similarly, the methods illustrated in each of Figures 2(a)-(c) can be used to detect exosomes.

[0205] The assays of the present invention may be used to determine the concentration of one or more, e.g., two or more, analytes in a sample. Thus, two or more analytes may be measured in the same sample. A panel of analytes that can be measured in the same sample includes, for example, a panel of assays for analytes or activities associated with a disease state or physiological condition. Certain such panels include cytokines and / or their receptors (e.g., one or more of TNF-α, TNF-β, ILL-α, ILL-β, IL2, IL4, IL6, IL-10, IL-L2, 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., one or more antibodies directed against Sm, RNP, SS-A, SS-α, J0-1, and Scl-70 antigens), allergen-specific antibodies, tumor markers (e.g., one or more of CEA, PSA, CA-125 II, CA 15-3, CA 19-9, CA 72-4, CYFRA, etc.), and / or other antibodies. 21-1, NSE, AFP, etc.), markers of cardiac 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., one or more of fibrin monomer, D-dimer, thrombin-antithrombin complex, prothrombin fragments 1 and 2, anti-factor Xa, etc.), markers of acute viral hepatitis infection (e.g., one or more 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, beta amyloid, Aβ 42, Aβ 40, Aβ 38, Aβ 39, Aβ 37, Aβ 34, tau protein, etc.) Markers of osteoporosis (e.g., one or more cross-linked NorC-telopeptide, total deoxypyridinoline, free deoxypyridinoline, osteocalcin, alkaline phosphatase, C-terminal propeptide of collagen type I, bone-specific alkaline phosphatase, etc.), 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.), thyroid disorders (e.g., one or more of thyroid stimulating hormone (TSH), total T3, free T3, total T4, free T4, and reverse T3), and a panel of markers for prostate cancer (e.g., one or more of total PSA, free PSA, complexed PSA, prostatic acid phosphatase, creatine kinase, etc.). Certain embodiments of the invention include, for example, the measurement of one or more, two or more, four or more, or ten or more analytes associated with a particular medical or physiological condition (e.g., analytes grouped together, such as those listed above in a panel; 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).

[0206] In a preferred embodiment, the panel includes one or more low abundance analytes in a conventional sample matrix, e.g., analytes with a concentration of less than about 100 fg / mL, preferably less than about 10 fg / mL. A non-limiting list of analytes that can 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, beta amyloid oligomers, C-peptide, IL-13, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-8, IL-12 / 23p40, IL-12p7, IL-12p8, IL-12p9, IL-12p10, IL-12p11, IL-12p12, IL-12p13, IL-12p14, IL-12p15, IL-12p16, IL-12p17, IL-12p18, IL-12p19, IL-13p16, IL-13p18, IL-13p19 ... 0, INF-g, PSA, PSAc, Tau, Phospho-Tau, TNFa, Troponin I, Cardiac 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 A, IgG2, IgG3, IgG4, AFP, CA125, Caspase-3 active active), CXCL11 / I-TAC, ErbB2 / HER2, HGFR / o-MET, IFN-β, MMP1, MMP2, MMP3, MMP9, β-NGF, TFF3, TIMP1, Kim-1, α-2 macroglobulin, D-dimer, ICAM-1, myeloperoxidase, myoglobin, PAI-1, PCSK9, plasminogen, renin / prorenin, tPA, CXCL1 / GRO-α, CCL2 / MCP1, CCL3 / MIP-1α, CCL4 / MIP-1β, C including CL5 / Rantes, CRP, CXCL9 / MIG, CXCL10 / IL-10, G-CSF, GM-CSF, IFN-α, IFN-γ, IL1α, IL-1β, 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, lactogenic hormone, HIV p24, HB-EGF, AKT, phospho-AKT, and combinations thereof.

[0207] In certain embodiments, the panel includes one or more low abundance analytes, e.g., analytes at a concentration of less than about 100 fg / mL, preferably less than about 10 fg / mL, in a conventional sample matrix, and the panel includes one or more of the following human targets: G-CDF, GM-CSF, IFN gamma, IL-1β, 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, TNFα, TSLP, VEGF, complexed PSA, free PSA, Aβ42, Aβ40, Aβ38, tau, cardiac troponin I, cardiac troponin T, HIV p24, C-peptide, and / or FGF21.

[0208] Additionally, the methods and kits described herein can be used in immunoassays to detect single organism susceptibility to antimicrobial resistance markers (PBP2a / mecA (S. aureus, Gram positive), TEM1 (E. coli, Gram negative)). These assays can be used for this class of analytes in both Gram positive and Gram negative. One or more of the following target proteins involved in antimicrobial resistance to vancomycin, beta-lactams, carbapenems, aminoglycosides and macrolide antibiotics may be included in the assay; ESBL genes of the erm family, vanA, vanB, aac(6')-aph(2''), KPC, NDM, OXA-48, VIM, OXA-23-like, OXA-40-like, OXA-58-like, CTX-M-1 and CTX-M-9 families, and combinations thereof. Additionally, one or more of the following proteins are included in the assay: 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 proteins L30, L31 and L32, and combinations thereof. Additionally, one or more of the following targets may be assessed, either alone or in combination with one or more of the markers defined herein: e.g., elongation factor EF-TU, ACP, acyl carrier protein, RplL, ribosomal protein GroS (MopB, 65,000), components of the chaperone system Gro-EL-Gro-ES and GapA, enzymes of the glycolytic pathway, and combinations thereof.

[0209] Furthermore, the methods and kits described herein can also be used in immunoassays to detect healthcare associated infections (HAI organisms), including but not limited to Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and extraintestinal pathogenic Escherichia coli. Resistance markers for each HAI organism are provided in the table below.

[0210] [Table 1]

[0211] Specific outer membrane proteins can be assessed in the immunoassays described herein.

[0212] [Table 2]

[0213] Additionally, the methods and kits described herein may be used in immunoassays to detect one or more of the following classes of biomarkers: cytokines, circulating tumor-specific proteins, one or more proteins associated with infectious diseases, intracellular markers, and the like, and combinations thereof.

[0214] Furthermore, the methods and kits described herein may be used to identify the following autoimmune diseases by assessing the presence or absence of one or more of the associated antigens listed below.

[0215] [Table 3]

[0216] In certain embodiments, the panel includes one or more low abundance analytes in a conventional sample matrix, e.g., analytes with a concentration of less than about 100 fg / mL, preferably less than about 10 fg / mL. Preferably, the panel 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 certain embodiments, the concentration of the analyte detected in the sample is within the range of 0.01 fM to 100 fM, 0.03 fM to 50 fM, or 0.03 fM to 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 an analyte molecule in a sample may 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 measured concentration of the analyte molecule in the sample is within about 10%, within about 3%, or within about 1% of the actual concentration of the analyte molecule in the sample. The detection limit of the assay is the concentration that gives a signal that is at least 2.5 standard deviations above the background signal, and preferably the assay is capable of detecting about 10-10,000 molecules in the sample, or 100-5,000 molecules in the sample, or 100-1000 molecules in the sample.

[0217] In further embodiments, the methods described herein can be used to detect low abundance analytes resulting from recent exposure and / or infection.Early diagnosis of various diseases or conditions, such as cancer, bacterial infections, such as Bacillus anthracis, viral infections, such as HIV, hepatitis, HPV, toxin exposure, such as ricin, botulinum toxin A, B, or E, is limited by the fact that the limit of detection (LOD) of available techniques, such as ELISA, is higher than the circulating concentration of low abundance proteins that may indicate the onset of disease.The panel can include low abundance analytes in one or more conventional sample matrices, such as analytes with concentrations of less than about 100 fg / mL, or 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 can include one or more of the following analytes that 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, beta amyloid oligomers, C-peptide, IL-13, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-8, INF-g, PSA, Tau, phospho-Ta u, TNFa, troponin I, cardiac 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 A, IgG2, IgG3, IgG4, AFP, CA125, caspase-3 active, CXCL11 / I-TAC, ErbB2 / HER2 , HGFR / o-MET, IFN-β, MMP1, MMP2, MMP3, MMP9, β-NGF, TFF3, TIMP1, Kim-1, α-2 macroglobulin, D-dimer, ICAM-1, myeloperoxidase, myoglobin, PAI-1, PCSK9, plasminogen, renin / prorenin, tPA, CXCL1 / GRO-α, CCL2 / MCP1, CCL3 / MIP-1α, CCL4 / MIP-1β, CCL5 / Rantes, CCL6 / MIP-1β, CCL7 / MIP-1β, CCL8 / MIP-1β, CCL9 / MIP-1β, CCL10 / MIP-1α, CCL11 / MIP-1β, CCL12 / MIP-1α, CCL13 / MIP-1β, CCL14 / MIP-1β, CCL15 / MIP-1α, CCL16 / MIP-1β, CCL17 / MIP-1α, CCL18 / MIP-1β, CCL19 / MIP-1α, CCL20 / MIP-1β, CCL21 / MIP-1α, CCL22 / MIP-1α, CCL23 / MIP-1α, CCL24 / MIP-1β, CCL25 / MIP-1α, CCL26 / MIP-1α, CCL27 / MIP-1β, CCL28 / MIP-1α, CCL29 / MIP-1α, CCL29 / MIP-1β, CCL29 / MIP-1α, CCL20 / MIP-1α, CCL21 / MIP-1α, CCL22 / MIP-1α, CCL23 / MIP-1α, CCL24 / MIP-1β, CCL25 / MIP-1α, CCL26 / MIP-1α, C RP, CXCL9 / MIG, CXCL10 / IL-10, G-CSF, GM-CSF, IFN-α, IFN-γ, IL1α, IL-1β, 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, lactogenic hormone, HB-EGF, AKT, phospho-AKT, and combinations thereof.

[0218] The method of the present invention is designed to allow for the detection of a wide variety of biological and biochemical agents, as described above. In one embodiment, the method may be used to detect pathogens and / or potentially pathogenic viruses, bacteria, including biological warfare agents ("BWA"), and toxins in a variety of relevant clinical and environmental matrices, including, but not limited to, blood, sputum, stool, filters, swabs, etc. A non-limiting list of pathogens and toxins that can be analyzed (singly or in combination) using the methods of the invention include Bacillus anthracis (anthrax), Yersinia pestis (plague), Vibrio cholerae (cholera), Francisella tularensis (tularemia), Brucella spp. (brucellosis), Coxiella burnetii (Q fever), Listeria, Salmonella, Shigella, V. cholera, Chlamydia trachomatis, Burkholderia pseudomallei, orthopoxviruses including variola virus (variola), and the like. viruses), viral encephalitis, Venezuelan equine encephalitis virus (VEE), western e quine encephalitis virus (WEE), eastern equine encephalitis virus (EEE), alphavirus, viral hemorrhagic fever, 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 fungus, Bacillus globigii, Serratiamarcescens, yellow rain, trichothecene mycotoxins, Salmonella typhimurium, aflatoxins, Indian rat flea, Diamanus montanus, smallpox, monkeypox virus, arenaviruses, hantaviruses, Lassa fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Rift Valley fever virus, Crimean-Congo virus, hantaviruses, Marburg hemorrhagic fever, yellow fever virus, dengue virus, influenza (including human and animal strains such as avian influenza H5N1, influenza A, influenza A, H1 specific, influenza A, H3 specific, influenza A, H5 specific, influenza A, 2009-H1N1 specific, and 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 pneumoniae, Borrelia burgdorferi, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Staphylococcus aureus, Staphylococcal enterotoxin B (SEB), Abrin, Shiga toxin 1, Shiga toxin 2, Moraxella catarrhalis, Streptococcus pyogenes, Clostridium These include C. 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.

[0219] The improved binding assay methods described herein can be used to expand the dynamic range of the binding assay, i.e., the range of concentrations of analyte molecules in a fluid sample or samples that can be quantified by the system or method without dilution, or to modify assay conditions (e.g., concentrations of reagents used, etc.) that produce similar results, where the measured concentration of the analyte molecule can be substantially accurately determined. The concentration of the analyte molecule in the fluid sample may be considered to be substantially accurately determined if the measured concentration of the analyte molecule in the fluid sample is within about 10% of the actual (e.g., true) concentration of the analyte molecule in the fluid sample. In certain embodiments, the measured concentration of the analyte molecule in the fluid sample is substantially accurately determined in embodiments where the measured concentration is within about 5%, 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%, within about 0.1% of the actual concentration of the analyte molecule in the fluid sample. In some cases, the measured determined concentration 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%, no more than about 0.5%. The accuracy of an assay method may, in some embodiments, be determined by determining the concentration of an analyte molecule in a fluid sample of known concentration using a selected assay method and comparing the measured concentration to the actual concentration.

[0220] In some embodiments, a system or method may be capable of measuring concentrations of analyte molecules in a fluid sample over a dynamic range of about 1000 (3 log), about 10,000 (4 log), about 100,000 (5 log), about 350,000 (5.5 log), 1,000,000 (6 log), about 3,500,000 (6.5 log), about 10,000,000 (7 log), about 35,000,000 (7.5 log), about 100,000,000 (8 log) or more.

[0221] In some embodiments, concentrations of analyte molecules in a fluid sample (e.g., unknown concentrations) that can be substantially accurately determined are less than about 5000 fM (femtomoles), 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 (attomoles), 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 (zeptomoles), 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 detection limit (e.g., the lowest concentration of an analyte molecule that can be determined 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 (attomoles), about 100 aM, about 50 aM, about 10 aM, about 5 aM, about 1 aM, about 0.1 aM, about 500 zM (zeptomoles), about 100 zM, about 50 zM, about 10 zM, about 5 zM, about 1 zM, about 0.1 zM or less. In some embodiments, the concentration of an analyte molecule or particle in a fluid sample that can be substantially accurately determined is between about 5000 fM and about 0.1 fM, between about 3000 fM and about 0.1 fM, between about 1000 fM and about 0.1 fM, between about 1000 fM and about 0.1 zM, between about 100 fM and about 1 zM, between about 100 aM and about 0.1 zM, or less. The upper limit of detection (e.g., the upper concentration of an analyte molecule that can be determined in a solution) is at least about 100 fM, at least about 1000 fM, at least about 10 pM (picomolar), at least about 100 pM, at least about 100 pM, at least about 10 nM (nanomolar), at least about 100 nM, at least about 1000 nM, at least about 10 uM, at least about 100 uM, at least about 1000 uM, at least about 10 mM, at least about 100 mM, at least about 1000 mM, or more.In some embodiments, the concentration of the analyte molecule or particle in the fluid sample that is determined is less than about 50x10-15 M, or less than about 40x10-15 M, or less than about 30x10-15 M, or less than about 20x10-15 M, or less than about 10x10-15 M, or less than about, or less than about 1x10-15 M.

[0222] In some embodiments, the concentration of an analyte molecule in a sample that may 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 an analyte molecule in a sample that may be substantially accurately determined is between about 5000 fM and about 0.1 fM, between about 3000 fM and about 0.1 fM, between about 1000 fM and about 0.1 fM, between about 1000 fM and about 1 fM, between about 100 fM and about 0.1 fM. The concentration of an analyte molecule in a sample may 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 measured concentration of an analyte molecule in a sample may be within about 10%, within about 3%, or within about 1% of the actual concentration of the analyte molecule in the sample. The accuracy of the assay method may, in some embodiments, be determined by determining the concentration of the analyte molecule in a sample of known concentration using a selected assay method. Preferably, the assay is capable of detecting about 10-10,000 molecules in a sample, preferably 100-5,000 molecules in a sample, and more preferably 100-1000 molecules in a sample.

[0223] For example, use of the assay format described herein may improve detection signal and assay sensitivity by as much as 10-fold, preferably 50-fold, 100-fold or 1000-fold, as compared to a conventional sandwich immunoassay technique measured using the same capture antibody and one of two detection antibodies, as well as the same label and detection technique. Preferably, use of the assay format described herein improves detection signal and assay sensitivity by as much as 100-fold, as compared to a standard sandwich immunoassay.

[0224] One advantageous aspect of the methods of the invention is that signal amplification allows individual binding events to be detected as bright spots of light, especially when combined with sensitive optical detection techniques. Quantification of the signal can be performed by counting individual events (which can increase sensitivity for low concentrations of analyte due to improved ability to distinguish binding events from background noise) or by integrating the signal of all binding events (which can provide a better dynamic range for measuring high concentrations of analyte).

[0225] The methods of the invention may be used in a variety of assay devices and / or formats. Assay devices include assay modules, such as assay plates, cartridges, multi-well assay plates, reaction vessels, test tubes, cuvettes, flow cells, assay chips, lateral flow devices, etc., that have assay reagents (which may include targeting agents or other binding reagents) that are added as the assay progresses or that are preloaded into wells, chambers, or assay regions of the assay modules. These devices may use a variety of assay formats for specific binding assays, such as immunoassays, immunochromatographic assays. Exemplary assay devices and formats are described later in this specification. In certain embodiments, the methods of the invention may use assay reagents stored in a dry state, and the assay device / kit may further include or be provided with a desiccant to keep the assay reagents dry. An assay device preloaded with assay reagents can significantly increase the speed and simplify the assay measurement while maintaining excellent stability during storage. The dried assay reagents may be any assay reagent that can be dried and subsequently reconstituted prior to use in an assay. Such reagents include, but are not limited to, binding reagents, enzymes, enzyme substrates, indicator dyes and other reactive compounds useful in binding assays that can be used to detect the analyte of interest. Assay reagents may further include substances that are not directly involved in the mechanism of detection but play an auxiliary role in the assay, including, but not limited to, blocking agents, stabilizers, detergents, salts, pH buffers, preservatives, etc. Reagents may be present in a free state or supported on a solid phase, including the surface of a compartment (e.g., a chamber, channel, flow cell, well, etc.) in an assay module, or the surface of a colloid, bead or other particulate support.

[0226] The method of the present invention can be used with a variety of methods for measuring the amount of an analyte, particularly with methods for measuring the amount of an analyte bound to a solid phase. Techniques that can be used include, but are not limited to, cell culture-based assays, binding assays (including agglutination tests, immunoassays, nucleic acid hybridization assays, etc.), enzymatic assays, colorimetric assays, and other techniques known in the art. Other suitable techniques will be readily apparent to a person of average skill in the art. Some measurement techniques allow the measurement to be visually inspected, which may require the use of an instrument to perform the measurement, which may be advantageous.

[0227] Methods for measuring the amount of an analyte include label-free techniques, including but not limited to: i) techniques that measure the change in mass or refractive index at a surface after the analyte binds to the surface (e.g., surface ultrasound techniques, surface plasmon resonance sensors, ellipsometry techniques, etc.); ii) mass spectrometry techniques (including techniques such as MALDI, SELDI, etc. that can measure the analyte on a surface); iii) chromatographic or electrophoretic techniques; iv) fluorescence techniques (based on the intrinsic fluorescence of the analyte).

[0228] Methods for measuring the amount of an analyte further include techniques for measuring the analyte through the detection of a label that can be directly or indirectly bound to the analyte (e.g., through the use of a binding partner of a labeled analyte). Suitable labels include labels that can be directly visualized (e.g., visually identifiable particles and labels that generate a measurable signal, such as light scattering, optical absorption, fluorescence, chemiluminescence, electrochemiluminescence, radiation, magnetic field, etc.). Labels that can be used also include enzymes or other chemically reactive species that have a chemical activity that results in a measurable signal, such as light scattering, absorption, fluorescence, etc. The use of enzymes as labels is established in enzyme-linked immunosorbent assays, called ELISA, enzyme immunoassays, or EIA. In the ELISA format, an unknown amount of antigen is immobilized on a surface and then a specific antibody is flowed over the surface to allow the specific antibody to bind to the antigen. This antibody binds to the enzyme, and in a final step, a substance is added that converts the enzyme into a product that causes a change in the detectable signal. The formation of the product is detectable by a difference from the substrate in a measurable characteristic, such as, for example, absorption, fluorescence, chemiluminescence, light scattering, etc. Some (but not all) assay methods that may be used with the solid phase binding methods according to the invention may benefit from, or even require, a washing step to remove unbound components (e.g., label) from the solid phase, and therefore the methods of the invention may include such a washing step.

[0229] In those embodiments using a pair of detectable labels, the labels are selected based on their ability to be detected independently and / or to cooperate to produce a detectable signal when the pair of labels are in close proximity to one another, i.e., directly or indirectly bound to the analyte of interest in a detection complex. In one embodiment, the first detectable label is a first enzyme of a coupled enzymatic reaction system and the second detectable label is a second enzyme of the coupled enzymatic reaction system, and the method further comprises the step of adding one or more substrates of the reaction system, thereby producing a detectable product of the enzymatic reaction system. Reaction vessels that contain detectable product and those that do not are distinguishable. In a preferred embodiment, detectable product is produced only when the first and second enzymes are in close proximity, e.g., less than 200 nm, ideally less than 50 nm. In one embodiment, the first enzyme is an oxidase, e.g., glucose oxidase, the second enzyme is a peroxidase, and the substrate comprises an oxidase substrate, e.g., glucose, and a labeled tyramide, Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine), or a 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 produce a product that reacts with the second enzyme, which produces a second product that reacts with the labeled reactive derivative to produce a detectable species. Preferably, this reaction is catalyzed by the first and second enzymes of the detection complex to immobilize the labeled reactive derivative on the surface. This derivative can be measured to determine the number of analyte molecules present on the surface. In one embodiment, the labeled reactive derivative is biotin-tyramide, and the method further comprises adding labeled streptavidin and measuring the label on the streptavidin.

[0230] Yet another proximity-dependent labeling system that can be used in the method is a FRET pair, for example, where 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, where excitation is transferred from the donor molecule to the acceptor molecule without emitting light. FRET is a useful system because its efficiency is inversely proportional to the sixth power of the distance between the molecules, which is a distance comparable to the size of a biopolymer. In this labeling system, the proximity-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, the absorption spectrum of the acceptor preferably overlaps with the fluorescence emission spectrum of the donor, and the transfer dipole orientations of the donor and acceptor should be approximately parallel. A non-limiting list of FRET pairs is shown in Table 1.

[0231] [Table 4]

[0232] A variety of FRET detection methods exist for light microscopy, including acceptor photobleaching, donor photobleaching, ratiometric imaging, sensitized emission and fluorescence lifetime measurements.

[0233] Another suitable labeling system using a pair of detection labels that can be used in the embodiment is a system that can measure the first and second detectable labels independently. For example, the first and second detectable labels can be luminescent labels that have different spectral characteristics. Alternatively, the first detectable label is a first enzyme that reacts with a first substrate to generate a first signal, and the second detectable label is a second enzyme that reacts with a second substrate to generate a different second signal, and the method further includes adding the first enzyme substrate and the second enzyme substrate, and counting the number of reaction vessels in which the first and second signals are generated. The first and second signals can have different optical absorption and / or emission signals due to different spectral characteristics.

[0234] When the first and second detectable labels comprise first and second enzymes, the enzymes may each be hydrolases, such as phosphatases, sulfatases, galactosidases, glucuronidases, or combinations thereof, and thus the first and second substrates are selected from phosphate, sulfate, galactosides and glucuronides modified stabilized dioxetanes, 4-methylumbelliferyl, fluorescein, or combinations thereof. Alternatively, the first and second enzymes are selected from horseradish peroxidase, β-galactosidase, and alkaline phosphatase.

[0235] Alternatively, the label used to detect the analyte molecule may be a fluorescent species that can be used in single molecule fluorescence detection, e.g., fluorescence correlation spectroscopy, and / or fluorescence cross-correlation spectroscopy. Single molecule fluorescence detection involves flowing an eluent containing the detectable species through the capillary, focusing a light source on the contents within the capillary to define a monitoring region, observing the monitoring region with a photodetector, and detecting fluorescent molecules that pass through the monitoring region.

[0236] In one embodiment, the analyte of interest in a sample may be measured using an electrochemiluminescence-based assay format, such as an electrochemiluminescence (ECL)-based immunoassay. The high sensitivity, wide dynamic range, and selectivity of ECL are important attributes in medical diagnostics. Commercially available ECL instruments have demonstrated exceptional performance and have been widely used for several reasons, including their excellent sensitivity, dynamic range, precision, and tolerance to complex sample substrates. Species capable of inducing the emission of ECL (ECL-active species) have been used as ECL labels, such as i) organometallic compounds in which the metal is, for example, from the Group VIII noble metals (Ru-containing and Os-containing organometallic compounds, including, for example, the tris-bipyridyl-ruthenium (RuBpy) moiety), and ii) luminol and related compounds. Species involved in ECL labeling in the ECL process are referred to herein as ECL coreactants. Commonly used coreactants include tertiary amines for RuBpy-derived ECL (see, e.g., U.S. Pat. No. 5,846,485), oxalate and persulfate, and hydrogen peroxide for luminol-derived ECL (see, e.g., U.S. Pat. No. 5,240,863). The light emitted by the ECL label can be used in diagnostic procedures as a reporter signal (Bard et al., U.S. Pat. No. 5,238,808, incorporated herein by reference). For example, the ECL label can be covalently attached 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 released from the ECL label. Alternatively, the ECL signal from an ECL-active compound can be an indicator of the chemical environment (see, e.g., U.S. Pat. No. 5,641,623, which describes an ECL assay that monitors the formation or decomposition of an ECL coreactant).For further background on ECL, ECL labels, ECL assays and apparatus for performing ECL assays, see U.S. Patent 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, 5,786,211, 5,786,121, 5,786,221, 5,786,312, 5,786,433, 5,786,512, 5,786,512, 5,786,312, 5,786,5 ... See, for example, WO 31,147, WO 6,066,448, WO 6,136,268, WO 5,776,672, WO 5,308,754, WO 5,240,863, WO 6,207,369, WO 6,214,552 and WO 5,589,136, as well as WO 99 / 63347, WO 00 / 03233, WO 99 / 58962, WO 99 / 32662, WO 99 / 14599, WO 98 / 12539, WO 97 / 36931 and WO 98 / 57154, all of which are incorporated herein by reference.

[0237] When multiple assay measurements are performed on a single sample, the method of the present invention can be applied to singleplex or multiplex formats. Multiplex measurements that can be used in the present invention include, but are not limited to, i) using multiple sensors; ii) using separate assay areas on a surface that are distinguishable by their location on the surface (e.g., an array); iii) using reagents coated on particles that are distinguishable by particle properties, such as size, shape, color, etc.; iv) generating distinguishable assay signals based on optical properties (e.g., absorbance or emission spectrum); or v) based on temporal properties of the assay signal (e.g., time, frequency or phase of the signal).

[0238] In some embodiments, the concentration measurement of the analyte molecule in the sample may be determined, at least in part, by comparing the measured parameter to a calibration standard. For example, a fraction of the binding surface containing the analyte molecule may be compared to a calibration curve to determine the concentration measurement of the analyte molecule in the sample. The calibration curve may be generated by completing an assay of multiple standardized samples of known concentrations under the conditions used to analyze the test samples. By reading the signal related to the detection / quantification of the analyte molecule in each standardized sample, a calibration curve may be generated that relates the detection of the analyte molecule to the known concentration of the analyte molecule. An assay may then be completed with a sample containing an unknown concentration of the analyte molecule, and the detection of the analyte molecule in this assay may be plotted on the calibration curve to determine the concentration measurement of the analyte molecule in the sample.

[0239] In the specific case of using imaging techniques to measure the optical signal (such as fluorescence, chemiluminescence or electrochemiluminescence), binding events can be detected as bright spots of the light source. If the surface density of point sources is low (e.g., the probability of finding a point source in the RxR region, -R being the spatial resolution in the detection system- is less than 10%), any observed point source is likely to be due to a single binding event. Under these conditions, counting of events may be the most sensitive measurement. As the surface density increases, it becomes increasingly difficult to resolve and count individual binding events. Under these conditions, integration of the optical signal of the binding surface allows for accurate measurements.

[0240] It will be clear to those skilled in the art that the methods described herein can be applied to many immunoassay platforms known to those skilled in the art. Various features of the immunoassay platform can be adjusted to suit a particular platform, which adjustments are well within the ability of those skilled in the art. For example, the methods described herein can be applied to bead-based formats using encoded particles. In such systems, the beads used can be magnetic or non-magnetic, and the surface of the beads is modified to have one or more copies of the capture reagent. The detection reagents used in the system are a pair of detection reagents. In one embodiment, the two detection reagents comprise distinguishable fluorescent labels. Alternatively, the two detection reagents are modified with a nucleic acid probe as described herein, in which case the immunoassay method comprises an extension process, e.g., RCA-PLA, to generate an amplification product indicative of the presence of each detectable detection reagent. When the detection reagent comprises two distinguishable fluorescent labels, the measurement step comprises the introduction of the beads into a flow cell, and when the beads are magnetic, the measurement comprises capturing the beads in the flow cell. If the detection reagent is modified with a nucleic acid probe, the measurement step includes forming a sandwich complex on the beads, performing RCA-PLA, and labeling the amplicons with a fluorescently labeled detection probe. The labeled beads are then introduced into a flow cell, and if the beads are magnetic, they are captured in the flow cell. In each embodiment, the assay can be multiplexed by spectrum based identification of the fluorescently labeled coded beads. An excitation light source and an emission light detector in multicolor detection can be used to detect the binding events in each embodiment, and quantification can be achieved by counting beads that have both detectable labels or that contain detectably labeled extension products, and quantification can also be achieved by detection by integrated intensity, for example, integration over the signal of all binding events.Accordingly, a kit is provided for use in the above-described methods, comprising in one or more vials, containers, or compartments one or more of the following: (a) magnetic or non-magnetic beads carrying a capture reagent; (b) two detection reagents carrying distinguishable fluorescent labels; and (c) any buffers and / or excipients for the assay protocol. Another kit can be used in the above-described method, which comprises, in one or more vials, containers, or compartments, one or more of the following: (a) magnetic or non-magnetic beads bearing a capture reagent; (b) two detection reagents modified with a nucleic acid probe (optionally the detection reagents are provided separately and proximity probes (1 and 2) are provided along with instructions for modifying the detection reagent with the probe); and (c) a fluorescently labeled probe; any reagents required for modifying the detection reagent with the proximity probe; assay diluent, calibrator, circularization oligonucleotide, a ligation mixture or components thereof, such as ligation buffer, ATP, BSA, Tween 20, T4 DNA ligase; an RCA mixture or components thereof, such as BSA, buffer, dNTPs, Tween 20, Phi29 DNA polymerase.

[0241] In another embodiment, the method described herein can be applied to flow cell analysis, bead-based format. In such a system, the beads used can be magnetic, and the surface of the beads is modified to contain one or more copies of the capture reagent. The detection reagent used in this system is a pair of detection reagents modified with a nucleic acid probe as described herein, in which case the immunoassay method includes an extension process, e.g., RCA-PLA, to generate an amplification product indicative of the presence of each detectable detection reagent. The measurement step includes forming a sandwich complex on the beads, performing RCA-PLA, and labeling the amplicon with an ECL-labeled detection probe. The labeled beads are then introduced into the flow cell, and the beads are captured within the flow cell. In particular, a magnetic field is applied to the electrode surface to attract magnetic particles, e.g., beads, which can include various metals, e.g., platinum. A voltage source is used to apply a voltage to the electrodes, and an emission photodetector can be used to detect the binding events; quantification can be achieved by counting the beads with detectably labeled extension products, and quantification can also be achieved by detection by integrated intensity, e.g., integration over the signal of all binding events. Kits that can be used in the above-described methods may contain, in one or more vials, containers, or compartments, one or more of the following: (a) magnetic beads with a capture reagent; (b) two detection reagents modified with a nucleic acid probe (optionally the detection reagents are provided separately and proximity probes (1 and 2) are provided along with instructions for modifying the detection reagent with the probe); and (c) an ECL-labeled probe; any reagents required for modifying the detection reagent with the proximity probe; assay diluent, calibrator, circularization oligonucleotide, a ligation mix or components thereof such as ligation buffer, ATP, BSA, Tween 20, T4 DNA ligase; an RCA mix or components thereof such as BSA, buffer, dNTPs, Tween 20, Phi29 DNA polymerase.

[0242] In a particular embodiment of the bead-based format of flow cell analysis, the sample is incubated with a mixture of biotinylated monoclonal analyte-specific capture antibodies and monoclonal analyte-specific antibodies, each of which is conjugated with an oligonucleotide, and reacted to form a sandwich complex. After the addition of streptavidin-coated microparticles, the complex is bound to the solid phase by the interaction between biotin and streptavidin. The ligation mixture is added to the mixture, and the mixture and the ligation mixture are incubated together, washed to remove excess circularized oligonucleotides, and incubated with the RCA mixture. The mixture is washed, and a mixture of biotin-labeled detection probes is added. To incorporate a suitable label, such as a luminescent label, a chemiluminescent label, or an electrochemiluminescent label such as SULFO-TAG, the detection probe is synthesized with a terminal biotin label and pre-bound to SULFO-TAG-labeled streptavidin. The reaction mixture is drawn into the measurement cell, and the microparticles are magnetically captured on the surface of an electrode, for example, a metal electrode, such as a platinum electrode. Unbound material is then removed with a suitable washing buffer, such as ProCell (TPA-containing buffer). A voltage is then applied to the electrodes to induce the emission of chemiluminescence, and the emission is measured by a photomultiplier tube. The application of the voltage and the measurement of the resulting emission can be performed in any suitable flow cell, such as, for example, Cobas and / or Elecsysinstrument (available from Hoffmann-La Roche LTD.).

[0243] In yet another embodiment, the method described herein can be applied to a bead-based format, allowing individual molecules to be digitally counted by capillary flow. In such a system, the beads used can be magnetic, and the surface of the beads 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 that include distinguishable fluorescent labels. The measurement step includes forming a sandwich complex including the capture reagent, the analyte, and the 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 in multicolor detection can be used to detect the binding events, and quantification can be achieved by correlating the detection of the two fluorophores in the flow cell, and quantification can also be achieved by detection by integrated intensity, for example, integration over the signal of all binding events. A kit that can be used in the above-mentioned method 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 labels; and (c) any buffers and / or excipients for the assay protocol.

[0244] Furthermore, the methods described herein can be applied to bead-based formats, which include separating beads into individual nanowells. In such systems, the beads used can be magnetic, and the surface of the beads is modified to contain one or more copies of the capture reagent. The detection reagents used in the system are a pair of detection reagents that contain distinguishable enzyme labels. The measurement step involves forming a sandwich complex containing the capture reagent, the analyte, and the detection reagent, and adding substrates for the two enzyme labels. The beads are then captured in individual nanowells. The assay can be multiplexed by spectroscopy based identification of enzyme products with different spectral properties. An excitation light source and an emission light detector in multicolor detection can be used to detect the binding events, and quantification can be achieved by counting the nanowells that contain both enzyme products, and quantification can also be achieved by detection by integrated intensity, for example, integrated over the signal of the entire nanowell. A kit that can be used in the above-described method can include, in one or more vials, containers, or compartments, one or more of the following: (a) magnetic beads with a capture reagent; (b) two detection reagents, each modified with a distinguishable enzyme label, such as a biotinylated detection reagent and a hapten-conjugated detection reagent; (c) streptavidi-β-galactosidase, an anti-hapten-conjugated enzyme, resorufin β-d-galactopyranoside; (d) an array, such as a Quanterix DVD format array; (e) fluorocarbon oil; and (f) any buffers and / or excipients for the assay protocol. In this particular embodiment, the detectable signal is enhanced by combining the use of a nanowell high sensitivity system with a proximity-based detection system. Although a particular proximity-based detection system is used to illustrate this particular embodiment, those skilled in the art will appreciate the fact that other proximity-based detection systems described herein can be used to enhance the detectable signal in the assay, such as FRET donor / acceptor systems; luminescent labels that have distinct spectral properties; or the use of first and second enzymes that are hydrolases as described above and appropriate associated substrates.

[0245] Furthermore, the methods described herein can be applied to bead array-based platforms. In such systems, the beads used can be non-magnetic and the surface of the beads is modified to contain one or more copies of the capture reagent. The detection reagent used in this system is a pair of detection reagents comprising a first and a second nucleic acid probe. The measurement step includes forming a sandwich complex comprising the capture reagent, the analyte and the detection reagent, extending one of the probes to form an extension sequence (wherein the extension depends on the co-localization of the first and second probes in the sandwich complex), labeling the extension sequence with a fluorescent probe, and releasing the extension sequence from the surface into an elution solution. An excitation light source and an emission light detector in multicolor detection can be used to detect the binding events, and quantification can be achieved by counting the individual detectably labeled extension products, and quantification can also be achieved by detection by integrated intensity, for example, integration over the signal of all binding events. Kits that can be used in the above-described methods may contain, in one or more vials, containers, or compartments, one or more of the following: (a) non-magnetic beads carrying a capture reagent; (b) two detection reagents modified with nucleic acid probes (optionally the detection reagents are provided separately and proximity probes (1 and 2) are provided along with instructions for modifying the detection reagent with the probes); and (c) a fluorescently labeled probe; any reagents required for modifying the detection reagent with the proximity probe; assay diluent, calibrator, circularization oligonucleotide, a ligation mix or components thereof such as ligation buffer, ATP, BSA, Tween 20, T4 DNA ligase; an RCA mix or components thereof such as BSA, buffer, dNTPs, Tween 20, Phi29 DNA polymerase.

[0246] The improved binding assay described herein can be carried out using one or more kits that contain a set of components used in the assay.For example, a kit used to detect an analyte in a sample includes a surface that contains a capture reagent and an immobilized reagent for the analyte in one or more vials, containers or compartments; and a detection reagent for the analyte that is linked to a nucleic acid probe.Such a kit can include an immobilized reagent that includes an immobilized oligonucleotide sequence.

[0247] Another kit that can be used to practice the methods described herein comprises, in one or more vials, containers or compartments, a surface comprising a capture reagent for the analyte and an immobilized reagent comprising an immobilized oligonucleotide sequence; a first detection reagent that binds to a first nucleic acid probe; and a second detection reagent that binds to a second nucleic acid probe.

[0248] Another kit that can be used to perform the binding assays described herein comprises, in one or more vials, containers or compartments, a surface comprising a capture reagent and an immobilization reagent for the analyte; a first detection reagent for the analyte comprising a first proximity probe; a second detection reagent for the analyte comprising a second proximity probe; and a connector sequence comprising (i) an internal sequence complementary to the second proximity probe and (ii) two terminal sequences complementary to the non-overlapping regions of the first proximity probe. Alternatively, the kit may comprise a surface comprising a capture reagent and an immobilised reagent for the analyte; a first detection reagent for the analyte comprising a first proximity probe; a second detection reagent for the analyte comprising a second proximity probe; and (i) a first connector oligonucleotide, and (ii) a second connector oligonucleotide, where (x) a first end of the first connector and a first end of the second connector are complementary to the two non-overlapping regions of the first proximity probe, and (y) a second end of the first connector and a second end of the second connector are complementary to the two non-overlapping regions of the first proximity probe. Additionally, the immobilised reagent in either or both of these kits may comprise an immobilised oligonucleotide sequence.

[0249] Additionally, the methods described herein can be performed using a kit comprising, in one or more vials, containers or compartments, a first detection reagent comprising a first detectable label; a second detection reagent comprising a second detectable label; a plurality of reaction vessels configured to contain one or none of the analyte molecules; and, optionally, a surface comprising the capture reagent.

[0250] Finally, a kit for analyte detection using the methods described herein includes, in one or more vials, containers or compartments, a surface comprising an immobilized capture reagent; a first detection reagent having a first detectable label; a second detection reagent having a second detectable label; and a crosslinker that reacts with the first and second detection reagents. The crosslinker can include a multifunctional crosslinker that links reactive moieties bound to the detection reagents, or a multivalent binding partner of a binding moiety bound to the detection reagents. Suitable multifunctional crosslinkers include, but are not limited to, amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. Similarly, an example of a multivalent binding partner is a multivalent anti-species antibody that targets the detection reagent, which is an antibody of that animal species. The crosslinker can also include streptavidin, avidin, or biotin when paired with one of the binding partners that bind to the detection reagent. The crosslinker may also be an oligonucleotide comprising a sequence complementary to the nucleic acid probe directly or indirectly bound to the kit component. In certain embodiments, the kit for use in the methods described herein comprises, in one or more vials, containers or compartments, an immobilized capture reagent; a first detection reagent having a first detectable label and a first nucleic acid probe; a second detection reagent having a second detectable label and a second nucleic acid probe; and a surface comprising a third nucleic acid having a region complementary to the first and second nucleic acid probes.

[0251] The surface of the kits described herein can include a plurality of capture reagents for one or more analyte molecules, the capture reagents being distributed across a plurality of resolvable binding regions or reaction vessels located on the surface, e.g., in an array, multi-well plate, or micro- or nano-well plate, and the surface can further include a plurality of particles, each of which includes a plurality of capture reagents for the analyte molecules.

[0252] The above-mentioned kits may further include one or more of the following: one or more additional reagents, buffers, polymerases, ligases, and / or dNTPs (labeled or unlabeled). Furthermore, if one or more detection reagents include a detectable label, the kit may also include a co-reactant for the detectable label used in the kit. Alternatively, if one or more detection reagents are components of a coupled enzyme reaction system, each of the detection reagents includes a first and a second enzyme, and the kit may further include, in one or more containers, vials, or compartments, one or more substrates for the coupled enzyme reaction system, and optionally a labeled component configured to bind to the product of the coupled enzyme reaction system. For example, the first enzyme may be an oxidase, the second enzyme may be a peroxidase, and the kit may further include an oxidase substrate and a labeled tyramide derivative. In another embodiment, the first and second detectable reagents may include components of a proximity-dependent detection system, such as a FRET donor, a FRET acceptor, or luminescent labels that differ from each other in terms of their spectral properties.

[0253] Additional Alternative Embodiments A further embodiment is illustrated in Figure 7. A portion of each proximity probe in the sandwich immunoassay complex of panel (a) is temporarily protected by a short strand of RNA hybridized to each segment. The RNA strand is enzymatically removed, each of the proximity probes hybridize to each other, and the strand is extended by polymerase extension using biotinylated dNTPs (panel (b)). Each biotinylated base incorporated into the strand binds to streptavidin labeled with a detectable label (panel (c)).

[0254] Yet another approach is illustrated in Figure 8. The proximity probes can be bound to an immobilization reagent and a detection reagent (as shown in panel (a)) or each proximity probe can be bound to two detection reagents as described above (not shown). In a manner similar to that illustrated in Figure 7, a portion of each proximity probe is temporarily protected by a short strand of RNA hybridized to each segment. The RNA strand is enzymatically removed, each of the proximity probes hybridize to each other, and the strand is extended by polymerase extension using biotinylated dNTPs (panel (b)). Each biotinylated base incorporated into the strand binds to streptavidin labeled with a detectable label (panel (c)).

[0255] A further embodiment is shown in FIG. 18. In this embodiment, multiple short oligonucleotides are used as clasp sequences (1801) that hybridize to a portion of the amplicon, thereby folding the product into a compact structure that is easily imaged. Each clasp sequence includes at least two sequences (1802 and 1803, respectively), each complementary to a sequence of the amplicon, and when each clasp sequence hybridizes to its complement, the amplicon folds on itself as shown in FIG. 18. Additionally, the amplicon is attached to a surface via an anchoring interaction with an anchoring reagent (1804) as described herein. The clasp process can be performed in situ by adding the clasp sequences to the reaction mixture during the amplification step. Thus, once each cycle of amplification is completed, the clasp sequences are free to hybridize to the newly formed amplicon and cause it to fold. The clasps may be added after the amplification process is completed, folding the linear amplicon into a coiled or planar shape, depending on the design of the clasp sequence and its corresponding complement. For example, labeling molecules such as ECL or fluorescein can be incorporated into the clasp sequence (or may be incorporated into the amplicon as described above). This embodiment provides a three-dimensional structure on the surface, resulting in smaller features with higher signal density that allow easier imaging of the amplicons on the surface. EXAMPLES

[0256] General protocol for proximity ligation and rolling circle amplification A pair of detection antibodies against the target analyte was modified by adding proximity probes 1 and 2 as follows: 200ug of the first detection antibody in 100uL buffer was added with 1.74uL, 23mM sulfo-SMCC, diluted with 150mM phosphate buffer, and incubated for 30 minutes at room temperature. Free sulfo-SMCC was removed by size exclusion chromatography. The final concentration of detection antibodies was 2mg / mL or slightly lower. 95uL of 300uM thiol-modified oligonucleotides (proximity probes 1 and 2) were reduced with 5uL of 1mM DTT, 0.5mM EDTA, pH 8.4 in 100mM phosphate buffer for 1 hour at room temperature. The sequences of proximity probes 1 and 2 are as follows: Thiol-modified proximity probe 1: SH-AAA AAA AAA AGA CGC TAA TAG TTA AGA CGC TTU UU (SEQ ID NO:1; the three U residues are 2'O-methyl RNA). Thiol-modified proximity probe 2: SH-AAA AAA AAA ATA TGA CAG AAC TAG ACA CTC TT (SEQ ID NO: 2).

[0257] Excess Sulfo-SMCC and DTT were removed, for example, using three spin column separators, and the antibody and DNA were pooled for covalent conjugation. The solution was incubated with mixing at room temperature for 1 hour. The antibody-proximity probe conjugate was purified, for example, by size exclusion chromatography to remove unconjugated antibody and oligonucleotides.

[0258] MSD MULTI-SPOT® plates were blocked with the appropriate MSD® blocking solution for 1 hour and washed. Each binding area on the plate contains a capture antibody and an immobilization moiety (immobilized as a BSA-oligonucleotide complex, the oligonucleotides selected to be specific for the rolling circle amplicon). The sequence of the immobilization oligonucleotide used in this example was 5'-AAGAGAGTAGTACAGCAGCCGTCAAAAAAAAAAAA- / 3ThioMC3-D / -3' (SEQ ID NO: 3). 25 μl each (for a total volume of 50 ul) of assay diluent and calibrator or sample (appropriately diluted) were added to each well. The plate was incubated with shaking for 1-3 hours and each well was washed. A solution of detection antibodies labeled with proximity probes 1 and 2, prepared as described above, was added to each well (25 uL per well) and incubated with shaking for 1-2 hours (alternatively, each individual detection antibody was added sequentially with 1 hour incubation after each antibody addition). A ligation mixture containing the following components was added to each well: (i) circularization oligonucleotide 1 (4 nM), circularization oligonucleotide 2 (4 nM), ligation buffer, ATP (1 mM), and T4 DNA ligase (0.15 U / uL). Each circularization oligonucleotide was: Circularization oligonucleotide 1: phosphate-CTA TTA GCG TCC AGT GAA TGC GAG TCC GTC TAA GAG AGT AGT AGA GCA GCC GTC AAG AGT GTC TA (SEQ ID NO: 4) Circularization oligonucleotide 2: phosphate-GTT CTG TCA TAT TTA A GC GTC TTA A (SEQ ID NO: 5).

[0259] The plate was incubated with the ligation mixture for 30 minutes at room temperature, washed to remove excess circularized oligonucleotides, and incubated with the RCA mixture for 1.5 hours at 37°C. The RCA mixture contained RCA buffer, dNTPs (250 uM each), Phi29 DNA polymerase (0.125 U / ml). The plate was washed and a mixture of detection probes was added and incubated for 30 minutes at 37°C, where the detection probe mixture contained: 20 mM Tris (1 mM SDTA, 250 mM NaCl, 0.01% Triton, BSA (200 ug / ml), Tween 20 (0.05%), detection probe (6.25 nM)). The detection probe was the sequence CAG TGA ATG CGA GTC CGT CT (SEQ ID NO: 6). To incorporate the electrochemiluminescent label SULFO-TAG (Meso Scale Diagnostics), detection probes were synthesized with a terminal biotin label and pre-bound to SULFO-TAG-labeled streptavidin. Plates were washed, 150 μl of MSD read buffer was added, and read immediately on an MSD SECTOR® 6000 reader (plates and reader provided by Meso Scale Discovery, Rockville, MD).

[0260] The general procedure was used to detect the following analytes: troponin I, Akt(total), phospho-Akt(473), phospho-Akt(308), influenza A nucleoprotein (NP), IL-12p40, IL-12p70, Abeta1-42, bridging and isotyping Ig assays using a TNF alpha model system, bridging and isotyping Ig assays using Hepatitis B surface antigen, and bridging and isotyping Ig assays using Lyme C6. Increases in ECL signal and assay sensitivity over standard sandwich immunoassays varied between assays, but as much as 100-fold improvements were observed. For some assays tested, e.g., troponin I, Akt(total), IL-12p40, IL-12p70, Abeta1-42, the presence of immobilized moieties improved signal and / or dilution linearity by preventing dissociation of the detection complex during the amplification step. Standard curves for IL-10 assays according to the above procedure are shown in Figure 9. Additionally, Table 2 (below) shows the LODs for one set of representative assays derived according to the above procedure (column 2, "3-AB RCA / PLA assay") compared to the LODs for a standard two-antibody immunoassay protocol from Meso Scale Diagnostics (MSD), Rockville, MD, www.mesoscale.com (column 3, "MSD V-Plex 2-AB immunoassay protocol").

[0261] [Table 5] EXAMPLES

[0262] Assay Protocol with and without Immobilization Reagent MSD 7-spot MULTI-SPOT plates were each coated with Troponin I capture antibody (220ug / mL) as described above in Example 1. The capture antibody was spotted with or without an immobilization moiety, i.e., BSA covalently coupled to amplicon-specific oligonucleotides (5ug / mL immobilization, if present). 25μl (50ul total) of assay diluent, calibrator, or sample (appropriately diluted) was added to each well. The plate was incubated with shaking for 2 hours and each well was washed. A solution of detection antibody labeled with proximity probes 1 and 2, prepared as described above, was added to each well (25uL per well) and incubated with shaking for 1 hour. Ligation mix was added to each well as described above in Example 1. The plate was incubated with ligation mix for 30 minutes at room temperature, washed to remove excess circularized oligonucleotides, and incubated with RCA mix at 37°C for 1.5 hours as described above in Example 1. Plates were washed and a mixture of detection probes was added and incubated for 30 minutes at 37°C as described in Example 1. Plates were washed and 150 μl of MSD read buffer was added and read immediately on an MSD SECTOR® 6000 reader. For fluorescent imaging, the MSD electrodes were removed from the top of the plates and wetted with PBS and a coverslip. Surfaces were viewed under a microscope using a Zyla camera, 20x objective, 2x2 binning, and customer filter set with a 2 second exposure.

[0263] As shown in Table 3 (below), ECL values ​​were 4-5 times higher in the presence of fixed reagents, and the detection limit was one-third lower (more sensitive).

[0264] [Table 6]

[0265] Figure 10(a) and (b) show fluorescent microscopy images of the plate surface with 5ug / mL fixation reagent (panel (a)) and without fixation reagent (panel (b)). The images show bright fluorescent spots associated with individual binding events, confirming that RCA amplification in the presence of fixation reagent was more effective at generating observable binding events. EXAMPLES

[0266] Comparison of one vs. two connector oligonucleotides The assay described in Example 2 was repeated, but instead of using two connector oligonucleotides with two separate ligation sites to form a circular template, a single linear connector oligonucleotide with one ligation site was used. As shown in Figure 11(a), a single linear connector oligonucleotide was prepared that was open at ligation site 1 or ligation site 2. Both single linear connector oligonucleotides were tested in parallel with the combination of oligonucleotides Circ-1 and Circ-2 used in Examples 1 and 2. Using the protocol described in Example 2, the single linear connector oligonucleotide was further tested at three concentrations, 125 nM, 62.5 nM, and 31 nM, while the standard assay using the combination of Circ-1 and Circ-2 oligonucleotides was tested at 125 nM. As shown in Figure 11(b), the two single linear connector oligonucleotides were successfully incorporated into the RCA amplification product with approximately the same efficiency as the two connector oligonucleotide mixture (Circ-1 and Circ-2). A single linear connector oligonucleotide that opened at ligation site 1 had comparable performance to the two connector mixture based on signal intensity, non-specific background, and overall sensitivity. As expected, a single linear connector oligonucleotide that opened at ligation site 2 had higher non-specific background and lower sensitivity. In this latter case, both ligation and priming were solely dependent on the presence of proximity probe #1; therefore, some of the specific advantages of the proximity amplification approach were compromised. EXAMPLES

[0267] Three antibody assays performed using alternative proximity probes, immobilized oligonucleotides, and connector sequences The assay was carried out using the protocol outlined in Example 1 with the following alternative sets of reagents:

[0268] [Table 7]

[0269] The results in Table 5 below are for a troponin assay, where the concentration of troponin was 500 pg / mL and each well of the MULTI-SPOT plate contained one capture spot with immobilized oligonucleotide from one of the sets listed in Table 4. As described in Example 1, the assay used one of the proximity probes (1) and one of the proximity probes (2) at the same concentrations. There was more non-specific binding for sets (a)-(c), which is due to the nine-fold higher concentration of detection oligonucleotide-SA-STAG compared to that described in Example 1. The higher concentration of detection oligonucleotide-SA-STAG is due to the co-titration of pre-bound complexes, rather than titration of SA-STAG alone as in Example 1.

[0270] [Table 8] EXAMPLES

[0271] Three antibody assays performed on additional immunoassay platforms: (a) Bead-based immunoassay format using coded particles. All assay steps were performed in 96-well filter plates. Liquid was removed from the plate with a vacuum manifold (not exceeding 10 inches Hg). Plates were not inverted. If clogging occurred, use the 15 ml tip of a conical tube to gently press the area under the clogged well, then use the rubber bulb of a 1 ml Pasteur pipette or gently press the clogged well with your thumb to clear the clog. After the final aspiration step, the bottom of the plate was tapped lightly on a stack of paper towels, then the bottom of the filter plate was wiped with a Kimwipe to remove any residual liquid / droplets.

[0272] Preparation of Wash Solution: Prepare 1x Wash Solution by diluting the entire volume of the 20x Wash Solution bottle with 285ml of deionized water.

[0273] Assay Standard Preparation: Lyophilized standards were reconstituted in 100% Assay Diluent (serum and plasma samples) or 50% Assay Diluent / 50% Tissue Culture Media (tissue culture supernatant); reconstitution volumes: (i) 1 vial: 1 ml; (ii) 2 vials: 0.5 ml per vial. Rehydrate for 8-10 minutes at room temperature. Gently invert vials several times and allow to sit for an additional 3-5 minutes to ensure complete hydration. If more than one standard was used, combine equal volumes of each standard and mix gently. 7-point calibration curves were prepared by serial 3-fold dilutions of the reconstituted standards.

[0274] Analyte capture: (1) Vortex (30 sec) and sonicate (30 sec) the 10x capture bead stock. In a foil-wrapped tube, dilute the 10x capture bead stock (2.5 μl per well) into wash solution (25 μl per well, approximately 2,000-5,000 beads / assay). For higher multiplexing, adjust the volume of wash solution to accommodate the additional volume of 10x capture bead stock that was retained.

[0275] (2) Pre-wet the standard and sample wells with 200 μl wash solution.

[0276] (3) Vortex (30 sec) and sonicate (30 sec) the diluted capture bead solution. Immediately add 25 μl to each assay well, followed by 200 μL of 1x wash solution. Aspirate and repeat wash with 200 μL of wash solution. Gently tap the bottom of the filter plate if necessary.

[0277] (4) 50 μl of incubation buffer was added to all assay wells.

[0278] (5) 100 μl of standard samples were added to the designated wells. To wells designated as samples, 50 μl assay diluent was added followed by 50 μl sample. Plates were covered and incubated at room temperature for 2 hours on a shaker (500-600 rpm). During the entire incubation, the assay plate was covered with an opaque lid to protect it from light. Speed ​​may need to be adjusted depending on the radius of the shaker.

[0279] Analyte Detection (6) Preparation of 1x mixture of fluorescently labeled detection antibodies: Dilute 10x detection antibody mixture (10 μl per well) in diluent (100 μl per well). The mixture contains a pair of detection antibodies specific for the analyte of interest, one labeled with Alexa Fluor 350 (blue fluorescent label) and the other labeled with Alexa Fluor 594 (red fluorescent label) (each of these fluorescent labels is available from Life Technologies, Grand Island, NY, www.lifetechnologies.com). For higher multiplexing, adjust the volume of diluent to prepare additional volumes of 10x antibody mixture required. Aspirate and wash assay wells twice with 200 μL of wash solution. Add 100 μl of diluted detection antibody mixture to each assay well. Cover plate and incubate for 1 hour on a plate shaker (500-600 rpm).

[0280] Assay readout (8) Aspirate and wash the assay wells three times with 200 μL of wash solution. Dry the bottom of the filter plate with a clean paper towel to completely remove any residual droplets. Add 100 μl wash solution to each assay well and place the plate on a plate shaker (500-600 rpm) for 2-3 minutes.

[0281] (9) The bead suspension is analyzed with a multicolor fluorescent particle analyzer (such as a FACS system or modified xMAP instrument) that contains color channels for each individual fluorescent label. To maximize sensitivity, the assay is run under the condition that any given particle may bind to one analyte or none at all, and the amount of analyte is specific for a given analyte (based on the particle's coding) and quantified by counting the number of particles that contain both fluorescent labels. Optionally, the assay can be run in a multiplex format using coded beads, where the code indicates that a capture antibody on the bead and an additional detection antibody pair for each analyte are specific for the analyte. If coding is determined using the addition of fluorescent colors incorporated into the beads, such as in the xMAP, the analyzer must have additional detection channels corresponding to the measurement of the added colors and the identification of the bead code.

[0282] (b) A bead-based immunoassay format using encoded particles containing an immobilization moiety and two detection reagents modified with nucleic acid probes. All assay steps were performed in 96-well filter plates as outlined in Example 5(a). Wash solutions and assay standards were prepared as described in Example 5(a) and a pair of detection antibodies against the target analytes was modified with the addition of proximity probes 1 and 2 as described in Example 1. The analytes were captured on capture beads as described in Example 5(a). The capture beads contain an immobilization moiety that is immobilized on the bead surface as a BSA-oligonucleotide complex, with the oligonucleotide selected to be specific for the rolling circle amplicon. The sequence of the immobilization oligonucleotide used is SEQ ID NO:3.

[0283] 25 μl of assay diluent, calibrator, or sample (appropriately diluted) was mixed with the mixture of capture beads. The mixture was incubated with shaking for 1-3 hours and washed. A solution of detection antibodies labeled with proximity probes 1 and 2, prepared as described above, was added to the mixture and incubated with shaking for 1-2 hours (alternatively, each individual detection antibody was added sequentially with 1 hour incubation after each addition). The ligation mixture described in Example 1 was added. This mixture was incubated with the ligation mixture for 30 minutes at 37°C, washed to remove excess circularized oligonucleotide, and incubated with the RCA mixture described above in Example 1 for 1.5 hours at 37°C. The mixture was washed and the mixture of fluorescein-labeled detection probes described above was added and incubated for 30 minutes at 37°C. The mixture was washed and the particles were aspirated into a multichannel fluorescent particle analyzer.

[0284] (c) Bead-based format and separation of captured analyte molecules into individual nanowells Samples were prepared in 100ul of 25% bovine serum (2-4x dilution) and 500K beads (paramagnetic 2.7μm, sometimes fluorescently coded) coated with capture antibody were added to the sample. Samples were incubated at 23°C for approximately 2 hours. Samples were washed 3 times with PBS (5X, 0.1% Tween-20) and a mixture of labeled detection antibodies was added (a mixture containing a first biotinylated detection antibody and a hapten-conjugated antibody). The mixture was incubated at 23°C for approximately 1 hour. The mixture was washed 3 times with PBS (5X, 0.1% Tween20) and enzyme labels were added, streptavidin-β-galactosidase (40pM), anti-hapten conjugated enzyme was also added and the mixture was incubated at 23°C for approximately 30 minutes (or run on a Simoa analyzer for 3 minutes). The mixture was washed seven times with PBS (5X, 0.1% Tween 20) and 15 ul of the enzyme substrate, resorufin-beta-d-galactopyranoside (100 uM in loading buffer) was added.

[0285] The mixture was drawn onto an array of nanowells (provided by Quanterix in DVD format, made from cyclic olefin polymer, 24 samples per disk) and allowed to sit for approximately 2 min. The array was rinsed with buffer, the array was sealed with fluorocarbon oil, incubated at 23 °C for 2-5 min, and the results were read on a multicolor fluorescent imager. Image analysis was used to count the number of nanowells encapsulating both fluorescent enzyme products, thereby obtaining a value related to the concentration of the analyte in the sample.

[0286] (d) Bead-based immunoassay format with flow cell analysis First incubation: 10 ul of sample, a biotinylated monoclonal analyte-specific capture antibody (working solution 2.6 mg / l), and a mixture of monoclonal analyte-specific antibodies each conjugated to an oligonucleotide (working solution 0.3 mg / l) were reacted to form a sandwich complex. The mixture of monoclonal analyte-specific antibodies was prepared as described in Example 1 and includes a pair of antibodies conjugated to proximity probes 1 and 2 as described above in Example 1.

[0287] Second incubation: After the addition of streptavidin-coated microparticles (Dynal M280, 2.8um, 0.72mg / ml, biotin binding capacity 470ng / mg), the complex was bound to the solid phase by the interaction between biotin and streptavidin. The ligation mixture prepared by the protocol described in Example 1 was added to this mixture. The mixture was incubated with the ligation mixture for 30 minutes at 37°C, washed to remove excess circularized oligonucleotides, and incubated with the RCA mixture described in Example 1. The mixture was washed, and a mixture of biotin-labeled detection probes was added and incubated for 30 minutes at 37°C. The detection probe mixture was prepared as described in Example 1. To incorporate electrochemiluminescent label SULFO-TAG (Meso Scale Diagnostics), the d...

Claims

1. 1. 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 the 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 comprising binding reagent, the analyte, and the first and second detection reagents; b. extending the second probe using an extension process that requires the first and second probes to be brought into proximity to form an extended sequence that includes an anchor sequence complement that is complementary to the anchor sequence; c. hybridizing the anchor sequence to the complement of the anchor sequence; and d. determining the amount of extended sequence bound to the surface; The method of any one of the preceding claims, wherein the analyte is G-CDF, GM-CSF, IFN gamma, IL-1β, 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, TNFα, TSLP, VEGF, complexed PSA, free PSA, Aβ42, Aβ40, Aβ38, tau, cardiac troponin I, cardiac troponin T, HIV p24, C-peptide, and / or FGF21.

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

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

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

7. 2. 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. 2. The method of claim 1, wherein the extending step comprises binding a probe to a template nucleic acid sequence, forming 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 localized on the surface after probe extension.

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

11. The method of claim 10, wherein the probe to be extended is bound to the anchor reagent at a position less than 100 um from the location of the complex on the surface.

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

13. The method of claim 12 , wherein the extension step comprises an isothermal amplification method.

14. The method of claim 13, wherein the isothermal amplification method is helicase-dependent amplification or rolling circle amplification (RCA).

15. 2. The method of claim 1, wherein the extension process comprises contacting the complex formed in step (a) with a connector sequence comprising (i) an internal sequence complementary to the second probe and (ii) two terminal sequences complementary to the non-overlapping regions of the first probe.

16. 16. The method of claim 15, further comprising ligating the two end sequences of the connector oligonucleotide to form a circular target sequence that hybridizes to both the first and second probes.

17. 2. The method of claim 1, wherein the extension process comprises contacting the complex formed in step (a) with a first connector oligonucleotide sequence comprising a first connector probe sequence complementary to a first region of the first probe and a first region in the second probe, and a second connector oligonucleotide comprising 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.

18. 18. The method of claim 17, further comprising ligating the first and second connector oligonucleotides to form a circular target sequence that hybridizes to both the first and second probes.

19. The method of claim 1 , wherein the surface comprises particles.

20. The method of claim 1 , wherein the surface comprises a well of a multi-well plate.

21. The method of claim 1 , wherein the surface comprises a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains on the surface.

22. 21. The method of claim 20, wherein the well comprises a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains within the well.

23. The method of claim 1 , wherein the surface comprises a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain on the surface.

24. 21. The method of claim 20, wherein the well comprises a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain within the well.

25. The method of claim 1 , wherein the capture reagent and the anchor reagent are less than 10 nm on the surface.

26. 10. The method of claim 1, wherein the surface comprises an electrode, and the measuring step further comprises applying a potential waveform to the electrode to generate an electrochemiluminescence signal.

27. 20. The method of claim 19, further comprising collecting the particles on an electrode and applying a potential waveform to the electrode to generate an electrochemiluminescence signal.

28. 2. The method of claim 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 that is complementary to a region of the extension sequence.

29. 29. The method of claim 28, wherein the detectable label is measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, a magnetic field, or a combination thereof.

30. 30. The method of claim 29, wherein the detectable label is an ECL label and the measuring step comprises measuring an ECL signal.

31. In one or more vials, containers, or compartments, a. a surface comprising (i) a capture reagent for an 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 kit for the detection of an analyte of interest in a sample comprising a second detection reagent for said analyte linked to a second nucleic acid probe, The above kits, wherein the analyte is G-CDF, GM-CSF, IFN gamma, IL-1 beta, 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, TNF alpha, TSLP, VEGF, complexed PSA, free PSA, Abeta 42, Abeta 40, Abeta 38, tau, cardiac troponin I, cardiac troponin T, HIV p24, C-peptide, and / or FGF21.

32. 32. The kit of claim 31, wherein the capture reagent comprises an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

33. 32. The kit of claim 31, wherein the first detection reagent comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer.

34. 32. The kit of claim 31, wherein the second detection reagent comprises an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

35. 32. The kit of claim 31, wherein the surface comprises a particle.

36. 32. The kit of claim 31, wherein the surface comprises a well of a multi-well plate.

37. 32. The kit of claim 31, wherein the surface comprises a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains on the surface.

38. 37. The kit of claim 36, wherein the well comprises a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at two distinct binding domains within the well.

39. 32. The kit of claim 31, wherein the surface comprises a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain on the surface.

40. 37. The kit of claim 36, wherein the well comprises a plurality of distinct binding domains, and the capture reagent and the anchor reagent are located at the same binding domain within the well.

41. The kit of claim 31 , wherein the capture reagent and the anchor reagent are within 10-100 nm on the surface.

42. 32. The kit of claim 31 , wherein the surface comprises an electrode.

43. 10. The method of claim 1, wherein the analyte is a healthcare associated infection selected from Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and extraintestinal pathogenic Escherichia coli.

44. 32. The kit of claim 31, wherein the marker is healthcare associated infection selected from Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and extraintestinal pathogenic Escherichia coli.

45. 2. The method of claim 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.

46. 2. The method of claim 1, wherein the analytes are elongation factor EF-TU, ACP, acyl carrier protein, RplL, ribosomal protein GroS (MopB, 65,000), components of the chaperone system Gro-EL-Gro-ES and GapA, enzymes of the glycolytic pathway, and combinations thereof.

47. The kit of claim 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.

48. 32. The kit of claim 31, wherein the analytes are elongation factor EF-TU, ACP, acyl carrier protein, RplL, ribosomal protein GroS (MopB, 65,000), components of the chaperone system Gro-EL-Gro-ES and GapA, enzymes of the glycolytic pathway, and combinations thereof.

49. 1. 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 linked to a first nucleic acid probe; b. binding the analyte complex formed in step (a) to: (i) a capture reagent on a 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 the first and second probes to be brought into proximity to form an extended sequence that includes an anchor sequence complement that is complementary to the anchor sequence; d. hybridizing the anchor sequence to the complement of the anchor sequence; and e. determining the amount of extended sequence bound to said surface.

50. The concentration step (a) comprises (i) contacting a sample containing the analyte with a solid phase linked to a targeting agent that is complementary to at least a portion of the first nucleic acid probe, thereby forming an enriched complex containing the analyte bound to the solid phase via a binding reaction between the first nucleic acid probe and the targeting agent; (ii) collecting the enriched complex; (iii) separating unbound components of the sample from the enriched complex; and 50. The method of claim 49, further comprising (iv) releasing the concentrated complex to separate the solid phase from the analyte to form an analyte complex.

51. 1. A kit for the detection of 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; c. a second detection reagent for the analyte linked to a second nucleic acid probe; and d. The kit further comprising a solid phase comprising a targeting agent complementary to at least a portion of said first nucleic acid probe.

52. 52. The kit of claim 51, wherein the solid phase is a particle.

53. 52. The kit of claim 51, wherein the capture reagent comprises an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

54. 52. The kit of claim 51, wherein the first detection reagent comprises an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

55. 52. The kit of claim 51, wherein the second detection reagent comprises an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

56. 52. The kit of claim 51, wherein the surface comprises a well of a multi-well plate.

57. 57. The kit of claim 56, wherein the surface comprises a plurality of separate binding domains and a capture reagent, and the anchor reagent is located on two separate binding domains on the surface.

58. 57. The kit of claim 56, wherein the well comprises a plurality of separate binding domains and a capture reagent, and the anchor reagent is located over two separate binding domains within the well.

59. 57. The kit of claim 56, wherein the surface comprises a plurality of separate binding domains and the capture reagent, and the anchor reagent is located on the same binding domain on the surface.

60. 60. The kit of claim 59, wherein the well comprises a plurality of separate binding domains and the capture reagent, and the anchor reagent is located over the same binding domain within the well.

61. 52. The kit of claim 51, wherein the capture reagent and the anchor reagent are within 10-100 nm on the surface.

62. 52. The kit of claim 51, wherein the surface comprises an electrode.

63. 1. A method for detecting an analyte of interest in a sample, comprising: a. binding the analyte to: (i) a capture reagent on a particle comprising the 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 comprising the binding reagent, the analyte, and the first and second detection reagents; b. extending the second probe to form an extension sequence that includes an anchor sequence complement that is complementary to the anchor sequence using an extension process that requires the first and second probes to be brought into proximity, wherein the extension process is performed during or after the particles are allowed to settle by gravity onto the surface of a well of a multiwell plate; c. hybridizing the anchor sequence to the anchor sequence complement; and d. determining the amount of extension sequence bound to the particle.

64. 64. The method of claim 63, wherein the capture reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

65. 65. The method of claim 64, wherein the first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

66. 66. The method of claim 65, wherein the first detection reagent is an antibody.

67. 64. The method of claim 63, wherein the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

68. 64. The method of claim 63, wherein the capture reagent and the first and second detection reagents are antibodies to the analyte.

69. 64. The method of claim 63, wherein the anchor oligonucleotide sequence comprises a single-stranded oligonucleotide sequence.

70. 64. The method of claim 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.

71. 64. The method of claim 63, wherein the extension step comprises PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (sustained synthesis reaction), or isothermal amplification.

72. 72. The method of claim 71, wherein the extension step comprises an isothermal amplification method.

73. 73. The method of claim 72, wherein the isothermal amplification method is helicase-dependent amplification or rolling circle amplification (RCA).

74. 1. A method for detecting an analyte of interest in a sample, comprising: a. binding the analyte to: (i) a capture reagent on a particle comprising the 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 comprising binding reagent, the analyte, and the first and second detection reagents; b. extending the second probe to form an extension sequence that includes an anchor sequence complement that is complementary to the anchor sequence using an extension process that requires the first and second probes to be brought into proximity, wherein the extension process is performed during or after the particles are allowed to settle by gravity onto the surface of a well of a multiwell plate; c. Measuring the amount of extension sequence bound to the particle. The method comprising:

75. 75. The method of claim 74, wherein the capture reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

76. 75. The method of claim 74, wherein the first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

77. 75. The method of claim 74, wherein the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

78. 75. The method of claim 74, wherein the capture reagent and the first and second detection reagents are antibodies to the analyte.

79. 75. The method of claim 74, wherein the extension sequence further comprises one or more detection sequences, and the measuring step further comprises contacting the extension sequence with the one or more detection sequences and a complementary plurality of labeled probes.

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

81. 81. The method of claim 80, wherein the extending step comprises an isothermal amplification method.

82. 82. The method of claim 81, wherein the isothermal amplification method is helicase-dependent amplification or rolling circle amplification (RCA).

83. 1. A method for detecting exosomes in a sample, comprising: a. binding the exosomes to (i) a capture reagent on a surface comprising the 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 comprising the binding reagent, the exosomes, and the first and second detection reagents; b. extending the second probe using an extension process that requires the first and second probes to be brought into proximity to form an extended sequence that includes an anchor sequence complement that is complementary to the anchor sequence; c. hybridizing the anchor sequence to the complement of the anchor sequence; and d. determining the amount of extended sequence bound to the surface.

84. 84. The method of claim 83, wherein the capture reagent comprises an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

85. 84. The method of claim 83, wherein the first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

86. 84. The method of claim 83, wherein the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

87. 84. The method of claim 83, wherein the capture reagent and the first and second detection reagents are antibodies to a target molecule within or on the surface of the exosome.

88. 88. The method of claim 87, wherein the capture reagent binds to a common exosome target protein selected from CD9, CD63, CD81, Hsp70, PDCD6IP or Tsg101.

89. 88. The method of claim 87, wherein at least one of the first or second detection reagents is an antibody against a disease-specific target molecule within or on the surface of the exosome.

90. 84. The method of claim 83, wherein the first and second detection reagents each bind to an interacting exosome target molecule.

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

92. 84. The method of claim 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.

93. 84. The method of claim 83, wherein the extending step comprises binding a probe to a template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification.

94. 84. The method of claim 83, wherein the extension probe remains located on the surface after extension of the probe.

95. 95. The method of claim 94, wherein the complex remains bound to the surface after the elongation step.

96. 95. The method of claim 94, wherein the extender probe is attached to an anchor reagent that is located within 10-100 um of the location of the complex on the surface.

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

98. 98. The method of claim 97, wherein the extension step comprises an isothermal amplification method.

99. 99. The method of claim 98, wherein the isothermal amplification method is helicase-dependent amplification or rolling circle amplification (RCA).

100. 84. The method of claim 83, wherein the extension process comprises contacting the complex formed in step (a) with a connector sequence comprising (i) an internal sequence complementary to the second probe and (ii) two terminal sequences complementary to the non-overlapping regions of the first probe.

101. 101. The method of claim 100, comprising ligating two end sequences of a connector oligonucleotide to form a circular target sequence hybridized to both the first and second probes.

102. 84. The method of claim 83, wherein the extension process comprises contacting the complex formed in step (a) with a first connector oligonucleotide sequence comprising a first connector probe sequence complementary to a first region of the first probe and a first region of the second probe, and a second connector oligonucleotide comprising 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.

103. 103. The method of claim 102, further comprising ligating the first and second connector oligonucleotides to form a circular target sequence that hybridizes to both the first and second probes.

104. 84. The method of claim 83, wherein the sample comprises purified exosomes.

105. 84. The method of claim 83, wherein the measuring step further comprises imaging the amplicons present on the surface.

106. The method of claim 105, wherein the amplicons are labeled with a plurality of fluorescent probes, and the subsequent measuring step comprises fluorescently staining the exosomes, imaging the fluorescently labeled amplicons and the stained exosomes, and comparing the results of the imaging steps relative to each other.

107. 1. A kit for detection of exosomes of interest in a sample, comprising in one or more vials, containers, or compartments: a. a surface comprising (i) a capture reagent for said exosome, and (ii) an anchor reagent comprising an anchor oligonucleotide sequence; b. a first detection reagent for the exosome linked to a first nucleic acid probe; and c. The kit further comprises a second detection reagent for the exosome linked to a second nucleic acid probe.

108. 108. The kit of claim 107, wherein the capture reagent comprises an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

109. 108. The kit of claim 107, wherein the first detection reagent comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimitope, or an aptamer.

110. 108. The kit of claim 107, wherein the second detection reagent comprises an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.

111. 108. The kit of claim 107, wherein the surface comprises a particle.

112. 108. The kit of claim 107, wherein the surface comprises a well of a multi-well plate.

113. 108. The kit of claim 107, wherein the surface comprises a plurality of separate binding domains, and the capture reagent and the anchor reagent are located on two separate binding domains on the surface.

114. 113. The kit of claim 112, wherein the well comprises a plurality of separate binding domains, and the capture reagent and the anchor reagent are located on two separate binding domains within the well.

115. 114. The kit of claim 113, wherein the surface comprises a plurality of separate binding domains, and the capture reagent and the anchor reagent are located on the same binding domain on the surface.

116. 113. The kit of claim 112, wherein the well comprises a plurality of separate binding domains, and the capture reagent and the anchor reagent are located on the same binding domain within the well.

117. The kit of claim 107, wherein the capture reagent and the anchor reagent are within 10-100 nm on the surface.

118. 108. The kit of claim 107, wherein the surface comprises an electrode.

119. The kit of claim 107, wherein the capture reagent binds a common exosome target protein selected from CD9, CD63, CD81, Hsp70, PDCD6IP or Tsg101.

120. The kit of claim 107, wherein the first and second detection reagents each bind to an interacting exosome target protein.

121. The kit of claim 102, wherein the interacting target proteins include ligand-receptor pairs and / or mRNA molecules and RNA binding proteins.

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