Electrochemiluminescent labeled probe for use in immunoassay, method using such probe, and kit comprising such probe

Abstract

To provide a non-naturally occurring nucleic acid probe for use in an immunoassay, to provide a conjugate compound comprising a detection reagent conjugated to the non-naturally occurring nucleic acid probe, to provide a kit for forming the conjugate, to provide a method for forming the conjugate, and to provide an anchor reagent.SOLUTION: Provided are a non-naturally occurring nucleic acid probe comprising an oligonucleotide, the oligonucleotide being 14 to 24 nucleotides in length and comprising 14 or 15 contiguous nucleotides of 5'-GACAGAACTAGACAC-3'; and a conjugated compound comprising a detection reagent conjugated to the non-naturally occurring nucleic acid probe, optionally, the detection reagent being an antigen-binding substance.SELECTED DRAWING: Figure 1(a)

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy created on February 26, 2020 is designated 0076-0010WO1_SL.txt and is 12,578 bytes in size.

[0002] The present invention relates to a method for performing immunoassays, which method is designed to amplify the signal of an immunoassay and anchor the immunoassay conjugates used therein. [Background technology]

[0003] A significant body of literature has been developed regarding 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 samples. The high degree of specificity in many biochemical binding systems has led to numerous assay methods and systems of value 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 analyte's participation in the binding reaction. In some approaches, this participation may be indicated by measuring an observable label attached to one or more binding materials.

[0004] While sandwich immunoassay formats offer excellent sensitivity and specificity in many applications, some analytes exist at concentrations that are too low for detection by conventional immunoassay techniques. Sandwich immunoassay performance can also be limited by nonspecific binding of the detection antibody and the instability of sandwich complexes containing high-off-rate antibodies. 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 significantly impact the sensitivity and specificity of the assay. For example, in complex assays requiring multiple binding events and / or reactions, if any one event or reaction is suboptimal, the sensitivity and specificity of the entire assay may be poor. Summary of the Invention

[0005] The present invention contemplates the following specific embodiments. Various modifications, additions, and variations 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 variations are intended to be included within the scope of the following claims.

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

[0007] In embodiment (1), the capture reagent can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer. In certain embodiments, the capture reagent is an antibody. The detection reagent can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in certain embodiments, the detection reagent is an antibody. In one specific example of embodiment (1), the capture reagent and the detection reagent are antibodies against the analyte. The anchor reagent can comprise an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, antigen, ligand, receptor, hapten, epitope, or mimotope, and optionally, the anchor region can comprise an aptamer, and the anchor reagent can comprise an aptamer ligand. The anchor region can comprise a nucleic acid sequence, and the anchor reagent can comprise a DNA-binding protein. The anchor region can comprise an oligonucleotide sequence, and the anchor reagent can 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 include forming a triple helix between the anchor region and the anchor reagent. The method may also include 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 include one or more hapten-modified bases, the anchor reagent may include one or more antibodies specific for the hapten, and / or the anchor region may include one or more ligand-modified bases, and the anchor reagent may include one or more receptors specific for the ligand. 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 mimotope, and the plurality of detectable moieties comprise binding partners for the one or more modified bases and detectable labels, respectively. The one or more modified bases may comprise streptavidin, and the plurality of detectable moieties each comprise biotin and a detectable label, and / or the one or more modified bases may comprise biotin, and the plurality of detectable moieties each comprise streptavidin and a detectable label, and / or the one or more modified bases may comprise avidin, and the plurality of detectable moieties each comprise biotin and a detectable label, and / or the one or more modified bases may comprise biotin, and the plurality of detectable moieties each comprise avidin and a detectable label.

[0009] The first step of embodiment (1) may involve 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, the first step of embodiment (1) may involve 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 involve 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.

[0010] The extension step of embodiment (1) can include binding a probe to a circular nucleic acid and extending the circular template by rolling circle amplification. 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 (e.g., by linking a linear template to form a circle), and extending the circular template by rolling circle amplification. In these embodiments, the extension probe can remain localized on the surface after probe extension. In such embodiments, the probe can be an optimized probe having a probe sequence of 14 to 24 nucleotides in length, and / or the template can be an optimized template having a template sequence of 53 to 76 nucleotides in length. Thus, the complex can remain bound to the surface after the extension step; for example, the extension probe can bind to the anchor reagent within 10 to 100 μm of the location of the complex on the surface. In one particular embodiment, the extender probe binds to the anchor reagent less than 100 μm, less than 50 μm, or more particularly less than 10 μm from the location of the complex on the surface.

[0011] The extension step of embodiment (1) may include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (sustained synthesis reaction), or an isothermal amplification method. In one embodiment, the extension step may 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 multiwell plate. The surface may include multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. When the surface is a well of a plate, the well may include multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well, and / or the surface may include multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain on the surface. In one embodiment, the well may include multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain within the well. The capture reagent and anchor reagent may be within 10 to 100 nm of each other on the surface. The surface may include an electrode, and the measuring step may further include applying a voltage waveform to the electrode to generate an electrochemiluminescence signal. Optionally, the method includes collecting particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescence signal.

[0013] The measuring step of embodiment (1) may 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, wherein the detection probe comprises a nucleic acid sequence 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 specific example of embodiment (1), the detectable label is an ECL label, and the measuring step may include measuring an ECL signal. The detection probe may have multiple ECL labels. The detection probe may be bound to multiple ECL label moieties via a bond at the 3' end of a probe nucleotide component.

[0014] Embodiment (2): A kit for detecting an analyte of interest in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an anchoring reagent; and (b) a detection reagent for the analyte linked to a nucleic acid probe.

[0015] The anchor reagent of embodiment (2) may comprise an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimotope, and the capture reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer. In certain embodiments, the capture reagent may comprise an antibody, and / or the detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, 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 particles and / or wells of a multiwell plate. The surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. When the surface of the kit is a well of a plate, the surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well, and / or the surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain on the surface. In a specific example of the kit, the surface is a well, with the well comprising multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain within the well. The capture reagent and anchor reagent may be within 10 to 100 nm of each other 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 capture reagent on 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, analyte, and detection reagent; (b) extending the probe to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence; (c) hybridizing the anchor sequence to the anchor sequence complement; and (d) measuring the amount of extended sequence bound to the surface.

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

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

[0020] The extension step of embodiment (3) 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 probe to a template circular nucleic acid and extending the circular template by rolling circle amplification. Alternatively, the extension step may include binding the probe to a template nucleic acid sequence, forming a circular nucleic acid template (e.g., by ligation), and extending the circular template by rolling circle amplification. In such embodiments, the probe may be an optimized probe having a probe sequence of 14 to 24 nucleotides in length, and / or the template may be an optimized template having a template sequence of 53 to 76 nucleotides in length. The extension probe may remain localized on the surface after probe extension, e.g., the complex remains bound to the surface after the extension step. In one example, the extension probe binds to the anchor reagent within 10 to 100 μm of the location of the complex on the surface. In a specific embodiment, the extension probe binds to the anchor reagent at a position less than 100 μm, less than 50 μm, or more specifically less than 10 μm from the location of the complex on the surface.In this specific embodiment, the extension step can include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (sustained synthesis reaction), or isothermal amplification.For example, the extension step can include isothermal amplification, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0021] The surface of embodiment (3) may comprise particles and / or wells of a multiwell plate. The surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. When the surface is a well of a plate, it may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well. The surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain on the surface. When the surface is a well, it may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain within the well. The capture reagent and anchor reagent may be within 10 to 100 nm of each other on the surface. In certain examples, the surface may comprise an electrode, and the measuring step may further comprise applying a voltage waveform to the electrode to generate an electrochemiluminescence signal. The method may include collecting particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescence signal. The measuring step may 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 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 may be an ECL label, and the measuring step may include measuring an ECL signal. The detection probe may have multiple ECL labels. The detection probe may be bound to multiple ECL label moieties via a bond at the 3' end of the probe nucleotide component.

[0022] Embodiment (4): A kit for detecting an analyte of interest in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an 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 comprising an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer. In a particular example, the capture reagent may comprise an antibody. Similarly, the detection reagent may comprise an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in particular, the detection reagent may comprise an antibody.

[0024] The kit of embodiment (4) includes a surface, which may include particles and / or wells of a multiwell plate. The surface may include multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. When the surface is a well, the well may include multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well. The surface may include multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain on the surface; for example, when the surface is a well, the well may include multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain within the well. For example, the capture reagent and anchor reagent are within 10 to 100 nm of each other on the surface. The surface of embodiment (4) may 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 an anchor reagent comprising a capture reagent for the analyte and 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 a first and second detection reagent; (b) extending the second probe using an extension process requiring proximity of the first and second probes 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) can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer. In a specific example, the capture reagent is an antibody. Similarly, the first detection reagent can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in a specific example, the first detection reagent is an antibody. The second detection reagent can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in a specific example, the second detection reagent is an antibody. More specifically, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0027] In embodiment (5), the anchor oligonucleotide sequence may comprise a single-stranded oligonucleotide sequence or a double-stranded oligonucleotide sequence. In this embodiment, 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. The extension sequence may also 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. The extension sequence 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, aptamer ligand, antibody, antigen, ligand, receptor, hapten, epitope, or mimotope, and the plurality of detectable moieties may each comprise a binding partner of the one or more modified bases and a detectable label. For example, one or more modified bases comprise streptavidin and the plurality of detectable moieties each comprise biotin and a detectable label; one or more modified bases comprise biotin and the plurality of detectable moieties each comprise streptavidin and a detectable label; one or more modified bases comprise avidin and the plurality of detectable moieties each comprise biotin and a detectable label; and / or one or more modified bases comprise biotin and the plurality of detectable moieties each comprise avidin and a detectable label.

[0028] Step (a) of embodiment (5) 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. Alternatively, step (a) may comprise binding the analyte to the following species in the following order: (i) a detection reagent for the analyte, and (ii) a capture reagent on the surface. Or, step (a) may 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) may include binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step may further include binding the probe to the template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extension probe may remain localized on the surface after probe extension; for example, the complex remains bound to the surface after the extension step. The extension probe may bind to the anchor reagent within 10 to 100 μm of the location of the complex on the surface. In a specific embodiment, the extension probe binds to the anchor reagent less than 100 μm, less than 50 μm, or more specifically less than 10 μm from the location of the complex on the surface. The extension step may include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (sustained synthesis reaction), or isothermal amplification. In certain instances, the extension step may involve 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 comprise 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 on 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) may comprise particles and / or wells of a multiwell plate. The surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. When the surface is a well of a plate, the well may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well. The surface may also comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain on the surface. When the surface is a well of a plate, the well may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain within the well. The capture reagent and anchor reagent may be within 10 to 100 nm of each other on the surface. In certain examples, the surface may comprise an electrode, and the measuring step may further comprise applying a voltage waveform to the electrode to generate an electrochemiluminescence signal. Optionally, the method of embodiment (5) further comprises collecting the particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescence signal.

[0032] The measuring step of embodiment (5) may 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, wherein the detection probe comprises a nucleic acid sequence 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 specific example, the detectable label is an ECL label, and the measuring step may include measuring an ECL signal. The detection probe may have multiple ECL labels. The detection probe may be bound to multiple ECL label moieties via a bond at the 3' end of a probe nucleotide component.

[0033] Embodiment (6): A kit for detecting an analyte of interest in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an anchor reagent comprising an anchor oligonucleotide sequence; (b) a first detection reagent for the analyte linked to a first nucleic acid probe; and (c) a second detection reagent for the analyte linked to a second nucleic acid probe.

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

[0035] The surface of embodiment (6) may comprise a particle and / or a well of a multiwell plate. The surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. If the surface is a well, the well may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well. The surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain on the surface, and / or if the surface is a well, the well may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain within the well. The capture reagent and anchor reagent may be within 10-100 nm of each other on the surface. In certain examples, 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 a first and second detection reagent; and (b) binding the detection complex formed in (c) to a surface 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 first and second proximity probes; (d) ligating the two end sequences of the 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 binding domain that binds to an anchor reagent; (f) binding the amplicon to the anchor reagent; and (g) 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 first and second detection reagents; and (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 first detection reagent for the analyte comprising a second proximity probe. (c) contacting two proximity probes, each of which has a second end of the connector and a second end of the second connector, the second end of which is 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 produce an amplicon comprising a binding domain that binds to an 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 embodiments (7) and (8) can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in a specific example, the capture reagent is an antibody. Similarly, the first detection reagent can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and for example, the first detection reagent is an antibody. Furthermore, the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and for example, the second detection reagent is an antibody. In a specific example of embodiments (7) and (8), the capture reagent and the first and second detection reagents are antibodies against the analyte.

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

[0040] The amplicon of embodiments (7) and (8) may further include one or more detection sequences, and the measuring step may further include contacting the extended sequence with a plurality of labeled probes complementary to the one or more detection sequences. Furthermore, the amplicon may further include one or more modified bases, and the measuring step may further include contacting the extended sequence with a plurality of detectable moieties capable of binding to the one or more modified bases. Furthermore, the amplicon may further include one or more labeled bases, and the measuring step may further include detecting the presence of the one or more labeled bases. The one or more modified bases may comprise an aptamer, aptamer ligand, antibody, antigen, ligand, receptor, hapten, epitope, or mimotope, and the plurality of detectable moieties may each comprise a binding partner of the one or more modified bases and a detectable label. The one or more modified bases may comprise streptavidin, and the plurality of detectable moieties each comprise biotin and a detectable label; the one or more modified bases may comprise biotin, and the plurality of detectable moieties each comprise streptavidin and a detectable label; the one or more modified bases may comprise avidin, and the plurality of detectable moieties each comprise biotin and a detectable label; and / or the one or more modified bases may comprise biotin, and the plurality of detectable moieties each comprise avidin and a detectable label.

[0041] Step (a) of embodiments (7) and (8) may comprise 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) may comprise 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) may comprise 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 anchor reagent within 10-100 μm of the location of the complex on the surface. In a specific embodiment, the extension probe binds to the anchor reagent less than 100 μm, less than 50 μm, or more specifically less than 10 μm from the location of the complex on the surface.

[0043] The surface of embodiments (7) and (8) may comprise a particle and / or a well of a multiwell plate. The surface may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. When the surface is a well of a plate, the well may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well. When the surface is a well of a plate, the well may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain on the surface. When the surface is a well of a plate, the well may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain within the well. In a specific example, the capture reagent and anchor reagent are within 10 to 100 nm of each other on the surface.

[0044] Furthermore, the surface may include an electrode, and the measuring step may include applying a voltage waveform to the electrode to generate an electrochemiluminescence signal. In these embodiments ((7) and (8)), the method may include collecting particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescence signal. The measuring step may 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 complementary to a region of the amplicon. The detectable label is measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, a magnetic field, or a combination thereof. For example, the detectable label may be an ECL label, and the measuring step may include measuring an ECL signal. The detection probe may have multiple ECL labels. The detection probe may be bound to multiple ECL label moieties via linkage at the 3' end of the probe nucleotide component.

[0045] Embodiment (9): A kit for detecting an analyte of interest in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an 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 non-overlapping regions of the first proximity probe. In an embodiment, the first and second proximity probes are nucleic acid probes.

[0046] Embodiment (10): A kit for detecting an analyte of interest in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte and (ii) an 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. In an embodiment, the first and second proximity probes are nucleic acid probes.

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

[0048] The surface of embodiments (9) and (10) may comprise a particle and / or a well of a multiwell plate. The surface may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. When the surface is a well of a plate, the well may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well. When the surface is a well, the well may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on 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 anchor reagent disposed on the same binding domain within the well. In a specific example, the capture reagent and anchor reagent are within 10 to 100 nm of each other on the surface.

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

[0050] Embodiment (11): 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 the 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 first and second detection reagents; and (b) binding the detection complex formed in (c) to a surface 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) an anchor sequence. and a sequence matching the target sequence of the first and second proximity probes, (c) hybridizing the connector sequence to first and second proximity probes, (d) ligating the two end sequences of the 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 complementary to the anchor sequence, (f) hybridizing the anchor sequence to one of the anchor sequence complements, and (g) measuring the amount of amplicon on the surface. In an embodiment, the first and second proximity probes are nucleic acid probes.

[0051] Embodiment (12): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to (i) a capture reagent for the analyte on a surface comprising the 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 first and second detection reagents; and (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, and (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. (iii) the first and / or second connector oligonucleotides also contain sequences matching the anchor sequences; (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 multiple anchor sequence complements complementary to the anchor sequences; (f) hybridizing the anchor sequence to one of the anchor sequence complements; and (g) measuring the amount of amplicon on the surface. In an embodiment, the first and second proximity probes are nucleic acid probes.

[0052] The capture reagent of embodiments (11) and (12) is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer. In a specific example, the capture reagent is an antibody. The first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in a specific example, the first detection reagent is an antibody. The second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in a specific 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 include one or more detection sequences, and the measuring step may include contacting the extended sequence with a plurality of labeled probes complementary to the one or more detection sequences. Furthermore, the amplicon may also include one or more modified bases, and the measuring step may include contacting the extended sequence with a plurality of detectable moieties capable of binding to the one or more modified bases. The amplicon may further include one or more labeled bases, and the measuring step may include detecting the presence of the one or more labeled bases. The one or more modified bases may include an aptamer, aptamer ligand, antibody, antigen, ligand, receptor, hapten, epitope, or mimotope, and the plurality of detectable moieties may each include a binding partner for the one or more modified bases and a detectable label. The one or more modified bases may comprise streptavidin, and the plurality of detectable moieties each comprise biotin and a detectable label; the one or more modified bases may comprise biotin, and the plurality of detectable moieties each comprise streptavidin and a detectable label; the one or more modified bases may comprise avidin, and the plurality of detectable moieties each comprise biotin and a detectable label; and / or the one or more modified bases may comprise biotin, and the plurality of detectable moieties each comprise avidin and a detectable label.

[0054] Step (a) of embodiments (11) and (12) may comprise 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) may comprise 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) may comprise 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 of 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 within 10-100 μm of the location of the complex on the surface. In a specific embodiment, the extension probe binds to the anchor reagent less than 100 μm, less than 50 μm, or more specifically less than 10 μm from the location of the complex on the surface.

[0056] The surface of embodiments (11) and (12) may comprise a particle and / or a well of a multiwell plate. Optionally, the surface may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on 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 anchor reagent disposed on two distinct binding domains within the well. When the surface is a well, the well may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on 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 anchor reagent disposed on the same binding domain within the well. The capture reagent and anchor reagent may be within 10-100 nm of each other on the surface.

[0057] In the surfaces of embodiments (11) and (12), the surface may include an electrode, and the measuring step may include applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. Optionally, embodiments (11) and (12) include collecting particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. The measuring step may also 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 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, a magnetic field, or a combination thereof. In one example, the detectable label is an ECL label, and the measuring step may include measuring an ECL signal. The detection probe may have multiple ECL labels. The detection probe may be bound to multiple ECL label moieties via linkages at the 3' ends of the probe nucleotide components.

[0058] In embodiments (11) and (12), the sample may contain one or more analyte molecules, and the surface may contain multiple capture reagents for the one or more analyte molecules distributed across multiple degradable binding regions located on the surface, and the method may include (x) binding 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 may include imaging the optical signal from the surface to create an image comprising multiple pixels, and each degradable binding region is mapped to one or more pixels in the image. The degradable binding regions may be elements of an array, and / or the degradable binding regions are configured to isolate individual particles. Each resolvable binding region can be an individual nanowell having a volume of less than 100 nL, e.g., at least 99% of the binding regions contain either zero or one analyte molecule, at least about 95% of the binding regions contain either zero or one analyte molecule, at least about 80% of the binding regions contain either zero or one analyte molecule, and / or at least about 50% of the binding regions contain either zero or one analyte molecule. The concentration of analyte molecules in the sample in embodiments (11) and (12) can be determined at least in part using a calibration curve, Poisson distribution analysis, and / or Gaussian distribution analysis of the number of binding regions containing at least one or more analyte molecules.

[0059] In embodiments (11) and (12), the sample may include one or more analyte molecules, the surface may include a plurality of particles each including a plurality of binding reagents for the analyte molecules, the plurality of particles distributed across a plurality of degradable binding regions, and the method may include (i) binding one or more analyte molecules to the one or more binding reagents on the surface, (ii) distributing the plurality of particles across the array of degradable binding regions, and (iii) determining the presence or absence of analyte molecules at each degradable binding region to identify the number of binding regions that include the analyte molecules and / or the number of binding regions that do not include the analyte molecules.

[0060] Embodiment (13): A kit for detecting an analyte of interest in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte, and (ii) an anchor reagent comprising an anchor oligonucleotide sequence; (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. In an embodiment, the first and second proximity probes are nucleic acid probes.

[0061] Embodiment (14): A kit for detecting an analyte of interest in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for the analyte and (ii) an anchor reagent comprising an anchor oligonucleotide sequence; (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. In an embodiment, the first and second proximity probes are nucleic acid probes.

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

[0063] The surface of embodiments (13) and (14) may comprise particles and / or wells of a multiwell plate. It may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. When the surface is a well, the well may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well. The surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain on the surface. When the surface is a well, the well may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain within the well. The capture reagent and anchor reagent may be within 10-100 nm of each other 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 may include: (a) binding the analyte to first and second detection reagents to form detection complexes, each detection complex including the analyte, a first detection reagent, and a second detection reagent, wherein the first detection reagent has a first detectable label and the second detection reagent has a second detectable label; (b) distributing the analyte among multiple reaction vessels such that a majority of the reaction vessels contain no more than one analyte; and (c) detecting the number of analyte molecules by counting the number of reaction vessels containing the first and second detectable labels. In this embodiment (15), the first detection reagent may be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, for example, the first detection reagent is an antibody. Similarly, the second detection reagent can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, 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) may further include forming a solution containing the analyte and the detection reagent, and step (b) may include distributing the solution among 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. Alternatively, step (a) of embodiment (15) may further include forming a solution containing the analyte and the detection reagent, and step (b) may include distributing the solution among 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 100. Furthermore, step (a) of embodiment (15) may further include forming a solution containing the analyte and the detection reagent, and step (b) may include distributing the solution among 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 1000. Furthermore, step (a) of embodiment (15) may further include forming a solution containing the analyte and the detection reagent, and step (b) may 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, the method comprising: (a) binding the analyte to a capture reagent and first and second detection reagents to form detection complexes, each detection complex comprising the capture reagent, the analyte, a first detection reagent, and a second detection reagent, (i) the first detection reagent has a first detectable label, the second detection reagent has a second detectable label, and (ii) the capture reagent is on a surface; (b) distributing the analyte among a plurality of reaction vessels such that a majority of the reaction vessels contain no more than one analyte; and (c) detecting the number of analyte molecules by counting the number of reaction vessels containing the first and second detectable labels. In this embodiment, the capture reagent can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, for example, the capture reagent is an antibody. Similarly, the first detection reagent can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, for example, the first detection reagent is an antibody.Furthermore, the second detection reagent can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, for example, the second detection reagent is an antibody.For example, the capture reagent, the first and second detection reagents are antibodies against the analyte.

[0067] Step (b) of embodiment (16) may further include distributing the solution across multiple reaction vessels so 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. Furthermore, step (b) of embodiment (16) may further include distributing the solution across multiple reaction vessels so 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 100. Step (b) of embodiment (16) may further include distributing the solution across multiple reaction vessels so 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 1000. Furthermore, step (b) of embodiment (16) may further include distributing the solution across multiple reaction vessels so 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.

[0068] The capture reagent in the detection complex of embodiment (16) can be present on the surface before binding of the capture reagent to the analyte, or the capture reagent in the detection complex can bind to the analyte before immobilizing the capture reagent on the surface. In one example, the capture reagent can include a targeting moiety and the surface can include the complement of the targeting moiety. The targeting moiety and targeting agent binding partner are selected from the following binding pairs: avidin-biotin, streptavidin-biotin, receptor-ligand, antibody-antigen, and nucleic acid-nucleic acid complement.

[0069] In embodiment (16), the surface 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 among 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 among a plurality of reaction vessels, and then binding the analyte to the capture reagent.

[0070] Embodiment (16) may further include distributing a plurality of particles into a plurality of reaction vessels, wherein the plurality of particles comprise a target moiety, the capture reagent comprises a complement of the target moiety, and distributing the analyte is achieved by binding the complement of the target moiety to the target moiety. Embodiment (16) may also include washing the particles before and / or after the distributing step.

[0071] The surface of embodiment (16) can be located within one of the reaction vessels. In this embodiment, a capture reagent can be immobilized on the 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 target moiety immobilized thereon, the capture reagent comprising a complement of the target moiety, and distribution of the analyte is achieved by binding the complement of the target moiety to the target moiety. In this particular example, the method can further include washing the reaction vessel prior to the detection step.

[0072] The plurality of reaction vessels of embodiment (16) can comprise an array of nanowells. The plurality of reaction vessels can comprise 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 in a coupled enzyme reaction system, and the second detectable label is a second enzyme in the coupled enzyme reaction system. Step (d) can include adding one or more substrates of the reaction system, producing a product of the enzyme reaction system, 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, e.g., 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 includes an oxidase substrate and a labeled Amplex Red or luminol derivative. In this embodiment, the oxidase can be glucose oxidase, and the oxidase substrate is glucose. In one embodiment, the reaction catalyzed by the first and second enzymes in the detection complex results in 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) can include measuring a 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 generated by PLA-RCA. For example, the first detectable label can be a FRET donor, the detectable label can be a FRET acceptor, and the proximity-dependent signal can be 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 differ 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 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. Step (d) of embodiment (16) can include 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 be changes in optical absorbance with different spectral characteristics. Optionally, the first and second signals can be 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 can be selected from phosphate-, sulfate-, galactoside-, and glucuronide-modified 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 detecting step of embodiment (16) can include detection by light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, luminescence, radioactivity, a magnetic field, or a combination thereof.

[0075] Embodiment (17): A kit for detecting an analyte in a sample, the kit comprising, in one or more vials, containers, or compartments: (a) a first detection reagent comprising a first detectable label; (b) a second detection reagent comprising a second detectable label; and (c) a plurality of reaction vessels configured to contain no more than one analyte molecule.

[0076] Embodiment (18): A kit for detecting an analyte in a sample, the kit comprising, in one or more vials, containers, or compartments: (a) a first detection reagent comprising a first detectable label; (b) a second detection reagent comprising a second detectable label; (c) a surface comprising a capture reagent; and (d) a plurality of reaction vessels configured to contain no more than one analyte molecule.

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

[0078] The surface in embodiments (17) and (18) can be particles, e.g., the capture reagent is immobilized on a plurality of particles. Alternatively, the surface is an arrangement within one of the reaction vessels, e.g., the capture reagent is immobilized on the surface of the plurality of reaction vessels. Optionally, the reaction vessel has a surface with a target moiety immobilized thereon, and the capture reagent comprises the complement of the target moiety. The plurality of reaction vessels can comprise an array of nanowells or aqueous droplets dispersed in a water-in-oil emulsion. The plurality of reaction vessels can comprise at least 10,000 reaction vessels, and optionally, 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 can be a first enzyme of a coupled enzyme reaction system, and the second detectable label can be a second enzyme of the coupled enzyme reaction system. The kit can include one or more substrates of the reaction system in one or more additional vials, containers, or compartments. For example, the first enzyme is an oxidase, the second enzyme is a peroxidase, and the substrate includes an oxidase substrate and a labeled Amplex Red or luminol derivative. In certain embodiments, the oxidase is glucose oxidase, and the oxidase substrate is glucose. The first and second detectable labels can be components of a proximity-dependent system, e.g., the first detectable label is a FRET donor and the detectable label is a FRET acceptor. The first and second detectable labels can be measured independently. Optionally, the first and second detectable labels are luminescent labels that differ from each other in terms of spectral properties.

[0080] In the kits of embodiments (17) and (18), 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. The kit may include the first enzyme substrate and the second enzyme substrate in one or more vials, containers, or compartments. Optionally, the first and second signals are changes in 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 may 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 may 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, wherein 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; and (d) counting the individual crosslinked products in the eluent containing both the first and second detectable labels. In this embodiment (19), the capture reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in a specific example, the capture reagent is an antibody. Similarly, the first detection reagent can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in a specific example, the first detection reagent is an antibody. Furthermore, the second detection reagent can be an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and specifically, the second detection reagent can be an antibody. In one specific example, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0082] Embodiment (19) may further include adding a crosslinker to crosslink the first and second detection reagents, e.g., the first and second detection reagents comprise reactive moieties, and the crosslinker is a multifunctional crosslinker that links the reactive moieties. For example, the reactive moieties include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The crosslinker may include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The first and second detection reagents may comprise binding moieties, and the crosslinker is a multivalent binding partner of the binding moieties. In one example, the first and second detection reagents are animal species antibodies, and the crosslinker is a multivalent anti-species antibody that targets the animal species antibodies. The first and second detection reagents may comprise biotin, the crosslinker is streptavidin, the first and second detection reagents comprise streptavidin, the crosslinker is biotin, the first and second detection reagents are linked to streptavidin, 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 which may comprise a sequence complementary to the first nucleic acid probe and a distinct sequence complementary to the second nucleic acid probe.

[0083] The surface of embodiment (19) may comprise particles, reaction vessels such as tubes or ampoules, and / or the surface may comprise wells of a multi-well plate. The method of embodiment (19) may further comprise collecting 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 specific example, the first and second detectable labels comprise ECL labels, and the counting step may comprise measuring the ECL signal.

[0084] Embodiment (20): A kit for detecting an analyte of interest in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising an immobilized capture reagent; (b) a first detection reagent having a first detectable label; (c) a second detection reagent having a second detectable label; and (d) a crosslinker reactive with the 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 crosslinker can include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The first and second detection reagents of embodiment (20) may 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 an animal species, and the crosslinker is a multivalent anti-species antibody targeting the antibodies 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, the crosslinker is biotin, and 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 may comprise a sequence complementary to the first nucleic acid probe and a distinct sequence complementary to the second nucleic acid probe.

[0086] The surface of embodiment (20) may comprise a particle, a well of a multi-well plate, or a reaction vessel, such as a tube or an ampoule. Furthermore, the surface may comprise a plurality of distinct binding domains, with the capture reagent disposed on the 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 disposed on the distinct binding domains within the well. The surface may also comprise an electrode.

[0087] Embodiment (21): A method for detecting an analyte of interest in a sample, the method 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 may comprise a first detectable label and a first nucleic acid probe, and the second detection reagent may comprise 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 regions 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 containing both the first and second detectable labels.

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

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

[0090] Embodiment (22): A kit for detecting an analyte of interest in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising an immobilized capture reagent; (b) a first detection reagent having a first detectable label and a first nucleic acid probe; (c) a second detection reagent having a second detectable label and a second nucleic acid probe; and (d) a third nucleic acid having a region complementary to the first and second nucleic acid probes.

[0091] The surface of embodiment (22) may comprise a particle, a well of a multiwell plate, or a reaction vessel such as a tube or ampoule. The surface may comprise a plurality of distinct binding domains, with the capture reagent disposed on the distinct binding domains on the surface; if the surface is a well, the well may comprise a plurality of distinct binding domains, with the capture reagent disposed on the distinct binding domains within the well. The surface may optionally comprise an electrode.

[0092] Embodiment (23): A method for detecting an analyte of interest in a sample, the method 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 may include a first nucleic acid probe and the second detection reagent may include a second nucleic acid probe, and the capture reagent in the complex is immobilized on a surface; (b) extending the second nucleic acid probe to form an extended sequence comprising a detectable label, where the extension depends on the colocalization 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 individual extended sequences in the eluent. In this embodiment, the capture reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer. In a specific example, the capture reagent is an antibody. Similarly, the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in a specific example, the first detection reagent is an antibody.The second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an epitope, a mimotope, or an aptamer, and in particular, the second detection reagent is an antibody.In a specific 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 collecting 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) may include binding a probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step may also include binding a 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 a first probe to the template nucleic acid sequence, binding a second probe to the template sequence, and ligating the first and second probes. Optionally, the label is a fluorescent label, and counting individual extension sequences may include detecting single-molecule fluorescence, such as fluorescence correlation spectroscopy and / or fluorescence cross-correlation spectroscopy. Detecting single-molecule fluorescence may include flowing an 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. Detection of single molecule fluorescence may also involve flowing an 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.

[0096] Embodiment (24): A method for detecting an analyte of interest in a sample, the method 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 containing both the first and second detectable labels. In this embodiment, the capture reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, e.g., the capture reagent is an antibody, and the first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, e.g., the first detection reagent is an antibody, and the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, e.g., 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.

[0097] The surface of embodiment (24) can include particles, reaction vessels such as tubes or ampoules, and / or wells of a multiwell plate. The method of embodiment (24) can include collecting 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, a magnetic field, or a combination thereof; 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 analyte's capture reagent, (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 a first and second detection reagent; (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; 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, mimotope, or aptamer, e.g., the capture reagent is an antibody, and the first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, e.g., the first detection reagent is an antibody, and the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, e.g., 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.

[0099] The extended sequence of embodiment (25) may include one or more detection sequences, and the measuring step may include contacting the extended sequence with a plurality of labeled probes complementary to the one or more detection sequences, the extended sequence may include one or more modified bases, and the measuring step may 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 may include one or more labeled bases, and the measuring step may include detecting the presence of the one or more labeled bases. The one or more modified bases may include aptamers, aptamer ligands, antibodies, antigens, ligands, receptors, haptens, epitopes, or mimotopes, and the plurality of detectable moieties may include binding partners of the one or more modified bases and detectable labels, respectively. 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 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.

[0100] Step (a) of embodiment (25) may include binding the analyte to the following species in the order of (i) a capture reagent on the surface and (ii) a detection reagent for the analyte; binding the analyte to the following species in the order of (i) a capture reagent for the analyte and (ii) a detection reagent for the analyte on the surface; or simultaneously or substantially simultaneously binding the analyte to the species of (i) a capture reagent on the surface and (ii) a detection reagent for the analyte. The extension step may include binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction, or binding the probe to the template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. In this embodiment, the extension probe may remain localized on the surface after probe extension; for example, the complex may remain bound to the surface after the extension step. The extension step may include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (sustained synthesis reaction), or isothermal amplification. In certain instances, the extension step may involve 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 regions of the first probe. The process may further comprise ligating the two terminal sequences of the connector oligonucleotide to form a circular target sequence that hybridizes to both the first and second probes. The extension process of embodiment (25) may also include 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 on 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 may also include ligating the first and second connector oligonucleotides to form a circular target sequence that hybridizes to both the first and second probes.

[0102] The surface of embodiment (25) may comprise particles or wells of a multiwell plate. The surface may comprise multiple distinct binding domains, with the capture reagent disposed on two distinct binding domains on the surface. When the surface is a well, the well may comprise multiple distinct binding domains, with the capture reagent disposed on two distinct binding domains within the well. The surface may comprise multiple distinct binding domains, with the capture reagent disposed on the same binding domain on the surface; when the surface is a well, the well may comprise multiple distinct binding domains, with the capture reagent disposed on the same binding domain within the well. The surface may comprise an electrode, and the measuring step may include applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. The method optionally includes collecting particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. The measuring step may further include binding the 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, with the detection probe comprising a nucleic acid sequence 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 specific example, the detectable label is an ECL label, and the measuring step can include measuring an ECL signal. The detection probe can have multiple ECL labels. The detection probe can be linked to multiple ECL label moieties via a linkage at the 3' end of the probe nucleotide component.

[0103] Embodiment (26): A kit for detecting an analyte of interest in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising a capture reagent for the analyte; (b) a first detection reagent for the analyte linked to a first nucleic acid probe; and (c) a second detection reagent for the analyte linked to a second nucleic acid probe.

[0104] In embodiment (26), the capture reagent may comprise an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, e.g., the capture reagent may comprise an antibody; the first detection reagent may comprise an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, e.g., the first detection reagent may comprise an antibody; the second detection reagent may comprise an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, e.g., the second detection reagent may comprise an antibody; the surface may comprise particles and / or wells of a multiwell plate. The surface may comprise multiple distinct binding domains, and the capture reagent may be disposed on two distinct binding domains on the surface; and if the surface is a well, the well may comprise multiple distinct binding domains, and the capture reagent may be disposed on two distinct binding domains within the well. Optionally, the surface may comprise a plurality of distinct binding domains, with the capture reagent disposed on the same binding domain on the surface, and if the surface is a well, the well may comprise a plurality of distinct binding domains, with the capture reagent disposed on the same binding domain within the well. The surface may comprise an electrode.

[0105] The surfaces of embodiments 1-26 can include the inner surfaces of assay vessels, such as test tubes, cuvettes, flow cells, FACS cell sorters, cartridges, or wells of multi-well plates. The 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 for detecting an analyte of interest in a sample containing one or more analyte molecules, the method comprising: (a) contacting the sample with a surface comprising a plurality of degradable binding regions positioned on the surface, each degradable binding region comprising a plurality of capture reagents for one or more analyte molecules in the sample; (b) binding the one or more analyte molecules to (i) one or more capture reagents on the surface, (ii) a first detection reagent for the analyte comprising a first detectable label, and (iii) a second detection reagent for the analyte comprising a second detectable label, thereby forming a detection complex on the degradable binding domains on the surface comprising the capture reagent, the analyte, and first and second detection reagents, wherein the first and second detectable labels are different label 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 create an image including 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 configured to isolate individual particles. Each resolvable binding region can be an individual nanowell having a volume of less than 100 nL, and / or at least 99% of the binding regions contain either zero or one analyte molecule, at least about 95% of the binding regions contain either zero or one analyte molecule, at least about 80% of the binding regions contain either zero or one analyte molecule, or at least about 50% of the binding regions contain either 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, Poisson distribution analysis, and / or Gaussian distribution analysis of the number of binding regions containing at least one or more analyte molecules.

[0107] The surface of embodiment (27) may include a plurality of particles, each including a plurality of capture reagents for an analyte molecule, the plurality of particles being distributed among a plurality of degradable binding regions, and the method may 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, the first and second detection reagents including first and second detectable labels, respectively; (ii) distributing the plurality of particles across the array of degradable binding regions; and (iii) determining the number and type of binding regions that include analyte molecules. and / or determining the presence or absence of analyte molecules in each resolvable binding region to identify the number of binding regions that do not contain analyte molecules, wherein optionally each resolvable binding region is an individual nanowell having a volume of less than 100 nL, and / or at least 99% of the binding regions contain either zero or one analyte molecule, at least about 95% of the binding regions contain either zero or one analyte molecule, at least about 80% of the binding regions contain either zero or one analyte molecule, and / or at least about 50% of the binding regions contain either zero or one analyte molecule.

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

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

[0110] The surface of embodiment (27) may comprise particles or wells of a multiwell plate. In a specific example, the surface may comprise an electrode, and the identifying step may comprise applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. The method of embodiment (27) may comprise collecting particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. The first detectable label is measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, a 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, a magnetic field, or a combination thereof. The first and second detectable labels can be measured independently; 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 inner 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, pin, probe, bead, or filtration medium, lateral flow medium, 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, the kit comprising: (a) a surface comprising a plurality of degradable linkage regions positioned thereon, each degradable linkage 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 degradable binding regions of embodiment (28) can be elements of an array and / or can be configured to isolate individual particles. Each degradable binding region is optionally an individual nanowell having a volume of less than 100 nL. The surface can include a plurality of particles, each including a plurality of capture reagents for an analyte molecule, the plurality of particles distributed among the degradable binding regions. The kit can include first and second detection reagents for each of the one or more analyte molecules, the first and second detection reagents including first and second detectable labels, respectively.

[0114] In embodiment (28), the capture reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, e.g., the capture reagent is an antibody, and the first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, e.g., the first detection reagent is an antibody, and the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, e.g., the second detection reagent is an antibody. In a specific example, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0115] The surface of embodiment (28) may comprise a particle or well of a multiwell plate. In a particular example, the surface may comprise an electrode, and the identifying step may comprise applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. The first detectable label is measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, a 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, a magnetic field, or a combination thereof. The first and second detectable labels may 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 properties.

[0116] The surface of embodiment (28) 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, pin, probe, bead, or filtration medium, lateral flow medium, such as 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 an HIV p24 capture reagent 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 the binding reagent, HIV p24, and the first and second detection reagents; (b) extending the second probe using an extension process requiring proximity of the first and second probes 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.

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

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

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

[0121] The extension step of embodiment (29) may include binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step may further include binding the probe to the template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extension probe may remain localized on the surface after probe extension; for example, the complex remains bound to the surface after the extension step. The extension probe may bind to the anchor reagent within 10 to 100 μm of the location of the complex on the surface. In a specific embodiment, the extension probe binds to the anchor reagent less than 100 μm, less than 50 μm, or more specifically less than 10 μm from the location of the complex on the surface. The extension step may include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (sustained synthesis reaction), or isothermal amplification. In certain instances, the extension step may involve an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0122] The extension process of embodiment (29) 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 comprise 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 (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 on 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) may comprise particles and / or wells of a multiwell plate. The surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. When the surface is a well of a plate, the well may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well. The surface may also comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain on the surface. When the surface is a well of a plate, the well may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain within the well. The capture reagent and anchor reagent may be within 10 to 100 nm of each other on the surface. In a specific example, the surface may comprise an electrode, and the measuring step may include applying a voltage waveform to the electrode to generate an electrochemiluminescence signal. Optionally, the method of embodiment (29) further includes collecting particles on the electrode and applying a voltage 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 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 specific example, the detectable label is an ECL label, and the measuring step can include measuring an ECL signal. The detection probe can have multiple ECL labels. The detection probe can be bound to multiple ECL label moieties via linkages at the 3' ends of the probe nucleotide components.

[0125] Embodiment (30): A kit for detecting HIV p24 in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising (i) a capture reagent for HIV p24, and (ii) an 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) may comprise an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in certain examples, the capture reagent may comprise an antibody. Similarly, the first detection reagent may comprise an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in certain examples, the first detection reagent may comprise an antibody. Similarly, the second detection reagent may comprise an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in certain examples, the second detection reagent may comprise an antibody.

[0127] The surface of embodiment (30) may comprise particles and / or wells of a multi-well plate. The surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. If the surface is a well, the well may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well. The surface may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain on the surface, and / or if the surface is a well, the well may comprise multiple distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain within the well. The capture reagent and anchor reagent may be within 10-100 nm of each other 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 a 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. (b) contacting the detection complex formed in (c) with a connector sequence comprising (i) an internal sequence complementary to a 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 the 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 a 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 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; and (c) contacting the first and second connector oligonucleotides. (d) hybridizing a first and second connector oligonucleotide 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 an anchor reagent; (f) binding the amplicon to the anchor reagent; and (g) measuring the amount of amplicon on the surface.

[0130] In embodiments (31) and (32), the capture reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and in certain examples, the capture reagent is an antibody. Similarly, the first detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and for example, the first detection reagent is an antibody. Furthermore, the second detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer, and for example, the second detection reagent is an antibody. In certain examples of embodiments (31) and (32), the capture reagent and the first and second detection reagents are antibodies against HIV p24.

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

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

[0133] Step (a) of embodiments (31) and (32) may comprise 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) may comprise 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) may comprise 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 may remain bound to the surface after the extension step. For example, the amplicons bind to the anchor reagent within 10-100 μm of the location of the complex on the surface. In a specific embodiment, the extension probe binds to the anchor reagent less than 100 μm, less than 50 μm, or more specifically less than 10 μm from the location of the complex on the surface.

[0135] The surface of embodiments (31) and (32) may comprise a particle and / or a well of a multiwell plate. The surface may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains on the surface. When the surface is a well of a plate, the well may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on two distinct binding domains within the well. When the surface is a well of a plate, the well may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain on the surface. When the surface is a well of a plate, the well may comprise a plurality of distinct binding domains, with the capture reagent and anchor reagent disposed on the same binding domain within the well. In certain examples, the capture reagent and anchor reagent are within 10-100 nm of each other on the surface.

[0136] Additionally, the surface may include an electrode, and the measuring step may include applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. In these embodiments ((31) and (32)), the method may further include collecting particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescent signal. The measuring step may 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 complementary to a region of the amplicon. The detectable label is measured by measuring light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, a magnetic field, or a combination thereof. For example, the detectable label may be an ECL label, and the measuring step may include measuring an ECL signal. The detection probe may have multiple ECL labels. The detection probe may be bound to multiple ECL label moieties via linkage at the 3' end of the probe nucleotide component.

[0137] Embodiment (33): A method for detecting an analyte of interest in a sample, the method comprising: (a) concentrating the sample under conditions sufficient to form an analyte complex comprising the analyte bound to a first detection reagent, wherein the first detection reagent is linked to a first nucleic acid probe; (b) binding the analyte complex formed in step (a) to (i) a capture reagent on the surface comprising the analyte's 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 requiring the first and second probes to be in close proximity to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence; (d) hybridizing the anchor sequence to the anchor sequence complement; and (e) measuring the amount of extended sequence bound to the surface. The enrichment step (a) may further include: (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 the analyte complex.

[0138] Embodiment (34): A kit for detecting an analyte of interest in a sample, comprising: 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, the method comprising: (a) binding exosomes to (i) a capture reagent on a surface comprising an exosomal capture reagent and an anchor reagent comprising an anchor oligonucleotide sequence, (ii) a first exosomal detection reagent linked to a first nucleic acid probe, and (iii) a second exosomal detection reagent linked to a second nucleic acid probe, thereby forming a complex on the surface comprising the binding reagent, the exosome, and the first and second detection reagents; (b) extending the second probe using an extension process requiring proximity of the first and second probes 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 detecting exosomes of interest in a sample, comprising, in one or more vials, containers, or compartments: (a) a surface comprising (i) an exosome capture reagent, and (ii) an anchor reagent comprising an anchor oligonucleotide sequence; (b) a first exosome detection reagent linked to a first nucleic acid probe; and (c) a second exosome detection reagent linked to a second nucleic acid probe.

[0141] Embodiment (37): A method for detecting an analyte of interest in a sample, comprising: binding the analyte to (i) a capture reagent on a surface comprising a capture reagent for the analyte and an anchor reagent comprising an anchor 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 first and second detection reagents; (b) extending the first and second nucleic acid probes to form extended sequences comprising anchor sequence complements that are complementary to the anchor sequences; (c) hybridizing the anchor sequences to the anchor sequence complements; and (d) measuring the amount of extended sequences bound to the surface using a labeled probe of Formula I. [ka] In the formula, B is a nucleotide base, R is an electrochemiluminescent label, L1 is a linking group, L2 is a linking group, j is an integer of 0 to 11, k is an integer of 0 to 1, m is an integer of 0 to 11, and n is an integer of 0 to 5.

[0142] Embodiment (38): A method for detecting an analyte of interest in a sample, comprising: binding the analyte to (i) a capture reagent on a surface comprising a capture reagent for the analyte and an anchor reagent comprising an anchor sequence, and (ii) a detection reagent for the analyte linked to a nucleic acid probe, thereby forming a complex on the surface comprising a binding reagent, the analyte, and the detection reagent; (b) extending the nucleic acid probe 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 using a labeled probe of Formula I.

[0143] In any of the above embodiments (1) through (38), the anchor reagent is attached to the surface before, during, or after binding of the analyte to the capture reagent. In embodiments including kits, the anchor reagent is provided separately from the surface and then immobilized on the surface, and the capture reagent is immobilized on the surface. In embodiments including kits, the anchor reagent and capture reagent are provided immobilized on the surface.

[0144] Any of the above embodiments (1) to (38) may include a labeled probe of Formula I, [ka] In the formula, B is a nucleotide base, R is an electrochemiluminescent label, L1 is a linking group, L2 is a linking group, j is an integer of 0 to 11, k is an integer of 0 to 1, m is an integer of 0 to 11, and n is an integer of 0 to 5.

[0145] Any of the above embodiments (1) to (38) may include a labeled probe of Formula II, [ka] In the formula, j is an integer of 0 to 11, k is an integer of 0 to 1, m is an integer of 0 to 11, n is an integer of 0 to 5, and R is an electrochemiluminescent label: [ka] is.

[0146] Any of the methods described in the embodiments herein may include a method of measuring electrochemiluminescence comprising: (a) applying a potential to an electrode under conditions such that a complex in proximity to the electrode emits electrochemiluminescence, wherein the complex comprises a target oligonucleotide and a labeled probe provided herein, and the labeled probe comprises an oligonucleotide complementary to the target oligonucleotide; and (b) measuring the emitted electrochemiluminescence.

[0147] The nucleic acid probe linked to, for example, a detection reagent according to any of embodiments (1) to (38) may comprise an oligonucleotide, the oligonucleotide being 14 to 24 nucleotides in length and comprising 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO: 33). In one embodiment, the present invention provides a method for conjugating a nucleic acid probe to a non-nucleic acid detection reagent to form a conjugate, the method comprising contacting the detection reagent and the nucleic acid probe with a heterobifunctional crosslinker to form a conjugate under conditions in which the detection reagent reacts with a first reactive group of the crosslinker and the nucleic acid reacts with a second reactive group of the crosslinker, the heterobifunctional crosslinker comprising (i) a first reactive group capable of reacting with the detection reagent to attach the crosslinker to the detection reagent, and (ii) a second reactive group that is substantially unreactive with the detection reagent but capable of reacting with the nucleic acid probe to attach the crosslinker to the nucleic acid probe, the method not comprising purifying the reaction product of the detection reagent and the crosslinker prior to the reaction of the crosslinker with the nucleic acid probe.

[0148] In embodiments, the present invention provides a method for conjugating a nucleic acid probe to a non-nucleic acid detection reagent to form a conjugate, the method comprising: (a) contacting the detection reagent with a heterobifunctional crosslinker to form a first composition under conditions in which the detection reagent reacts with a first reactive group of the crosslinker, the heterobifunctional crosslinker comprising (i) a first reactive group capable of reacting with the detection reagent to attach the crosslinker to the detection reagent, and (ii) a second reactive group that is substantially unreactive with the detection reagent but is capable of reacting with the nucleic acid probe to attach the crosslinker to the nucleic acid probe; and (b) contacting the first composition with the nucleic acid probe under conditions in which the second reactive group of the crosslinker reacts with the nucleic acid probe to form a conjugate, the method not comprising purifying the reaction product of the detection reagent and the crosslinker prior to reacting the crosslinker with the nucleic acid probe.

[0149] In embodiments, the present invention provides a kit for conjugating a nucleic acid probe to a non-nucleic acid detection reagent to form a conjugate, the kit comprising: (a) a heterobifunctional crosslinker comprising (i) a first reactive group capable of reacting with the detection reagent to attach the crosslinker to the detection reagent, and (ii) a second reactive group that is substantially unreactive with the detection reagent but capable of reacting with the nucleic acid probe to attach the crosslinker to the nucleic acid probe; (b) a first size separation device capable of separating the conjugate from unreacted nucleic acid probe; and (c) a nucleic acid-binding fluorophore, wherein the fluorescence intensity of the fluorophore increases when the fluorophore binds to a nucleic acid. In embodiments, the present invention provides a method for conjugating a nucleic acid probe to a non-nucleic acid detection reagent to form a conjugate, the method comprising: (a) reacting the detection reagent and the nucleic acid probe to form a conjugate; (b) separating the conjugate from unreacted nucleic acid probe using a size separation device to form a purified conjugate; (c) forming a test composition comprising a sample of the purified conjugate and a nucleic acid-binding fluorophore selected to have a fluorescence intensity that increases upon binding of the fluorophore to a nucleic acid; and (d) measuring the fluorescence of the test composition to determine the amount of nucleic acid probe in the purified conjugate. [Brief explanation of the drawings]

[0150] [Figure 1(a)-(c)] The use of anchor reagents in immunoassays is illustrated. Figure 1(a) illustrates the use of anchor 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 extended and binds to the anchor reagent. In Figure 1(b), the anchor reagent comprises an oligonucleotide sequence comprising a region complementary to a portion of the extension sequence formed on the detection reagent. Figure 1(c) illustrates a specific embodiment in which two detection reagents are used to bind the analyte, each comprising a nucleic acid probe. The probes of the detection reagents are subjected to an amplification process that allows hybridization of one extension probe to the anchor oligonucleotide sequence. [Figure 2(a)] A particular embodiment is shown in which immune complexes formed on a surface bearing an anchoring reagent are subjected to a PLA-RCA process to incorporate multiple detectable species into the extended sequences attached to the immune complex. [Figure 2(b)-2(c)] 1A-1C are two alternative configurations of connecting oligonucleotides that can be used in the methods of the present invention. [Figure 3] One method for attaching oligonucleotides to proteins is shown. [Figure 4(a)] Figure 4(a) shows a preferred embodiment of the invention in which a surface-bound complex is formed between a capture reagent, an analyte, and two detection reagents, each attached to a first and second proximity probe, respectively, which are ligated 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 complementary to the sequence of the amplicon formed as the assay proceeds. [Figure 4(b)] An exemplary sequence of the first circular DNA template Circ-1, the detection oligonucleotide sequence, the inactive region of the amplicon, and the portion PP2 designed to hybridize to the second proximity probe is shown in Figure 4(c). An alternative embodiment is shown in Figure 4(c). [Figure 5] 1 illustrates an alternative method for generating amplicons that can be amplified by rolling circle amplification. [Figure 6(a)-(b)] 1 illustrates an alternative method for generating amplicons that can be amplified by rolling circle amplification. [Figure 7] An alternative embodiment is shown in which a portion of each proximity probe in the sandwich complex is temporarily protected by short strands of RNA hybridized to each segment. These strands are enzymatically removed, allowing the proximity probes to hybridize to each other and the strands to be extended. [Figure 8] As described above with reference to Figure 8, a further embodiment is shown in which proximity probes are attached to the capture and detection reagents, with a portion of each proximity probe being temporarily protected by a short strand of RNA hybridised to it. [Figure 9]1 shows the calibration curve for an IL-10 assay performed using the method described in Example 1. [Figure 10(a)-(b)] Fluorescence microscope images are shown with (a) and without (b) the use of an anchoring reagent. [Figure 11(a)] 1 shows the configuration of single linear connector oligonucleotide sequences containing either ligation site 1 or 2, and the use of these connectors in the assays of the present invention. [Figure 11(b)] 1 shows comparative performance data for assays using a combination of Circ-1 and Circ-2 with either a single linear connector oligonucleotide sequence containing linkage site 1 or a single linear connector oligonucleotide sequence containing linkage site 2. [Figure 12] 1 shows a calibration curve for an HIV p24 assay performed using the methods described in Examples 1 and 6. [Figure 13] 1 shows the results of an analysis of a seroconversion panel using the methods described in Examples 1 and 6. [Figure 14(a)-(c)] 1 shows the results of an HIVp24 assay including an analyte concentration step. [Figure 15] 1 shows a calibration curve for the HIV p24 assay including analyte concentration steps. [Figure 16] FIG. 1 is a schematic diagram of the assay method described herein, including an analyte enrichment step. [Figure 17] FIG. 1 is a schematic diagram of an 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 diagram of an assay in which multiple staple sequences are used to attach amplicons, thereby forming more compact structures on the surface. [Figure 19] FIG. 1 is a schematic diagram of a bridging immunoassay format incorporating 3AB PLA-RCA technology and the use of a targeting moiety and its complement to bind a capture reagent to a surface. [Figure 20(a)-(b)] FIG. 1 is a schematic diagram of a biosynthetic method for template formation. [Figure 21]FIG. 1 is a schematic diagram of sample multiplexing. [Figure 22] FIG. 1 is a schematic diagram of the detection of lipoprotein complexes using the methods described herein. [Figure 23] (a) Comparison of the average label per protein (L / P) ratio of antibody-oligonucleotide conjugates measured by fluorescent dye method and gel electrophoresis, and (b) signal measured by fluorescent dye method as a function of oligonucleotide concentration for free oligonucleotide and oligonucleotide in antibody-oligonucleotide conjugates. [Figure 24] FIG. 1 shows a schematic diagram of a two-antibody amplified ECL immunoassay performed on a streptavidin-coated surface. [Figure 25] (a) Elution profile of crude antibody-oligonucleotide conjugation reaction product from a G-100 Superfine column, along with profiles for unconjugated antibody, unconjugated oligonucleotide, and purified conjugate; (b) elution profile and collected fractions for IL-6 detection antibody-oligonucleotide conjugate; and (c) calibration curve for an amplified ECL immunoassay using G-100 purified IL-6 detection antibody-oligonucleotide conjugate and results when the conjugate was purified by centrifugal ultrafiltration. [Figure 26] (a) Relative fluorescence signals after binding to free versus antibody-conjugated oligonucleotides for six nucleic acid-sensitive fluorescent dyes (left), the percentage of signal from antibody-oligonucleotide conjugates associated with dye-protein interaction (center), and the signal-to-background ratios obtained during measurements of 100 ng of conjugates (right), and (b) fluorescence signals measured using SYBR Green I dye for three antibody-oligonucleotide conjugates as a function of oligonucleotide concentration (and a comparison of these signals with the signal obtained with unconjugated oligonucleotides). [Figure 27]The performance of four different labeled detection oligonucleotide constructs in an amplified ECL assay for three different analytes in (a) sequential and (b) simultaneous assay formats is compared. [Figure 28] The performance of two different labeled detection oligonucleotide constructs in an amplified ECL assay for three different analytes in sequential and simultaneous assay formats is compared. [Figure 29] The effect of crosslinker challenge ratio and conjugation protocol on the production and performance of antibody-oligonucleotide conjugates is shown, providing (a) label per protein (L / P) and performance in an amplified ECL assay, and (b) characterization of conjugate formation by gel electrophoresis. [Figure 30] The performance of different biotin-anchor oligonucleotide constructs in an amplified ECL assay for two analytes is compared. [Figure 31] (a) The effect of probe length of a detection antibody-oligonucleotide probe conjugate on the signal, background, and detection limit obtained in an assay using the conjugate, and (b) the effect of GC content of the probe on the sensitivity of the ligation step in the assay to different temperatures. [Figure 32] The performance of different Circ oligonucleotide constructs in an amplified ECL assay for the analyte is compared. [Figure 33] FIG. 1 is a schematic diagram of the improvements made during optimization of reagents for performing an amplified ECL assay. [Figure 34] (a) Calibration curves for assays of three analytes comparing the signals obtained in conventional and amplified formats, and (b) comparison of the detection limits obtained in the two formats for 41 different assays targeting different analytes. DETAILED DESCRIPTION OF THE INVENTION

[0151] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless the context otherwise requires, the singular shall include the plural and the plural shall include the singular. The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0152] As used herein, the term "about" is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value.

[0153] As used herein, "between" is a range that includes the endpoints of the range. For example, a number between x and y specifically includes the numbers x and y, as well as any number that falls within the range between x and y.

[0154] As used herein, a "kit" refers to a set of components provided or assembled together for use, for example, to make a composition, manufacture a device, or perform a method. A kit may include one or more components. The components of a kit may be provided in one package or multiple packages, each package containing one or more components. The listed components of a kit may then be provided as a single physical component or as multiple components that are combined for use in the kit. For example, instrument components of a kit may be provided fully assembled or as multiple instrument components that are assembled before use. Similarly, liquid reagent components of a kit may be provided as a complete liquid formulation in a container, as one or more dry reagents and one or more liquid diluents that are combined to provide a complete liquid formulation, or as two or more liquid solutions that are combined to provide a complete liquid formulation. As is known in the art, assay kit components are often shipped and stored separately due to different storage needs, such as storage temperatures of 4°C versus -70°C.

[0155] In the context of the analyte to be measured in an assay, or the grade of reagents used in an assay, the term "multiple" refers to multiple structurally and / or functionally distinct analytes or reagents (e.g., capture antibody A and capture antibody B), rather than simply multiple copies of an analyte or reagent (e.g., capture antibody A and another copy of capture antibody A). For example, the term "multiple immobilized antigens" refers to multiple structurally or functionally distinct antigens being immobilized and does not describe a situation in which multiple copies of only a single antigen are present. However, the use of the term "multiple" in this context does not exclude the possibility of multiple copies of either the multiple analytes or reagents being present. For example, multiple immobilized antigens may refer to immobilized antigens that include one or more copies of antigen A and one or more copies of antigen B.

[0156] As used herein, the term "polypeptide" is intended to encompass a single "polypeptide" as well as multiple "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (i.e., peptide bonds). The term "polypeptide" refers to any chain or chains of amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or other terms used to refer to a chain or chains of amino acids are included in the definition of "polypeptide," and the term "polypeptide" can be used in place of or synonymously with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-natural amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a designated nucleic acid sequence. Polypeptides can be generated in any manner, including chemical synthesis. In the context of a polypeptide, a "linear sequence" or "sequence" is the order of amino acids in a polypeptide from amino to carboxyl terminal, in which residues adjacent to each other in the sequence are adjacent in the primary structure of the polypeptide.

[0157] A "binding reagent" or "binding substance" refers to a reagent or substance characterized by its ability to bind to another substance (sometimes referred to as a "binding partner"). The binding reagents, binding substances, and binding partners of the present invention include "antigen-binding substances," a term that refers to antibodies, antibody fragments, antibody derivatives, antibody analogs, antibody variants, engineered antibodies, and other substances that bind to antigens in a manner similar to antibodies. Antigen-binding substances include substances that contain at least one heavy or light chain complementarity-determining region (CDR) of an antibody. Antigen-binding substances include substances that contain at least two CDRs from one or more antibodies. Antigen-binding substances include substances that contain at least three CDRs from one or more antibodies. Antigen-binding substances include substances that contain at least four CDRs from one or more antibodies. Antigen-binding substances include substances that contain at least five CDRs from one or more antibodies. Antigen-binding substances include substances that contain at least six CDRs from one or more antibodies.

[0158] Antigen-binding agents derived from antibodies or other antigen-binding agents may include adding, removing, or substituting one or more antibodies to the antibody sequence to improve the affinity and / or specificity of the antibody for its desired target (e.g., by using established methods for "affinity maturation" of antibodies) and / or to improve other properties of the reagent (e.g., improving stability or reducing interactions with potentially interfering components in the sample, such as complement, rheumatoid factor, or anti-species antibodies). In one embodiment, the antigen-binding agent is the Fab portion of an antibody, reducing potential interference with Fc-binding components of the sample. In one embodiment for measuring an analyte in a sample from a particular species, the antigen-binding agent is a modified form of an antibody designed to match the antibody class of that species (e.g., a murine antibody may be humanized for use in assays performed on human samples to avoid interference from human anti-mouse antibodies (i.e., human antibodies targeting murine antibodies) that are often present in human samples).

[0159] As used herein, a "human" or "fully human" antibody includes antibodies having the amino acid sequence of a human immunoglobulin, as described below and, for example, in U.S. Pat. No. 5,939,598, and includes antibodies isolated from a human immunoglobulin library or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins. A "human" or "fully human" antibody also includes antibodies comprising at least a heavy chain variable domain, or at least heavy and light chain variable domains, wherein the variable domains have the amino acid sequence of a human immunoglobulin variable domain. A "humanized" antibody is an antibody from another species whose constant and framework sequences have been modified to conform to the class of human antibodies while retaining the ability of the original antibody to bind to its target antigen.

[0160] The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin contains at least a heavy chain variable domain, and usually contains at least heavy and light chain variable domains. The structure of basic immunoglobulins in vertebrate systems is relatively well understood. See, e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press).

[0161] The term "immunoglobulin" encompasses a wide variety of biochemically distinguishable polypeptide classes. Those skilled in the art will understand that heavy chains produced by animal species can be classified into different classes, such as gamma, mu, alpha, delta, or epsilon, and that these classes can be further divided into subclasses (e.g., gamma 1 through gamma 4). It is the nature of this chain that determines the "class" of an antibody, such as IgG, IgM, IgA, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and are known to confer functional differentiation. Modified versions of each of these classes and isotypes are readily discernible to those skilled in the art in light of the present disclosure and, therefore, are within the scope of the present invention. All immunoglobulin classes are clearly within the scope of the present invention. The following discussion generally relates to immunoglobulins of the IgG class. Most forms of IgG produced in mammals contain two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are usually linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains, which begin at the mouth of the "Y" and continue through the variable region. The exact molecular weight can vary between species and subclasses. Some species, such as camelids, may also produce IgG forms lacking light chains.

[0162] Light chains may be produced in different, classifiable forms, such as kappa (Vκ) or lambda (Vλ). Each heavy chain class can bind to either kappa or lambda light chains. Generally, light and heavy chains are covalently linked to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the base of each chain.

[0163] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light chain portion (Vκ or Vλ—collectively "VL") and the heavy chain portion (VH) determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases as they become more distal from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 and CL domains typically comprise the carboxy termini of the heavy and light chains, respectively.

[0164] As mentioned above, the variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL and VH domains of an antibody, or a subset of the complementarity-determining regions (CDRs) within these variable domains, combine to form the variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site at the end of each arm of a Y. More specifically, the antigen-binding site is usually defined by three CDRs from each of the VH and VL chains. As used herein, the terms HCDR1, HCDR2, and HCDR3 refer to VH CDR1, VH CDR2, and VH CDR3, respectively. Similarly, as used herein, the terms LCDR1, LCDR2, and LCDR3 refer to VL CDR1, VL CDR2, and VL CDR3, respectively. In some cases, for example, in certain immunoglobulins derived from camelid species or engineered based on camelid immunoglobulins, the complete immunoglobulin may consist only of heavy chains, excluding light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0165] In natural antibodies, each antigen-binding domain typically contains six "complementarity-determining regions" or "CDRs," short, noncontiguous sequences of amino acids specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, referred to as "framework" regions, exhibit less inter-molecular variability. The framework regions primarily adopt a sheet conformation, while the CDRs form loops connecting, and in some cases, part of, the beta-sheet structure. Thus, the framework regions function to form a scaffold that orients the CDRs through interchain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and framework regions, respectively, can be readily identified for a given heavy or light chain variable domain because they have been precisely defined (see below).

[0166] Where there are more than one definition for a term used and / or accepted in the art, the definition of the term used herein is intended to encompass all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. This region is described in Kabat et al. (1983) U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest," Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), and recently updated by Kunik et al., Nucl. Acids Res. 40:W521-W524 (2012), which are incorporated herein by reference, and the definitions include overlapping or subsets of amino acid residues relative to each other. Nevertheless, application of any definition to refer to the CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. One of ordinary skill in the art can routinely determine which residues comprise a particular CDR, given the variable region amino acid sequence of an antibody.

[0167] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence without reliance on experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al. (1983) US Department of Health and Human Services, "Sequence of Proteins of Immunological Interest," as disclosed in Paratome, an online tool for systematic identification of antigen-binding regions of antibodies based on sequence or structure.

[0168] Refers to the numbering system specified by Kunik et al., Nucl. Acids Res. 40:W521-W524 (2012). Paratome-based analyses typically conform to Kabat numbering, but may also include residues adjacent to conventional CDRs.

[0169] Antibodies or antigen-binding fragments, variants, or derivatives thereof of the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab'), Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), fragments comprising either the VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. scFv constructs are known in the art and are described, for example, in U.S. Patent No. 5,892,019. Immunoglobulins or antibodies of the present invention can be of any type (IgG, IgE, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.), or subclass of immunoglobulin molecule. Many approaches for producing antibodies and other antigen-binding substances are known in the art, including production from B-cell cultures, hybridoma cell cultures, cultures or transiently or permanently transfected host cell lines, bacteria, yeast, plant cells and insect cells.

[0170] As used herein, the term "heavy chain portion" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion comprises at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide for use in the present invention may comprise a polypeptide chain comprising a CH1 domain, a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain, a polypeptide chain comprising a CH1 domain and a CH3 domain, a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present invention comprises a polypeptide chain comprising a CH3 domain. Furthermore, a binding polypeptide for use in the present invention may lack at least a portion of a CH2 domain (e.g., all or part of the CH2 domain). As noted above, it will be understood by those skilled in the art that these domains (eg, heavy chain portions) may be engineered to differ in amino acid sequence from that of a native immunoglobulin molecule.

[0171] In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof, disclosed herein, the heavy chain portion of one polypeptide chain of the multimer is identical to that of the second polypeptide chain of the multimer. Alternatively, the monomers comprising the heavy chain portions of the present invention are not identical. For example, each monomer may comprise a different target binding site, for example, to form a bispecific antibody.

[0172] As used herein, the term "light chain portion" includes amino acid sequences derived from an immunoglobulin light chain, e.g., a kappa or lambda light chain. Preferably, the light chain portion includes at least one of a VL or CL domain.

[0173] The "antigen-binding substance" defined above can be described or specified in terms of the epitope or portion of the substance that it recognizes or specifically binds to. The portion of a target polypeptide that specifically interacts with the antigen-binding domain of an antibody is an "epitope" or "antigenic determinant." A target polypeptide can contain a single epitope, but typically contains at least two epitopes, and can contain any number of epitopes depending on the size, conformation, and type of antigen. Furthermore, it should be noted that an "epitope" on a target polypeptide can be or can include non-polypeptide elements; for example, an epitope can include a carbohydrate side chain.

[0174] The minimum size of a peptide or polypeptide epitope of an antigen-binding substance is believed to be about 4 to 5 amino acids. A peptide or polypeptide epitope preferably contains at least 7, more preferably at least 9, and most preferably at least about 15 to about 30 amino acids. Because CDRs can recognize antigenic peptides or polypeptides in their tertiary forms, the amino acids comprising the epitope need not be contiguous, and in some cases may not even be on the same peptide chain. A peptide or polypeptide epitope recognized by an antigen-binding molecule of the present invention may contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or about 15 to about 30 contiguous or noncontiguous amino acids.

[0175] By "antigen" is meant a substance capable of specifically or preferentially binding to an antibody-binding substance, such as an antibody or antigen-binding fragment thereof.

[0176] In the context of antibodies (and, by analogy, other antigen-binding substances), "preferentially binds" means that the antibody specifically binds to an epitope more readily than it binds to a reference epitope (which may be a related, similar, homologous, or analogous epitope). Thus, an antibody that "preferentially binds" to a given epitope will bind that epitope more readily than the reference epitope, even if such an antibody may cross-react with the reference epitope.

[0177] In the context of antibodies (and, by analogy, other antigen-binding substances), "specifically binds" means that the antibody binds to an epitope via its antigen-binding domain, and that the binding involves complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it preferentially binds to that epitope via its antigen-binding domain compared to a random, unrelated epitope.

[0178] As a non-limiting example, an antibody can be considered to bind preferentially to a first epitope relative to a second epitope if, under the experimental conditions (e.g., conditions for performing the assay), the amount of binding to the first epitope relative to the amount of binding to the second epitope (expressed as a ratio) is greater than 1, 10, 100, 1,000, or 10,000. In another non-limiting example, an antibody can be considered to bind preferentially to a first epitope relative to a second epitope if the equilibrium dissociation constant (KD) for binding to the first epitope is less than 1, 0.1, 0.01, 0.001, or 0.0001 relative to the antibody's KD for the second epitope (expressed as a ratio). In another non-limiting example, an antibody can be considered to bind a first epitope preferentially relative to a second epitope if the association rate constant (also called on-rate or k) for binding to the first epitope, relative to the antibody's k (expressed as a ratio), is greater than 1, 10, 100, 1,000, or 10,000. In another non-limiting example, an antibody can be considered to bind a first epitope preferentially relative to a second epitope if the dissociation rate constant (also called off-rate or k) for dissociating from the first epitope, relative to the antibody's k (expressed as a ratio), is less than 1, 0.1, 0.01, 0.001, or 0.0001.

[0179] When comparing the preference of two antibodies for a first epitope over a second epitope, the antibody with a stronger preference for the first epitope can be said to be more specific for the first epitope. The strength of preference can be determined, for example, by the ratio of the amounts of binding to the two epitopes under the selected experimental conditions, by the ratio of the KD values, by the ratio of the kon values, and / or by the ratio of the koff values ​​(these ratios are determined as described in the last paragraph).

[0180] Generally, the sensitivity and robustness of an assay are improved by using an antibody with high affinity and a slow off-rate. In embodiments, the antibodies or other antigen-binding substances disclosed herein bind to a target antigen with a KD of 10 nM, 1 nM, 500 pM, 200 pM, 100 pM, 30 pM, or 10 pM or less. In embodiments, the antibodies or other antigen-binding substances disclosed herein bind to a target polypeptide with a KD of 10 nM, 1 nM, 500 pM, 200 pM, 100 pM, 30 pM, or 10 pM or less. In embodiments, the antibodies or other antigen-binding substances disclosed herein dissociate from a target antigen with a koff of 5 x 10 sec, 10 sec, 5 x 10 sec, or 10 sec or less. In embodiments, an antibody or other antigen-binding agent of the invention disclosed herein dissociates from a target polypeptide with a koff of 5×10 −4 s −1 , 10 −4 s −1 , 5×10 −5 s −1 , or 10 −5 s −1 , 5×10 −6 s −1 , 10 −6 s −1 , 5×10 −7 s −1 or 10 −7 s −1 or less.

[0181] Antigen-binding substances of the present invention may be "multispecific," bispecific, trispecific, or of higher multispecificity, meaning that they simultaneously recognize and bind to two or more different epitopes present on one or more different antigens (e.g., proteins). Thus, whether an antigen-binding substance is "monospecific" or "multispecific," e.g., "bispecific," refers to the number of different epitopes with which the binding polypeptide reacts. Multispecific antibodies may be specific for different epitopes of a target, or may be specific for heterologous epitopes, such as a target polypeptide as well as a heterologous polypeptide.

[0182] As previously mentioned, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of the immunoglobulin heavy chain.

[0183] As used herein, the term "hinge region" includes the portion of the heavy chain that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol. 161:4083 (1998)).

[0184] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a second thiol group. In most native IgGs, the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system).

[0185] As used herein, the terms "linked," "fused," and "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components by any means, including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous, longer ORF in a manner that maintains the correct translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more segments corresponding to polypeptides encoded by the original ORFs (these segments are not normally so joined in nature). Thus, although the reading frame is made continuous throughout the fused segments, the segments may be physically or spatially separated, for example, by in-frame linker sequences. For example, polynucleotides encoding CDRs of immunoglobulin variable regions may be fused in-frame but separated by polynucleotides encoding at least one immunoglobulin framework region or additional CDR regions, so long as the "fused" CDRs are co-translated as part of a continuous polypeptide.

[0186] As used herein, the term "oligonucleotide" refers to a short polymer of nucleic acid, such as DNA or RNA. In embodiments, oligonucleotides are about 5 to about 150 nucleotides in length. Oligonucleotides can be designed to specifically hybridize to DNA or RNA sequences, for example, as probes for detecting specific sequences complementary to the oligonucleotide. Oligonucleotides can be single-stranded or double-stranded. The oligonucleotides described herein can be produced in any manner, including chemical synthesis.

[0187] The term "oligonucleotide" may include structural analogs, including non-natural chemical structures. In embodiments, the oligonucleotide includes modifications at its 5' or 3' end, internal modifications, or a combination thereof. Methods for modifying nucleotides and / or nucleic acids are known in the art. Examples of oligonucleotide modifications include, but are not limited to, attachment modifications that can be used to attach the oligonucleotide to another substance or surface, fluorophores and fluorescence quenchers, modified bases, phosphorylation modifications, for example, when the oligonucleotide is used as a ligase substrate, spacers, for example, to create distance within the oligonucleotide between the nucleic acid sequence and the reactive functional group, and phosphorothioate linkages, for example, to increase the resistance of the oligonucleotide to nuclease degradation. Exemplary modifications are provided in Table A below. [Table 1]

[0188] In embodiments, the modifications include biotin, streptavidin, avidin, amino groups, thiol groups, aldehyde groups, hydrazide groups, azide groups, alkyne groups, maleimide groups, and / or iodoacetamide groups.

[0189] In one example, a nucleotide and / or nucleic acid may contain a chemical modification that provides a reactive functional group that links it to another substance, such as a label, or that can be linked to another substance, such as a label, for example, through the use of an amine- or thiol-modified nucleotide base, phosphate, or sugar. The term "reactive functional group" refers to an atom or related group of atoms that can undergo further chemical reaction, for example, to form a covalent bond with another functional group. Examples of reactive functional groups include, but are not limited to, amino, thiol, hydroxy, and carbonyl groups. In one embodiment, a reactive functional group includes a reactive thiol group. Labels that can be linked to a nucleotide or nucleic acid via these chemical modifications include, but are not limited to, detectable moieties such as biotin, haptens, fluorophores, and electrochemiluminescence (ECL) labels.

[0190] In another embodiment, the nucleotides of an oligonucleotide can be modified to prevent enzymatic or chemical elongation of the nucleic acid chain into which they are incorporated, for example, by replacing the ribose or deoxyribose group with dideoxyribose. In another example, the backbone components linking the nucleotide bases (e.g., sugar and / or phosphate groups) together can be modified or replaced, for example, by using peptide nucleic acids (PNAs) or by incorporating ribose analogs, such as those found in 2'-O-methyl-substituted RNA, locked nucleic acids, bridged nucleic acids, and morpholino nucleic acids. These "backbone" analogs can be present in one, some, or all of the backbone linkages of a nucleic acid and / or oligonucleotide and can provide certain advantages, such as hybridization products with improved binding stability and / or stability of linkages to nucleases. Another example of structural analogs of nucleotides and nucleic acids can include unnatural nucleotide bases. An unnatural (also called a "non-canonical" base) may hybridize with a natural (canonical) base or with another unnatural base.

[0191] A "probe" in the context of nucleic acids generally refers to an oligonucleotide (usually 5-50 bases) that contains a sequence that may be complementary to another nucleic acid sequence. In some applications, a probe that hybridizes to a complementary region of a target sequence allows for primed extension of the probe by a polymerase, serving as an initiation point for replication of an adjacent single-stranded region on the target sequence (in such cases, the probe may also be referred to as a "primer").

[0192] As used herein, a "nucleic acid probe" includes (i) an oligonucleotide modified with one or more reactive moieties that can be used to react and thereby link to another substance, or (ii) an oligonucleotide that is linked to another substance (e.g., by a reaction described in (i)). In embodiments, the nucleic acid probe is linked to a detection reagent. In embodiments, the nucleic acid probe is linked to a polypeptide. In embodiments, the nucleic acid probe is linked to an antibody or other antigen-binding substance. In embodiments, the reactive moiety is a reactive functional group. In embodiments, the reactive functional group is an alkene, strained alkene, alkyne, halide, alcohol, thiol, amine, phosphate, aldehyde, ketone, carboxylic acid, carboxylate, amide, ester, thioester, acylphosphate, acid halide, nitrile, acid anhydride, hydrazine, tetrazine, or azide. In embodiments, the reactive moiety is a member of a binding reagent-binding partner pair, e.g., biotin or streptavidin. In embodiments, the nucleic acid probe comprises a sequence complementary to a template oligonucleotide for amplification. In embodiments, the nucleic acid probe binds to the circular template oligonucleotide for rolling circle amplification (RCA) of the circular template oligonucleotide. In embodiments, the RCA generates an extended sequence. In embodiments, the nucleic acid probe is a primer for RCA.

[0193] A "labeled probe" refers to a compound comprising an oligonucleotide and a detectable moiety (also referred to as a "label"). A detectable moiety (or label) refers to a chemical group or moiety that has a detectable physical property or is capable of exhibiting a detectable physical property to a chemical group or moiety, including, for example, an enzyme that catalyzes the conversion of a substrate to a detectable product. Labels can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other methods. Examples of labels include, but are not limited to, radioisotopes, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, electrochemiluminescent moieties, magnetic particles, and bioluminescent moieties. In another embodiment, the label is a compound that is a member of a binding pair, where a first member of the binding pair (which can be referred to as the "primary binding reagent") is attached to a substrate, e.g., an oligonucleotide, and the other member of the binding pair (which can be referred to as the "secondary binding reagent") has a detectable physical property. Non-limiting examples of binding pairs include biotin and streptavidin, or avidin, complementary oligonucleotides, and antibody / antigen binding pairs. In embodiments, the "labeled probe" comprises an oligonucleotide and an electrochemiluminescent moiety. In embodiments, the oligonucleotide of the labeled probe comprises a sequence complementary to the extended sequence of the methods described herein. In embodiments, the labeled probe binds to the extended sequence, and the detectable label functions to allow for measurement of the amount of the extended sequence. In embodiments of the methods described herein, the amount of analyte in the sample is determined by measuring the amount of the labeled probe, i.e., the extended sequence.

[0194] "Complementary" refers to nucleic acid molecules or sequences of nucleic acid molecules that bind (or "hybridize") to each other through the formation of hydrogen bonds, for example, according to the Watson-Crick base pairing model. For example, hybridization can occur between two complementary DNA molecules (DNA-DNA hybridization), two RNA molecules (RNA-RNA hybridization), or complementary DNA and RNA molecules (DNA-RNA hybridization). Hybridization can occur between short nucleotide sequences that are complementary to a portion of a longer nucleotide sequence. Hybridization can occur between sequences that do not have 100% "sequence complementarity" (i.e., sequences in which less than 100% of the nucleotides align based on a base pairing model, such as the Watson-Crick base pairing model), but sequences with low sequence complementarity are less stable and less likely to hybridize than sequences with high sequence complementarity. In one embodiment, the nucleotides of complementary sequences have 100% sequence complementarity based on the Watson-Crick model. In another embodiment, the nucleotides of the complementary sequences have at least about 90%, 95%, 97%, or 99% sequence complementarity based on the Watson-Crick model.

[0195] Two nucleic acids are "hybridizable" or "hybridized" if they are capable of hybridizing or have hybridized, respectively. Whether two complementary sequences hybridize can depend on the stringency of the hybridization conditions, which can vary depending on conditions such as temperature, solvent, ionic strength, and other parameters (approaches to creating stringent hybridization conditions are well known and are exemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein). The stringency of the hybridization conditions can be selected to provide for the selective formation or maintenance of the desired hybridization product of two complementary nucleic acid sequences in the presence of other potentially cross-reactive or interfering sequences. Stringent conditions are sequence-dependent—typically, longer complementary sequences hybridize specifically at higher temperatures than shorter complementary sequences. Generally, stringent hybridization conditions are about 5°C to about 10°C lower than the thermal melting point (Tm) of a particular nucleotide sequence (i.e., the temperature at which 50% of the sequence hybridizes to a substantially complementary sequence) at a defined ionic strength, concentration of chemical denaturant, pH, and concentration of hybridization partner. Generally, nucleotide sequences with a higher proportion of G and C bases hybridize under more stringent conditions than nucleotide sequences with a lower proportion of G and C bases. Generally, stringency can be increased by increasing the temperature, increasing the pH, decreasing the ionic strength, and / or increasing the concentration of chemical nucleic acid denaturants (e.g., formamide, dimethylformamide, dimethyl sulfoxide, ethylene glycol, propylene glycol, ethylene carbonate, etc.).Stringent hybridization conditions typically include salt concentrations or ionic strengths of less than about 1 M, 500 mM, 200 mM, 100 mM, or 50 mM; hybridization temperatures of greater than about 20° C., 30° C., 40° C., 60° C., or 80° C.; and chemical denaturant concentrations of greater than about 10%, 20%, 30%, 40%, or 50%. Because many factors can affect hybridization stringency, the combination of parameters may be more important than the absolute value of any single parameter.

[0196] Exemplary hybridization conditions include a buffer solution having an ionic strength of about 20-200 mM (e.g., phosphate, Tris, or HEPES buffer, with about 20-200 mM buffer components) at a temperature of about 15-40°C, a pH of about 6.5-8.5. For example, the buffer solution may contain a salt concentration of about 10 mM to about 1 M, about 20 mM to about 500 mM, about 30 mM to about 100 mM, about 40 mM to about 80 mM, or about 50 mM. Exemplary salts include NaCl, KCl, (NH4)2SO4, Na2SO4, and CH3COONH4. A specific example is 50 M Tris-HCl, pH 7.4, at room temperature (about 18-25°C). Another specific example is 66 mM potassium acetate, 50 mM potassium chloride, 10 mM magnesium acetate, 33 mM Tris buffer, pH 8.2, at 22°C to 37°C (or about 27°C).

[0197] The term "sequence identity" or "% identity" in the context of nucleic acid or amino acid sequences refers to the percentage of residues in the compared sequences that are the same when the sequences are aligned over a particular comparison window. In some embodiments, only certain portions of two or more sequences are aligned to determine sequence identity. In some embodiments, only certain domains of two or more sequences are aligned to determine sequence similarity. The comparison window can be a segment of at least 10 to over 1000 residues, at least 20 to about 1000 residues, or at least 50 to 500 residues, over which sequences can be aligned and compared. Alignment methods for determining sequence identity are well known and can be performed using publicly available databases such as BLAST. "Percent identity" or "% identity" when referring to amino acid sequences can be determined by methods known in the art. For example, in some embodiments, the "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul, Proceedings of the National Academy of Sciences USA 87:2264-2268 (1990), modified as in Karlin and Altschul, Proceedings of the National Academy of Sciences USA 90:5873-5877 (1993). Such an algorithm is incorporated into BLAST programs, such as BLAST+ or the NBLAST and XBLAST programs described in Altschul et al., Journal of Molecular Biology, 215:403-410 (1990). BLAST protein searches can be performed, for example, using a program such as the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules of the present disclosure. If gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Research 25(17):3389-3402 (1997).When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0198] In some embodiments, a polypeptide or nucleic acid molecule has 70%, at least 70%, 75%, at least 75%, 80%, at least 80%, 85%, at least 85%, 90%, at least 90%, 95%, at least 95%, 97%, at least 97%, 98%, at least 98%, 99%, or at least 99% or 100% sequence identity to a reference polypeptide or nucleic acid molecule (or a fragment of a reference polypeptide or nucleic acid molecule), respectively. In some embodiments, a polypeptide or nucleic acid molecule has about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% sequence identity to a reference polypeptide or nucleic acid molecule (or a fragment of a reference polypeptide or nucleic acid molecule), respectively.

[0199] Hybridization occurs when two nucleic acids contain complementary sequences, although mismatches between bases may occur depending on the stringency of hybridization. In embodiments, a sequence capable of hybridizing to the complement of a second sequence is substantially similar to the second sequence. In embodiments, a sequence capable of hybridizing to the complement of a second sequence has 70%, at least 70%, 75%, at least 75%, 80%, at least 80%, 85%, at least 85%, 90%, at least 90%, 95%, at least 95%, 97%, at least 97%, 98%, at least 98%, 99%, or at least 99% or 100% sequence identity with the second sequence.

[0200] The present invention includes immunoassays that involve (i) anchoring detection complexes formed between a target analyte and one or more analyte-binding reagents used in the assay, and / or (ii) amplifying signals from labeled detection complexes. Anchoring can be used to stabilize complexes containing low-binding affinity interactions and / or high-molecular-weight labels or label moieties. Signal amplification can be achieved by amplifying the detectable signal for each individual detection complex by attaching an extender probe to the binding complex containing multiple labels or detection label moieties. In a preferred embodiment, the method involves attaching an extender probe containing 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 few binding events, even individual analyte-binding reagent complexes. The basic approach is not limited to immunoassays and can be used to perform binding assays using other classes of binding reagents.

[0201] In embodiments, the present invention provides binding assays that use surface-bound anchor reagents to attach and stabilize detection complexes containing the analyte of interest to a surface. This approach can 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 before subsequent processing. The use of anchor reagents in binding assays is illustrated in Figure 1(a). A surface (101) includes a capture reagent (102) that binds to analyte A, and an anchor reagent (103). In one or more steps, the analyte binds to a capture reagent and a detection reagent (104) that also binds to the analyte, and 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 each of the capture reagent and the detection reagent sequentially (in either order). Thus, a complex (106) is formed on the surface containing the capture reagent, analyte, and detection reagent. The probe is extended to form an extension sequence (107) that includes an anchor region that binds to the anchor reagent. The extension sequence binds to the anchor reagent and the amount of extension sequence bound to the surface is measured.

[0202] Those skilled in the art of binding assays will readily appreciate the range of capture reagents and companion binding partners that can be used in the present methods. 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, mimotope / antibody pairs, aptamer / target molecule pairs, hybridization partners, and intercalator / target molecule pairs. In one embodiment, the binding assay uses antibodies or other receptor proteins as capture and / or detection reagents for the analyte of interest. The term "antibody" includes intact antibody molecules (including hybrid antibodies assembled by the in vitro reassociation of antibody subunits), antibody fragments, and recombinant protein constructs comprising the antigen-binding domain of an antibody (e.g., as described in Porter, R.R. and Weir, R.C.J. Cell Physiol., 67(Suppl); 51-64 (1966) and Hochman, I. Inbar, D. and Givol, D. Biochemistry 12:1130 (1973)), as well as antibody constructs that have been chemically modified, for example, by the introduction of a detectable label.

[0203] Similarly, the anchor reagent and the corresponding anchor member or anchor region can be any suitable binding pair, such as a receptor / ligand pair, an antibody / antigen, a natural or synthetic receptor / ligand pair, a hapten / antibody pair, an antigen / antibody pair, an epitope / antibody pair, a mimotope / antibody pair, an aptamer / target molecule pair, a hybridization partner, an intercalator / target molecule pair, and the use of a surface and anchor reagent that binds via electrostatic charge. For example, the anchor reagent can be an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimotope, and the corresponding anchor region comprises a complementary oligonucleotide sequence, an aptamer ligand, an aptamer, an antigen, an antibody, a receptor, a ligand, or an antibody, respectively. In a specific embodiment, the anchor region is an oligonucleotide sequence, and the anchor reagent comprises a DNA-binding protein. Alternatively, if the anchor region is a double-stranded oligonucleotide sequence, the anchor reagent can comprise an intercalator. In additional embodiments, the anchor region may contain one or more modified oligonucleotide bases, and the corresponding anchor reagent may contain one or more moieties that bind to the modified bases on the anchor region. For example, the modified base may contain a hapten or a ligand, and the corresponding anchor reagent may contain one or more antibodies or ligands specific to the hapten or ligand, respectively. Furthermore, the anchor region may contain multiple labeled nucleotide bases that can be used to detect the detection complex.

[0204] In a specific embodiment shown in FIG. 1(b), the anchor reagent bound to the surface comprises an oligonucleotide used to anchor the detection complex to the surface. The anchor oligonucleotide sequence binds to a complementary oligonucleotide sequence attached to the detection complex. In this embodiment, the surface (108) comprises a capture reagent (109) that binds to analyte A and an anchor reagent (110) comprising an anchor oligonucleotide sequence (111). In one or more steps, the analyte binds to the capture reagent and detection reagent (112), which also binds to the analyte, and the detection reagent is linked to the nucleic acid probe (113). As described above with reference to FIG. 1(a), the analyte can bind to the capture reagent and detection reagent simultaneously or substantially simultaneously, or the analyte can bind to each of the capture reagent and detection reagent sequentially (in either order). Thus, a complex (114) is formed on the surface comprising the binding reagent, analyte, and detection reagent. The probe is extended to form an extension sequence (115) comprising 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. The extension sequence may also include a detection sequence, in which case a detection probe complementary to the detection sequence may be added and bound to the extension sequence, and the label may be measured to determine the amount of extension sequence on the surface.

[0205] In certain embodiments of the method shown in Figure 1(b), an anchor reagent is used to attach the detection complex to the surface, and the probe attached to the detection complex is extended to generate an extension region that binds to the anchor reagent. The embodiment further includes binding the nucleic acid probe on the detection reagent to either (i) a circular oligonucleotide that is then subjected to rolling circle amplification to generate an amplicon that binds to the anchor reagent, or (ii) a linear oligonucleotide whose ends bind to the nucleic acid probe and link to form a circular oligonucleotide, which is then subjected to rolling circle amplification to generate an amplicon that binds to the anchor reagent. The surface includes a capture reagent and an anchor reagent. In one or more steps, the analyte forms a detection complex on the surface by binding to the detection reagent that includes the capture reagent and the nucleic acid probe. The detection complex is contacted with either (i) a circular oligonucleotide that includes a sequence complementary to the nucleic acid probe, or (ii) a linear oligonucleotide that has a first terminal sequence and a second terminal sequence that correspond to the non-overlapping region of the nucleic acid probe. A circular or linear oligonucleotide hybridizes to the nucleic acid probe, and when a linear oligonucleotide is used, the terminal sequence of the linear oligonucleotide is ligated from the circular target sequence hybridized to the nucleic acid probe. The nucleic acid probe is extended by rolling circle hybridization to generate an extended sequence containing a binding reagent that binds to the anchor reagent, and the amount of extended sequence bound to the surface is measured. The extended sequence may also contain one or more detection sequences complementary to labeled detection probes, which hybridize to amplicons and are used to measure the amount of amplicons bound to the surface. In an alternative embodiment, the extension process incorporates labeled nucleotide bases into the extended sequence, which are used to directly detect amplicons on the surface without adding one or more labeled probes complementary to the amplicons. The generation of an extended sequence complementary to the sequence of the anchor oligonucleotide and / or detection probe can be achieved by incorporating a circular or linear oligonucleotide sequence from the anchor and / or detection probe.

[0206] A detection complex can contain one or more detection reagents, for example, to increase the specificity of the assay for the analyte. For example, if the assay is designed to emit a detectable signal, the use of multiple detection reagents can increase the specificity of the assay if each detection reagent is in close proximity to the analyte, or if the signal from a single detection reagent bound to the analyte is distinguishable from the signal emitted from multiple detection reagents bound to the analyte. One embodiment of such an assay is shown in Figure 1(c). 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), which bind to the analyte, and each of the first and second detection reagents binds to a nucleic acid probe (122 and 123, respectively, the first and second nucleic acid probes). The analyte can bind to the capture reagent and detection reagent simultaneously, substantially simultaneously, or in sequential steps. 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 contains a complement of the anchor sequence that is complementary to the anchor sequence. In the penultimate step, the anchor sequence hybridizes to the complement of the anchor sequence, and the amount of extended sequence bound to the surface is measured.

[0207] A specific embodiment of the method shown in Figure 1(c) is shown in Figure 2(a), in which 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 bound to proximity probes. The method further includes binding the detection reagent to a connector sequence, which is then ligated to form a circular target sequence, and subjecting it to rolling circle amplification to generate an amplicon that binds to the anchor reagent. The surface (201) includes a capture reagent (202) and an anchor reagent (203). In one or more steps, the analyte forms a detection complex (208) on the surface by binding to the capture reagent, a first detection reagent (204) comprising a first proximity probe (205), and a second detection reagent (206) comprising a second proximity probe (207). The detection complex contacts two connector sequences (209a and 209b), each of which contains 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 hybridize to the first and second proximity probes, and the terminal sequence of the connector oligonucleotide is ligated from a circular target sequence (210) that hybridizes to both the first and second proximity probes. The second proximity probe is extended by rolling circle hybridization to produce an amplicon containing the 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 an alternative embodiment, the first proximity probe is extended and the second proximity probe may be capped or otherwise modified to prevent extension.) In the embodiment shown in Figure 2(a), the amplicon also contains two or more detection sequences complementary to labeled detection probes, which hybridize to the amplicon and are used to measure the amount of amplicon bound to the surface.In an alternative embodiment (not shown in Figure 2(a)), the extension process incorporates labeled nucleotide bases into the amplicon that are used to directly detect the amplicon on a surface without adding one or more labeled probes complementary to the amplicon. Figure 2(b) is a schematic diagram of the components of a connector sequence showing first and second connector oligonucleotides (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 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 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 link to form a circular target sequence that hybridizes to both the first and second proximity probes.

[0208] 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 terminal sequences (CE1 and CE2, respectively) complementary to non-overlapping regions of the first proximity probe. In this embodiment, only one ligation event is required to form the circular target sequence for rolling circle amplification (i.e., ligation of the terminals CE1 and CE2 hybridized to the first proximity probe), but priming / extension is from the second proximity probe, thus maintaining the proximity requirement of the two proximity probes. Preferably, the first proximity probe is capped or otherwise modified to prevent extension of the first probe.

[0209] The second proximity probe is then extended by rolling circle amplification of the circular target sequence to generate an amplicon containing a binding region that binds to the anchor reagent, and the amount of amplicon bound to the surface is measured.

[0210] 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 approximately 20-50 bases in length, preferably 25-40 bases in length, and most preferably approximately 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 connector terminal sequence. Alternatively, only one connector can 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 contains non-overlapping regions complementary to the two terminal sequences of the connector, CE1 and CE2, respectively, and the second proximity probe contains 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 is extended and the second proximity probe may be capped or otherwise modified to prevent extension.)

[0211] Thus, the embodiments shown in Figures 1-2 demonstrate that binding assays can be modified to incorporate anchoring reagents and / or amplify the signal from the detection complex. In preferred embodiments, anchoring reagents and signal amplification methods are used in the binding assays. In embodiments involving the use of anchoring reagents, the concentration of anchoring reagent present on the surface is 0.2-200 μg / mL, specifically 1.0-50 μg / mL, and more specifically 3.0-10 μg / mL. Alternatively, enhancement of the binding assay can be achieved using only one or the other method. Thus, the present invention includes assays that omit the anchoring reagent and use signal amplification methods such as those described in Figures 1-2.

[0212] In these embodiments, where the anchor reagent comprises an anchor sequence bound directly or indirectly (e.g., via a binding reaction) to a surface, methods established in the art for immobilizing oligonucleotides can be used to generate the anchor reagent, including covalent and non-covalent methods. The anchor reagent can be directly immobilized to the solid phase or indirectly immobilized via a secondary binding reagent, such as the targeting reagent described below. For example, the anchor reagent can be linked to or comprise a targeting reagent that binds to the complement of the targeting reagent immobilized on the solid phase. 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 via a cross-linking agent (e.g., the targeting reagent and the complement can be biotin and the cross-linking reagent can be a multivalent biotin-binding receptor such as streptavidin). In one embodiment, the targeting agent and its complement comprise a first oligonucleotide and a complementary oligonucleotide, a receptor-ligand pair, an antigen-antibody pair, a hapten-antibody pair, an epitope-antibody pair, a mimotope-antibody pair, an aptamer-target molecule pair, a hybridization partner, or an intercalator-target molecule pair. The targeting agents and complements used in a multiplex assay for multiple analytes are selected so that the targeting agent and complement associated with the capture or detection reagent for the analyte being 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 analytes being measured by the assay. For example, binding of a binding reagent to its associated binding domain (via its associated targeting agent and targeting agent complement) should be substantially greater than binding to binding domains associated with other analytes (and displaying different targeting agent complements). Preferably, the cross-reactivity of the binding of the analyte's capture or detection reagent to the binding domain associated with the other analyte compared to binding to the correct binding domain is <1%, more preferably <0.1%, and even more preferably <0.01%. In a preferred embodiment, the targeting agent / targeting agent complement comprises a pair of oligonucleotides comprising complementary sequences, and the targeting agent and its complement are contacted under conditions sufficient to hybridize the targeting agent to its complement.

[0213] When a targeting agent is used, there is some flexibility in immobilizing the anchor reagent used in the assay to a solid phase. In one embodiment, the anchor reagent is provided to the user pre-immobilized to the solid phase via targeting agent-targeting agent complement interaction. In another embodiment, the anchor reagent linked to the targeting agent and the solid phase supporting the immobilized targeting agent complement are provided as separate components. Thus, the assay further includes a step of immobilizing the anchor reagent to the solid phase by binding the targeting agent to its complement (directly or through the use of a cross-linking agent). This step can be performed before, simultaneously with, or after the step related to the formation of the detection complex.

[0214] 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 (covalently or non-covalently) to a surface 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. The protein can be modified with the anchor oligonucleotide and attached to the surface using known methods, for example, using the well-established heterobifunctional crosslinker sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), as shown in Figure 3. By reacting the N-hydroxysuccinimide (NHS) group of SMCC with bovine serum albumin (BSA), the BSA is labeled with a thiol-reactive maleimide group. The maleimide group then reacts with the thiol-modified oligonucleotide to form a BSA-oligonucleotide conjugate linked via a stable thioether bond. In one particular example, the array is formed by printing a series of BSA-oligonucleotide conjugates onto a graphitic carbon surface, preferably a screen-printed carbon ink electrode. Alternatively, if the protein is avidin or streptavidin, the anchor sequence can be linked to biotin and bound to the immobilized avidin or streptavidin via a biotin-avidin or biotin-streptavidin interaction.

[0215] The anchor oligonucleotide attached to the anchor reagent can be any sequence that hybridizes to the elongation sequence (or amplicon) developing during the elongation process. The anchor oligonucleotide can also contain a non-complementary region (e.g., a poly(A) sequence), which serves as a linker sequence between the surface and the complementary (hybridizing) region, with the complementary region extending away from the surface. In one embodiment, the hybridization sequence is selected for a region of the amplicon that is not involved in binding to the proximity probe or detection probe (the "inactive" region). In a more specific embodiment, the hybridization sequence is complementary to the entire length of the inactive 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 length complement to the inactive region of the amplicon, 25 nucleotides in length) - (20 nucleotide poly(A) arm), or (ii) (complement to a portion of the inactive region of the amplicon, 15 nucleotides in length) - (30 nucleotide poly(A) arm).

[0216] 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), a complex containing two proximity probes is contacted with one or more connector oligonucleotides (209a-209b or 211). 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 one of the connectors contains the same sequence as that used in the anchor reagent. During extension of the second proximity probe, the extension 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 including a labeled nucleotide in the RCA extension reaction). Alternatively, multiple labeled probes containing detectable labels are added and hybridized to the extended second proximity probes to measure the amount of analyte bound to the surface.

[0217] Any suitable amplification technique can be used to generate the extended sequence (or amplicon), including, but not limited to, PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (sustained synthesis reaction), and isothermal amplification methods, such as helicase-dependent amplification and rolling circle amplification (RCA). In a preferred embodiment, RCA is used because it offers significant advantages in terms of sensitivity, multiplexing, dynamic range, and scalability. The technique of RCA is 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 119, 2000; Faruqi et al., BMC Genomics 2:4, 2000; Nallur et al., Nucl. Acids Res. 29:e118, 2001; Dean et al., Genome Res. 11:1095 1099, 2001; Schweitzer et al., Nature Biotech. 20:359 365, 2002; 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 copy strand attached to the original target DNA, allowing for spatial resolution of the target and rapid amplification of the signal. RCA facilitates (i) detection of a single target molecule, (ii) amplification of signals from proteins as well as DNA and RNA, (iii) localization of amplified molecules on a solid surface, (iv) measurement of many different targets simultaneously, and (v) analysis of one or more targets in solution or solid phase. Spatial localization of RCA products with detection complexes is particularly advantageous when performing multiplex binding assays in array or particle-based formats.

[0218] A specific embodiment of the invention is shown in Figure 4(a) in which both the anchor reagent and signal amplification processes are used. A complex is formed on a surface (401) between two detection reagents (304 and 305) containing a capture reagent (402), an analyte (403), and first and second proximity probes (406 and 407), respectively. First and second connector oligonucleotides (Circ-1 (408) and Circ-2 (409), respectively, in Figure 4(a)) are added, and if both proximity probes are present in the complex, each hybridizes to and crosslinks 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 containing one or more detection sequences (413) and anchor oligonucleotide sequence complements (414) (including partial anchor sequence complements (415)). The anchor oligonucleotide sequence (416) (attached to a capture moiety (417)) and its complement hybridize, multiple detection probes hybridize to multiple detection probe sequences, and the amount of analyte bound to the surface is measured (not shown but illustrated in Figure 1(a)). Figure 4(b) shows an exemplary sequence of the first circular DNA template Circ-1 (408) (designed to hybridize to the first proximity probe (PP1)), the detection oligonucleotide sequence, the inactive region of the amplicon (which can be used in whole or in part to bind to the anchor oligonucleotide sequence), and the portion PP2 (designed to hybridize to the second proximity probe). An additional embodiment is shown in Figure 4(c), in which a circular DNA template is 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 a free 3' end. In this embodiment, the second proximity probe comprises a sequence complementary to the detection sequence (413).

[0219] In one embodiment, the assay format described herein utilizes a detection reagent attached to a detection sequence at its 5' end with an exposed 3' end to facilitate ligation to a connector probe to form a circular DNA template, which is then amplified by rolling circle amplification to extend a second proximity probe (PP2). In this embodiment, PP1 is potentially independently available for extension by a polymerase, but this issue can be addressed by adding modified bases to prevent priming by the polymerase. Alternatively, the ligation template PP1 can be directly attached to the detection reagent via its 3' end, preventing this oligonucleotide from serving as a primer for DNA polymerase even if it is degraded.

[0220] Another approach for generating target sequences to be amplified by RCA or any suitable amplification method is shown in Figure 5. In this embodiment, each of the proximity probes can fold into a looped hairpin structure. The formation of these hairpin structures generates a single-stranded loop and a double-stranded portion containing the 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 recombinase's ability to catalyze site-specific recombination of DNA containing sequence-specific recombination sites. For example, Cre recombinase from bacteriophage P1 catalyzes recombination at sites containing loxP sites; other non-limiting examples include, but are not limited to, flippase (flp, from Yeast), Hin (Salmonella), and Tre, an 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 shown in Figure 5, along with the light and dark shading of the recombination sites.

[0221] Figure 6(a) further illustrates another method for generating a target sequence to be amplified by RCA or any suitable amplification method. Each proximity probe attached to the detection reagent contains a loxP site, which allows for site-specific recombination between the two oligonucleotides by Cre recombinase, resulting in the formation of a new oligonucleotide sequence consisting of the 5' portion of one proximity probe and the 3' portion of the other proximity probe flanked by the loxP site. The newly created target sequence can then be amplified by any suitable method, labeled, optionally anchored, and detected as described above. Figure 6(a) illustrates this embodiment, which uses a T7 RNA polymerase promoter as the operable element for amplification. It will be understood that other RNA polymerase sites, such as T3 and SP6, linked at either the 3' or 5' portion of the proximity probe are equally suitable for use in this method. In this embodiment, the loxP (recombination) site is 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 a circular DNA template that can be used in RCA.

[0222] The present invention includes a method for detecting an analyte, comprising binding the analyte to a capture reagent and two detection reagents on a surface to form a detection complex. The method includes measuring the detection complex, which preferentially measures a complex containing both detection reagents compared to a complex containing only one of the two detection reagents. In one embodiment, the method includes forming a complex, then cross-linking the detection reagents, and detecting the cross-linking reagent. Any suitable cross-linking chemistry can be used to link the components of the detection complex. For example, the first and second detection reagents can contain reactive moieties that are reacted and linked by the addition of a multifunctional cross-linking agent that links the reactive moieties. In this embodiment, the reactive moieties and cross-linking agents 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 contain binding moieties, and the cross-linking agent 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 animal species antibodies and the crosslinker is a multivalent anti-species antibody that targets the animal species antibodies; (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 a distinct sequence complementary to the second nucleic acid probe.

[0223] In certain embodiments, an analyte of interest in a sample can be detected by binding the analyte to an immobilized capture reagent, a first detection reagent, and a second detection reagent to form a complex, 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 regions complementary to the first and second probes, or (iii) ligating the first and second probes.

[0224] The crosslinked products can be detected once they bind to the surface, or optionally, the crosslinked products can be released from the surface into an eluent and detected. In this regard, only individual crosslinked products in the eluent containing 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 detecting single-molecule fluorescence, for example, fluorescence correlation spectroscopy and / or fluorescence cross-correlation spectroscopy. In this embodiment, detecting single-molecule fluorescence includes flowing the eluent through a 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 can 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 where both signals are detected from the interrogation zone. Alternatively, one label may be a fluorescence resonance energy transfer (FRET) donor and the other label may be a FRET acceptor, and the detection method may further comprise exciting the FRET donor at the interrogation zone and detecting a fluorescent signal from the FRET acceptor.

[0225] In certain embodiments, an analyte in a sample can be detected by binding the analyte to an immobilized capture reagent, a first detection reagent, and a second detection reagent to form a complex, where the first detection reagent comprises a first nucleic acid probe and the second detection reagent comprises a second nucleic acid probe; extending the second nucleic acid probe to form an extended sequence comprising a detectable label, where the extension depends on the colocalization 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 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 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 also include binding a first probe to a template nucleic acid sequence, binding a second probe to the template sequence, and ligating the first and second probes.

[0226] In the methods of the present invention using a capture reagent, the capture reagent can be directly immobilized on a solid phase or indirectly immobilized via a secondary binding reagent, such as the targeting reagent described below. For example, the capture reagent can be linked to or contain a targeting reagent that binds to the complement of the targeting reagent immobilized on the solid phase. 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 via a cross-linking agent (e.g., the targeting reagent and complement can be biotin and the cross-linking reagent can be a multivalent biotin-binding receptor such as streptavidin). In one embodiment, the targeting agent and its complement comprise a first oligonucleotide and a complementary oligonucleotide, a receptor-ligand pair, an antigen-antibody pair, a hapten-antibody pair, an epitope-antibody pair, a mimotope-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 being 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 analytes being measured by the assay. For example, binding of the binding reagent to its associated binding domain (via the associated targeting agent and its complement) should be substantially greater than binding to binding domains associated with other analytes (and presenting complements of different targeting agents). Preferably, the cross-reactivity of the binding of the analyte's capture or detection reagent to the binding domain associated with the other analyte compared to binding to the correct binding domain is <1%, more preferably <0.1%, more preferably <0.01%. In a preferred embodiment, the targeting agent / targeting agent complement comprises a pair of oligonucleotides containing complementary sequences, and the targeting agent and its complement are contacted under conditions sufficient to hybridize the targeting agent to its complement.

[0227] When a targeting agent is used, there is some flexibility in immobilizing the capture reagent used in the assay on a solid phase. In one embodiment, the capture reagent is provided to the user pre-immobilized on a solid phase via targeting agent-targeting agent complement interaction. In another embodiment, the capture reagent linked to the targeting agent and the solid phase supporting the immobilized targeting agent complement are provided as separate components. Thus, the assay further includes a step of immobilizing the capture reagent on the solid phase by binding the targeting agent to its complement (directly or through the use of a cross-linking agent). This step can be performed before, simultaneously with, or after the step related to the formation of the detection complex.

[0228] In certain embodiments, multifunctional targeting agents can be used in the assay methods and components described herein. The multifunctional targeting agent can include (a) a first segment designed to bind to a capture reagent via a first segment complement (i.e., the capture reagent comprises a targeting agent complement 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 functions as an anchor reagent on the surface). Thus, in this embodiment, the surface includes a multifunctional targeting agent in contact with a capture reagent that binds to the targeting agent via binding between the first segment and the first segment complement. The 3-AB RCA / PLA assay proceeds as described herein, with the amplicon binding to the anchor segment of the multifunctional targeting agent prior to the measurement step. This method can be used to ensure a 1:1 ratio of capture agent to anchor reagent is used in the assay method.

[0229] A wide variety of surfaces are suitable for use in the methods of the present invention, including conventional surfaces from binding assay techniques. Surfaces can be made from a variety of different materials, including polymers (e.g., polystyrene and polypropylene), ceramics, glass, and composites (e.g., carbon-polymer composites such as carbon-based inks). Suitable surfaces include the surfaces of macroscopic objects such as assay vessels (e.g., test tubes, cuvettes, flow cells, microfluidic channels, the inner surfaces of capillaries (e.g., BioTechne's ELLA glass nanoreactor), FACS cell sorters, cartridges, and wells of multiwell plates), slides, assay chips (e.g., those used for gene or protein chip measurements), pins or probes, beads, filtration media, and lateral flow media (e.g., filtration membranes used in lateral flow test strips).

[0230] Suitable surfaces also include particles (including, but not limited to, colloids or beads) commonly used in other types of particle-based assays, such as magnetic, polypropylene, and latex particles; materials commonly used in solid-phase synthesis, such as polystyrene and polyacrylamide particles; and materials commonly used in chromatography, such as silica, alumina, polyacrylamide, and polystyrene. Materials can also be fibers, such as carbon fibrils. Microparticles can be inanimate or, alternatively, can include living biological entities such as cells, viruses, bacteria, and the like. The particles used in the present methods can be composed of any material suitable for attachment to one or more capture or detection reagents and can be collected, for example, by centrifugation, gravity, filtration, or magnetic collection. A wide variety of different types of particles capable of attaching to capture or detection reagents are commercially available for use in binding assays. These include nonmagnetic particles and particles containing magnetizable materials that allow the particles to be collected in a magnetic field. In one embodiment, the particles are composed of conductive and / or semiconductive materials, such as colloidal gold particles. Microparticles can have a wide variety of sizes and shapes. By way of example and not limitation, the microparticles can be between 5 nanometers and 100 micrometers. Preferably, the microparticles have a size between 20 nm and 10 micrometers. The particles can be spherical, rectangular, rod-like, etc., or irregular in shape.

[0231] The particles used in the present methods can be coded to allow identification of specific particles or subpopulations of particles in a mixture of particles. The use of such coded particles has been used to enable multiplexing of assays using particles as solid phase supports for binding assays. In one approach, particles are manufactured to contain one or more fluorescent dyes, and specific populations of particles are identified based on the intensity and / or relative intensity of their fluorescence emission at one or more wavelengths. This approach has been used in the Luminex XMAP system (see, e.g., U.S. Pat. No. 6,939,720) and the BECTON DICKINSON cytometric bead array system. Alternatively, particles can be coded based on differences in other physical properties, such as size, shape, embedded optical pattern, etc. One or more particles provided in a mixture or set of particles can be coded to distinguish them from other particles in the mixture based on their optical properties, size, shape, embedded optical pattern, etc.

[0232] 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, and the capture analytes are immobilized on coded beads, such that the coding identifies the capture reagent (and analyte target) of a particular bead. The method can further include counting the number of beads with bound analytes (using the detection approaches described herein).

[0233] Alternatively or additionally, the detection complexes and / or capture reagents can be directly or indirectly bound to different, individual binding domains on one or more solid phases, such as in the case of a binding array where the binding domains are individual array elements, or in the case of a set of beads where the binding domains are individual beads, such that a distinct assay signal is generated at and measured from each binding domain. When capture reagents for different analytes are immobilized on different binding domains, the different analytes bound to those domains can be measured separately. In one example of such an embodiment, the binding domains are prepared by immobilizing separate domains of capture reagents that bind to the analytes of interest on one or more surfaces. Optionally, the surface can partially define one or more boundaries of a vessel (e.g., a flow cell, well, cuvette, etc.) that holds or through which the sample is passed. In a preferred embodiment, the individual binding domains are formed on electrodes for use in electrochemical or electrochemiluminescent assays. Multiplexed measurement of analytes on surfaces containing multiple binding domains using electrochemiluminescence is used by Meso Scale Diagnostics, LLC, in their MULTI-ARRAY® and SECTOR® imager line of products (see, 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).

[0234] Furthermore, the detection complex and / or capture reagent can be directly or indirectly bound to an electrode surface, which optionally contains distinct individual binding domains, as described above. The electrode surface can be a component of a multiwell plate and / or a flow cell. The electrodes can comprise conductive materials, such as metals such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, and conductive materials. They can also comprise metals coated with oxides, such as aluminum oxide-coated aluminum. The electrodes can include a working electrode and a counter electrode, which can be made of the same or different materials, such as a metal counter electrode and a carbon working electrode. In one specific embodiment, the electrodes comprise 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 comprise elemental carbon, such as graphite, carbon black, carbon nanotubes, and the like. Advantageously, they may comprise 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 multiwell plate, having electrodes (e.g., working and / or counter electrodes) comprising carbon, e.g., a carbon layer, and / or a screen-printed layer of carbon ink.

[0235] The present invention includes a method for detecting and counting individual detection complexes. In certain embodiments, a surface can contain multiple capture reagents for one or more analyte molecules present in a sample, the multiple capture reagents being distributed across multiple degradable binding regions located on the surface. Under the conditions used to perform and analyze the measurement, a "degradable binding region" is the smallest surface area associated with an individual binding event that can be resolved and distinguished from other areas where additional individual binding events occur. Thus, the method comprises binding one or more analyte molecules to one or more capture reagents on the surface, determining the presence or absence of the analyte molecules at the multiple degradable binding regions on the surface, and identifying the number of degradable binding regions containing the analyte molecules and / or the number of analyte domains that do not contain the analyte molecules.

[0236] The degradable binding regions can be optically interrogated in whole or in part; i.e., individual degradable binding regions can be optically interrogated individually, and / or the entire surface containing multiple degradable binding regions can be imaged, with one or more pixels or clusters of pixels in the image being mapped to individual degradable binding regions. The degradable binding regions can also be microparticles within multiple microparticles. Degradable binding regions that exhibit changes in their optical characteristics can be identified by conventional optical detection systems. Depending on the species detected (e.g., the type of fluorescent entity) and the operating wavelength, optical filters designed for specific wavelengths can be used for optical interrogation of the degradable binding regions. In embodiments where optical interrogation is used, the system can include two or more 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 degradable 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 semiconductor (CMOS) 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) can be used for imaging.

[0237] The measuring step of the method may include imaging the optical signal from the surface (or a portion thereof) to create an image consisting of multiple pixels, with each resolvable binding region being mapped to one or more pixels or groups of pixels in the image. Image analysis to identify a pixel or set of pixels having a signal indicative of a binding event (detection complex) can be accomplished using art-recognized methods, such as the numerous image analysis algorithms and software available for identifying and counting labeled biological structures in fluorescence 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. Resolvable binding regions are found by identifying adjacent regions with intensity above a threshold. Binding domains are classified as binding events if they meet size and intensity requirements.

[0238] In one embodiment, the degradable binding regions are elements of an array. In a preferred embodiment, the array is an array of microwells or nanowells, e.g., individual depressions or wells in a single substrate. Preferably, the well volume is less than 100 nL, preferably less than 50 nL. In one embodiment, the well volume is in the range of about 10 aL to 100 pL. Optionally, the wells can be configured to hold microparticles.

[0239] In one embodiment, at least 50% of the degradable binding regions located on the substrate and addressed during the assay contain either zero or one analyte molecule. Preferably, at least 80%, more preferably at least 95%, and most preferably at least 99% of the degradable binding regions contain zero or more analyte molecules. The concentration of analyte molecules 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 more analyte molecules. In certain embodiments, a surface includes a plurality of particles, each containing a plurality of capture reagents for an analyte molecule, the plurality of particles distributed across a plurality of degradable binding regions (e.g., an array of microwells or nanowells). Thus, the method includes (i) binding one or more analyte molecules to one or more capture reagents on the surface, (ii) distributing the plurality of particles across the array of degradable binding regions, and (iii) determining the presence or absence of analyte molecules in each degradable binding region to identify the number of binding domains containing analyte molecules and / or the number of binding domains not containing analyte molecules.

[0240] It may also be advantageous to detect analytes in a limited volume using one or more of the methods of the present invention. In these embodiments, analyte molecules in a sample bind to a pair of detection reagents, each bearing a distinguishable label, and the analyte is distributed to multiple locations, e.g., wells or reaction vessels (referred to herein as "reaction vessels") on a substrate, such as a plate, dish, chip, fiber optics, grid, etc., such that the majority of the reaction vessels contain no more than one analyte. This method allows a user to detect analyte molecules by counting the number of reaction vessels containing each distinguishable label attached to the analyte. In some cases, the multiple reaction vessels addressed represent a portion or essentially all of the total volume of reaction vessels that may contain at least one analyte molecule (e.g., associated with at least one analyte molecule or no analyte molecule). See the following published U.S. patent applications: U.S. Patent Application No. 2007 / 0259448, U.S. Patent Application No. 2007 / 0259385, U.S. Patent Application No. 2007 / 0259381, and 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 can be addressed, and a measurement can be made indicating the number / percentage of reaction vessels containing 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 can be determined.

[0241] In certain embodiments that allow for the detection of analyte molecules in a limited volume, the 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 contains the analyte, a first detection reagent, and a second detection reagent, with the first and second detection reagents having a first and a second detectable label, respectively. The detection complexes can be formed simultaneously, substantially simultaneously, or sequentially. The detection complexes are distributed among multiple reaction vessels so that the majority of the reaction vessels contain no more than one detection complex, and the number of analyte molecules is detected by counting the number of reaction vessels containing each of the first and second detectable labels. Preferably, the detection complexes are distributed among multiple reaction vessels so that the probability of detecting unbound first and second detection reagents 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 distributed among a plurality of reaction vessels, i.e., divided or separated into portions or parts, for example, by dispensing portions of the detection complex into a plurality of reaction vessels and / or by flowing a solution containing the detection complex through a plurality of reaction vessels, such that the detection complex is separated into individual reaction vessels on a support.

[0242] In a further embodiment, an analyte in a sample can be detected by (a) binding the analyte to a surface-bound capture reagent and first and second detection reagents to form a detection complex, (i) each detection complex comprising 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, simultaneously or substantially simultaneously, or by sequentially adding each component to build the detection complex in a stepwise manner. The detection complex is distributed among multiple reaction vessels such that the majority of the reaction vessels contain no more than one analyte, and the number of analyte molecules is detected by counting the number of reaction vessels containing the first and second detectable labels. The method can be performed with or without washing after each step and before the detection step.

[0243] The surface can be a particle, and optionally, a plurality of capture reagents are immobilized on the particle or particles. In this embodiment, the distribution step can be performed in several ways: (i) 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 among a plurality of reaction vessels; (ii) the capture reagent is immobilized on a plurality of particles, and distribution of the analyte is achieved by distributing the particles among a plurality of reaction vessels, and then binding the analyte to the capture reagent.

[0244] The multiple reaction vessels can also contain aqueous droplets dispersed in a water-in-oil emulsion. The emulsion can be made with droplets up to 100 μm in diameter and approximately 1 nL in volume. The high capacity (>10 droplets per mL of emulsion), ease of emulsion preparation, and high stability over a wide range of conditions make it an ideal means of compartmentalizing biochemical assays. Each droplet functions as an independent reaction vessel, and optional particle-attached detection complexes can be distributed among the droplets.

[0245] Alternatively, the surface may be a location within one of the reaction vessels; for example, if the reaction vessels are wells of a plate, the surface may be a domain or region within one of the wells of the plate. In this embodiment, the capture reagent may 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 to which target moieties are immobilized, and the capture reagent includes the complement of the target moiety, and the partitioning step is achieved by binding the complement of the target moiety to the target moiety located in the multiple reaction vessels.

[0246] In additional or alternative embodiments, the binding assays described herein may also include a pre-enrichment step to improve assay performance, for example, by increasing the concentration of the analyte in the sample and / or reducing the concentration of contaminants present in the sample that may interfere with assay performance. This can be accomplished by (a) contacting a sample containing the analyte of interest with a solid phase, e.g., particles, linked to a first binding reagent that binds to the analyte to form a complex containing the analyte bound to the first binding reagent; (b) collecting the complex; (c) separating unbound components of the sample from the complex; and (d) releasing the complex. This method of concentrating the analyte can be performed prior to performing the binding assays described herein to remove impurities that may interfere with assay performance. In this regard, reference is made to U.S. Application Publication No. 2010 / 0261292, the disclosure of which is incorporated herein by reference.

[0247] Specifically, the enrichment step involves subjecting a sample containing the analyte under conditions sufficient to form an analyte complex comprising the analyte bound to a first detection reagent, the first detection reagent being linked to a first nucleic acid probe. The analyte complex formed at the end of the enrichment step then binds to (i) a capture reagent on the surface comprising the analyte's 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. The surface-bound complex is subjected to an extension process that requires the first and second probes to be in close proximity, extending the second probe to form an extended 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 extended 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 bound to a targeting agent complementary to at least a portion of the first nucleic acid probe, thereby forming an enriched complex containing the analyte that binds 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 the analyte complex.

[0248] As used herein, collection refers to the physical localization of materials in a mixture. Collection includes localization of materials by binding reaction or adsorption. For example, materials in a mixture can be collected on a solid phase by adsorption of the material to 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 can also include techniques in the art for localizing materials at a location / volume within a larger fluid volume, such as optical tweezers (which use light to manipulate microscopic objects as small as a single atom, and radiation pressure from a focused laser beam can trap small particles), electric or magnetic fields, focusing flow, density gradient centrifugation, etc.

[0249] Certain embodiments of the present invention involve collections of microparticles or materials bound to microparticles. Suitable collection methods include many methods known in the field of microparticle-based assays that achieve localization of microparticles from suspension. These include sedimentation under gravity or by centrifugation, filtration through 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.

[0250] As used herein, release refers to the delocalization of previously collected material. Materials held in a localized location by chemical bonds or specific or nonspecific binding interactions can be delocalized by cleaving the bonds or interactions, allowing the materials to diffuse or mix into the surrounding medium. There are many well-established cleavable chemical linkers available, providing covalent bonds that can be cleaved without requiring harsh conditions. For example, disulfide-containing linkers can be cleaved using thiols or other reducing agents, cis-diol-containing linkers can be cleaved using periodate, and metal-ligand interactions (such as nickel-histidine) can be cleaved by changing the pH or introducing a competing ligand. Similarly, there are many established reversible binding pairs that can be used (including those identified in the field of affinity chromatography). For example, the binding of many antibody-ligand pairs can be reversed by changing the pH, adding protein denaturants or chaotropic agents, adding a competing ligand, etc. Other suitable reversible binding pairs include complementary nucleic acid sequences whose hybridization can be reversed under various conditions, including changes in pH, decreases in salt concentration, increases in temperature above the melting temperature of the pair, and / or the addition of nucleic acid denaturants (such as formamide). 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 to an 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 to the solid phase.

[0251] Release also includes physical delocalization of materials, for example, by mixing, shaking, vortexing, convective fluid flow, or mixing by application of magnetic, electrical, or optical forces. When microparticles or materials bound to microparticles are collected, such physical methods can be used to resuspend the particles in the surrounding matrix. Release can simply be the reverse of the previous collection step (e.g., by one of the mechanisms described above), or collection and release can proceed by two different mechanisms. In one such example, collection of particle-bound material (e.g., an analyte or a complex containing the analyte) can be achieved by physical collection of the particle. The material is then released by cleaving the bond or reversing the binding reaction that retains the material on the particle. In a second such example, material (e.g., the analyte of an analyte-containing complex) is collected on a surface via a binding interaction with a binding reagent linked to the surface. The material is then released by cleaving the bond or second binding interaction linking the binding reagent to the surface.

[0252] Collection followed by release can be used to concentrate and / or purify analytes in a sample. By collecting in a first volume and releasing into a second, smaller volume, the analytes of the sample can be concentrated. Concentration can often significantly improve the sensitivity of the subsequent measurement step. By collecting and removing some or all of the uncollected sample from the sample, potential assay interferences in the sample can be reduced or eliminated. Optionally, removing unbound sample can include washing the collected material with a defined liquid reagent (e.g., an assay or wash buffer) and releasing the collected material into the liquid reagent to provide a uniform matrix for the subsequent assay step.

[0253] As shown in Figure 3(a) of US 2010 / 0261292, which is incorporated herein by reference, this method includes contacting a sample containing a target analyte with particles bound 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 particles are linked via a binding reaction between the first and second targeting agents to form a complex containing the target analyte bound to the first binding reagent. The complex is then collected, and unbound components of the sample are separated from the complex. The complex is then 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 specific embodiment is shown in Figure 16. 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 that binds to a detection antibody 1604. The particles are mixed with the proximity probes, and the capture sequence hybridizes to the probe sequence, forming a complex 1605. The complex is then mixed with a sample containing an analyte 1606 and, optionally, one or more contaminants 1607-1608. The analyte binds to the detection antibody (1609), and the contaminants are removed (1610). The particles are removed from the complex containing the bound analyte under appropriate conditions to form an enriched solution of analyte bound to the proximity probes (1611), which can be used as described herein in an immunoassay in which 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.

[0254] In another embodiment, an immunoassay complex between a detection antibody and an analyte is formed in solution, followed by amplification, and then the amplified product is attached to particles via a capture reagent and / or anchor. Optionally, the amplified product can be filtered and then captured on the particles via a capture reagent and / or anchor. This method is shown in Figure 17. Analyte A (1701) binds to detection antibodies (1702 and 1703, respectively) that are each bound to a proximity probe (1704 and 1705, respectively), forming a detection complex containing the analyte bound to each detection antibody (1706). The detection complex is contacted with two connector sequences (1707a and 1707b), each of which contains a terminal sequence complementary to the non-overlapping region of a first proximity probe and a terminal sequence complementary to the non-overlapping region of a second proximity probe. The connector sequence hybridizes to the first and second proximity probes, and the terminal sequence of the connector oligonucleotide is ligated from a circular target sequence (1708) that hybridizes to both the first and second proximity probes. The second proximity probe is extended by rolling circle hybridization to generate an amplicon containing a binding reagent complementary to the anchor reagent. The amplicon is contacted with a surface (1709) containing a capture reagent (1710) and an anchor reagent (1711), and the amount of amplicon bound to the surface is measured by labeling using multiple labeled probes (1712).

[0255] Labeled probes In embodiments, the present disclosure provides a labeled probe comprising an oligonucleotide and at least one electrochemiluminescent moiety. In embodiments, the present disclosure provides a labeled probe comprising an oligonucleotide and at least two electrochemiluminescent moieties. In embodiments, the electroluminescent moiety is an electrochemiluminescent label. In embodiments, the labeled probe is used in the methods and assays described herein to measure the amount of an extended sequence. In embodiments, the extended sequence is an amplification product (or amplicon) of the RCA process. In embodiments, the labeled probe comprises an oligonucleotide complementary to a detection sequence in the extended sequence. In embodiments, multiple labeled probes are used to measure the amount of extended sequence bound to a surface in the methods described herein. In embodiments, the amount of an analyte in a sample is determined by measuring the labeled probe.

[0256] Detectable luminescent labels, such as fluorophores, are known to have a self-quenching effect. Self-quenching is the reduction of the luminescence intensity of one label by another, and typically increases with higher label concentrations or label densities. Therefore, to reduce the self-quenching effect, multiple luminescent labels in close proximity are generally avoided. Surprisingly, the inventors discovered that the labeled probes of the present disclosure, which contain structures that hold multiple electrochemiluminescent labels in close proximity, still provide efficient generation of ECL from the labels and improve signal compared to single-labeled probes. Thus, in embodiments, the labeled probes of the present disclosure contain two or more electrochemiluminescent labels. In embodiments, the labeled probes contain 2 to 10 electrochemiluminescent labels. In embodiments, the labeled probes contain 2 to 5 electrochemiluminescent labels. In embodiments, the labeled probes contain 3 electrochemiluminescent labels. In embodiments, the labeled probes contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 electrochemiluminescent labels.

[0257] In embodiments, the present disclosure provides a labeled probe comprising an oligonucleotide and a plurality of electrochemiluminescent labels. The probe may comprise (i) one or more (or more) labels linked to modified nucleotide bases of the oligonucleotide, (ii) a labeling moiety having one or more (or more) labels linked to the 5' end of the oligonucleotide, (iii) a labeling moiety having one or more (or more) labels linked to the 3' end of the oligonucleotide, or (iv) a combination of two or more of (i), (ii), and (iii).

[0258] In embodiments, the present disclosure provides a labeled probe of formula I: [ka] In the formula, B is a nucleotide base, R is an electrochemiluminescent label, L1 is a linking group, L2 is a linking group, j is an integer of 0 to 11, k is an integer of 0 to 1, m is an integer of 0 to 11, and n is an integer of 0 to 5.

[0259] The nucleotide base B in Formula I can be any nucleotide base as long as it does not interfere with the electrochemiluminescence ability of the labeled probe. In embodiments, the nucleotide base is a natural nucleotide base. In embodiments, the nucleotide base is a synthetic nucleotide base. In embodiments, the nucleotide base is a purine or pyrimidine. In embodiments, the nucleotide base is adenine, cytosine, guanine, thymine, or uracil. In embodiments, the nucleotide base is xanthine, hypoxanthine, 2,6-diaminopurine, 6,8-diaminopurine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, isoguanine, or isocytosine.

[0260] R in Formula I can be any suitable electrochemiluminescent label. Suitable electrochemiluminescent labels include electrochemiluminescent organometallic complexes of ruthenium, osmium, iridium, rhenium, and lanthanide metals. Suitable electrochemiluminescent labels include electrochemiluminescent organometallic complexes of these metals containing bipyridine or phenanthroline ligands (substituted or unsubstituted). Examples of suitable electrochemiluminescent labels can be found in U.S. Patent Nos. 5,714,089, 6,316,607, 6,808,939, 9,499,573, 6,468,741, 6,479,233, and 6,136,268. In an embodiment, the electrochemiluminescent label is [ka] is.

[0261] In embodiments, L1 and L2 are independently alkyl, haloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl-substituted cycloalkyl, heterosubstituted cycloalkyl, heteroalkyl, cyanoalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, phenyl, or a combination thereof, optionally having a carbon chain substituted with a heteroatom. In embodiments, L1 and L2 are independently alkyl linkers having a carbon chain substituted with a heteroatom. In embodiments, the one or more heteroatoms are independently nitrogen, sulfur, phosphate, or oxygen. In embodiments, L1 is from about 1 to about 20 carbon and / or heteroatoms in length. In embodiments, L1 is from about 4 to about 15 carbon and / or heteroatoms in length. In embodiments, L1 is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 carbon and / or heteroatoms in length. In embodiments, L2 is about 1 to about 30 carbon and / or heteroatoms in length. In embodiments, L2 is about 7 to about 26 carbon and / or heteroatoms in length. In embodiments, L2 is about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, or about 26 carbon and / or heteroatoms in length.

[0262] As used herein, "between" refers to a range that includes the endpoints of the range. For example, the integers 0 to 11 specifically include the integers 0 and 11, and any integers between 0 and 11, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The integers 0 to 5 specifically include the integers 0 and 5, and any integers between 0 and 5, i.e., 1, 2, 3, and 4.

[0263] In embodiments, R comprises a ruthenium complex RP1P2P3, where each of P1, P2, and P3 is independently a bipyridine, a substituted bipyridine, a phenanthroline, or a substituted phenanthroline. [ka] is.

[0264] In an embodiment, B is a uracil attached to L1 at position 5 of the uracil.

[0265] In an embodiment, L1 is [ka] , or a combination thereof, and p is an integer of 1 to 12.

[0266] In an embodiment, L2 is [ka] , or a combination thereof, and q is an integer of 0 to 11.

[0267] In embodiments, the present disclosure provides a labeled probe of Formula II: [ka] In the formula, j is an integer of 0 to 11, k is an integer of 0 to 1, m is an integer of 0 to 11, n is an integer of 0 to 5, and R is an electrochemiluminescent label: [ka] In an embodiment, j is an integer of 0 to 5, k is 0, m is an integer of 0 to 5, and n is an integer of 2 to 7. In an embodiment, k is 0, j is 0, m is 1, and n is 5.

[0268] In embodiments, the oligonucleotide of the labeled probe comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO:31). In embodiments, the oligonucleotide of the labeled probe comprises a sequence having at least 85% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO:31). In embodiments, the labeled probe oligonucleotide comprises a sequence having at least 88% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31). In embodiments, the labeled probe oligonucleotide comprises a sequence having at least 90% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31). In embodiments, the labeled probe oligonucleotide comprises a sequence having at least 95% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31). In embodiments, the labeled probe oligonucleotide comprises a sequence having at least 98% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31). In embodiments, the labeled probe oligonucleotide comprises a sequence having at least 99% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31).

[0269] In embodiments, the labeled probe oligonucleotide comprises 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31). In embodiments, the labeled probe oligonucleotide comprises 5'-CAGTGAATGCGAGTCCGTCTAAG-3' (SEQ ID NO: 32). In embodiments, the labeled probe oligonucleotide comprises one or more modifications described herein. In embodiments, the labeled probe comprises an amino modifier. In embodiments, the labeled probe comprises an internal amino-modified dT base (iAmMC6T). In embodiments, the labeled probe comprises an internal spacer 18 (iSp18). In embodiments, the labeled probe comprises a 3' amino modifier (3AmMO). In embodiments, the labeled probe oligonucleotide comprises 5'-CAGTGAATGCGAGTCCGTCTAAG / iAmMC6T / iSp18 / iAmMC6T / iSp18 / 3AmMO / -3' (SEQ ID NO: 44 with modification).

[0270] In embodiments, the present disclosure provides a method of measuring electrochemiluminescence, comprising: (a) applying a potential to an electrode under conditions such that a complex in proximity to the electrode emits electrochemiluminescence, the complex comprising a target oligonucleotide and a labeled probe provided herein, the labeled probe comprising an oligonucleotide complementary to the target oligonucleotide; and (b) measuring the emitted electrochemiluminescence. Exemplary electrodes and methods of measuring electrochemiluminescence are described herein.

[0271] In embodiments, the labeled probe is used in the measurement step of the assay described herein, i.e., in measuring the amount of extended sequence bound to the surface. The labeled probe can be used in all methods described herein, for example, methods using one detection reagent, two detection reagents, or two or more detection reagents. In embodiments, a method for measuring electrochemiluminescence includes forming a composition containing (i) a target nucleic acid containing a target sequence and (ii) a labeled probe, where the labeled probe comprises an oligonucleotide complementary to the target sequence; incubating the composition under conditions in which the labeled probe hybridizes to the target nucleic acid to form a complex; bringing the complex into proximity with an electrode; applying a potential to the electrode under conditions in which the complex emits electrochemiluminescence; and measuring the emitted electrochemiluminescence.

[0272] In embodiments, the labeled probe is used in the detection step of the assays described herein, i.e., in measuring the amount of surface-bound extension sequence. The labeled probe can be used in all of the methods described herein, for example, methods using one detection reagent, two detection reagents, or more than two detection reagents.

[0273] In embodiments, the target nucleic acid is an extended sequence generated from an RCA reaction as described herein. In embodiments, the target nucleic acid is immobilized on an electrode. In embodiments, the target nucleic acid is immobilized on an electrode such that complex formation brings the complex into proximity with the electrode. In embodiments, the target nucleic acid is immobilized directly on the electrode or indirectly via a binding reagent as provided herein. In embodiments, the complex further comprises a binding reagent capable of binding to the target nucleic acid, the binding reagent being immobilized on the electrode. In embodiments, the complex further comprises a binding reagent capable of binding to the target nucleic acid, the binding reagent being immobilized on the electrode, and bringing the complex into proximity with the electrode comprises incubating the composition with the electrode under conditions in which the labeled nucleic acid binds to the binding reagent. In embodiments, the binding reagent is an anchor reagent. In embodiments, the binding reagent comprises a sequence complementary to the target nucleic acid.

[0274] In embodiments, the target nucleic acid is immobilized on a solid phase carrier. In embodiments, the target nucleic acid is immobilized directly on the solid phase carrier or indirectly via a binding reagent as provided herein. In embodiments, a binding reagent capable of binding to the target nucleic acid is immobilized on the solid phase carrier, and the solid phase carrier is immobilized on an electrode. In embodiments, the target nucleic acid is immobilized on the solid phase carrier, and bringing the complex into proximity with the electrode comprises incubating the composition with the electrode under conditions in which the labeled nucleic acid binds to the binding reagent. In embodiments, the binding reagent is immobilized on the solid phase carrier, and bringing the complex into proximity with the electrode comprises incubating the composition with the solid phase carrier under conditions in which the labeled nucleic acid binds to the binding reagent, and collecting the solid phase carrier.

[0275] In embodiments, incubation is for about 9 minutes, 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, or about 8 hours. In embodiments, incubation is at about 15°C, about 18°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, or about 30°C. In embodiments, incubation is at about 15°C to about 30°C for about 10 minutes to about 8 hours. In embodiments, incubation is at about 15°C to about 30°C for about 10 minutes to about 8 hours. In embodiments, incubation is at about 18°C ​​to about 29°C for about 20 minutes to about 6 hours. In one embodiment, incubation is at about 20°C to about 28°C for about 20 minutes to about 6 hours. In one embodiment, incubation is at about 21°C to about 26°C for about 30 minutes to about 4 hours. In one embodiment, incubation is at about 22°C to about 24°C for about 40 minutes to about 2 hours. In one embodiment, incubation is at about 23°C for about 1 hour.

[0276] Exemplary solid phase carriers are described herein. In embodiments, the binding reagent comprises a sequence complementary to the target oligonucleotide. In embodiments, the solid phase carrier is a particle, and the particle is collected on the electrode using gravity, centrifugation, filtration, or application of a magnetic field.

[0277] In embodiments, the present disclosure provides kits for measuring electrochemiluminescence, including a labeled probe provided herein and electrodes, an ECL read buffer, a nucleic acid polymerase, a nucleic acid ligase, an assay diluent, additional nucleic acid reagents, assay consumables, or a combination thereof. Examples of additional nucleic acid reagents include buffers and reagents for solubilizing, diluting, and / or stabilizing nucleic acids. Examples of assay consumables that may be included in the kit include assay modules designed to contain samples and / or reagents during one or more steps of the assay, pipette tips and other consumables for transferring liquid samples and reagents, covers and seals for the assay modules and other consumables used in the assay, racks for holding other assay consumables, labels (including human-readable or machine-readable formats such as barcodes, RFID tags, etc.) for identifying samples or other assay consumables, and media (including paper and electronic media) for providing information about the assay and / or instructions for performing the assay.

[0278] In embodiments, the kit includes an electrode, wherein the electrode is a carbon-based electrode. In embodiments, the kit includes an assay consumable, wherein the assay consumable is a multi-well plate assay consumable, wherein each well of the plate includes a carbon ink electrode. In embodiments, the kit includes a multi-well assay plate having a plurality of wells, wherein the assay plate is used as a container for at least one binding reagent. In embodiments, the binding reagent is immobilized on the plate. Multiple wells in the plate may have binding reagents immobilized therein. The binding reagent in each of these wells may be the same as in all, some, or none of these wells. In embodiments, multiple binding reagents are immobilized as an array of binding reagents in each of these wells. The immobilized binding reagent and / or the array of immobilized binding reagents may be immobilized on an electrode (which may be a carbon-based electrode, or more specifically, a carbon ink electrode) in the well.

[0279] In embodiments, the kit includes an ECL read buffer, wherein the ECL read buffer comprises tripropylamine. In embodiments, the kit includes an ECL read buffer, wherein the ECL read buffer comprises butyldiethanolamine. Exemplary ECL read buffers are described, for example, in US 62 / 787,892, filed January 3, 2019.

[0280] Method for producing labeled probes The present disclosure includes a method for producing the labeled probe described herein. In an embodiment, a modified oligonucleotide having two or more alkylamine moieties is reacted with an excess of an amine-reactive labeling reagent, which comprises an electrochemiluminescent label. The reaction mixture is then purified to isolate a product in which each amine moiety is linked to an electrochemiluminescent label.

[0281] In embodiments, the modified oligonucleotide having a reactive amine group has the structure shown in Formula VIII, the product has the structure shown in Formula I, and R is an electrochemiluminescent label. In embodiments, the modified oligonucleotide has the structure shown in Formula IX, the product has the structure shown in Formula II, and R is an electrochemiluminescent label. The other components of the formula (L1, L2, k, m, etc.) are as described for Formulas I and II above. [ka]

[0282] In embodiments, the amine-reactive labeling reagent comprises an active ester form of an electrochemiluminescent label, which reacts with the amine moiety to form an amide bond between the modified oligonucleotide and the label. In embodiments, the active ester is an NHS ester. In embodiments, the label is [ka] and the amine-reactive labeling reagent is [ka] is.

[0283] In embodiments, the modified oligonucleotides are prepared by solid phase synthesis. In embodiments, the final product is purified by ion exchange chromatography. In embodiments, the final product is purified by anion exchange chromatography. In embodiments, the final product is purified by gel electrophoresis.

[0284] Nucleic acid probes In embodiments, the nucleic acid probe linked to the detection reagent is an oligonucleotide that is crosslinked or conjugated to the detection reagent. "Conjugation," "bioconjugation," or variations thereof are used herein to refer to the formation of a stable covalent bond between two molecules, at least one of which is a biomolecule, such as a protein, polypeptide, polynucleotide, etc. The linked molecule may be referred to as a "conjugate" or a "bioconjugate." In embodiments, the conjugate comprises a nucleic acid probe and a detection reagent.

[0285] In embodiments, the nucleic acid probe comprises one or more regions complementary to a template nucleic acid. In embodiments, the template nucleic acid is a template for amplification, e.g., by PCR. In embodiments, the template nucleic acid is a circular nucleic acid template, or one or more linear nucleic acid templates that are ligated to form a circular nucleic acid template, e.g., for RCA. In embodiments, the template nucleic acid sequence is a connector oligonucleotide used in the electrochemiluminescence assay methods described herein. In embodiments, the connector oligonucleotide is a linear oligonucleotide, the 5' and 3' ends of which can be ligated to generate a circular nucleic acid template. In embodiments, the connector oligonucleotide comprises a sequence at its 5' end that is complementary to a sequence at its 3' end, such that a ligase can ligate the 5' and 3' ends together to form a circular nucleic acid template. In embodiments, the circular nucleic acid template is a template for rolling circle amplification (RCA). In embodiments, the nucleic acid probe is a primer for the RCA reaction, i.e., it extends the circular nucleic acid template to form an extended sequence.

[0286] The present inventors have unexpectedly discovered that shorter nucleic acid probes improve the performance of the electrochemiluminescence measurement and assay methods of the present invention. Conventional thinking has been that relatively long oligonucleotides would be necessary to achieve adequate performance. Shorter nucleic acid probes offer the additional advantage of simplifying conjugation protocols (especially enabling the use of simpler, less labor-intensive, and faster approaches to separate protein-probe conjugates from unconjugated probes) and are also easier and less expensive to synthesize and purify. Thus, in embodiments, nucleic acid probes of the present disclosure include oligonucleotides about 10 to about 30 nucleotides in length. In embodiments, nucleic acid probes include oligonucleotides about 12 to about 28 nucleotides in length. In embodiments, nucleic acid probes include oligonucleotides about 13 to about 26 nucleotides in length. In embodiments, nucleic acid probes include oligonucleotides about 14 to about 24 nucleotides in length. In embodiments, nucleic acid probes include oligonucleotides about 11 to about 22 nucleotides in length. In embodiments, nucleic acid probes include oligonucleotides about 12 to about 21 nucleotides in length. In embodiments, the nucleic acid probe comprises an oligonucleotide about 13 to about 20 nucleotides in length. In embodiments, the nucleic acid probe comprises an oligonucleotide about 13 to about 18 nucleotides in length. In embodiments, the nucleic acid probe comprises an oligonucleotide about 14 to about 19 nucleotides in length. In embodiments, the nucleic acid probe comprises an oligonucleotide about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length. In embodiments, the nucleic acid probe comprises an oligonucleotide about 14 nucleotides. In embodiments, the nucleic acid probe comprises an oligonucleotide about 15 nucleotides. In embodiments, the nucleic acid probe comprises an oligonucleotide comprising 5'-GACAGAACTAGACAC-3' (SEQ ID NO: 33). In embodiments, the nucleic acid probe comprises an oligonucleotide comprising 5'-ACAGAACTAGACAC-3' (SEQ ID NO: 40).In embodiments, the nucleic acid probe comprises an oligonucleotide comprising 5'-GACAGAACTAGACA-3' (SEQ ID NO: 41). In embodiments, the nucleic acid probe comprises an oligonucleotide comprising 5'-TGCACAGCTCGACGC-3' (SEQ ID NO: 42). In embodiments, the nucleic acid probe comprises an oligonucleotide, the oligonucleotide being 14 to 24 nucleotides in length and comprising 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO: 33).

[0287] In embodiments, the nucleic acid probe comprises one or more nucleic acid modifications to enable conjugation to a detection reagent. In embodiments, conjugation of the nucleic acid probe to the detection reagent is achieved using a heterobifunctional crosslinker. In embodiments, the nucleic acid probe comprises a non-natural 5' modification comprising a reactive functional group. Non-limiting examples of functional groups include, for example, alkenes and strained alkenes, alkynes, halides, alcohols, thiols, amines, phosphates, aldehydes, ketones, carboxylic acids, carboxylates, amides, esters, thioesters, acylphosphates, acid halides, nitriles, acid anhydrides, hydrazines, tetrazines, azides, and the like. In embodiments, the reactive functional group is a thiol, amine, carboxylic acid, active ester, hydrazine, aldehyde, ketone, alkyne, strained alkene, azide, or tetrazine. In embodiments, the reactive functional group is a thiol. In embodiments, the reactive functional group is a tetrazine. In embodiments, the reactive functional group is a vinyl or strained alkene. In embodiments, the reactive functional group is an azide. In embodiments, the reactive functional group is an alkyne or a strained alkyne. In embodiments, the reactive functional group is 4-formylbenzamide. In embodiments, the reactive functional group is hydrazinonicotinamide.

[0288] In embodiments, the non-natural 5' modification can react with a heterobifunctional crosslinker of the present disclosure. In embodiments, the non-natural 5' modification can react with maleimide, iodoacetamide, or activated disulfide. In embodiments, the non-natural 5' modification can react with tetrazine. In embodiments, the non-natural 5' modification can react with vinyl or strained alkene. In embodiments, the non-natural 5' modification can react with azide. In embodiments, the non-natural 5' modification can react with alkyne or strained alkyne. In embodiments, the non-natural 5' modification can react with hydrazinonicotinamide. In embodiments, the non-natural 5' modification can react with 4-formylbenzamide.

[0289] In embodiments, the non-natural nucleic acid probe is of Formula III: [ka] It comprises a reactive functional group (R), which is a thiol, amine, carboxylic acid, active ester, hydrazine, aldehyde, ketone, alkyne, strained alkene, azide, or tetrazine. In one embodiment, the reactive functional group is a thiol (-R is -SH).

[0290] In embodiments, the non-natural nucleic acid probe further comprises a non-natural 5' modification comprising a hapten or biotin. In embodiments, the hapten comprises fluorescein, dinitrophenyl, or digoxigenin. In embodiments, the modification comprises biotin. In embodiments, the modification comprises a thiol. In embodiments, the modification is the 5' thiol modifier C6 SS (5ThioMC6-D).

[0291] In embodiments, the non-natural nucleic acid probe is of Formula IV: [ka]

[0292] In embodiments, the present disclosure provides conjugate compounds comprising a detection reagent conjugated to a non-natural nucleic acid probe described herein. In embodiments, the oligonucleotide of the conjugated compound is 14-24 nucleotides in length and comprises 14 or 15 consecutive nucleotides of SEQ ID NO: 33. In embodiments, the oligonucleotide of the conjugated compound is 14-24 nucleotides in length, comprises 14 or 15 consecutive nucleotides of SEQ ID NO: 33, and further comprises a non-natural 5' modification comprising a reactive functional group. In embodiments, the oligonucleotide of the conjugated compound is 14-24 nucleotides in length, comprises 14 or 15 consecutive nucleotides of SEQ ID NO: 33, and further comprises a thiol, amine, carboxylic acid, active ester, hydrazine, aldehyde, ketone, alkyne, strained alkene, azide, or tetrazine. In embodiments, the oligonucleotide of the conjugated compound is 14-24 nucleotides in length, is of Formula III, and comprises 14 or 15 consecutive nucleotides of SEQ ID NO: 33. [ka] wherein R is a reactive group. In embodiments, the reactive group R is a thiol, amine, carboxylic acid, active ester, hydrazine, aldehyde, ketone, alkyne, strained alkene, azide, or tetrazine. In embodiments, the reactive group R is a thiol (-R is -SH). In embodiments, the nucleic acid probe comprises an oligonucleotide comprising 5'-GACAGAACTAGACAC-3' (SEQ ID NO: 33). In embodiments, the nucleic acid probe comprises an oligonucleotide comprising 5'-ACAGAACTAGACAC-3' (SEQ ID NO: 40). In embodiments, the nucleic acid probe comprises an oligonucleotide comprising 5'-GACAGAACTAGACA-3' (SEQ ID NO: 41). In embodiments, the nucleic acid probe comprises an oligonucleotide comprising 5'-TGCACAGCTCGACGC-3' (SEQ ID NO: 42).

[0293] In embodiments, the detection reagent of the conjugate compound is a binding reagent. In embodiments, the detection reagent of the conjugate compound is an antigen-binding agent. In embodiments, the detection reagent is an antibody. In embodiments, the non-natural nucleic acid probe oligonucleotide is 10-30 nucleotides in length. In embodiments, the non-natural nucleic acid probe oligonucleotide is 14-19 nucleotides in length. In embodiments, the non-natural nucleic acid probe oligonucleotide is about 14, about 15, about 16, about 17, about 18, or about 19 nucleotides in length. In embodiments, the non-natural nucleic acid probe oligonucleotide is 14 nucleotides in length. In embodiments, the non-natural nucleic acid probe oligonucleotide is 15 nucleotides in length.

[0294] In embodiments, the nucleic acid probe comprises a region complementary to the template nucleic acid sequence. In embodiments, the template nucleic acid sequence is a circular nucleic acid template or a linear nucleic acid template that can be linked to form a circular nucleic acid template. In embodiments, the circular nucleic acid template is a template for rolling circle amplification (RCA). In embodiments, the nucleic acid probe is a primer for the RCA reaction, i.e., it extends the circular nucleic acid template to form an extension sequence.

[0295] In embodiments, the non-natural oligonucleotides of the present disclosure are shorter than conventional RCA template oligonucleotides. Similar to nucleic acid probes, shorter template oligonucleotides offer the advantage of being easier and less expensive to synthesize and purify. Because more copies of a shorter oligonucleotide can be amplified per RCA reaction per unit time compared to a longer oligonucleotide, shorter template oligonucleotides can also increase assay sensitivity. In embodiments, the present disclosure provides non-natural oligonucleotides about 40 to about 100 nucleotides in length, comprising the sequence 5'-GTTCTGTC-3' at their 5' end and 5'-GTGTCTA-3' at their 3' end. The 5' and 3' end sequences of an oligonucleotide are sometimes referred to as the 5' and 3' terminal sequences, respectively. In embodiments, the non-natural oligonucleotides comprise 5'-CAGTGAATGCGAGTCCGTCTAAG-3' (SEQ ID NO: 34) and 5'-AAGAGAGTAGTACAGCA-3' (SEQ ID NO: 35). In embodiments, the non-naturally occurring oligonucleotide is about 50 to about 78 nucleotides in length. In embodiments, the non-naturally occurring oligonucleotide is about 53 to about 76 nucleotides in length. In embodiments, the non-naturally occurring oligonucleotide is about 50 to about 70 nucleotides in length. In embodiments, the non-naturally occurring oligonucleotide is about 53 to about 61 nucleotides in length. In embodiments, the non-naturally occurring oligonucleotide is about 54 to about 61 nucleotides in length. In embodiments, the non-naturally occurring oligonucleotide is 61 nucleotides in length. In embodiments, the non-natural oligonucleotide comprises about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, or about 76 nucleotides and comprises at its 5' terminal sequence 5'-GTTCTGTC-3' and at its 3' terminal sequence 5'-GTGTCTA-3'. In embodiments, the oligonucleotide further comprises a 5' terminal phosphate group.In an embodiment, the oligonucleotide comprises at its 5' end sequence 5'-GTTCTGTC-3' and at its 3' end sequence 5'-GTGTCTA-3'.

[0296] In embodiments, the oligonucleotide consists of 5'-GTTCTGTCATATTTCAGTGAATGCGAGTCCGTCTAAGAGAGTAGTACAGCAAGAGTGTCTA-3' (SEQ ID NO: 36). In embodiments, the oligonucleotide consists of 5'-GCTGTGCAATATTTCAGTGAATGCGAGTCCGTCTAAGAGAGTAGTACAGCAAGAGCGTCGA-3' (SEQ ID NO: 43). In embodiments, the oligonucleotide further comprises a 5'-terminal phosphate group. In embodiments, the oligonucleotide is a linear oligonucleotide that can be ligated to form a circular oligonucleotide, e.g., a circular RCA template.

[0297] Oligonucleotide sequence Table B is a summary of the oligonucleotide sequences provided herein. [Table 2-1] [Table 2-2] [Table 2-3]

[0298] Any of the oligonucleotides provided in Table B can contain one or more modifications. Oligonucleotide modifications are described herein. In embodiments, the oligonucleotides of Table B contain one or more modifications for attachment to another substance, such as a label, a protein, or a surface. In embodiments, the oligonucleotides of Table B contain biotin, streptavidin, avidin, an amino group, a thiol group, an aldehyde group, a hydrazide group, an azide group, an alkyn...

Claims

1. A labeled probe of formula I, 【Chemistry 1】 where B is a nucleotide base, R is an electrochemiluminescent label, and L １ is a linking group, and L ２ is a linking group, j is an integer from 0 to 11, k is an integer from 0 to 1, m is an integer from 0 to 11, and n is an integer from 0 to 5.

2. R is a ruthenium complex RP １ P ２ P ３ P １ , P ２ and P ３ 2. The labeled probe of claim 1, wherein each of is independently a bipyridine, a substituted bipyridine, a phenanthroline, or a substituted phenanthroline.

3. The electrochemiluminescent label R is 【Chemistry 2】 The labeled probe according to claim 2 ,

4. B is uracil 5th position L １ The labeled probe of any one of claims 1 to 3, wherein the nucleotide is uracil attached to the nucleotide.

5. L １ but, 【Chemistry 3】 、 or including combinations thereof, The labeled probe according to any one of claims 1 to 4, wherein p is an integer from 1 to 12.

6. L ２ but, 【Chemistry 4】 、 or including combinations thereof, The labeled probe according to any one of claims 1 to 5, wherein q is an integer of 0 to 11.

7. A labeled probe of formula II, 【Chemistry 5】 where j is an integer from 0 to 11, k is an integer from 0 to 1, m is an integer from 0 to 11, n is an integer from 0 to 5, and R is an electrochemiluminescent label: 【Chemistry 6】 , a labeled probe.

8. 2. The labeled probe of claim 1, wherein k is 0, j is 0, m is 1, and n is 5.

9. The oligonucleotide is The labeled probe of any one of claims 1 to 8, comprising a sequence having at least 90% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO:31).

10. The oligonucleotide is The labeled probe of any one of claims 1 to 8, comprising 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO:31).

11. The oligonucleotide is The labeled probe of any one of claims 1 to 8, comprising 5'-CAGTGAATGCGAGTCCGTCTAAG-3' (SEQ ID NO:32).

12. 1. A method for measuring electrochemiluminescence, comprising: (a) applying a potential to an electrode under conditions such that a complex in proximity to the electrode emits electrochemiluminescence, the complex comprising a target oligonucleotide and a labeled probe according to any one of claims 1 to 11, the labeled probe comprising an oligonucleotide complementary to the target oligonucleotide; (b) measuring the emitted electrochemiluminescence.

13. The method of claim 12 , wherein the target oligonucleotide is immobilized on the electrode.

14. The method of claim 12, wherein the complex further comprises a binding reagent capable of binding to the target oligonucleotide, the binding reagent being immobilized on the electrode.

15. The method of claim 12, wherein the target oligonucleotide is immobilized on a solid support.

16. The method of claim 12, wherein a binding reagent capable of binding to the target oligonucleotide is immobilized on a solid support, and the solid support is immobilized on the electrode.

17. 17. The method of claim 15 or 16, wherein the solid support is a particle, and the particle is collected on the electrode using gravity, centrifugation, filtration, or application of a magnetic field.

18. A kit for measuring electrochemiluminescence, comprising: a labeled probe according to any one of claims 1 to 11; (a) electrode, (b) ECL reading buffer; (c) a nucleic acid polymerase; (d) a nucleic acid ligase; (e) assay diluent; (f) additional nucleic acid reagents; (g) Assay consumables; or (h) any combination thereof.

19. 20. The kit of claim 18, wherein the kit comprises the electrode, the electrode being a carbon-based electrode.

20. 20. The kit of claim 18, wherein the kit comprises the assay consumable, the assay consumable being a multi-well plate assay consumable, each well of the plate comprising a carbon ink electrode.

21. The kit of any one of claims 18 to 20, wherein the kit comprises the ECL read buffer, and the ECL read buffer comprises tripropylamine.

22. The kit of any one of claims 18 to 20, wherein the kit comprises the ECL read buffer, and the ECL read buffer comprises butyldiethanolamine.

23. A non-naturally occurring nucleic acid probe comprising an oligonucleotide, said oligonucleotide being 14-24 nucleotides in length and comprising 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

24. 24. The non-natural nucleic acid probe of claim 23, further comprising a non-natural 5' modification comprising a reactive functional group.

25. 25. The non-natural nucleic acid probe of claim 24, wherein the reactive functional group is a thiol, amine, carboxylic acid, active ester, hydrazine, aldehyde, ketone, alkyne, strained alkene, azide, or tetrazine.

26. 26. The non-natural nucleic acid probe of claim 25 having formula IIIA. 【Chemistry 7】

27. 24. The non-natural nucleic acid probe of claim 23, further comprising a non-natural 5' modification comprising a hapten or biotin.

28. 28. The non-natural nucleic acid probe of claim 27, wherein the hapten comprises fluorescein, dinitrophenyl, or digoxigenin.

29. 28. The non-natural nucleic acid probe of claim 27, wherein the modification comprises biotin.

30. 30. The non-natural nucleic acid probe of claim 29 having formula IV: 【Chemistry 8】

31. The non-natural nucleic acid probe of any one of claims 23 to 30, wherein the oligonucleotide is 14 to 19 nucleotides in length.

32. 32. The non-natural nucleic acid of claim 31 , wherein the oligonucleotide is 15 nucleotides in length.

33. A conjugate compound comprising a detection reagent conjugated to the non-natural nucleic acid probe of any one of claims 23 to 32.

34. The conjugate compound of claim 33, wherein the detection reagent is an antigen-binding agent.

35. A non-naturally occurring oligonucleotide comprising from 53 to 76 nucleotides and comprising at its 5' end the sequence 5'-GTTCTGTC-3' and at its 3' end the sequence 5'-GTGTCTA-3'.

36. The oligonucleotide of claim 35, wherein the oligonucleotide sequence further comprises 5'-CAGTGAATGCGAGTCCGTCTAAG-3' (SEQ ID NO:34) and 5'-AAGAGAGTAGTACGCA-3' (SEQ ID NO:35).

37. The oligonucleotide of claim 36, wherein the oligonucleotide is 53 to 61 nucleotides in length.

38. 36. The oligonucleotide of claim 35, wherein the oligonucleotide consists of 5'-GTTCTGTCATATTTCAGTGAATGCGAGTCCGTCTAAGAGAGTAGTACAGCAAGAGTGTCTA-3' (SEQ ID NO:36).

39. The oligonucleotide of any one of claims 35 to 38, further comprising a 5' terminal phosphate group.

40. 1. A kit for conjugating a nucleic acid probe to a non-nucleic acid detection reagent to form a conjugate, comprising: (a) a heterobifunctional crosslinker, (i) a first reactive group capable of reacting with the detection reagent to attach the crosslinker to the detection reagent; and (ii) a heterobifunctional crosslinker that is substantially non-reactive towards the detection reagent but includes a second reactive group capable of reacting with the nucleic acid probe to attach the crosslinker to the nucleic acid probe; (b) a first size separation device capable of separating the conjugate from unreacted nucleic acid probes; (c) a nucleic acid binding fluorophore, wherein a fluorescence intensity of the fluorophore increases when the fluorophore binds to a nucleic acid.

41. 41. The kit of claim 40, wherein the first reactive group comprises an amine-reactive moiety.

42. 42. The kit of claim 41, wherein the first reactive group comprises an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester.

43. (i) the nucleic acid probe comprises a thiol moiety and the second reactive group comprises a maleimide, iodoacetamide, or activated disulfide moiety; (ii) the nucleic acid probe comprises an alkene or strained alkene moiety and the second reactive group comprises a tetrazine moiety; (iii) the nucleic acid probe comprises a tetrazine moiety and the second reactive group comprises a vinyl or strained alkene moiety; (iv) the nucleic acid probe comprises an alkyne or strained alkyne moiety and the second reactive group comprises an azide moiety; (v) the nucleic acid probe comprises an azide moiety and the second reactive group comprises an alkyne or strained alkyne moiety; (vi) the nucleic acid probe comprises a 4-formylbenzamide moiety and the second reactive group comprises a hydrazinonicotinamide moiety; or (vii) The kit of any one of claims 40 to 42, wherein the nucleic acid probe comprises a hydrazinonicotinamide moiety and the second reactive group comprises a 4-formylbenzamide moiety.

44. The kit according to any one of claims 40 to 42, wherein the nucleic acid probe is a thiol-modified oligonucleotide and the second reactive group is a thiol-reactive group.

45. 45. The kit of claim 44, wherein the second reactive group comprises a maleimide or iodoacetamide moiety.

46. The nucleic acid probe is a thiol-modified oligonucleotide, the first reactive group is an amine-reactive group, the second reactive group is a thiol-reactive group, and the heterobifunctional crosslinker is a compound of formula V: 【Chemistry 9】 The kit of claim 40, wherein r is an integer from 0 to 24.

47. 47. The kit of claim 46, wherein r is 4.

48. The kit according to any one of claims 40 to 43, further comprising the nucleic acid probe.

49. 49. The kit of claim 48, wherein the nucleic acid probe comprises 14 or 15 consecutive oligonucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

50. The kit according to any one of claims 44 to 47, further comprising the nucleic acid probe.

51. 51. The kit of claim 50, wherein the nucleic acid probe is modified at the 5' end with the thiol moiety.

52. 52. The kit of claim 51, wherein the nucleic acid probe is a compound of formula IIIA: 【Chemistry 10】

53. The kit of any one of claims 50 to 52, wherein the nucleic acid probe comprises 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

54. 54. The kit of any one of claims 40 to 53, wherein the first size separation device is a dialysis device, an ultrafiltration device or a size exclusion column, and the separation device has a molecular weight cut-off suitable for separating oligonucleotides having a molecular weight of about 5,000 daltons or less from conjugates having a molecular weight of more than 50,000 daltons.

55. 55. The kit of claim 54, wherein the separation device is a column comprising a size-exclusion chromatography matrix.

56. 56. The kit of claim 55, wherein the size-exclusion chromatography matrix comprises SEPHADEX G100 beads.

57. 55. The kit of any one of claims 38 to 54, wherein the fluorophore is a QUANT-IT OLIGREEN dye, a QUANTI-IT RIBOGREEN dye, a QUANTIFLUOR ssDNA dye, a SYBR Green I dye, or a SYBR Green II dye.

58. 56. The kit of claim 55, wherein the fluorophore is SYBR Green I dye.

59. The kit of any one of claims 40 to 58, wherein the kit further comprises a calibration oligonucleotide.

60. The kit of any one of claims 40 to 59, wherein the kit further comprises a second size separation device for desalting the detection reagent prior to conjugation.

61. The kit of any one of claims 40 to 60, wherein the detection reagent is a protein.

62. 62. The kit of claim 61, wherein the detection reagent is an antigen-binding agent.

63. The kit of any one of claims 40 to 62, wherein the kit components are in a single package.

64. 1. A method for conjugating a nucleic acid probe to a non-nucleic acid detection reagent to form a conjugate, comprising: (a) reacting a detection reagent and a nucleic acid probe to form a conjugate; (b) separating the conjugate from unreacted nucleic acid probe using a size separation device to form a purified conjugate; (c) forming a test composition comprising a sample of the purified conjugate and a nucleic acid binding fluorophore selected to have a fluorescence intensity that increases upon binding to a nucleic acid; (d) measuring the fluorescence of the test composition to determine the amount of nucleic acid probe in the purified conjugate.

65. 65. The method of claim 64, wherein the fluorophore is a QUANT-IT OLIGREEN dye, a QUANTI-IT RIBOGREEN dye, a QUANTIFLUOR ssDNA dye, a SYBR Green I dye, or a SYBR Green II dye.

66. 66. The method of claim 65, wherein the fluorophore is SYBR Green I dye.

67. 67. The method of any one of claims 64 to 66, wherein the size separation device is a dialysis device, an ultrafiltration device or a size exclusion column, and the separation device has a molecular weight cut-off suitable for separating oligonucleotides having a molecular weight of about 5,000 daltons or less from conjugates having a molecular weight of more than 50,000 daltons.

68. 68. The method of claim 67, wherein the separation device is a column comprising a size exclusion chromatography matrix.

69. 69. The method of claim 68, wherein the size exclusion chromatography matrix comprises SEPHADEX G100 beads.

70. 70. The method of any one of claims 64 to 69, further comprising forming at least one calibration composition comprising a known amount of a calibration oligonucleotide and said fluorophore, and measuring said fluorescence.

71. 71. The method of claim 70, wherein determining the amount of the oligonucleotide in the purified conjugate comprises comparing the fluorescence measured from the test composition to the fluorescence measured with the one or more calibration compositions.

72. 72. The method of claim 71, further comprising measuring the concentration of a detection reagent of the purified conjugate.

73. 73. The method of claim 72, wherein the concentration of the detection reagent is measured using a BCA protein assay.

74. 74. The method of claim 73, further comprising calculating the average number of bound nucleic acid probes per detection reagent in the purified conjugate.

75. The method of any one of claims 64 to 74, wherein the detection reagent is an antigen-binding substance.

76. 1. A method for conjugating a nucleic acid probe to a non-nucleic acid detection reagent to form a conjugate, comprising: contacting the detection reagent and the nucleic acid probe with the heterobifunctional crosslinker under conditions in which the detection reagent reacts with a first reactive group of the heterobifunctional crosslinker and the nucleic acid reacts with a second reactive group of the crosslinker to form the conjugate, the heterobifunctional crosslinker comprising: (i) a first reactive group capable of reacting with the detection reagent to attach the crosslinker to the detection reagent; (ii) a second reactive group that is substantially non-reactive to the detection reagent but capable of reacting with the nucleic acid probe to attach the crosslinker to the nucleic acid probe; The method does not include purifying the reaction product of the detection reagent and the cross-linking agent prior to the reaction of the cross-linking agent with the nucleic acid probe.

77. 1. A method for conjugating a nucleic acid probe to a non-nucleic acid detection reagent to form a conjugate, comprising: (a) contacting the detection reagent with the heterobifunctional crosslinker under conditions where the detection reagent reacts with a first reactive group of the heterobifunctional crosslinker to form a first composition, the heterobifunctional crosslinker comprising: (i) a first reactive group capable of reacting with the detection reagent to attach the crosslinker to the detection reagent; and (ii) forming a first composition that is substantially non-reactive toward the detection reagent but includes a second reactive group capable of reacting with the nucleic acid probe to attach the crosslinker to the nucleic acid probe; (b) contacting the first composition with the nucleic acid probe under conditions in which the second reactive group of the crosslinker reacts with the nucleic acid probe to form the conjugate; The method does not include purifying the reaction product of the detection reagent and the cross-linking agent prior to the reaction of the cross-linking agent with the nucleic acid probe.

78. 78. The method of claim 76 or 77, further comprising separating the conjugates from unconjugated nucleic acid probes using a size separation device.

79. (a) separating the conjugate from unreacted nucleic acid probe using a size separation device to produce a purified conjugate; (b) forming a test composition comprising the purified conjugate and a nucleic acid binding fluorophore selected to have a fluorescence intensity that increases upon binding to a nucleic acid; 77. The method of claim 76, further comprising: (c) measuring the fluorescence of the test composition to determine the amount of the nucleic acid probe in the purified conjugate.

80. (a) separating the conjugate from unreacted nucleic acid probe using a size separation device to produce a purified conjugate; (b) forming a test composition comprising the purified conjugate and a nucleic acid binding fluorophore selected to have a fluorescence intensity that increases upon binding to a nucleic acid; 78. The method of claim 77, further comprising: (c) measuring the fluorescence of the test composition to determine the amount of the nucleic acid probe in the purified conjugate.

81. 81. The method of claim 79 or 80, wherein the fluorophore is a QUANT-IT OLIGREEN dye, a QUANTI-IT RIBOGREEN dye, a QUANTIFLUOR ssDNA dye, a SYBR Green I dye, or a SYBR Green II dye.

82. 82. The method of claim 81 , wherein the fluorophore is SYBR Green I dye.

83. 83. The method of any one of claims 73 to 82, further comprising forming at least one calibration composition comprising a known amount of a calibration oligonucleotide and said fluorophore, and measuring said fluorescence.

84. 84. The method of claim 83, wherein determining the amount of the nucleic acid probe in the purified conjugate comprises comparing the fluorescence measured from the test composition to the fluorescence measured with the one or more calibration compositions.

85. 81. The method of claim 80, further comprising assaying the concentration of a detection reagent in the purified conjugate sample.

86. 82. The method of claim 81, wherein the assay is a BCA protein assay.

87. 83. The method of claim 82, further comprising calculating the average number of bound nucleic acid probes per detection reagent in the purified conjugate.

88. 88. The method of any one of claims 76-87, wherein the first reactive group comprises an amine-reactive moiety.

89. 89. The method of claim 88, wherein the first reactive group comprises an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester.

90. (i) the nucleic acid probe comprises a thiol moiety and the second reactive group comprises a maleimide, iodoacetamide, or activated disulfide moiety; (ii) the nucleic acid probe comprises an alkene or strained alkene moiety and the second reactive group comprises a tetrazine moiety; (iii) the nucleic acid probe comprises a tetrazine moiety and the second reactive group comprises a vinyl or strained alkene moiety; (iv) the nucleic acid probe comprises an alkyne or strained alkyne moiety and the second reactive group comprises an azide moiety; (v) the nucleic acid probe comprises an azide moiety and the second reactive group comprises an alkyne or strained alkyne moiety; (vi) the nucleic acid probe comprises a 4-formylbenzamide moiety and the second reactive group comprises a hydrazinonicotinamide moiety; or (vii) The method of any one of claims 76 to 87, wherein the nucleic acid probe comprises a hydrazinonicotinamide moiety and the second reactive group comprises a 4-formylbenzamide moiety.

91. 88. The method of any one of claims 76 to 87, wherein the nucleic acid probe comprises a thiol and the second reactive group is a thiol reactive group.

92. 92. The method of claim 91 , wherein the second reactive group comprises a maleimide or iodoacetamide moiety.

93. the nucleic acid probe comprises a thiol, the first reactive group is an amine reactive group, the second reactive group is a thiol reactive group, and the heterobifunctional crosslinker is a compound of formula V: 【Chemistry 11】 88. The method of any one of claims 76 to 87, wherein r is an integer from 0 to 24.

94. 94. The method of claim 93, wherein r is 4.

95. 91. The method of any one of claims 76 to 90, wherein the nucleic acid probe comprises 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

96. The method of any one of claims 91 to 94, wherein the nucleic acid probe is modified at the 5' end with a thiol moiety.

97. 97. The method of claim 96, wherein the nucleic acid probe is a compound of formula IIIA. 【Chemistry 12】

98. 98. The method of claim 96 or 97, wherein the oligonucleotide comprises 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

99. 99. The method of any one of claims 76 to 98, wherein the size separation device is a dialysis device, an ultrafiltration device or a size exclusion column, and the separation device has a molecular weight cut-off suitable for separating oligonucleotides having a molecular weight of about 5,000 daltons or less from conjugates having a molecular weight of more than 50,000 daltons.

100. 100. The method of claim 99, wherein the separation device is a column comprising a size exclusion chromatography matrix.

101. 101. The method of claim 100, wherein the size exclusion chromatography matrix comprises SEPHADEX G100 beads.

102. The method of any one of claims 76 to 101, wherein the detection reagent is an antigen-binding substance.

103. 103. The method of any one of claims 76 to 102, wherein the cross-linking agent is in molar excess relative to the amount of the detection reagent and the amount of the nucleic acid probe is in molar excess relative to the amount of the cross-linking agent.

104. 104. The method of claim 103, wherein the amount of said crosslinker is at least a four-fold molar excess relative to the amount of said detection reagent.

105. 105. The method of claim 104, wherein the molar amount of said nucleic acid probe is at least 1.1 times the molar amount of said crosslinker.

106. The method of any one of claims 76 to 105, wherein said method results in at least 95% of said detection reagent being conjugated to a nucleic acid probe.

107. The method of any one of claims 76 to 105, wherein the average number of said nucleic acid probes attached to each detection reagent of said conjugate is about 2 or more.

108. A kit for carrying out an assay, comprising: (a) an anchor reagent comprising an anchor oligonucleotide; (b) a labeled probe according to any one of claims 1 to 7; and (c) a connector oligonucleotide comprising a 5' terminal nucleotide sequence, a 3' terminal nucleotide sequence, said 5' and 3' terminal nucleotide sequences being capable of hybridizing to a nucleic acid probe, a first internal nucleotide sequence capable of hybridizing to a complement of said anchor oligonucleotide, and a second internal nucleotide sequence capable of hybridizing to a complement of a detection oligonucleotide of said labeled probe; (d) a nucleic acid ligase; and (e) a nucleic acid polymerase.

109. A kit for carrying out an assay, comprising: (a) an anchor reagent comprising an anchor oligonucleotide; (b) a labeled probe; (c) a connector oligonucleotide comprising a 5' terminal nucleotide sequence, a 3' terminal nucleotide sequence, said 5' and 3' terminal nucleotide sequences being capable of hybridizing to a nucleic acid probe, a first internal nucleotide sequence capable of hybridizing to a complement of said anchor oligonucleotide, and a second internal nucleotide sequence capable of hybridizing to a complement of a detection oligonucleotide of said labeled probe; (d) a nucleic acid ligase; and (e) a nucleic acid polymerase, (i) the 5' and 3' terminal nucleotide sequences do not overlap with the first and second internal sequences; (ii) the combined length of the 5' and 3' terminal sequences is between 14 and 24 nucleotides in length; (iii) the connector oligonucleotide is 53 to 76 nucleotides in length.

110. The kit according to claim 109, wherein the labeled probe is a labeled probe according to any one of claims 1 to 7.

111. The labeled probe is The kit according to any one of claims 108 to 110, comprising a sequence having at least 90% sequence identity with 14 or 15 consecutive nucleotides of 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO:31).

112. The labeled probe is The kit of any one of claims 108 to 110, comprising 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31).

113. The labeled probe is The kit of any one of claims 108 to 110, comprising 5'-CAGTGAATGCGAGTCCGTCTAAG-3' (SEQ ID NO: 32).

114. The kit of any one of claims 108 to 113, wherein the anchor oligonucleotide is 10 to 30 nucleic acids in length.

115. The kit of any one of claims 104 to 110, wherein the anchor oligonucleotide comprises 5'-AAGAGAGTAGTACAGCA-3' (SEQ ID NO:35).

116. 112. The kit of claim 111, wherein the anchor oligonucleotide consists of 5'-AAGAGAGTAGTACAGCAGCCGTCAA-3' (SEQ ID NO:37).

117. The kit of any one of claims 108 to 113, wherein the anchor oligonucleotide is 17 or 25 oligonucleotides in length.

118. The kit of claim 117, wherein the anchor oligonucleotide comprises 5'-AAGAGAGTAGTACAGCA-3' (SEQ ID NO:35).

119. The kit of any one of claims 108 to 118, wherein the connector oligonucleotide further comprises a 5' terminal phosphate group.

120. 120. The kit of any one of claims 108-119, wherein the connector oligonucleotide is 53-61 nucleotides in length.

121. The kit according to any one of claims 108 to 120, wherein the sum of the lengths of the 3' and 5' terminal sequences is 14 to 19 nucleotides in length.

122. 122. The kit of claim 121, wherein the combined length of the 3' and 5' terminal sequences is 14 or 15 nucleotides in length.

123. The kit according to any one of claims 108 to 122, wherein the 5' terminal sequence is GTTCTGTC and the 3' terminal sequence is GTGTCTA.

124. 124. The kit of claim 123, wherein the connector oligonucleotide sequence consists of 5'-GTTCTGTCATATTTCAGTGAATGCGAGTCCGTCTAAGAGAGTAGTACAGCAAGAGTGTCTA-3' (SEQ ID NO:36).

125. 125. The kit of any one of claims 108-124, wherein the kit further comprises a nucleic acid probe comprising a first sequence that is complementary to the 5' terminal sequence of the connector oligonucleotide and an adjacent second sequence that is complementary to the 3' terminal sequence of the connector oligonucleotide.

126. 126. The kit of claim 125, wherein the nucleic acid probe comprises the 14 or 15 contiguous nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

127. 127. The kit of claim 125 or 126, wherein the nucleic acid probe further comprises a non-natural modification that presents a reactive functional group.

128. 128. The kit of claim 127, wherein the nucleic acid probe is a compound of formula IIIA. 【Chemistry 13】

129. 127. The kit of claim 125 or 126, wherein the nucleic acid probe is conjugated to a detection reagent.

130. The detection reagent is a protein and the conjugate is a compound of formula VI: 【Chemistry 14】 130. The kit of claim 129, wherein r is an integer from 0 to 24, x is an integer from 1 to 20, and protein-NH- is the conjugated form of an amino group derived from the protein.

131. 131. The kit of claim 130, wherein r is 4.

132. The kit comprises: A capture reagent for the analyte; a detection reagent for said analyte; 125. The kit of any one of claims 108 to 124, wherein the capture and detection reagents are capable of binding to the analyte to form a complex, and the detection reagent is linked to a nucleic acid probe comprising a first sequence that is complementary to the 5' terminal sequence of the connector oligonucleotide and an adjacent second sequence that is complementary to the 3' terminal sequence of the connector oligonucleotide.

133. 133. The kit of claim 132, wherein the nucleic acid probe comprises 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

134. The detection reagent is a protein and the conjugate is a compound of formula VI: 【Chemistry 15】 134. The kit of claim 132 or 133, wherein r is an integer from 0 to 24, x is an integer from 1 to 20, and protein-NH- is the conjugated form of an amino group derived from the protein.

135. 135. The kit of claim 134, wherein r is 4.

136. The kit of any one of claims 132 to 135, wherein the capture agent and the detection agent are proteins.

137. The kit of claim 136, wherein the capture reagent and the detection reagent are antigen-binding agents.

138. The kit comprises: (a) a plurality of capture reagents for a plurality of analytes; (b) one or more detection reagents for said plurality of analytes; 125. The kit of any one of claims 108 to 124, wherein for each analyte, the kit comprises a capture reagent and a detection reagent capable of binding to the analyte to form a complex, wherein the one or more detection reagents are each linked to a nucleic acid probe comprising a first sequence that is complementary to the 5' terminal sequence of the connector oligonucleotide and an adjacent second sequence that is complementary to the 3' terminal sequence of the connector oligonucleotide.

139. 139. The kit of claim 138, wherein the nucleic acid probe comprises 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

140. Each detection reagent is a protein and is linked to a nucleic acid probe to form a conjugate of formula VI: 【Chemistry 16】 140. The kit of claim 138 or 139, wherein r is an integer from 0 to 24, x is an integer from 1 to 20, and protein-NH- is the conjugated form of an amino group derived from the protein.

141. 141. The kit of claim 140, wherein r is 4.

142. The kit of any one of claims 138 to 141, wherein the capture and detection agents are proteins.

143. The kit of claim 141, wherein the capture and detection reagents are antigen-binding agents.

144. 144. The kit of any one of claims 108 to 143, wherein the anchor reagent further comprises a targeting reagent capable of binding to the complement of the targeting reagent.

145. A kit according to any one of claims 132 to 137, wherein the anchor reagent and the capture reagent each comprise a targeting reagent capable of binding to the complement of the targeting reagent.

146. 144. The kit of any one of claims 138 to 143, wherein the anchor reagent and capture reagent each comprise a targeting reagent capable of binding to the complement of the targeting reagent.

147. The kit of any one of claims 144 to 146, further comprising a solid support having immobilized thereon the complement of the targeting reagent.

148. The kit of claim 147, wherein the solid support is an electrode.

149. The kit of claim 148, wherein the electrode is a carbon-based electrode.

150. 150. The kit of claim 149, wherein the kit comprises consumables for a multi-well plate assay, each well of the plate comprising a carbon ink electrode.

151. The kit of claim 147, wherein the solid support is a bead.

152. 152. The kit of any one of claims 144-151, wherein the targeting reagent and the complement of the targeting reagent are two members of a binding partner pair selected from avidin-biotin, streptavidin-biotin, antibody-hapten, antibody-antigen, antibody-epitope tag, nucleic acid-complementary nucleic acid, aptamer-aptamer target, and receptor-ligand.

153. 153. The kit of claim 152, wherein the targeting reagent is biotin and the complement of the targeting reagent is streptavidin.

154. The anchoring reagent is 【Chemistry 17】 The kit of claim 153,

155. The kit of any one of claims 108 to 143, wherein the kit further comprises a solid phase support, and the anchoring reagent is immobilized on the solid phase support.

156. The kit of any one of claims 133 to 138, wherein the kit further comprises a solid phase support, and the anchor reagent and capture reagent are immobilized on the solid phase support.

157. 157. The kit of claim 155 or 156, wherein the solid support is an electrode.

158. The kit of claim 157, wherein the electrode is a carbon-based electrode.

159. 159. The kit of claim 158, wherein the kit comprises consumables for a multi-well plate assay, each well of the plate comprising a carbon ink electrode.

160. 157. The kit of claim 155 or 156, wherein the solid support is a bead.

161. The kit of any one of claims 139 to 144, further comprising a solid phase support, the anchor reagent and the capture reagent being immobilized on the solid phase support to form an array, each array element comprising one of the plurality of capture reagents and at least one of the anchor reagents.

162. 157. The kit of claim 155 or 156, wherein the solid support is an electrode.

163. The kit of claim 157, wherein the electrode is a carbon-based electrode.

164. 159. The kit of claim 158, wherein the kit comprises consumables for a multi-well plate assay, each well of the plate containing a carbon ink electrode, and the electrode is one of a carbon ink electrode.

165. The kit of any one of claims 139 to 144, further comprising one or more solid supports, wherein the anchor reagent and capture reagent are immobilized on the one or more solid supports to form an array, each array element comprising one of the plurality of capture reagents and at least one of the anchor reagents.

166. 166. The kit of claim 165, wherein the kit comprises a plurality of commercially available solid supports, each solid support having at least one array element.

167. 167. The kit of claim 166, wherein each solid support has only one array element.

168. The kit according to any one of claims 165 to 167, wherein the solid support is an electrode.

169. The kit of claim 168, wherein the electrode is a carbon-based electrode.

170. 170. The kit of claim 169, wherein the kit comprises consumables for a multi-well plate assay, each well of the plate comprising one of the electrodes.

171. 168. The kit of claim 167, wherein the solid support is a bead in a bead array.

172. (i) an array having a plurality of complements of different target reagents immobilized on one or more solid supports, each array element comprising a complement of a different target reagent; 140. The kit of any one of claims 134 to 139, further comprising (ii) a different targeting reagent for each of the complements of the different targeting reagents, each targeting reagent being a binding partner for the complement of the corresponding targeting reagent.

173. (i) the capture reagents are coupled to the target reagents, each of the capture reagents being coupled to a different target reagent; 173. The kit of claim 172, wherein (ii) the anchor reagent is divided into a plurality of portions each containing at least one of the anchor reagents, and the at least one of the anchor reagents of each portion is connected to a different target reagent.

174. (i) the capture reagent comprises an additional linking reagent; (ii) the anchor reagent comprises the additional linking reagent; (iii) the targeting reagent is connected to a linking reagent; (iv) the linking reagent is a binding partner of the additional linking agent.

175. The kit of any one of claims 172 to 174, wherein each targeting reagent and its corresponding targeting reagent complement are complementary oligonucleotides.

176. 176. The kit of any one of claims 172 to 175, wherein the array is on a solid support, the solid support being an electrode.

177. The kit of claim 176, wherein the electrode is a carbon-based electrode.

178. 178. The kit of claim 177, wherein the kit comprises consumables for a multi-well plate assay, each well of the plate comprising a carbon ink electrode.

179. 179. The kit of claim 178, wherein each element of the array is on a different solid support, and the solid support is a bead.

180. 180. The kit of any one of claims 172 to 179, wherein the linking reagent and further linking reagent are the two members of a binding partner pair selected from avidin-biotin, streptavidin-biotin, antibody-hapten, antibody-antigen, antibody-epitope tag, nucleic acid-complementary nucleic acid, aptamer-aptamer target, and receptor-ligand.

181. 180. The kit of claim 179, wherein the linking reagent is biotin and the additional linking reagent is streptavidin.

182. 182. The kit of any one of claims 109-181, further comprising one or more of a blocking reagent, a binding assay reaction buffer, a ligase reaction buffer, a polymerase reaction buffer, an ECL read buffer and / or a unique product identifier.

183. 1. A method for measuring an analyte, comprising: (a) binding the analyte to (i) a capture reagent at a binding domain on a surface, the binding domain further comprising an anchor reagent comprising an anchor oligonucleotide, and (ii) a conjugate comprising a detection reagent and a nucleic acid probe, thereby forming a complex comprising the capture reagent, the analyte, and the conjugate at the binding domain; (b) extending the nucleic acid probe of the conjugate to form an extended sequence comprising an anchor oligonucleotide complement complementary to the anchor oligonucleotide and a detection sequence complement complementary to a detection oligonucleotide of a labeled probe; (c) binding the labeled probe comprising the detection oligonucleotide to the extension sequence; (d) measuring the amount of the labeled probe bound to the binding domain; The method, wherein the labeled probe is a labeled probe according to any one of claims 1 to 10.

184. 1. A method for measuring an analyte, comprising: (a) binding the analyte to (i) a capture reagent at a binding domain on a surface, the binding domain further comprising an anchor reagent comprising an anchor oligonucleotide, and (ii) a conjugate comprising a detection reagent and a nucleic acid probe comprising an oligonucleotide having a first probe sequence and an adjacent second probe sequence, thereby forming a complex comprising the capture reagent, the analyte, and the conjugate at the binding domain; (b) attaching the nucleic acid probe of the complex to a connector oligonucleotide, the connector oligonucleotide comprising a 5' terminal sequence complementary to the first probe sequence, a 3' terminal sequence complementary to the second probe sequence, a first internal sequence capable of hybridizing to a complement of the anchor oligonucleotide, and a second internal sequence capable of hybridizing to a complement of a detection oligonucleotide of a labeled probe; (c) ligating the connector oligonucleotides to form a circular template oligonucleotide; (d) extending the nucleic acid probe by rolling circle amplification to form an extended sequence; (e) binding a labeled probe comprising the detection oligonucleotide to the extension sequence; (f) measuring the amount of the labeled probe bound to the binding domain, The method, wherein the labeled probe is a labeled probe according to any one of claims 1 to 10.

185. 1. A method for measuring an analyte, comprising: (a) coupling the analyte to (i) a capture reagent having a binding domain on a surface, the binding domain further comprising an anchor oligonucleotide, and (ii) a conjugate of Formula VI, 【Chemistry 18】 In the formula, r is an integer of 0 to 24, x is an integer of 1 to 20, and protein-NH- is a conjugate type of an amino group derived from the protein. forming a complex comprising said capture reagent, said analyte, and said conjugate by binding to a conjugate of formula VI at said binding domain; (b) extending the nucleic acid probe of the conjugate to form an extended sequence comprising an anchor oligonucleotide complement that is complementary to the anchor oligonucleotide; (c) determining the amount of said extension sequence bound to said binding domain.

186. 1. A method for measuring an analyte, comprising: (a) coupling the analyte to (i) a capture reagent having a binding domain on a surface, the binding domain further comprising an anchor reagent comprising an anchor oligonucleotide, and (ii) a conjugate of Formula VI, 【Chemistry 19】 wherein the protein is a detection reagent, r is an integer from 0 to 24, x is an integer from 1 to 20, and protein-NH- is a conjugate type of an amino group derived from the protein; the conjugate comprises a detection reagent and a nucleic acid probe comprising an oligonucleotide having a first probe sequence and an adjacent second probe sequence; forming a complex comprising said capture reagent, said analyte, and said conjugate by binding to a conjugate of formula VI at said binding domain; (b) attaching the nucleic acid probe of the complex to a connector oligonucleotide, the connector oligonucleotide comprising a 5' terminal sequence complementary to the first probe sequence, a 3' terminal sequence complementary to the second probe sequence, a first internal sequence capable of hybridizing to a complement of the anchor oligonucleotide, and a second internal sequence capable of hybridizing to a complement of a detection oligonucleotide of a labeled probe; (c) ligating the connector oligonucleotides to form a circular template oligonucleotide; (d) extending the nucleic acid probe by rolling circle amplification to form an extended sequence; (e) binding the labeled probe comprising the detection oligonucleotide to the extension sequence; (f) measuring the amount of said labeled probe bound to said binding domain.

187. 187. The method of claim 185 or 186, wherein r is 4.

188. 1. A method for measuring an analyte, comprising: (a) binding the analyte to (i) a capture reagent at a binding domain on a surface, the binding domain further comprising an anchor reagent having an anchor oligonucleotide, and (ii) a conjugate comprising a detection reagent and a nucleic acid probe comprising an oligonucleotide having a first probe sequence and an adjacent second probe sequence, thereby forming a complex comprising the capture reagent, the analyte, and the conjugate at the binding domain; (b) attaching the nucleic acid probe of the complex to a connector oligonucleotide, the connector oligonucleotide comprising a 5' terminal sequence complementary to the first probe sequence, a 3' terminal sequence complementary to the second probe sequence, and an internal sequence capable of hybridizing to the complement of the anchor oligonucleotide; (c) ligating the connector oligonucleotides to form a circular template oligonucleotide; (d) extending the nucleic acid probe by rolling circle amplification to form an extended sequence; (e) measuring the amount of the extension sequence bound to the binding domain, The method, wherein the nucleotide probe is 14-24 nucleotides in length.

189. 1. A method for measuring an analyte, comprising: (a) binding the analyte to (i) a capture reagent at a binding domain on a surface, the binding domain further comprising an anchor reagent having an anchor oligonucleotide, and (ii) a conjugate comprising a detection reagent and a nucleic acid probe comprising an oligonucleotide having a first probe sequence and an adjacent second probe sequence, thereby forming a complex comprising the capture reagent, the analyte, and the conjugate at the binding domain; (b) attaching the first and second nucleic acid probes of the complex to a connector oligonucleotide, the connector oligonucleotide comprising a 5' terminal sequence complementary to the first nucleic acid probe, a 3' terminal sequence complementary to the second nucleic acid probe, a first internal sequence capable of hybridizing to a complement of the anchor oligonucleotide, and a second internal sequence capable of hybridizing to a complement of a detection oligonucleotide of a labeled probe; (c) ligating the connector oligonucleotides to form a circular template oligonucleotide; (d) extending the nucleic acid probe by rolling circle amplification to form an extended sequence; (e) binding the labeled probe comprising the detection oligonucleotide to the extension sequence; (f) measuring the amount of the labeled probe bound to the binding domain, The method, wherein the nucleic acid probe is 14 to 24 nucleotides in length.

190. The labeled probe is a compound of formula I, 【Chemistry 20】 where B is a nucleotide base, R is an electrochemiluminescent label, and L １ is a linking group, and L ２ is a linking group, j is an integer from 0 to 11, k is an integer from 0 to 1, m is an integer from 0 to 11, and n is an integer from 0 to 5.

191. 191. The method of claim 186, 187, 189 or 190, wherein the labeled probe comprises 14 or 15 consecutive oligonucleotides of 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO:31).

192. 192. The method of any one of claims 184 or 186-191, wherein the connector oligonucleotide further comprises a 5' terminal phosphate group.

193. 193. The method of any one of claims 184 or 186-192, wherein the connector oligonucleotide is 53-76 nucleotides in length.

194. 193. The method of any one of claims 184 or 188-192, wherein the connector oligonucleotide is 61 nucleotides in length.

195. 194. The method of any one of claims 184 or 186 to 193, wherein the 5' terminal sequence is GTTCTGTC and the 3' terminal sequence is GTGTCTA.

196. 195. The method of any one of claims 184 or 186-194, wherein the connector oligonucleotide consists of 5'-GTTCTGTCATATTTCAGTGAATGCGAGTCCGTCTAAGAGAGTAGTACAGCAAGAGTGTCTA-3' (SEQ ID NO:36).

197. 197. The method of any one of claims 183 to 196, wherein the nucleic acid probe is 14 to 24 nucleotides in length.

198. 200. The method of claim 196, wherein the nucleic acid probe is 14 or 15 nucleotides in length.

199. 202. The method of any one of claims 184 to 197, wherein the nucleic acid probe comprises 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

200. 200. The method of any one of claims 183-198, wherein the anchor oligonucleotide is 17-25 nucleotides in length.

201. The method of any one of claims 183 to 200, wherein the anchor oligonucleotide comprises 5'-AAGAGAGTAGTACAGCA-3' (SEQ ID NO:35).

202. The method of any one of claims 183 to 201, wherein the capture reagent and / or the detection reagent is a protein.

203. The method of any one of claims 183 to 202, wherein the capture reagent and / or the detection reagent is an antigen-binding agent.

204. The method of any one of claims 183 to 202, wherein the capture and detection reagents are antigen-binding agents.

205. 202. The method of any one of claims 183 to 201, wherein the anchor reagent and capture reagent each comprise a targeting reagent capable of binding to the complement of the targeting reagent, the binding domain comprises the complement of the targeting reagent immobilized thereon, and the method further comprises binding the anchor reagent and capture reagent to the binding domain.

206. 206. The method of claim 205, wherein the targeting reagent and the complement of the targeting reagent are the two members of a binding partner pair selected from avidin-biotin, streptavidin-biotin, antibody-hapten, antibody-antigen, antibody-epitope tag, nucleic acid-complementary nucleic acid, aptamer-aptamer target, and receptor-ligand.

207. 207. The method of claim 206, wherein the targeting reagent is biotin and the complement of the targeting reagent is streptavidin.

208. The method of any one of claims 183 to 207, wherein the surface is a bead.

209. 209. The method of any one of claims 183 to 208, wherein the surface comprises an electrode, and the detecting step comprises applying a potential to the electrode and measuring electrochemiluminescence.

210. 184. The method of claim 183, further comprising repeating steps (a)-(d) for one or more additional analytes, wherein each analyte binds to a different capture reagent in a different binding domain on the same surface or a different surface.

211. 185. The method of claim 184, further comprising repeating steps (a)-(f) for one or more additional analytes, wherein each analyte binds to a different capture reagent in a different binding domain on the same surface or a different surface.

212. 186. The method of claim 185, further comprising repeating steps (a)-(c) for one or more additional analytes, wherein each analyte binds to a different capture reagent in a different binding domain on the same surface or a different surface.

213. The method of claim 186 or 187, further comprising repeating steps (a)-(f) for one or more additional analytes, wherein each analyte binds to a different capture reagent in a different binding domain on the same surface or a different surface.

214. 189. The method of claim 188, further comprising repeating steps (a)-(e) for one or more additional analytes, wherein each analyte binds to a different capture reagent in a different binding domain on the same surface or a different surface.

215. 190. The method of claim 189, further comprising repeating steps (a)-(f) for one or more additional analytes, wherein each analyte binds to a different capture reagent in a different binding domain on the same surface or a different surface.

216. The labeled probe for each analyte is a compound of formula I, 【Chemistry 21】 where B is a nucleotide base, R is an electrochemiluminescent label, and L １ is a linking group, and L ２ is a linking group, j is an integer from 0 to 11, k is an integer from 0 to 1, m is an integer from 0 to 11, and n is an integer from 0 to 5.

217. 217. The method of claim 213, 215 or 216, wherein the labeled probes used for each analyte comprise the same oligonucleotide.

218. 218. The method of claim 217, wherein the oligonucleotide consists of 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO:31).

219. 219. The method of any one of claims 211 and 213-218, wherein the connector oligonucleotide used for each analyte further comprises a 5' terminal phosphate group.

220. 220. The method of any one of claims 211 and 213-219, wherein the connector oligonucleotides used for each analyte are 53-76 nucleotides in length.

221. 220. The method of any one of claims 211 and 213-219, wherein the connector oligonucleotide for each analyte is 61 nucleotides in length.

222. 221. The method of any one of claims 211 and 213-220, wherein the connector oligonucleotides used for each analyte have the same sequence.

223. 223. The method of claim 222, wherein the 5' terminal sequence is GTTCTGTC and the 3' terminal sequence is GTGTCTA.

224. 224. The method of claim 223, wherein the connector oligonucleotide sequence is 5'-GTTCTGTCATATTTCAGTGAATGCGAGTCCGTCTAAGAGAGTAGTACAGCAAGAGTGTCTA-3' (SEQ ID NO:36).

225. The method of any one of claims 210 to 224, wherein the nucleic acid probe used for each analyte is 14 to 24 nucleotides in length.

226. 226. The method of claim 225, wherein the nucleic acid probe used for each analyte is 14 or 15 nucleotides in length.

227. 228. The method of any one of claims 210 to 227, wherein the conjugates used for each analyte have the same nucleic acid probe sequence.

228. 228. The method of claim 227, wherein said nucleic acid probe comprises 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

229. 229. The method of any one of claims 210-228, wherein the anchor oligonucleotides used for each analyte are between 17 and 25 oligonucleotides in length.

230. 230. The method of any one of claims 210 to 229, wherein the same anchor oligonucleotide is used for each analyte.

231. 231. The method of claim 230, wherein the anchor oligonucleotide comprises 5'-AAGAGAGTAGTACAGCA-3' (SEQ ID NO:35).

232. The method of any one of claims 210 to 231, wherein a different detection reagent is used for each analyte.

233. The method of any one of claims 210 to 231, wherein the same detection reagent is used for each analyte.

234. The method of any one of claims 210 to 233, wherein the capture reagent and / or the detection reagent comprises a protein.

235. The method of any one of claims 210 to 234, wherein the capture reagent and / or the detection reagent comprises an antigen-binding substance.

236. 236. The method of claim 235, wherein the capture and detection reagents are antigen-binding agents.

237. The method of any one of claims 210 to 23, wherein each binding domain is on a separate surface, said surfaces being beads in a bead array.

238. 237. The method of any one of claims 210 to 236, wherein each binding domain is on a single surface, said binding domains forming said elements of an array of capture reagents on said surface.

239. 239. The method of claim 238, wherein the surface comprises an electrode and the detecting step comprises applying a potential to the electrode and measuring electrochemiluminescence.

240. 240. The method of any one of claims 210 to 239, wherein the binding of each analyte with its corresponding capture reagent is carried out in parallel by contacting the one or more surfaces with a single liquid volume containing multiple analytes.

241. 241. The method of claim 240, wherein each method step is performed in parallel for each analyte.

242. each binding domain comprises a complement of a target reagent capable of binding to the target reagent, each anchor reagent and capture reagent comprises an additional linking reagent capable of binding to the linking reagent; (1) binding the capture reagent and anchor reagent to a target reagent connected to the linking reagent via the additional linking reagent; and (2) binding the product of (1) to said binding domain containing the complement of said targeting reagent, thereby immobilizing said capture reagent and anchoring agent to each binding domain, (i) each binding domain comprises the complement of a different targeting agent; 242. The method of any one of claims 210-241, wherein (ii) the complement of each targeting reagent selectively binds to one of the targeting reagents.

243. the linking reagent has multiple binding sites for additional linking reagents, and the immobilization of the capture reagent and anchor reagent comprises: The method of claim 240, further comprising forming the target reagent connected to the linking reagent by binding the complement of the target reagent connected to an additional linking agent to a binding site of the linking reagent under conditions such that at least one other binding site of the linking reagent remains unbound.

244. 242. The method of claim 240 or 241, wherein the linking reagent is avidin or streptavidin and the additional linking reagent is biotin.

245. 243. The method of claim 240, 241 or 242, wherein the targeting reagent and the complement of the corresponding targeting reagent are complementary oligonucleotides.

246. The non-natural nucleic acid probe of any one of claims 23 to 34, wherein the oligonucleotide is 14 or 15 nucleotides in length.

247. The non-natural nucleic acid probe of any one of claims 23 to 34, wherein the oligonucleotide comprises 5'-ACAGAACTAGACAC-3' (SEQ ID NO:40).

248. The non-natural nucleic acid probe of any one of claims 23 to 34, wherein the oligonucleotide comprises 5'-GACAGAACTAGACA-3' (SEQ ID NO:41).

249. The non-natural nucleic acid probe of any one of claims 23 to 34, wherein the oligonucleotide comprises 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

250. 64. The kit of any one of claims 40 to 63, wherein the fluorescence intensity of the fluorophore increases when the fluorophore binds to single-stranded nucleic acid.

251. 99. The method of any one of claims 95 to 98, wherein the oligonucleotide comprises 5'-ACAGAACTAGACAC-3' (SEQ ID NO:40).

252. 99. The method of any one of claims 95 to 98, wherein the oligonucleotide comprises 5'-GACAGAACTAGACA-3' (SEQ ID NO:41).

253. 99. The method of any one of claims 95 to 98, wherein the oligonucleotide comprises 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

254. The kit of any one of claims 125 to 182, wherein the nucleic acid probe comprises an oligonucleotide, the oligonucleotide comprising 5'-ACAGAACTAGACAC-3' (SEQ ID NO:40).

255. The kit of any one of claims 125 to 182, wherein the nucleic acid probe comprises an oligonucleotide, the oligonucleotide comprising 5'-GACAGAACTAGACA-3' (SEQ ID NO:41).

256. The kit of any one of claims 125 to 182, wherein the nucleic acid probe comprises an oligonucleotide, the oligonucleotide comprising 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

257. 246. The method of any one of claims 183-245, wherein the nucleic acid probe comprises an oligonucleotide, the oligonucleotide comprising 5'-ACAGAACTAGACAC-3' (SEQ ID NO:40).

258. The kit of any one of claims 183 to 245, wherein the nucleic acid probe comprises an oligonucleotide, the oligonucleotide comprising 5'-GACAGAACTAGACA-3' (SEQ ID NO:41).

259. The kit of any one of claims 183 to 245, wherein the nucleic acid probe comprises an oligonucleotide, the oligonucleotide comprising 5'-GACAGAACTAGACAC-3' (SEQ ID NO:33).

260. 81. The method of any one of claims 64-75, 79 or 80, wherein the fluorescence intensity of the fluorophore increases when the fluorophore binds to single stranded nucleic acid.

261. An anchoring reagent of formula X, 【Chemical 22】 The anchor reagent wherein the oligonucleotide comprises 5'-AAGAGAGTAGTACAGCA-3' (SEQ ID NO:35).

262. 1. A method for measuring an analyte, comprising: (a) binding the analyte to a first capture reagent within a binding domain on a surface, the binding domain further comprising an anchor reagent comprising an anchor oligonucleotide; (b) binding a conjugate comprising a detection reagent and a nucleic acid probe to a second capture reagent in the binding domain to form a complex comprising the second capture reagent and the conjugate, wherein the detection reagent is a competitor of the analyte for binding to the first and second capture reagents; (c) extending the nucleic acid probe of the conjugate in the complex to form an extended sequence comprising an anchor oligonucleotide complement complementary to the anchor oligonucleotide and a detection sequence complement complementary to a detection oligonucleotide of a labeled probe; (d) binding the labeled probe comprising the detection oligonucleotide to the extension sequence; (e) measuring the amount of the labeled probe bound to the binding domain, The method, wherein the labeled probe is a labeled probe according to any one of claims 1 to 10.

263. 1. A method for measuring an analyte, comprising: (a) binding the analyte to a first conjugate comprising a first detection reagent and a nucleic acid probe; (b) binding the capture reagent of the binding domain on the surface to a second conjugate comprising a second detection reagent and a nucleic acid probe to form a complex comprising the capture reagent and the second conjugate, wherein (i) the binding domain further comprises an anchor reagent comprising an anchor oligonucleotide, and (ii) the capture reagent is a competitor of the analyte for binding to the first and second detection reagents; (c) extending the nucleic acid probe of the second conjugate in the complex to form an extended sequence comprising an anchor oligonucleotide complement complementary to the anchor oligonucleotide and a detection sequence complement complementary to a detection oligonucleotide of a labeled probe; (d) binding the labeled probe comprising the detection oligonucleotide to the extension sequence; (e) measuring the amount of the labeled probe bound to the binding domain, The method, wherein the labeled probe is a labeled probe according to any one of claims 1 to 10.

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