Electrochemiluminescence labeled probes for immune methods, methods of using such probes and kits comprising such probes

Abstract

The invention relates to a labeled probe. The invention also relates to analytical methods and compounds and kits for use in such assays.

Description

[0001] This application is a divisional application of CN202080032283.1. Technical Field

[0002] Sequence Listing

[0003] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on February 26, 2020 is named 0076-0010WO1_SL.txt and is 12,578 bytes in size.

[0004] The present invention relates to methods for performing immunoassays. The methods are designed to amplify immunoassay signals and anchor immunoassay complexes employed therein. Background Art

[0005] A large amount of literature has been developed about the technology of sensitive measurement of analytes of interest in samples using binding reactions, such as antigen-antibody reactions, nucleic acid hybridization, and receptor-ligand reactions. The high degree of specificity in many biochemical binding systems has produced many valuable analytical methods and systems in a variety of markets including basic research, human and veterinary diagnostics, environmental monitoring, and industrial testing. The presence of the analyte of interest can be measured by directly measuring the participation of the analyte in the binding reaction. In some methods, this participation can be indicated by measuring one or more observable markers connected to the binding material.

[0006] Although the sandwich immunoassay format provides excellent sensitivity and specificity in many applications, some analytes are present in concentrations that are too low for detection by conventional immunoassay techniques. The performance of sandwich immunoassays can also be limited by nonspecific binding of the detection antibody and the instability of the sandwich complex including the high dissociation rate antibody. However, efforts to improve conventional immunoassay techniques to increase sensitivity and specificity generally produce more complex, labor-intensive protocols that may be hampered by inefficiencies in each step that can greatly affect the sensitivity and specificity of the analysis. For example, in complex analyses that require multiple binding events and / or reactions, if any one event or reaction is less than ideal, the sensitivity and specificity of the overall analysis may be compromised. Summary of the invention

[0007] The present invention covers the following specific embodiments. Those skilled in the art may make various modifications, additions and changes to the embodiments described herein without departing from the spirit and scope of the present invention. Such modifications, additions and changes are intended to fall within the scope of the claims.

[0008] Embodiment (1): A method for detecting an analyte of interest in a sample, comprising: binding the analyte to: (i) a capture reagent on a surface comprising a capture reagent and an anchoring reagent for the analyte; and (ii) a detection reagent for the analyte attached to a nucleic acid probe; thereby forming a complex on the surface comprising the capture reagent, the analyte and the detection reagent; extending the probe to form an extended sequence comprising an anchor region bound to the anchoring reagent; binding the extended sequence to the anchoring reagent; and measuring the amount of the extended sequence bound to the surface.

[0009] In embodiment (1), the capture reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer. In a specific embodiment, the capture reagent is an antibody. The detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer, and in a specific embodiment, the detection reagent is an antibody. In a specific example of embodiment (1), the capture and detection reagents are antibodies against the analyte. The anchoring reagent can comprise an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an antigenic determinant or a mimic antigenic determinant; and optionally, the anchoring region can comprise an aptamer and the anchoring reagent can comprise an aptamer ligand. The anchoring region can comprise a nucleic acid sequence and the anchoring reagent can comprise a DNA binding protein. The anchoring region can comprise an oligonucleotide sequence and the anchoring reagent can comprise a complementary oligonucleotide sequence. The anchoring region can comprise a single-stranded oligonucleotide sequence or a double-stranded oligonucleotide sequence.

[0010] The binding step of embodiment (1) may further comprise forming a triple helix between the anchor region and the anchoring agent. The method may further comprise denaturing the anchor region to expose the single strand sequence prior to the binding step; exposing the anchor region to helicase activity prior to the binding step; and / or exposing the anchor region to nuclease treatment prior to the binding step. In this embodiment, the anchor region may comprise one or more bases modified by a hapten and the anchoring agent may comprise one or more antibodies specific for the hapten; and / or the anchor region may comprise one or more bases modified by a ligand and the anchoring agent may comprise one or more receptors specific for the ligand. The extension sequence may further comprise one or more detection sequences and the measuring step may comprise contacting the extension sequence with a plurality of labeled probes complementary to the one or more detection sequences; the extension sequence may comprise one or more modified bases and the measuring step may comprise contacting the extension sequence with a plurality of detectable moieties capable of binding to the one or more modified bases; and / or the extension sequence may comprise one or more labeled bases and the measuring step may further comprise detecting the presence of the one or more labeled bases. In this embodiment, one or more modified bases include an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an antigenic determinant, or a mimic antigenic determinant, and each of the multiple detectable moieties includes a binding partner of the one or more modified bases and a detectable label. One or more modified bases may include streptavidin and each of the multiple detectable moieties includes biotin and a detectable label; and / or one or more modified bases may include biotin and each of the multiple detectable moieties includes streptavidin and a detectable label; and / or one or more modified bases may include avidin and each of the multiple detectable moieties includes biotin and a detectable label; and / or one or more modified bases may include biotin and each of the multiple detectable moieties includes avidin and a detectable label.

[0011] The first step of embodiment (1) may include binding the analyte to the following substances in the following order: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte; or the first step of embodiment (1) may include binding the analyte to the following substances in the following order: (i) a detection reagent for the analyte; and (ii) a capture reagent on a surface; and / or the first step may include binding the analyte to the following substances simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte.

[0012] The extension step of embodiment (1) may include binding the probe to a circular nucleic acid and extending the circular template by rolling circle amplification. The extension step of embodiment (1) may include binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction; and / or binding the probe to a template nucleic acid sequence to form a circular nucleic acid template (e.g., by connecting a linear template to form a ring), and extending the circular template by rolling circle amplification. In these embodiments, the extended probe may remain confined to the surface after the probe is extended. 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. Thus, the complex may remain bound to the surface after the extension step, for example, the extended probe is bound to the anchoring agent at a position within 10-100 μm of the position of the complex on the surface. In a specific embodiment, the extended probe is bound to the anchoring agent at a position less than 100 μm, less than 50 μm, or more specifically, less than 10 μm of the position of the complex on the surface.

[0013] The extension step of embodiment (1) may include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustained synthesis reaction) or isothermal amplification method. In one embodiment, the extension step may include isothermal amplification methods, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0014] The surface mentioned in Example (1) may include particles and / or holes of a porous disk. The surface may include multiple different binding domains and the capture reagent and the anchoring reagent are located on two different binding domains on the surface. If the surface is a hole in a disk, the hole may include multiple different binding domains and the capture reagent and the anchoring reagent are located on two different binding domains in the hole; and / or the surface may contain multiple different binding domains and the capture reagent and the anchoring reagent are located on the same binding domain on the surface. In one embodiment, the hole may contain multiple different binding domains and the capture reagent and the anchoring reagent are located on the same binding domain in the hole. The capture reagent and the anchoring reagent may be within 10-100nm on the surface. The surface may include electrodes and the measuring step may further include applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal, and optionally, the method includes collecting particles on the electrodes and applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal.

[0015] The measuring step of embodiment (1) may further include combining the extended sequence with a detection probe having a detectable label, measuring the detectable label and associating the measured value with the amount of analyte in the sample, wherein the detection probe includes a nucleic acid sequence complementary to the region of the extended sequence. The detectable label can be measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field or a combination thereof. In the specific example of embodiment (1), the detectable label is an ECL label and the measuring step may include measuring the ECL signal. The detection probe may have multiple ECL labels. The detection probe may be connected to multiple ECL label portions via a bond at the 3' end of the probe nucleotide component.

[0016] Embodiment (2): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising (i) a capture reagent for the analyte and (ii) an anchoring reagent; and (b) a detection reagent for the analyte attached to a nucleic acid probe.

[0017] The anchoring agent of embodiment (2) may include an oligonucleotide sequence, an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an antigenic determinant, or a mimic antigenic determinant, and the capture agent may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer. In a specific embodiment, the capture agent may include an antibody and / or the detection agent may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer. In a specific embodiment of the kit, the detection agent is an antibody.

[0018] The surface of the test kit of embodiment (2) may comprise particles and / or holes of a porous disk. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface of the test kit is a hole of a disk, then the surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains in the hole; and / or the surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain on the surface. In a specific example of the test kit, the surface is a hole and the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain in the hole. The capture agent and the anchoring agent may be within 10-100 nm on the surface. In addition, the surface of the test kit may comprise an electrode.

[0019] 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 anchoring reagent comprising an anchoring oligonucleotide sequence; and (ii) a detection reagent for the analyte linked to a nucleic acid probe; thereby forming a complex comprising the capture reagent, the analyte and the detection reagent on the surface; (b) extending the probe to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence; (c) hybridizing the anchor sequence with the anchor sequence complement; and (d) measuring the amount of the extended sequence bound to the surface.

[0020] In embodiment (3), the capture reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer, and in a specific example, the capture reagent is an antibody. Similarly, the detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer, and in a specific example of embodiment (3), the detection reagent is an antibody. In an example of embodiment (3), the capture and detection reagents 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 a plurality of labeled probes complementary to the one or more detection sequences; or or in addition, the extension sequence can further include one or more modified bases and the measuring step can further include contacting the extension sequence with a plurality of detectable moieties capable of binding to the one or more modified bases. In a specific example, the extension sequence can further include one or more labeled bases and the measuring step can further include detecting the presence of the one or more labeled bases. One or more modified bases include an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an antigenic determinant, or a mimicking antigenic determinant, and each of the plurality of detectable moieties includes a binding partner of the one or more modified bases and a detectable label. One or more modified bases may include streptavidin and each of the plurality of detectable moieties includes biotin and a detectable label; one or more modified bases include biotin and each of the plurality of detectable moieties includes streptavidin and a detectable label; one or more modified bases include avidin and each of the plurality of detectable moieties includes biotin and a detectable label; and / or one or more modified bases include biotin and each of the plurality of detectable moieties includes avidin and a detectable label.

[0021] Step (a) of embodiment (3) may comprise binding the analyte to the following substances in the following order: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte. Alternatively, step (a) may comprise binding the analyte to the following substances in the following order: (i) a detection reagent for the analyte; and (ii) a capture reagent on a surface. In another example, step (a) may comprise binding the analyte to the following substances simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte.

[0022] The extension step of embodiment (3) may include binding the probe to the template nucleic acid sequence and extending the probe by polymerase chain reaction. Alternatively, the extension step may include binding the probe to the template circular nucleic acid and extending the circular template by rolling circle amplification. Alternatively, the extension step may include binding the probe to the template nucleic acid sequence, forming a circular nucleic acid template (e.g., by connection) 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 confined to the surface after the probe is extended, for example, the complex remains bound to the surface after the extension step. In one example, the extension probe is bound to an anchoring agent at a position within 10-100 μm of the position of the complex on the surface. In a specific embodiment, the extension probe is bound to an anchoring agent at a position less than 100 μm, less than 50 μm, or more specifically, less than 10 μm of the position of the complex on the surface. In this embodiment, the extension step may comprise PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustained synthesis reaction) or an isothermal amplification method. For example, the extension step may comprise an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0023] The surface of embodiment (3) may include particles and / or holes of a porous disk. The surface may include multiple different binding domains and the capture reagent and anchoring reagent are located on two different binding domains on the surface. If the surface is a hole of a disk, it may include multiple different binding domains and the capture reagent and anchoring reagent are located on two different binding domains in the hole. The surface may include multiple different binding domains and the capture reagent and anchoring reagent are located on the same binding domain on the surface. If the surface is a hole, it may include multiple different binding domains and the capture reagent and anchoring reagent are located on the same binding domain in the hole. The capture reagent and anchoring reagent may be within 10-100nm on the surface. In a specific example, the surface may include electrodes and the measuring step may further include applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The method may further include collecting particles on the electrodes and applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The measuring step may further include combining the extended sequence with a detection probe having a detectable label, measuring the detectable label and correlating the measured value with the amount of analyte in the sample, wherein the detection probe includes a nucleic acid sequence complementary to a region of the extended sequence. In this embodiment, the detectable label is measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field or a combination thereof. For example, the detectable label is an ECL label and the measuring step can include measuring the ECL signal. The detection probe can have multiple ECL labels. The detection probe can be connected to the multiple ECL label portion through a bond at the 3' end of the probe nucleotide component.

[0024] Embodiment (4): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising (i) a capture reagent for the analyte and (ii) an anchoring reagent comprising an anchoring oligonucleotide sequence; and (b) a detection reagent for the analyte attached to a nucleic acid probe.

[0025] The kit of embodiment (4) comprises a capture reagent, which comprises an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer. In a specific example, the capture reagent may comprise an antibody. Similarly, the detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer, and specifically, the detection reagent may comprise an antibody.

[0026] The kit of embodiment (4) comprises a surface that may include holes of particles and / or porous disks. The surface may comprise a plurality of different binding domains and the capture reagent and anchoring reagent are located on two different binding domains on the surface. If the surface is a hole, then the hole may comprise a plurality of different binding domains and the capture reagent and anchoring reagent are located on two different binding domains in the hole. The surface may comprise a plurality of different binding domains and the capture reagent and anchoring reagent are located on the same binding domain on the surface, for example, if the surface is a hole, then the hole may comprise a plurality of different binding domains and the capture reagent and anchoring reagent are located on the same binding domain in the hole. For example, the capture reagent and anchoring reagent are within 10-100nm on the surface. The surface of embodiment (4) may comprise an electrode.

[0027] 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 a capture reagent for the analyte and an anchor reagent comprising an anchor oligonucleotide sequence; (ii) a first detection reagent for the analyte linked to a first nucleic acid probe; and (iii) a second detection reagent for the analyte linked to a second nucleic acid probe; thereby forming a complex on the surface comprising a binding reagent, the analyte, and the first and second detection reagents; (b) extending the second probe using an extension method that requires the first and second probes to be adjacent to each other 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 the extended sequence bound to the surface.

[0028] The capture reagent of embodiment (5) can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer. In a specific example, the capture reagent is an antibody. Similarly, the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, and in a specific example, the first detection reagent is an antibody. The second detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, and in a specific example, the second detection reagent is an antibody. More specifically, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0029] 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, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an antigenic determinant or a mimicking antigenic determinant, and the plurality of detectable moieties each comprise a binding partner of one or more modified bases and a detectable label. For example, one or more modified bases include streptavidin and the multiple detectable moieties each include biotin and a detectable label; one or more modified bases include biotin and the multiple detectable moieties each include streptavidin and a detectable label; one or more modified bases include avidin and the multiple detectable moieties each include biotin and a detectable label; and / or one or more modified bases include biotin and the multiple detectable moieties each include avidin and a detectable label.

[0030] Step (a) of Example (5) may comprise binding the analyte to the following substances in the following order: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte. Alternatively, step (a) may comprise binding the analyte to the following substances in the following order: (i) a detection reagent for the analyte; and (ii) a capture reagent on a surface; or step (a) may comprise binding the analyte to the following substances simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte.

[0031] The extension step of embodiment (5) may include binding the probe to the template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step may 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 confined to the surface after the probe is extended, for example, the complex remains bound to the surface after the extension step. The extension probe may be bound to the anchoring agent at a position within 10-100 μm of the position of the complex on the surface. In a specific embodiment, the extension probe is bound to the anchoring agent at a position less than 100 μm, less than 50 μm, or more specifically, less than 10 μm from the position of the complex on the surface. The extension step may include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustaining synthesis reaction) or isothermal amplification method. In a specific example, the extension step may include an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0032] The extension method of embodiment (5) may include contacting the complex formed in step (a) with a connection sequence comprising: (i) an internal sequence complementary to the second probe and (ii) two terminal sequences complementary to the non-overlapping region of the first probe. The method may further include connecting the two terminal sequences of the connecting oligonucleotide to form a circular target sequence that hybridizes with both the first and second probes. Alternatively, the extension method may include contacting the complex formed in step (a) of embodiment (5) with the following: a first connecting oligonucleotide sequence comprising a first connecting probe sequence complementary to the first region of the first probe and the first region on the second probe, and a second connecting oligonucleotide comprising a second probe sequence complementary to the second non-overlapping region of the first probe and the second non-overlapping region of the second probe; and optionally, connecting the first and second connecting oligonucleotides to form a circular target sequence that hybridizes with both the first and second probes.

[0033] The surface of embodiment (5) may comprise particles and / or holes of a porous disk. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole of a disk, the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may also comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain on the surface. If the surface is a hole of a disk, the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain in the hole. The capture agent and the anchoring agent may be within 10-100 nm on the surface. In a specific example, the surface may comprise an electrode and the measuring step may further comprise applying a voltage waveform to the electrode to generate an electrochemiluminescent signal, and optionally, the method of embodiment (5) further comprises collecting particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescent signal.

[0034] The measuring step of embodiment (5) can further comprise combining the extended sequence with a detection probe having a detectable label, measuring the detectable label and associating the measured value with the amount of analyte in the sample, wherein the detection probe comprises a nucleic acid sequence complementary to the region of the extended sequence. The detectable label can be measured by measuring light scattering, 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 comprise measuring the ECL signal. The detection probe can have multiple ECL labels. The detection probe can be connected to multiple ECL label portions through a key at the 3' end of the probe nucleotide component.

[0035] 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 anchoring reagent comprising an anchoring 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.

[0036] The capture reagent of embodiment (6) may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, and in a specific example, the capture reagent may comprise an antibody. Similarly, the first detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, and in a specific example, the first detection reagent may comprise an antibody. Similarly, the second detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, and in a specific example, the second detection reagent may comprise an antibody.

[0037] The surface of embodiment (6) may include particles and / or holes of a porous disk. The surface may include multiple different binding domains and the capture reagent and anchoring reagent are located on two different binding domains on the surface. If the surface is a hole, the hole may include multiple different binding domains and the capture reagent and anchoring reagent are located on two different binding domains in the hole. The surface may include multiple different binding domains and the capture reagent and anchoring reagent are located on the same binding domain on the surface; and / or if the surface is a hole, the hole may include multiple different binding domains and the capture reagent and anchoring reagent are located on the same binding domain in the hole. The capture reagent and anchoring reagent may be within 10-100nm on the surface. In a specific example, the surface may include an electrode.

[0038] Embodiment (7): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent for the analyte on a surface comprising a capture reagent and an anchoring reagent; (ii) a first detection reagent for the analyte comprising a first proximity probe, and (iii) a second detection reagent for the analyte comprising a second proximity probe; thereby forming a detection complex on the surface comprising the capture reagent, the analyte, and the first and second detection reagents; (b) contacting the detection complex formed in (c) with a connecting sequence comprising: (i) an internal sequence complementary to the second proximity probe and (ii) two terminal sequences complementary to non-overlapping regions of the first proximity probe; (c) hybridizing the connecting sequence to the first and second proximity probes; (d) connecting the two terminal sequences of the connecting 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 anchoring reagent; (f) binding the amplicon to the anchoring reagent; and (g) measuring the amount of the amplicon on the surface.

[0039] Embodiment (8): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent for the analyte on a surface comprising a capture reagent and an anchoring 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 comprising the capture reagent, the analyte, and the first and second detection reagents on the surface; (b) contacting the detection complex formed in (c) with a first linker oligonucleotide and a second linker oligonucleotide, wherein (i) the first end of the first linker and the second end of the first linker are connected to the surface of the analyte. The first end of the second linker is complementary to two non-overlapping regions of the first proximity probe, and (ii) the second end of the first linker and the second end of the second linker are complementary to two non-overlapping regions of the first proximity probe; (c) hybridizing the first and second linker oligonucleotides to the first and second proximity probes; (d) connecting the first and second linker 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 including a binding domain that binds an anchoring agent; (f) binding the amplicon to the anchoring agent; and (g) measuring the amount of the amplicon on the surface.

[0040] The capture reagent of embodiments (7) and (8) can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, and in a specific example, the capture reagent is an antibody. Similarly, the first detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, for example, the first detection reagent is an antibody. In addition, the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, for example, the second detection reagent is an antibody. In the specific examples of embodiments (7) and (8), the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0041] The anchoring agent 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 antigenic determinant, or a mimicking antigenic determinant. In one example, the binding domain may comprise an aptamer and the anchoring agent may comprise an aptamer ligand. The binding domain may comprise a nucleic acid sequence and the anchoring agent may comprise a DNA binding protein; and / or the anchoring agent may comprise an oligonucleotide sequence and the amplicon may comprise a complementary oligonucleotide sequence.

[0042] The amplicons 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. In addition, the amplicons 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. In addition, the amplicons 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 include an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an antigenic determinant, or a mimicking antigenic determinant, and the plurality of detectable moieties each include a binding partner of the one or more modified bases and a detectable label. One or more modified bases may include streptavidin and the multiple detectable moieties each include biotin and a detectable label; one or more modified bases may include biotin and the multiple detectable moieties each include streptavidin and a detectable label; one or more modified bases may include avidin and the multiple detectable moieties each include biotin and a detectable label; and / or one or more modified bases may include biotin and the multiple detectable moieties each include avidin and a detectable label.

[0043] Step (a) of embodiments (7) and (8) may comprise binding the analyte to: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte in the following order. Alternatively, step (a) may comprise binding the analyte to: (i) a first and a second detection reagent for the analyte; and (ii) a capture reagent on a surface. Additionally, step (a) may comprise binding the analyte to: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte simultaneously or substantially simultaneously.

[0044] The amplicons of embodiments (7) and (8) can remain confined to the surface after the probe is extended. The complex can remain bound to the surface after the extension step. For example, the amplicons are bound to the anchoring agent at a position within 10-100 μm of the position of the complex on the surface. In a specific embodiment, the extension probe is bound to the anchoring agent at a position less than 100 μm, less than 50 μm, or more specifically, less than 10 μm of the position of the complex on the surface.

[0045] The surface of embodiments (7) and (8) may comprise a particle and / or a hole of a porous disk. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole of a disk, then the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain on the surface. If the surface is a hole of a disk, then the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain in the hole. In a specific example, the capture agent and the anchoring agent are within 10-100 nm on the surface.

[0046] In addition, the surface may include electrodes and the measuring step may include applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. In these embodiments ((7) and (8)), the method may further include collecting particles on the electrodes and applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The measuring step may include combining the amplicon with a detection probe having a detectable label, measuring the detectable label, and correlating the measured value with the amount of analyte in the sample, wherein the detection probe includes a nucleic acid sequence complementary to the region of the amplicon. The detectable label is measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. For example, the detectable label is an ECL label and the measuring step may include measuring the ECL signal. The detection probe may have multiple ECL labels. The detection probe may be connected to multiple ECL label portions via a bond at the 3' end of the probe nucleotide component.

[0047] Embodiment (9): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising (i) a capture reagent for the analyte and (ii) an anchoring reagent; (b) a first detection reagent for the analyte comprising a first proximity probe; (c) a second detection reagent for the analyte comprising a second proximity probe; and (d) a connecting 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 embodiments, the first and second proximity probes are nucleic acid probes.

[0048] Embodiment (10): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising (i) a capture reagent for the analyte and (ii) an anchoring reagent; and (b) a first detection reagent for the analyte comprising a first proximity probe; (c) a second detection reagent for the analyte comprising a second proximity probe; and (d) (i) a first linking oligonucleotide and (ii) a second linking oligonucleotide, wherein (x) the first end of the first linker and the first end of the second linker are complementary to two non-overlapping regions of the first proximity probe, and (y) the second end of the first linker and the second end of the second linker are complementary to two non-overlapping regions of the first proximity probe. In an embodiment, the first and second proximity probes are nucleic acid probes.

[0049] The capture reagents of embodiments (9) and (10) may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant, or an aptamer. In a specific example, the capture reagent may comprise an antibody. The first detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant, or an aptamer, and in a specific example, the first detection reagent may comprise an antibody. The second detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant, or an aptamer, and in a specific example, the second detection reagent may comprise an antibody.

[0050] The surface of embodiments (9) and (10) may comprise a particle and / or a hole of a porous disk. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole of a disk, then the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain on the surface. If the surface is a hole, then the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain in the hole. In a specific example, the capture agent and the anchoring agent are within 10-100 nm on the surface.

[0051] The surfaces of embodiments (9) and (10) may comprise electrodes.

[0052] Embodiment (11): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent for the analyte on a surface comprising a capture reagent and an anchoring reagent comprising an anchoring 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 comprising the capture reagent, the analyte, and the first and second detection reagents on the surface; (b) contacting the detection complex formed in (c) with a linker sequence comprising: (i) 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. The invention relates to a method for preparing an oligonucleotide that is complementary to an internal sequence of the two adjacent probes, (ii) two terminal sequences complementary to the non-overlapping region of the first adjacent probe and (iii) a sequence that matches the anchor sequence; (c) hybridizing the connecting sequence to the first and second adjacent probes; (d) connecting the two terminal sequences of the connecting oligonucleotide to form a circular target sequence that hybridizes to both the first and second adjacent probes; (e) extending the second adjacent probe by rolling circle amplification of the target sequence to produce an amplicon including a plurality of anchor sequence complements that are complementary to the anchor sequence; (f) hybridizing the anchor sequence to one of the anchor sequence complements; and (g) measuring the amount of the amplicon on the surface. In an embodiment, the first and second adjacent probes are nucleic acid probes.

[0053] 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 a capture reagent and an anchoring reagent comprising an anchoring 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 comprising the capture reagent, the analyte, and the first and second detection reagents on the surface; (b) contacting the detection complex formed in (a) with a first linking oligonucleotide and a second linking oligonucleotide, wherein (i) the first end of the first linker and the first end of the second linker are contacted with the first proximity probe. The invention relates to a method for preparing a target sequence of the present invention, wherein the first and second proximity probes are provided with a first linker and a second linker having two non-overlapping regions complementary to each other, (ii) the second end of the first linker and the second end of the second linker are complementary to the two non-overlapping regions of the first proximity probe, and (iii) the first and / or second linkers further comprise a sequence matching the anchor sequence; (c) hybridizing the first and second linker oligonucleotides to the first and second proximity probes; (d) connecting the first and second linker oligonucleotides to form a circular target sequence hybridized 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 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 the amplicon on the surface. In an embodiment, the first and second proximity probes are nucleic acid probes.

[0054] The capture reagent of embodiments (11) and (12) is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer. In a specific example, the capture reagent is an antibody. The first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, and in a specific example, the first detection reagent is an antibody. Similarly, the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an 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.

[0055] The amplicons 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. In addition, the amplicons 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 amplicons 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 include aptamers, aptamer ligands, antibodies, antigens, ligands, receptors, haptens, antigenic determinants or mimic antigenic determinants, and the plurality of detectable moieties each include a combination partner of one or more modified bases and a detectable label. One or more modified bases may include streptavidin and the multiple detectable moieties each include biotin and a detectable label; one or more modified bases may include biotin and the multiple detectable moieties each include streptavidin and a detectable label; one or more modified bases may include avidin and the multiple detectable moieties each include biotin and a detectable label; and / or one or more modified bases may comprise biotin and the multiple detectable moieties each include avidin and a detectable label.

[0056] Step (a) of embodiments (11) and (12) may comprise binding the analyte to: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte in the following order. Alternatively, step (a) may comprise binding the analyte to: (i) a first and a second detection reagent for the analyte; and (ii) a capture reagent on a surface. Additionally, step (a) may comprise binding the analyte to: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte simultaneously or substantially simultaneously.

[0057] The amplicons in embodiments (11) and (12) can remain confined to the surface after the probe is extended, and optionally, the complex remains bound to the surface after the extension step. For example, the amplicons are bound to the anchoring agent at a location within 10-100 μm of the location of the complex on the surface. In a specific embodiment, the extension probe is bound to the anchoring agent at a location less than 100 μm, less than 50 μm, or more specifically, less than 10 μm from the location of the complex on the surface.

[0058] The surface of embodiments (11) and (12) may comprise a particle and / or a hole of a porous disk. Optionally, the surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole, the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain on the surface. If the surface is a hole, the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain in the hole. The capture agent and the anchoring agent may be within 10-100 nm on the surface.

[0059] The surface of embodiments (11) and (12) may include electrodes and the measuring step may include applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. Optionally, embodiments (11) and (12) further include collecting particles on the electrodes and applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The measuring step may also include combining the amplicon with a detection probe having a detectable label, measuring the detectable label and associating the measured value with the amount of analyte in the sample, wherein the detection probe includes a nucleic acid sequence complementary to the region of the amplicon. The detectable label can be measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field or a combination thereof. In one example, the detectable label is an ECL label and the measuring step may include measuring the ECL signal. The detection probe may have multiple ECL labels. The detection probe may be connected to a multiple ECL label portion via a bond at the 3' end of the probe nucleotide component.

[0060] The sample of embodiments (11) and (12) may include one or more analyte molecules, and the surface may include multiple capture reagents for the one or more analyte molecules, which are distributed across multiple resolvable binding zones located on the surface, and the method may include: (x) allowing one or more analyte molecules to bind to one or more capture reagents on the surface; (y) determining the presence or absence of analyte molecules in each binding zone; and (z) identifying the number of binding zones containing analyte molecules and / or the number of analyte domains that do not contain analyte molecules. The measuring step may include imaging the optical signal from the surface to produce an image including multiple pixels, and each resolvable binding zone is mapped to one or more pixels in the image. The resolvable binding zone can be a unit of an array and / or the resolvable binding zone is configured to separate individual particles. Each distinguishable binding zone can be an individual nanopore with a volume <100 nL, for example, wherein at least 99% of the binding zones contain zero or one analyte molecule, wherein at least about 95% of the binding zones contain zero or one analyte molecule, wherein at least about 80% of the binding zones contain zero or one analyte molecule, and / or wherein at least about 50% of the binding zones contain zero or one analyte molecule. The concentration of analyte molecules in the samples of Examples (11) and (12) can be determined at least in part using a calibration curve of the number of binding zones containing at least one or one analyte molecule, a Poisson distribution analysis, and / or a Gaussian distribution analysis.

[0061] In embodiments (11) and (12), the sample may include one or more analyte molecules, the surface may include multiple particles, each of which includes multiple binding reagents for the analyte molecules, wherein the multiple particles are distributed across multiple distinguishable binding zones, and the method may include: (i) allowing one or more analyte molecules to bind to one or more binding reagents on the surface, and (ii) distributing the multiple particles across an array of distinguishable binding zones; and (iii) determining the presence or absence of analyte molecules in each distinguishable binding zone to identify the number of binding zones containing analyte molecules and / or the number of binding zones not containing analyte molecules.

[0062] 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 anchoring reagent comprising an anchoring 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 connecting 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 embodiments, the first and second proximity probes are nucleic acid probes.

[0063] 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 anchoring reagent comprising an anchoring oligonucleotide sequence; and (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 linking oligonucleotide and (ii) a second linking oligonucleotide, wherein (x) the first end of the first linker and the first end of the second linker are complementary to two non-overlapping regions of the first proximity probe, and (y) the second end of the first linker and the second end of the second linker are complementary to two non-overlapping regions of the first proximity probe. In an embodiment, the first and second proximity probes are nucleic acid probes.

[0064] The capture reagent of embodiments (13) and (14) may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, for example, the capture reagent may include an antibody. Similarly, the first detection reagent may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, for example, the first detection reagent may include an antibody. Similarly, the second detection reagent may include an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, for example, the second detection reagent may include an antibody.

[0065] The surface of embodiments (13) and (14) may comprise a particle and / or a hole of a porous disk. It may comprise a plurality of different binding domains and the capture agent and anchoring agent are located on two different binding domains on the surface. If the surface is a hole, the hole may comprise a plurality of different binding domains and the capture agent and anchoring agent are located on two different binding domains in the hole. The surface may comprise a plurality of different binding domains and the capture agent and anchoring agent are located on the same binding domain on the surface. If the surface is a hole, the hole may comprise a plurality of different binding domains and the capture agent and anchoring agent are located on the same binding domain in the hole. The capture agent and anchoring agent may be within 10-100 nm on the surface, and optionally, the surface may comprise an electrode.

[0066] Embodiment (15): A method for detecting an analyte in a sample, wherein the method may comprise: (a) combining the analyte with a first and a second detection reagent to form a detection complex, each detection complex comprising the analyte, a first detection reagent, and a second detection reagent, wherein the first detection reagent has a first detectable label and the second detection reagent has a second detectable label, (b) distributing the analyte across a plurality of reaction vessels so that a majority of the reaction vessels contain one or fewer analytes; and (c) detecting the number of analyte molecules by counting the number of reaction vessels containing the first and second detectable labels. In this embodiment (15), the first detection reagent may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, for example, the first detection reagent is an antibody. Similarly, the second detection reagent may be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, for example, the second detection reagent is an antibody. In a specific example, the first and second detection reagents are antibodies against the analyte.

[0067] Step (a) of embodiment (15) may further include forming a solution comprising the analyte and the detection reagent, and step (b) may include distributing the solution across multiple reaction vessels so that the probability of finding the unbound first detection reagent and the unbound second detection reagent in the same container is less than 1 / 10. Alternatively, step (a) of embodiment (15) may further include forming a solution comprising the analyte and the detection reagent, and step (b) may include distributing the solution across multiple reaction vessels so that the probability of finding the unbound first detection reagent and the unbound second detection reagent in the same container is less than 1 / 100. In addition, step (a) of embodiment (15) may further include forming a solution comprising the analyte and the detection reagent, and step (b) may include distributing the solution across multiple reaction vessels so that the probability of finding the unbound first detection reagent and the unbound second detection reagent in the same container is less than 1 / 1000. In addition, step (a) of embodiment (15) may further include forming a solution comprising the analyte and the detection reagent, and step (b) may include distributing the solution across multiple reaction vessels so that the probability of finding unbound first detection reagent and unbound second detection reagent in the same container is less than 1 / 10000.

[0068] Embodiment (16): A method for detecting an analyte in a sample, the method comprising: (a) combining the analyte with a capture reagent and a first and a second detection reagent to form a detection complex, each detection complex comprising a capture reagent, an analyte, a first detection reagent, and a second detection reagent, wherein (i) the first detection reagent has a first detectable label and the second detection reagent has a second detectable label, (ii) the capture reagent is on a surface; (b) distributing the analyte across a plurality of reaction vessels so that a majority of the reaction vessels contain one or fewer analytes; and (c) detecting the number of analyte molecules by counting the number of reaction vessels containing the first and second detectable labels. In this embodiment, the capture reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, for example, the capture reagent is an antibody. Similarly, the first detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, for example, the first detection reagent is an antibody. In addition, the second detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer, for example, the second detection reagent is an antibody. For example, the capture reagent, the first and second detection reagents are antibodies against the analyte.

[0069] Step (b) of Example (16) may further include distributing the solution across multiple reaction vessels so that the probability of finding the unbound first detection reagent and the unbound second detection reagent in the same container is less than 1 / 10. In addition, step (b) of Example (16) may further include distributing the solution across multiple reaction vessels so that the probability of finding the unbound first detection reagent and the unbound second detection reagent in the same container is less than 1 / 100. Step (b) of Example (16) may further include distributing the solution across multiple reaction vessels so that the probability of finding the unbound first detection reagent and the unbound second detection reagent in the same container is less than 1 / 1000. In addition, step (b) of Example (16) may further include distributing the solution across multiple reaction vessels so that the probability of finding the unbound first detection reagent and the unbound second detection reagent in the same container is less than 1 / 10000.

[0070] The capture reagent in the detection complex of embodiment (16) can be on the surface before the capture reagent binds to the analyte; or the capture reagent in the detection complex binds to the analyte before the capture reagent is fixed to the surface. In one example, the capture reagent can include a targeting moiety and the surface can include a targeting moiety complement. The targeting moiety and the targeting agent binding partner are selected from the following binding pairs: avidin-biotin, streptavidin-biotin, receptor-ligand, antibody-antigen, nucleic acid-nucleic acid complement.

[0071] The surface of embodiment (16) is a particle, and optionally, the capture reagent is fixed on a plurality of particles, and the distribution of the analyte is achieved by combining the analyte with the capture reagent and distributing the particles into a plurality of reaction vessels. The capture reagent can be fixed on a plurality of particles, and the distribution of the analyte is achieved by distributing the particles into a plurality of reaction vessels and then combining the analyte with the capture reagent.

[0072] Embodiment (16) may further include distributing the plurality of particles into a plurality of reaction vessels, wherein the plurality of particles include a targeting moiety, the capture reagent includes a complement of the targeting moiety, and the distribution of the analyte is achieved by binding the complement of the targeting moiety to the targeting moiety. Embodiment (16) may also include washing the particles before and / or after the distributing step.

[0073] The surface of embodiment (16) can be a position within one of the reaction vessels. In this embodiment, the capture reagent can be fixed on the surface of multiple reaction vessels and the distribution of the analyte is achieved by combining the analyte with the capture reagent. Optionally, the reaction vessel has a surface having a targeting moiety fixed thereon, the capture reagent includes a targeting moiety complement, and the distribution of the analyte is achieved by combining the targeting moiety complement with the targeting moiety. In this specific example, the method can further include washing the reaction vessel before the detection step.

[0074] The plurality of reaction vessels of embodiment (16) may comprise a nanopore array. The plurality of reaction vessels may comprise at least 10,000 reaction vessels. In one embodiment, the volume of the reaction vessel is less than 100 nL. Optionally, less than 50% of the reaction vessels contain analyte upon detection, less than 10% of the reaction vessels contain analyte upon detection, less than 1% of the reaction vessels contain analyte upon detection, and / or less than 0.1% of the reaction vessels contain analyte upon detection.

[0075] In one aspect of embodiment (16), the first detectable label is a first enzyme of a coupled enzyme reaction system and the second detectable label is a second enzyme of the coupled enzyme reaction system, and step (d) may comprise adding one or more substrates of the reaction system, generating a product of the enzyme reaction system, and counting reaction vessels containing the product. In this embodiment, the product can only be generated when the first enzyme and the second enzyme are in close proximity, such as when the first and second enzymes are within 200 nM of each other, or when the first and second enzymes are within 50 nM of each other. For example, the first enzyme is an oxidase, the second enzyme is a peroxidase, and the substrate comprises an oxidase substrate and a labeled Amplex Red or luminol derivative. In this embodiment, the oxidase may be glucose oxidase and the oxidase substrate is glucose. In one embodiment, the labeled Amplex Red or luminol is fixed to a surface by detecting a reaction catalyzed by the first and second enzymes in the complex, and optionally, the method may comprise 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 may comprise adding labeled streptavidin and measuring the label on the streptavidin.

[0076] The step (d) of embodiment (16) may include measuring the proximity-dependent signal generated when the first and second detectable labels are bound to the same analyte molecule, and counting the number of reaction vessels that generate the proximity-dependent signal, such as the proximity-dependent signal is generated by PLA-RCA. For example, the first detectable label may be a FRET donor and the detectable label is a FRET acceptor, and the proximity-dependent signal is measured by exciting the FRET donor and measuring the emission from the FRET acceptor. In one example, the first and second detectable labels may be measured independently. Optionally, the first and second detectable labels are luminescent labels that are different from each other in terms of spectral characteristics. 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, and the step (d) of embodiment (16) may include adding the first enzyme substrate and the second enzyme substrate and counting the number of reaction vessels that generate the first and second signals. The first and second signals may be changes in absorbance with different spectral characteristics. Optionally, the first and second signals are luminescent signals with different spectral characteristics. The first and second enzymes can be hydrolases, for example selected from phosphatases, sulfatases, beta-galactosidases, glucuronidases or combinations thereof, and the first and second substrates are selected from phosphate, sulfate, beta-galactosidase and glucuronide modified stable dioxetanes, 4-methylcoumarinyl, fluorescein or combinations thereof. In a specific example, the first and second enzymes are selected from horseradish peroxidase, beta-galactosidase and alkaline phosphatase. The detection step of embodiment (16) can include detection by light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, luminescence, radioactivity, magnetic field or combinations thereof.

[0077] Embodiment (17): A kit for detecting an analyte in a sample, the kit comprising in one or more vials, containers or compartments: (a) a first detection reagent comprising a first detectable marker; (b) a second detection reagent comprising a second detectable marker; and (c) a plurality of reaction containers configured to contain one or fewer analyte molecules.

[0078] Embodiment (18): A kit for detecting an analyte in a sample, the kit comprising in one or more vials, containers or compartments: (a) a first detection reagent comprising a first detectable label; (b) a second detection reagent comprising a second detectable label; (c) a surface comprising a capture reagent; and (d) a plurality of reaction vessels configured to contain one or fewer analyte molecules.

[0079] The first and second detection reagents of embodiments (17) and (18) may include antibodies, antigens, ligands, receptors, oligonucleotides, haptens, antigenic determinants, mimic antigenic determinants, aptamers, or combinations thereof. In one example, the first and second detection reagents include antibodies. The capture antibody may include antibodies, antigens, ligands, receptors, oligonucleotides, haptens, antigenic determinants, mimic antigenic determinants, or aptamers, for example, the capture antibody may include antibodies. In one example, the capture reagent may include a targeting moiety and the surface may include a targeting moiety complement, for example, the targeting moiety and the targeting agent binding partner are selected from the following binding pairs: avidin-biotin, streptavidin-biotin, receptor-ligand, antibody-antigen, nucleic acid-nucleic acid complement.

[0080] The surface of embodiments (17) and (18) can be a particle, and for example, the capture reagent is fixed on a plurality of particles. Alternatively, the surface is a location within one of the reaction vessels and for example, the capture reagent is fixed on the surface of a plurality of reaction vessels. Optionally, the reaction vessel has a surface having a targeting moiety fixed thereon and the capture reagent comprises a targeting moiety complement. The plurality of reaction vessels can include a nanopore array or water droplets dispersed in an oil-in-water emulsion. The plurality of reaction vessels can include at least 10,000 reaction vessels, and optionally, one of the plurality of reaction vessels has a volume of less than 100 nL.

[0081] In the kits of embodiments (17) and (18), the first detectable label can be the first enzyme of the coupled enzyme reaction system and the second detectable label is the second enzyme of the coupled enzyme reaction system, and the kit can include one or more substrates of the reaction system in one or more additional vials, containers or compartments. For example, the first enzyme is an oxidase, the second enzyme is a peroxidase, and the substrate includes an oxidase substrate and a labeled Amplex Red or luminol derivative. In a specific embodiment, the oxidase is glucose oxidase and the oxidase substrate is glucose. The first and second detectable labels can be components of a proximity-dependent system, for example, the first detectable label is a FRET donor and the detectable label is a FRET acceptor. The first and second detectable labels can be measured independently. Optionally, the first and second detectable labels are luminescent labels that are different from each other in terms of spectral characteristics.

[0082] In the kits of embodiments (17) and (18), the first detectable label is a first enzyme that reacts with a first substrate to produce a first signal, and the second detectable label is a second enzyme that reacts with a second substrate to produce a different second signal, and the kit may include a first enzyme substrate and a second enzyme substrate in one or more vials, containers or compartments. Optionally, the first and second signals are changes in absorbance with different spectral characteristics. In one example, the first and second signals are luminescent 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, beta-galactosidases, glucuronidases or a combination thereof. The first and second substrates may be selected from stable dioxetanes, 4-methylcoumarinyl, fluorescein or a combination thereof modified by phosphate, sulfate, beta-galactosidase and glucuronidase. Optionally, the first and second enzymes are selected from horseradish peroxidase, beta-galactosidase and alkaline phosphatase.

[0083] Embodiment (19): A method for detecting an analyte of interest in a sample, comprising: (a) binding and forming a complex between the analyte and a capture reagent, a first detection reagent having a first detectable label, and a second detection reagent having a second detectable label, wherein the capture reagent in the complex is fixed to a surface; (b) cross-linking the first and second detection reagents to form a cross-linked product; (c) releasing the cross-linked product from the surface into an eluent; (d) counting each cross-linked product in the eluent that includes both the first and second detectable labels. In this embodiment (19), the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, and in a specific embodiment, the capture reagent is an antibody. Similarly, the first detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, and in a specific embodiment, the first detection reagent is an antibody. In addition, the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, and in particular, the second detection reagent can be an antibody. In one embodiment, the capture reagent and the first and second detection reagents are antibodies directed against the analyte.

[0084] Embodiment (19) can further comprise adding a cross-linking agent to cross-link the first and second detection reagents, for example, the first and second detection reagents comprise reactive moieties and the cross-linking agent is a multifunctional cross-linking agent connected to the reactive moieties. For example, the reactive moieties comprise amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The cross-linking agent can comprise amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The first and second detection reagents can comprise binding moieties and the cross-linking agent is a multivalent binding partner of the binding moiety. In one example, the first and second detection reagents are antibodies of an animal species and the cross-linking agent is a multivalent anti-species antibody targeting antibodies of the animal species. The first and second detection reagents may include biotin and the cross-linker is streptavidin; the first and second detection reagents contain streptavidin and the cross-linker is biotin; the first and second detection reagents are linked to streptavidin and the cross-linker is a polymer comprising multiple biotin molecules; and / or the first and second detection reagents include first and second nucleic acid probes, respectively, and the cross-linker is an oligonucleotide that may include a sequence complementary to the first nucleic acid probe and an independent sequence complementary to the second nucleic acid probe.

[0085] The surface of embodiment (19) may include particles, reaction vessels, such as tubes or ampoules, and / or the surface may include holes of a porous disk. The method of embodiment (19) may further include collecting the particles and washing the particles to remove impurities, and optionally, the first and second detectable labels are measured by measuring light scattering, 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.

[0086] Embodiment (20): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers, or compartments: (a) a surface comprising an immobilized capture reagent; (b) a first detection reagent having a first detectable label; (c) a second detection reagent having a second detectable label; and (d) a cross-linker reactive to the first and second detection reagents.

[0087] The first and second detection reagents of embodiment (20) may include a reactive moiety and the cross-linker is a multifunctional cross-linker attached to the reactive moiety. The reactive moiety may include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof; and the cross-linker may include amines, thiols, hydrazides, aldehydes, esters, iodoacetamides, maleimides, click chemistry reagents, and combinations thereof. The first and second detection reagents of embodiment (20) may include a binding portion and the cross-linker is a multivalent binding partner of the binding portion, for example, the first and second detection reagents are antibodies of animal species and the cross-linker is a multivalent anti-species antibody targeting the antibody of the animal species; the first and second detection reagents include biotin and the cross-linker is streptavidin; the first and second detection reagents include streptavidin and the cross-linker is biotin; the first and second detection reagents are connected to streptavidin and the cross-linker is a polymer including multiple biotin molecules; and / or the first and second detection reagents include first and second nucleic acid probes, respectively, and the cross-linker is an oligonucleotide that can contain a sequence complementary to the first nucleic acid probe and an independent sequence complementary to the second nucleic acid probe.

[0088] The surface of embodiment (20) may comprise a particle, a hole of a porous disk, or a reaction vessel, such as a tube or an ampoule. In addition, the surface may comprise a plurality of different binding domains and the capture reagents are located on different binding domains on the surface. If the surface is a hole, then the hole may comprise a plurality of different binding domains and the capture reagents are located on different binding domains within the hole. The surface may also comprise an electrode.

[0089] Embodiment (21): A method for detecting an analyte of interest in a sample, comprising: (a) combining the analyte with a capture reagent, a first detection reagent, and a second detection reagent to form a complex, wherein the first detection reagent may comprise a first detectable label and a first nucleic acid probe, the second detection reagent may comprise a second detectable label and a second nucleic acid probe, and the capture reagent in the complex is fixed to a surface; (b) cross-linking the first and second detection reagents as follows: (i) hybridizing the first probe with the second probe, (ii) hybridizing the first and second probes with a third nucleic acid having a region complementary to the first and second probes, or (iii) connecting the first and second probes; (c) releasing the cross-linked products from the surface into an eluent; and (d) counting the individual cross-linked products in the eluent that include both the first and second detectable labels.

[0090] The capture reagent of embodiment (21) can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer, for example, the capture reagent is an antibody. Similarly, the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer, for example, the first detection reagent is an antibody; the second detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an 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.

[0091] The surface of embodiment (21) may comprise a particle, a reaction vessel (e.g., a tube or an ampoule), or a hole of a porous disk. The method of embodiment (21) may further comprise collecting the particles and washing the particles to remove impurities. The first and second detectable labels may be measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, a magnetic field, or a combination thereof. In a specific example, the first and second detectable labels comprise an ECL label and the counting step may comprise measuring an ECL signal.

[0092] Embodiment (22): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising an immobilized capture reagent; (b) a first detection reagent having a first detectable label and a first nucleic acid probe; (c) a second detection reagent having a second detectable label and a second nucleic acid probe; and (d) a third nucleic acid having regions complementary to the first and second nucleic acid probes.

[0093] The surface of embodiment (22) may comprise a particle, a well of a porous disk, or a reaction vessel, such as a tube or ampoule. The surface may comprise a plurality of different binding domains and the capture reagents are located on different binding domains on the surface, and if the surface is a well, the well may comprise a plurality of different binding domains and the capture reagents are located on different binding domains within the well. The surface may optionally comprise an electrode.

[0094] Embodiment (23): A method for detecting an analyte of interest in a sample, comprising: (a) combining the analyte with a capture reagent, a first detection reagent, and a second detection reagent to form a complex, wherein the first detection reagent may include a first nucleic acid probe, the second detection reagent may include a second nucleic acid probe, and the capture reagent in the complex is fixed to a surface; (b) extending the second nucleic acid probe to form an extended sequence including a detectable label, wherein 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 each extended sequence in the eluent. In this embodiment, the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer. In a specific example, the capture reagent is an antibody. Similarly, the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, 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 antigenic determinant, a mimetic antigenic determinant 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.

[0095] The surface of embodiment (23) may comprise particles, reaction vessels (eg, tubes or ampoules); or wells of a porous disk. The method of embodiment (23) may further comprise collecting the particles and washing the particles to remove impurities.

[0096] The label of embodiment (23) can be measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field or a combination thereof. In a specific example, the label can include an ECL label and the counting step can include measuring the ECL signal.

[0097] The extension step of embodiment (23) may include binding the probe to the template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step may also include binding the first probe to the template nucleic acid sequence, forming a circular nucleic acid template and extending the circular template by rolling circle amplification. The extension step may include binding the first probe to the template nucleic acid sequence, binding the second probe to the template sequence and connecting the first and second probes. Optionally, the label is a fluorescent label and counting each extended sequence may include single molecule fluorescence detection, for example, it may include fluorescence correlation spectroscopy and / or fluorescence cross-correlation spectroscopy. Single molecule fluorescence detection may include flowing an eluent through a capillary, focusing a light source on a volume within the capillary to produce an interrogation zone, and observing the interrogation zone with a light detector to detect fluorescent molecules passing through the interrogation zone. Single molecule fluorescence detection may also include flowing an eluent through a capillary, focusing a light source on a volume within the capillary to produce an interrogation zone, and observing the interrogation zone with a light detector to detect fluorescent molecules passing through the interrogation zone.

[0098] Embodiment (24): A method for detecting an analyte of interest in a sample, comprising: (a) allowing the analyte to bind and form a complex with a capture reagent, a first detection reagent having a first detectable label, and a second detection reagent having a second detectable label, 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 each product in the eluent that includes both the first and second detectable labels. In this embodiment, the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, such as the capture reagent is an antibody; the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, such as 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 antigenic determinant, a mimic antigenic determinant, or an aptamer, such as the second detection reagent is an antibody; and in a specific example, the capture reagent and the first and second detection reagents are antibodies against the analyte.

[0099] The surface of embodiment (24) may include particles, reaction vessels (e.g., tubes or ampoules), and / or holes of a porous disk. The method of embodiment (24) may include collecting the particles and washing the particles to remove impurities. The first and second detectable labels may be measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof, and in specific embodiments, the first and second detectable labels include ECL labels and the counting step may include measuring the ECL signal.

[0100] Embodiment (25): A method for detecting an analyte of interest in a sample, comprising: (a) binding the analyte to: (i) a capture reagent on a surface comprising a capture reagent for the analyte; (ii) a first detection reagent for the analyte linked to a first nucleic acid probe; and (iii) a second detection reagent for the analyte linked to a second nucleic acid probe; thereby forming a complex on the surface comprising a binding reagent, the analyte, and the first and second detection reagents; (b) extending the second probe to form an extended sequence using an extension method that requires the first and second probes to be in proximity; and (c) measuring the amount of the extended sequence bound to the surface. In this embodiment, the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, for example, the capture reagent is an antibody; the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, for example, the first detection reagent is an antibody; the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer; for example, the second detection reagent is an antibody; and in a specific example, the capture reagent and the first and second detection reagents are antibodies to the analyte.

[0101] 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 include an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an antigenic determinant, or a mimic antigenic determinant, and the plurality of detectable moieties each include a binding partner of the one or more modified bases and a detectable label. One or more modified bases may 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.

[0102] Step (a) of embodiment (25) may comprise binding the analyte to the following substances in the following order: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte; binding the analyte to the following substances in the following order: (i) a detection reagent for the analyte; and (ii) a capture reagent on a surface; or binding the analyte to the following substances simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a detection reagent for the analyte. The extension step may comprise binding the probe to a template nucleic acid sequence and extending the probe by polymerase chain reaction; or binding the probe to a template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. In this embodiment, the extended probe may remain confined to the surface after the probe is extended, for example, the complex remains bound to the surface after the extension step. The extension step may comprise PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustained synthesis reaction) or an isothermal amplification method. In a specific example, the extension step may comprise an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0103] The extension method of embodiment (25) may include contacting the complex formed in step (a) with a connection sequence comprising: (i) an internal sequence complementary to the second probe, and (ii) two terminal sequences complementary to the non-overlapping region of the first probe. The method may further include connecting the two terminal sequences of the connection oligonucleotide to form a circular target sequence that hybridizes with both the first and second probes. The extension method of embodiment (25) may also include contacting the complex formed in step (a) with the following: a first connection oligonucleotide sequence comprising a first connection probe sequence complementary to the first region of the first probe and the first region on the second probe, and a second connection oligonucleotide comprising a second probe sequence complementary to the second non-overlapping region of the first probe and the second non-overlapping region of the second probe. The method may also include connecting the first and second connection oligonucleotides to form a circular target sequence that hybridizes with both the first and second probes.

[0104] The surface of embodiment (25) may include a particle or a hole of a porous disk. The surface may include a plurality of different binding domains and the capture reagent is located on two different binding domains on the surface. If the surface is a hole, the hole may include a plurality of different binding domains and the capture reagent is located on two different binding domains in the hole. The surface may include a plurality of different binding domains and the capture reagent is located on the same binding domain on the surface, and if the surface is a hole, the hole may include a plurality of different binding domains and the capture reagent is located on the same binding domain in the hole. The surface may include electrodes and the measuring step may include applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The method optionally includes collecting particles on the electrodes and applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The measuring step may further include combining the extended sequence with a detection probe having a detectable label, measuring the detectable label and correlating the measured value with the amount of analyte in the sample, wherein the detection probe includes a nucleic acid sequence complementary to the region of the extended sequence. The detectable label can be measured by measuring light scattering, 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 comprise measuring the ECL signal. The detection probe may have multiple ECL labels. The detection probe may be linked to the multiple ECL label moiety via a bond at the 3' end of the probe nucleotide component.

[0105] 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.

[0106] The capture reagent of embodiment (26) may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, for example, the capture reagent may comprise an antibody; the first detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, for example, the first detection reagent may comprise an antibody; the second detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant, or an aptamer, for example, the second detection reagent may comprise an antibody; and the surface may comprise a particle and / or a well of a porous disk. The surface may comprise a plurality of different binding domains and the capture reagent is located on two identical binding domains on the surface; and if the surface is a well, the well may comprise a plurality of different binding domains and the capture reagent is located on two different binding domains within the well. Optionally, the surface may comprise a plurality of different binding domains and the capture reagent is located on the same binding domain on the surface, and if the surface is a well, the well may comprise a plurality of different binding domains and the capture reagent is located on the same binding domain within the well. The surface may comprise an electrode.

[0107] The surface of Examples 1-26 may comprise the inner surface of an analytical container (e.g., a test tube, a colorimetric tube, a flow cell, a FACS cell sorter, a filter cartridge, or a well of a multi-well plate). The surface may also include a slide, an analytical chip, or an analytical array; a pin, a probe, a bead, or a filter medium; a lateral flow medium, such as a filter membrane.

[0108] Embodiment (27): A method for detecting an analyte of interest in a sample comprising one or more analyte molecules, the method comprising: (a) contacting the sample with a surface comprising a plurality of distinguishable binding zones on the surface, each distinguishable binding zone 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 comprising the capture reagent, the analyte, and the first and second detection reagents on the distinguishable binding domains on the surface, wherein the first and second detectable labels are different labeled compounds; (c) determining the presence or absence of analyte molecules in each binding zone; and (d) identifying the number of binding zones containing analyte molecules and / or the number of binding zones not containing analyte molecules. The identification step may comprise imaging the optical signal from the surface to produce an image comprising a plurality of pixels, and each distinguishable binding zone is mapped to one or more pixels in the image. The distinguishable binding zones may be units of an array and / or configured to separate individual particles. Each resolvable binding zone can be an individual nanopore with a volume <100 nL and / or at least 99% of the binding zones contain zero or one analyte molecule; at least about 95% of the binding zones contain zero or one analyte molecule; at least about 80% of the binding zones contain zero or one analyte molecule; or at least about 50% of the binding zones contain zero or one analyte molecule. The concentration of analyte molecules in the sample can be determined at least in part using a calibration curve of the number of binding zones containing at least one or one analyte molecule, Poisson distribution analysis, and / or Gaussian step analysis.

[0109] The surface of embodiment (27) may include multiple particles, each of which includes multiple capture reagents for analyte molecules, wherein the multiple particles are distributed across multiple distinguishable binding zones, and the method may include: (i) allowing one or more analyte molecules to bind to one or more capture reagents on the surface and a first and a second detection reagent for each of the one or more analyte molecules, wherein the first and the second detection reagents respectively include a first and a second detectable label; (ii) distributing the multiple particles across an array of distinguishable binding zones; and (iii) determining the presence or absence of analyte molecules in each distinguishable binding zone to identify the number of binding zones containing analyte molecules and / or the number of binding zones not containing analyte molecules, wherein optionally, each distinguishable binding zone is an individual nanopore with a volume of <100nL, and / or at least 99% of the binding zones contain zero or one analyte molecule; at least about 95% of the binding zones contain zero or one analyte molecule; at least about 80% of the binding zones contain zero or one analyte molecule; and / or at least about 50% of the binding zones contain zero or one analyte molecule.

[0110] In embodiment (27), the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer, for example, the capture reagent is an antibody; the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer, for example, the first detection reagent is an antibody; the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an 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.

[0111] Step (a) of Example (27) may include binding the analyte to the following substances in the following order: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte; binding the analyte to the following substances in the following order: (i) a first and a second detection reagent for the analyte; and (ii) a capture reagent on a surface; or binding the analyte to the following substances simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for the analyte.

[0112] The surface of embodiment (27) may comprise particles or holes of a porous disk. In a specific example, the surface may comprise electrodes and the identification step may comprise applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The method of embodiment (27) may further comprise collecting particles on the electrodes and applying a voltage waveform to the electrodes to generate an electrochemiluminescent signal. The first detectable marker is measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof; and / or the second detectable marker is measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. The first and second detectable markers can be measured independently, and in one example, the first and second detectable markers are luminescent markers that are different from each other in terms of spectral characteristics.

[0113] The surface of Example (27) may comprise the inner surface of an analytical container (e.g., a test tube, a colorimetric tube, a flow cell, a FACS cell sorter, a filter cartridge, or a well of a multi-well plate). The surface may also include a slide, an analytical chip, or an analytical array; a pin, a probe, a bead, or a filter medium; a lateral flow medium, such as a filter membrane.

[0114] Embodiment (28): A kit for detecting an analyte of interest in a sample comprising one or more analyte molecules, the kit comprising: (a) a surface comprising a plurality of distinguishable binding regions disposed on the surface, each distinguishable binding region comprising a plurality of capture reagents for one or more analyte molecules in the sample; (b) a first detection reagent for the analyte comprising a first detectable label, and (c) a second detection reagent for the analyte comprising a second detectable label; wherein the first and second detectable labels are different labeled compounds.

[0115] The resolvable binding zones of embodiment (28) can be elements of an array and / or configured to separate individual particles. Each resolvable binding zone is optionally an individual nanopore having a volume <100 nL. The surface can comprise a plurality of particles, each of which comprises a plurality of capture reagents for an analyte molecule, wherein the plurality of particles are distributed across a plurality of resolvable binding zones, and the kit can comprise: a first and a second detection reagent for each of the one or more analyte molecules, wherein the first and the second detection reagents comprise a first and a second detectable label, respectively.

[0116] In embodiment (28), the capture reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer, for example, the capture reagent is an antibody; the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an aptamer, for example, the first detection reagent is an antibody; the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a mimic antigenic determinant or an 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.

[0117] The surface of embodiment (28) may comprise a particle or a hole of a porous disk. In a specific example, the surface may comprise an electrode and the identification step may comprise applying a voltage waveform to the electrode to produce an electrochemiluminescent signal. The first detectable label is measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof; and / or the second detectable label is measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field, or a combination thereof. The first and second detectable labels can be measured independently, and in one example, the first and second detectable labels are luminescent labels that are different from each other in terms of spectral characteristics.

[0118] The surface of Example (28) may comprise the inner surface of an analytical container (e.g., a test tube, a colorimetric tube, a flow cell, a FACS cell sorter, a filter cartridge, or a well of a multi-well plate). The surface may also include a slide, an analytical chip, or an analytical array; a pin, a probe, a bead, or a filter medium; a lateral flow medium, such as a filter membrane.

[0119] 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 a capture reagent for HIV p24 and an anchor reagent comprising an anchor oligonucleotide sequence; (ii) a first detection reagent for HIV p24 linked to a first nucleic acid probe; and (iii) a second detection reagent for HIV p24 linked to a second nucleic acid probe; thereby forming a complex on the surface comprising a binding reagent, HIV p24, and the first and second detection reagents; (b) extending the second probe using an extension method that requires the first and second probes to be adjacent to each other 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 the extended sequence bound to the surface.

[0120] The capture reagent of embodiment (29) can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer. In a specific example, the capture reagent is an antibody. Similarly, the first detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, and in a specific example, the first detection reagent is an antibody. The second detection reagent can be an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, and in a specific example, the second detection reagent is an antibody. More specifically, the capture reagent and the first and second detection reagents are antibodies against HIV p24.

[0121] 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, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an antigenic determinant or a mimicking antigenic determinant, and the plurality of detectable moieties each comprise a binding partner of one or more modified bases and a detectable label. For example, one or more modified bases include streptavidin and the multiple detectable moieties each include biotin and a detectable label; one or more modified bases include biotin and the multiple detectable moieties each include streptavidin and a detectable label; one or more modified bases include avidin and the multiple detectable moieties each include biotin and a detectable label; and / or one or more modified bases include biotin and the multiple detectable moieties each include avidin and a detectable label.

[0122] Step (a) of Example (29) may comprise binding HIV p24 to the following substances 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 substances in the following order: (i) a detection reagent for HIV p24; and (ii) a capture reagent on a surface; or step (a) may comprise binding HIV p24 to the following substances simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a detection reagent for HIV p24.

[0123] The extension step of embodiment (29) may include binding the probe to the template nucleic acid sequence and extending the probe by polymerase chain reaction. The extension step may further include binding the probe to the template nucleic acid sequence, forming a circular nucleic acid template and extending the circular template by rolling circle amplification. The extension probe may remain confined to the surface after the probe is extended, for example, the complex remains bound to the surface after the extension step. The extension probe may be bound to the anchoring agent at a position within 10-100 μm of the position of the complex on the surface. In a specific embodiment, the extension probe is bound to the anchoring agent at a position less than 100 μm, less than 50 μm, or more specifically, less than 10 μm of the position of the complex on the surface. The extension step may include PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-sustained synthesis reaction) or isothermal amplification method. In a specific example, the extension step may include an isothermal amplification method, such as helicase-dependent amplification or rolling circle amplification (RCA).

[0124] The extension method of embodiment (29) may include contacting the complex formed in step (a) with a connection sequence comprising: (i) an internal sequence complementary to the second probe and (ii) two terminal sequences complementary to the non-overlapping region of the first probe. The method may further include connecting the two terminal sequences of the connecting oligonucleotide to form a circular target sequence that hybridizes with both the first and second probes. Alternatively, the extension method may include contacting the complex formed in step (a) of embodiment (29) with: a first connecting oligonucleotide sequence comprising a first connecting probe sequence complementary to the first region of the first probe and the first region on the second probe, and a second connecting oligonucleotide comprising a second probe sequence complementary to the second non-overlapping region of the first probe and the second non-overlapping region of the second probe; and optionally, connecting the first and second connecting oligonucleotides to form a circular target sequence that hybridizes with both the first and second probes.

[0125] The surface of embodiment (29) may comprise particles and / or holes of a porous disk. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole of a disk, then the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may also comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain on the surface. If the surface is a hole of a disk, then the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain in the hole. The capture agent and the anchoring agent may be within 10-100 nm on the surface. In a specific example, the surface may comprise an electrode and the measuring step may comprise applying a voltage waveform to the electrode to generate an electrochemiluminescent signal, and optionally, the method of embodiment (29) further comprises collecting particles on the electrode and applying a voltage waveform to the electrode to generate an electrochemiluminescent signal.

[0126] The measuring step of embodiment (29) can include combining the extended sequence with a detection probe having a detectable label, measuring the detectable label and correlating the measured value with the amount of p24 in the sample, wherein the detection probe includes a nucleic acid sequence complementary to the region of the extended sequence. The detectable label can be measured by measuring light scattering, 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 the ECL signal. The detection probe can have multiple ECL labels. The detection probe can be connected to multiple ECL label portions via a bond at the 3' end of the probe nucleotide component.

[0127] Embodiment (30): A kit for detecting HIV p24 in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising (i) a capture reagent for HIV p24 and (ii) an anchoring reagent comprising an anchoring oligonucleotide sequence; (b) a first detection reagent for HIV p24 linked to a first nucleic acid probe; and (c) a second detection reagent for HIV p24 linked to a second nucleic acid probe.

[0128] The capture reagent of embodiment (30) may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant, or an aptamer, and in a specific example, the capture reagent may comprise an antibody. Similarly, the first detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant, or an aptamer, and in a specific example, the first detection reagent may comprise an antibody. Similarly, the second detection reagent may comprise an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant, or an aptamer, and in a specific example, the second detection reagent may comprise an antibody.

[0129] The surface of embodiment (30) may comprise a particle and / or a hole of a porous disk. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole, then the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains within the hole. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain on the surface; and / or if the surface is a hole, then the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain within the hole. The capture agent and the anchoring agent may be within 10-100 nm on the surface. In a specific example, the surface may comprise an electrode.

[0130] 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 anchoring 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 comprising a capture reagent, HIV p24 and the first and second detection reagents on the surface; (b) contacting the detection complex formed in (c) with a connecting sequence comprising: (i) an internal sequence complementary to the second proximity probe and (ii) two terminal sequences complementary to the non-overlapping region of the first proximity probe; (c) hybridizing the connecting sequence with the first and second proximity probes; (d) connecting the two terminal sequences of the connecting oligonucleotide to form a circular target sequence hybridized with 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 anchoring reagent; (f) binding the amplicon to the anchoring reagent; and (g) measuring the amount of the amplicon on the surface.

[0131] 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 anchoring 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 surface comprising the capture reagent, HIV p24, and an anchoring reagent; p24 and a detection complex of first and second detection reagents; (b) contacting the detection complex formed in (c) with a first linker oligonucleotide and a second linker oligonucleotide, wherein (i) the first end of the first linker and the first end of the second linker are complementary to two non-overlapping regions of the first proximity probe, and (ii) the second end of the first linker and the second end of the second linker are complementary to two non-overlapping regions of the first proximity probe; (c) hybridizing the first and second linker oligonucleotides with the first and second proximity probes; (d) connecting the first and second linker 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 including a binding domain that binds to an anchoring reagent; (f) binding the amplicon to the anchoring reagent; and (g) measuring the amount of the amplicon on the surface.

[0132] The capture reagent of embodiments (31) and (32) is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, and 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 antigenic determinant, a simulated antigenic determinant or an aptamer, for example, the first detection reagent is an antibody. In addition, the second detection reagent is an antibody, an antigen, a ligand, a receptor, an oligonucleotide, a hapten, an antigenic determinant, a simulated antigenic determinant or an aptamer, for example, the second detection reagent is an antibody. In the specific examples of embodiments (31) and (32), the capture reagent and the first and second detection reagents are antibodies against HIV p24.

[0133] The anchoring agent 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 antigenic determinant, or a mimicking antigenic determinant. In one example, the binding domain may comprise an aptamer and the anchoring agent may comprise an aptamer ligand. The binding domain may comprise a nucleic acid sequence and the anchoring agent may comprise a DNA binding protein; and / or the anchoring agent may comprise an oligonucleotide sequence and the amplicon may comprise a complementary oligonucleotide sequence.

[0134] The amplicons of embodiments (31) and (32) may 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. In addition, the amplicons may further comprise one or more modified bases and the measuring step may comprise contacting the extended sequence with a plurality of detectable moieties capable of binding to the one or more modified bases. In addition, the amplicons may further comprise one or more labeled bases and the measuring step may comprise detecting the presence of the one or more labeled bases. The one or more modified bases may comprise an aptamer, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an antigenic determinant, or a mimicking antigenic determinant, and the plurality of detectable moieties each comprise a combination partner of the one or more modified bases and a detectable label. One or more modified bases may include streptavidin and the multiple detectable moieties each include biotin and a detectable label; one or more modified bases may include biotin and the multiple detectable moieties each include streptavidin and a detectable label; one or more modified bases may include avidin and the multiple detectable moieties each include biotin and a detectable label; and / or one or more modified bases may include biotin and the multiple detectable moieties each include avidin and a detectable label.

[0135] Step (a) of Examples (31) and (32) may comprise binding HIV p24 to the following substances in the following order: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for HIV p24. Alternatively, step (a) may comprise binding HIV p24 to the following substances in the following order: (i) a first and a second detection reagent for HIV p24; and (ii) a capture reagent on a surface. Additionally, step (a) may comprise binding HIV p24 to the following substances simultaneously or substantially simultaneously: (i) a capture reagent on a surface; and (ii) a first and a second detection reagent for HIV p24.

[0136] The amplicons of embodiments (31) and (32) remain confined to the surface after the probe is extended. The complex can remain bound to the surface after the extension step. For example, the amplicons are bound to the anchoring agent at a position within 10-100 μm of the position of the complex on the surface. In a specific embodiment, the extension probe is bound to the anchoring agent at a position less than 100 μm, less than 50 μm, or more specifically, less than 10 μm of the position of the complex on the surface.

[0137] The surface of embodiments (31) and (32) may comprise a particle and / or a hole of a porous disk. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains on the surface. If the surface is a hole of a disk, then the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on two different binding domains in the hole. The surface may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain on the surface. If the surface is a hole of a disk, then the hole may comprise a plurality of different binding domains and the capture agent and the anchoring agent are located on the same binding domain in the hole. In a specific example, the capture agent and the anchoring agent are within 10-100 nm on the surface.

[0138] In addition, the surface may include electrodes and the measuring step may include applying a voltage waveform to the electrodes to produce an electrochemiluminescent signal. In these embodiments ((31) and (32)), the method may further include collecting particles on the electrodes and applying a voltage waveform to the electrodes to produce an electrochemiluminescent signal. The measuring step may include combining the amplicon with a detection probe having a detectable label, measuring the detectable label and correlating the measured value with the amount of analyte in the sample, wherein the detection probe includes a nucleic acid sequence complementary to the region of the amplicon. The detectable label is measured by measuring light scattering, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, bioluminescence, phosphorescence, radioactivity, magnetic field or a combination thereof. For example, the detectable label is an ECL label and the measuring step may include measuring the ECL signal. The detection probe may have multiple ECL labels. The detection probe may be connected to the multiple ECL label portions via a bond at the 3' end of the probe nucleotide component.

[0139] Embodiment (33): A method for detecting an analyte of interest in a sample, comprising: (a) concentrating the sample under conditions sufficient to form an analyte complex comprising the analyte bound to a first detection reagent, wherein the first detection reagent is linked to a first nucleic acid probe; (b) binding the analyte complex formed in step (a) to: (i) a capture reagent on a surface comprising a capture reagent for the analyte and an anchor reagent comprising an anchor oligonucleotide sequence; and (ii) a second detection reagent for the analyte linked to a second nucleic acid probe; thereby forming a complex on the surface comprising the capture reagent, the analyte, and the first and second detection reagents; (c) extending the second probe using an extension method that requires the first and second probes to be in 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 the extended sequence bound to the surface. The concentration step (a) may further include (i) contacting a sample containing the analyte with a solid phase, wherein the solid phase is linked to a targeting agent complementary to at least a portion of the first nucleic acid probe, thereby forming a concentrated complex comprising the analyte bound to the solid phase through a binding reaction between the first nucleic acid probe and the targeting agent; (ii) collecting the concentrated complex; (iii) separating unbound components of the sample from the concentrated complex; and (iv) releasing the concentrated complex to separate the solid phase from the analyte to form an analyte complex.

[0140] Embodiment (34): A kit for detecting an analyte of interest in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising (i) a capture reagent for the analyte and (ii) an anchoring reagent comprising an anchoring oligonucleotide sequence; (b) a first detection reagent for the analyte linked to a first nucleic acid probe; (c) a second detection reagent for the analyte linked to a second nucleic acid probe; and (d) a solid phase comprising a targeting agent complementary to at least a portion of the first nucleic acid probe.

[0141] Embodiment (35): A method for detecting exosomes in a sample, comprising: (a) binding exosomes to: (i) a capture reagent on a surface comprising a capture reagent for exosomes and an anchoring reagent comprising an anchoring oligonucleotide sequence; (ii) a first detection reagent for exosomes linked to a first nucleic acid probe; and (iii) a second detection reagent for exosomes linked to a second nucleic acid probe; thereby forming a complex comprising a binding reagent, exosomes, and the first and second detection reagents on the surface; (b) extending the second probe using an extension method that requires the 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 with the anchor sequence complement; and (d) measuring the amount of the extended sequence bound to the surface.

[0142] Embodiment (36): A kit for detecting exosomes of interest in a sample, comprising in one or more vials, containers or compartments: (a) a surface comprising (i) a capture reagent for exosomes and (ii) an anchoring reagent comprising an anchoring oligonucleotide sequence; (b) a first detection reagent for exosomes linked to a first nucleic acid probe; and (c) a second detection reagent for exosomes linked to a second nucleic acid probe.

[0143] 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 the binding reagent, the analyte, and the first and second detection reagents; (b) extending the first and second nucleic acid 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 the extended sequence bound to the surface using a labeled probe of Formula I:

[0144]

[0145] Where B is a nucleotide base, R is an electrochemiluminescent label, and L 1 is a linking group, L 2 is a linking group, j is an integer between 0 and 11, k is an integer between 0 and 1, m is an integer between 0 and 11, and n is an integer between 0 and 5.

[0146] 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 the 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 with the anchor sequence complement; and (d) measuring the amount of the extended sequence bound to the surface using a labeled probe of Formula I.

[0147] In any of the above embodiments (1) to (38), the anchoring agent is attached to the surface before, during or after the analyte binds to the capture agent. In embodiments comprising a kit, the anchoring agent is provided separately from the surface and then fixed to the surface, wherein the capture agent is fixed to the surface. In embodiments comprising a kit, the anchoring agent and the capture agent are provided fixed to the surface.

[0148] Any of the above embodiments (1) to (38) may comprise a labeled probe of Formula I:

[0149]

[0150] Where B is a nucleotide base, R is an electrochemiluminescent label, and L 1 is a linking group, L 2 is a linking group, j is an integer between 0 and 11, k is an integer between 0 and 1, m is an integer between 0 and 11, and n is an integer between 0 and 5.

[0151] Any of the above embodiments (1) to (38) may comprise a labeled probe of formula II:

[0152]

[0153] wherein j is an integer between 0 and 11, k is an integer between 0 and 1, m is an integer between 0 and 11, n is an integer between 0 and 5, and R is an electrochemiluminescent label:

[0154]

[0155] Any of the methods described in the embodiments herein may comprise a method of measuring electrochemiluminescence, comprising: (a) applying a potential to an electrode under conditions where a complex in proximity to the electrode will emit electrochemiluminescence, wherein the complex comprises a target oligonucleotide provided herein and a labeled probe, wherein the labeled probe comprises an oligonucleotide complementary to the target oligonucleotide; and (b) measuring the emitted electrochemiluminescence.

[0156] The nucleic acid probe described in any one of embodiments (1) to (38) (e.g., linked to a detection reagent) may include an oligonucleotide, wherein the oligonucleotide is 14-24 nucleotides in length and includes 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO: 33). In an embodiment, the present invention provides a method for combining a nucleic acid probe with a non-nucleic acid detection reagent to form a conjugate, comprising contacting a detection reagent and a nucleic acid probe with a heterobifunctional crosslinker 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 to form a conjugate, wherein the heterobifunctional crosslinker includes (i) a first reactive group capable of reacting with the detection reagent to link the crosslinker to the detection reagent, and (ii) a second reactive group capable of reacting with the nucleic acid probe to link the crosslinker to the nucleic acid probe while being substantially non-reactive with the detection reagent, wherein the method does not include purifying the reaction product of the detection reagent and the crosslinker before the crosslinker reacts with the nucleic acid probe.

[0157] In an embodiment, the present invention provides a method for combining a nucleic acid probe with a non-nucleic acid detection reagent to form a conjugate, which comprises (a) contacting the detection reagent with a heterobifunctional cross-linker under conditions in which the detection reagent reacts with a first reactive group of the cross-linker to form a first composition, wherein the heterobifunctional cross-linker comprises (i) a first reactive group capable of reacting with the detection reagent to connect the cross-linker to the detection reagent, and (ii) a second reactive group capable of reacting with the nucleic acid probe to connect the cross-linker to the nucleic acid probe while being substantially non-reactive with the detection reagent; and (b) contacting the first composition with the nucleic acid probe under conditions in which the second reactive group in the cross-linker reacts with the nucleic acid probe to form a conjugate, wherein the method does not include purifying the reaction product of the detection reagent and the cross-linker before the cross-linker reacts with the nucleic acid probe.

[0158] In an embodiment, the present invention provides a kit for combining a nucleic acid probe with a non-nucleic acid detection reagent to form a conjugate, comprising: (a) a heterobifunctional cross-linker comprising: (i) a first reactive group capable of reacting with a detection reagent to connect the cross-linker to the detection reagent; and (ii) a second reactive group capable of reacting with the nucleic acid probe to connect the cross-linker to the nucleic acid probe while being substantially non-reactive to the detection reagent; (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 the nucleic acid. In an embodiment, the present invention provides a method for combining a nucleic acid probe with a non-nucleic acid detection reagent to form a conjugate, which comprises: (a) reacting a detection reagent with 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 of a sample comprising a purified conjugate and a nucleic acid-binding fluorophore, wherein the nucleic acid-binding fluorophore is selected for having a fluorescence intensity that increases when the fluorophore binds 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 DESCRIPTION OF THE DRAWINGS

[0159] Figures 1(a)-(c) illustrate the use of anchoring reagents in immunoassays. Figure 1(a) shows the use of an anchoring reagent to bind to and stabilize a detection complex comprising a capture reagent, an analyte of interest, and a detection reagent comprising a nucleic acid probe. The nucleic acid probe is extended to bind to the anchoring reagent. In Figure 1(b), the anchoring reagent comprises an oligonucleotide sequence comprising a region complementary to a portion of an extended sequence formed on the detection reagent. Figure 1(c) shows a specific embodiment in which two detection reagents are used to bind the analyte, each detection reagent comprising a nucleic acid probe. The probes on the detection reagents are subjected to an amplification method that enables hybridization of one extended probe to the anchoring oligonucleotide sequence.

[0160] FIG. 2( a ) shows a specific embodiment in which the immune complex formed on the surface carrying the anchoring agent is subjected to the PLA-RCA method to incorporate a plurality of detectable substances in the extended sequence linked to the immune complex. Figure 2(b) and 2(c) are two alternative configurations of linker oligonucleotides that can be used in the methods of the invention.

[0161] Figure 3 Demonstrating a method for attaching oligonucleotides to proteins.

[0162] FIG4(a) illustrates a preferred embodiment of the invention, wherein a surface-bound complex is formed between a capture reagent, an analyte, and two detection reagents, each of which is respectively linked to a first and a second proximity probe, which are linked to a ligation probe to form a circular DNA template that is amplified by rolling circle amplification. FIG4(a) also includes an amplification reagent, which includes an anchor oligonucleotide sequence complementary to the sequence of an amplicon formed as the analytical method progresses. FIG4(b) shows an exemplary sequence of a first circular DNA template Circ-1, a detection oligonucleotide sequence, an inert region of the amplicon, and a portion PP2 that is designed to hybridize with a second proximity probe. An alternative embodiment is depicted in FIG4(c).

[0163] Figure 5 and 6(a) - (b) illustrates an alternative method for generating amplicons that can be amplified by rolling circle amplification.

[0164] Figure 7 An alternative embodiment is described in which a portion of each proximity probe in the sandwich complex is temporarily protected by a short strand of RNA to which each fragment hybridizes. These strands are enzymatically removed to allow the proximity probes to hybridize to each other and extend the strand.

[0165] Figure 8 Another embodiment is shown in which proximity probes are attached to capture reagents and detection reagents, and a portion of each proximity probe is temporarily protected by a short strand of RNA to which it hybridizes, as shown in FIG. Figure 8 As mentioned above.

[0166] Fig. 9 The calibration curve for the IL-10 assay performed using the method described in Example 1 is shown.

[0167] FIG. 10 ( a )-( b ) show fluorescence microscopy images with (a) and without (b) an anchoring agent.

[0168] Figure 11 (a) shows the configuration of a single linear linker oligonucleotide sequence comprising ligation site 1 or 2, and the use of these linkers in the assays of the present invention. Figure 11 (b) shows comparative performance data for assays using a combination of Circ-1 and Circ-2 relative to a single linear linker oligonucleotide sequence comprising ligation site 1 or a single linear linker oligonucleotide sequence comprising ligation site 2.

[0169] Fig.12 Calibration curves for HIV p24 assays performed using the methods described in Examples 1 and 6 are shown.

[0170] Fig.13 Results of seroconversion panel analysis using the methods described in Examples 1 and 6 are presented.

[0171] Figures 14(a)-(c) show the results of HIV p24 analysis including an analyte concentration step.

[0172] Fig.15 Shown is the calibration curve for the HIV p24 assay including an analyte concentration step.

[0173] Fig.16 is a schematic diagram of an analytical method as described herein comprising an analyte concentration step.

[0174] Fig.17 is a schematic diagram of an analytical method as described herein, wherein amplicons are formed in solution prior to being bound to a surface by a capture reagent and / or an anchoring reagent.

[0175] Fig.18 is a schematic diagram of an analytical method that incorporates the use of multiple staple sequences to attach to the amplicons, thereby forming a more compact structure on a surface.

[0176] Fig.19 Schematic diagram of a bridged 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.

[0177] 20(a)-(b) are schematic diagrams of the biosynthetic method for template formation.

[0178] Fig.21 is a schematic diagram of sample multiplexing.

[0179] Fig. 22 is a schematic diagram of detecting lipoprotein complexes using the method described herein.

[0180] Fig.23a and Figure 23bShown are (a) a comparison of the average label / protein (L / P) ratios of antibody-oligonucleotide conjugates measured by the fluorescent dye method and gel electrophoresis, and (b) the signal measured by the fluorescent dye method as a function of oligonucleotide concentration for free oligonucleotide and oligonucleotide in antibody-oligonucleotide conjugates.

[0181] Fig.24 Shown is a schematic depiction of a double antibody amplified ECL immunoassay performed on a streptavidin-coated surface.

[0182] Figures 25(a)-(c) show (a) the elution profile from the G-100 Superfine column for the crude product of the antibody-oligonucleotide binding reaction relative to the profiles for unbound antibody, unbound oligonucleotide and purified conjugate; (b) the elution profile and collected eluate fractions for the IL-6 detection antibody-oligonucleotide conjugate; and (c) the calibration curve of the amplified ECL immunoassay using the G-100 purified IL-6 detection antibody-oligonucleotide conjugate relative to the results when the conjugate was purified by centrifugal ultrafiltration.

[0183] Figure 26 (a)-(b) shows (a) relative fluorescence signals after binding to free oligonucleotides relative to oligonucleotides bound to antibodies for six different nucleic acid-sensitive fluorescent dyes (left); (middle) % of signal from antibody-oligonucleotide conjugates associated with dye-protein interactions and (right) signal: background ratio obtained during measurement of 100 ng of conjugate; and (b) fluorescence signals measured using SYBR Green I dye for triple antibody-oligonucleotide conjugates as a function of oligonucleotide concentration (and comparison of these signals with those obtained with unbound oligonucleotides).

[0184] 27(a)-(b) compare 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.

[0185] Fig.28 The performance of two different labeled detection oligonucleotide constructs was compared in an amplified ECL assay for three different analytes in both sequential and simultaneous assay formats.

[0186] Figure 29(a)-(b) demonstrates the effects of cross-linker excitation ratio and binding scheme on the production and performance of antibody-oligonucleotide conjugates, providing (a) label / protein (L / P) and performance in amplified ECL analysis, and (b) characterization of conjugate formation by gel electrophoresis.

[0187] Figure 30(a)-(b) compares the performance of different biotin-anchor oligonucleotide constructs in an amplified ECL assay for two analytes.

[0188] Figure 31 (a)-(b) shows (a) the effect of probe length of the detection antibody-oligonucleotide probe conjugate on the signal, background and detection limit obtained in the analysis using the conjugate, and (b) the effect of GC content in the probe on the sensitivity of the ligation step in the analysis to changing temperature.

[0189] Fig.32 Comparison of the performance of different Circ oligonucleotide constructs in amplification ECL assays for analytes.

[0190] Fig.33 is a schematic depiction of the modifications made during optimization of reagents for amplified ECL analysis.

[0191] Figures 34(a)-(b) show (a) calibration curves for assays of three analytes comparing signals obtained in conventional and amplified formats; and (b) comparison of detection limits obtained in the two formats for 41 different assays targeting different analytes. DETAILED DESCRIPTION

[0192] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include the singular. The article "a / an" is used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an element" means one element or more than one element.

[0193] As used herein, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method being employed to determine the value.

[0194] As used herein, "between" is a range that includes the ends of the range. For example, numbers between x and y specifically include the numbers x and y, and any number between x and y.

[0195] As used herein, "kit" refers to a group of components provided or gathered for use together, such as producing a composition, manufacturing a device, or performing 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 of which may contain one or more of the components. The listed components of a kit may in turn also be provided as a single physical part or a plurality of parts combined for use with a kit. For example, the instrument components of a kit may be provided fully assembled or in the form of a plurality of instrument components assembled before use. Similarly, the liquid reagent components of a kit may be provided as a complete liquid formulation in a container, one or more dry reagents to be combined to provide a complete liquid formulation, and one or more liquid diluents or two or more liquid solutions to be combined to provide a complete liquid formulation. As known in the art, kit components for analysis are usually shipped and stored separately due to having different storage requirements, such as 4°C relative to a storage temperature of -70°C.

[0196] In the case of an analyte measured in an analysis or a class of reagents used in an analysis, the term "multiple" means more than one structurally and / or functionally different analyte or reagent (e.g., capture antibody A and capture antibody B), rather than just more than one copy of the analyte or reagent (e.g., capture antibody A and another copy of capture antibody A). For example, the term "multiple fixed antigens" means that more than one structurally or functionally different antigen is fixed, and does not describe the situation where there are multiple copies of only one single antigen. However, the use of the term "multiple" in this context does not exclude the possibility of multiple copies of any of the multiple analytes or reagents. For example, a plurality of fixed antigens may refer to fixed antigens including one or more copies of antigen A and one or more copies of antigen B.

[0197] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides", and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (i.e., peptide bonds). The term "polypeptide" refers to any one or more chains of amino acids, and does not refer to the specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to one or more chains of amino acids are included in the definition of "polypeptide", and the term "polypeptide" can be used to replace any of these terms or can be used interchangeably with them. The term "polypeptide" also means a post-expression modification product of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, proteolytic cleavage or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or made by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any manner, including chemical synthesis. In the context of polypeptides, a "linear sequence" or "sequence" is the order of amino acids in the polypeptide in the amino to carboxyl terminal direction wherein residues adjacent to each other in the sequence are contiguous in the primary structure of the polypeptide.

[0198] "Binding agent" or "binding substance" refers to an agent or substance characterized by the ability to bind to another substance (which may be referred to as a "binding partner"). The binding agents, binding substances and binding partners of the present invention include "antigen binding substances", which are terms referring 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 include at least one heavy chain or light chain complementary determining region (CDR) of an antibody. Antigen binding substances include substances that include at least two CDRs from one or more antibodies. Antigen binding substances include substances that include at least three CDRs from one or more antibodies. Antigen binding substances include substances that include at least four CDRs from one or more antibodies. Antigen binding substances include substances that include at least five CDRs from one or more antibodies. Antigen binding substances include substances that include at least six CDRs from one or more antibodies.

[0199] Antigen binding substances derived from antibodies or other antigen binding substances may include, for example, addition, removal or substitution of one or more antibodies in 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 of potentially interfering components in the sample, such as complement, rheumatoid factor, or anti-species antibodies). In one embodiment, the antigen binding substance is a Fab portion of an antibody that reduces potential interference from the Fc binding component of the sample. In one embodiment of measuring an analyte in a sample from a specific species, the antigen binding substance is a modified form of an antibody designed to match the antibody class of the species (e.g., a mouse antibody can be humanized for analysis of human samples to avoid interference from human anti-mouse antibodies (i.e., human antibodies targeting mouse antibodies) that are typically present in human samples.

[0200] As used herein, "human" or "fully human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or from an animal transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described below and, for example, in U.S. Pat. No. 5,939,598. "Human" or "fully human" antibodies also include antibodies comprising at least a heavy chain variable domain or at least a heavy chain and a light chain variable domain, wherein the variable domain has the amino acid sequence of a human immunoglobulin variable domain. "Humanized" antibodies are antibodies from other species whose constant and framework sequences have been modified in an attempt to match a class of human antibodies while maintaining the ability to bind to the target antigen of the original antibody.

[0201] The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin comprises at least the variable domain of a heavy chain, and typically comprises at least the variable domain of a heavy chain and a light chain. The basic immunoglobulin structure 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).

[0202] The term "immunoglobulin" includes various broad classes of polypeptides that can be distinguished biochemically. It will be appreciated by those skilled in the art that heavy chains produced by animal species can be classified into different classes, such as γ, μ, α, δ or ε, and these classes can be further divided into subclasses (e.g., γ1-γ4). The properties of this chain determine the "class" of the antibody as IgG, IgM, IgA IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and known to confer functional specialization. In view of the present invention, modified forms of each of these classes and isotypes can be easily distinguished by those skilled in the art, and are therefore within the scope of the present invention. All immunoglobulin classes are obviously within the scope of the present invention. The following discussion will generally be directed to the IgG class of immunoglobulins. Most forms of IgG produced in mammals include 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 joined by disulfide bonds in a "Y" configuration, with the light chains beginning at the mouth of the "Y" to support the heavy chains and continuing throughout the variable region. The exact molecular weight can vary between species and between subclasses. Some species, such as camelid species, can also produce IgG forms without light chains.

[0203] Light chains can also be produced in different classifiable forms, such as kappa (Vκ) or lambda (Vλ) forms. Each heavy chain class can be associated with a kappa or lambda light chain. In general, the light chain and heavy chain are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0204] Both the light chain and the heavy chain are divided into regions with structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of the light chain (Vκ or Vλ-collectively referred to as "VL") and the heavy chain (VH) portion determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region increases as it is farther away from the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains usually include the carboxyl termini of the heavy chain and the light chain, respectively.

[0205] As noted above, variable regions allow antibodies to selectively recognize and specifically bind antigenic determinants on antigens. That is, the VL domains and VH domains of antibodies or a subset of complementary determining regions (CDRs) in these variable domains combine to form variable regions that define three-dimensional antigen binding sites. This quaternary antibody structure forms an antigen binding site present at each arm end of Y. More specifically, the antigen binding site is generally defined by three CDRs on 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, such as certain immunoglobulins derived from camelid species or based on camelid immunoglobulin engineering, complete immunoglobulins can be composed of only heavy chains without light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0206] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" that are usually present in each antigen binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form an antigen binding domain because the antibody assumes its three-dimensional configuration in an aqueous environment. The rest of the amino acids in the antigen binding domain are called "framework" regions, showing less intermolecular variability. The framework region mainly adopts a folded configuration and the CDRs form loops that connect the β-sheet structure and form a part of the β-sheet structure in some cases. Therefore, the framework region is used to form a skeleton that provides the positioning of the CDRs in the correct direction through interchain non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to the antigenic determinant on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate antigenic determinant. A person of ordinary skill in the art can easily identify any given heavy or light chain variable domain of amino acids that include CDRs and framework regions, respectively, because they have been precisely defined (see below).

[0207] In the case of two or more definitions of terms used and / or accepted in the art, unless explicitly stated otherwise, the definitions of terms as used herein are intended to include all such meanings. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen combining sites seen in the variable regions of heavy and light chain polypeptides. This region has been described by Kabat et al. (1983) U. S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest", Chothia and Lesk, "Journal of Molecular Biology (J. Mol. Biol.)" 196: 901-917 (1987), and by Kunik et al., "Nucl. Acids Res. (Nucl. Acids Res.)" 40: W521-W524 (2012) recently updated, the document is incorporated herein by reference, wherein when compared to each other, the definition includes overlaps or subgroups of amino acid residues. However, the application of any definition of CDRs involving antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The exact number of residues encompassing a particular CDR will vary depending on the sequence and size of the CDR. Where the variable region amino acid sequence of an antibody is specified, one skilled in the art can determine in a conventional manner which residues comprise a particular CDR.

[0208] Kabat et al. also define a variable domain sequence numbering system applicable to any antibody. A person of ordinary skill in the art can assign this "Kabat numbering" system to any variable domain sequence unambiguously, without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al. (1983) US Pat. of Health and Human Services, Sequences of Proteins of Interest in Immunology.

[0209] Kunik et al., Nucleic Acids Research 40:W521-W524 (2012) disclose an online tool Paratome for systematically identifying antigen binding regions in antibodies based on sequence or structure. Typically, analysis based on Paratome matches Kabat numbering, but may also include residues adjacent to conventional CDRs.

[0210] The 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, antigen-binding fragments (e.g., Fab, Fab' and F(ab') 2, Fd, Fv, single-chain Fv (scFv), disulfide-linked Fv (sdFv)), fragments including VL or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. scFv constructs are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. The immunoglobulins or antibodies of the present invention can be any type of immunoglobulin molecule, IgG, IgE, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, etc.) or subclass. Many methods for producing antibodies and other antigen-binding substances are well known in the art and include production from B cell cultures, from fusion tumor cell cultures, from cultures or transiently or permanently transfected host cell strains, from bacteria, from yeast, from plant cells, and from insect cells.

[0211] As used herein, the term "heavy chain portion" comprises an amino acid sequence derived from an immunoglobulin heavy chain. The polypeptide comprising the heavy chain portion includes at least one of the following: a CH1 domain, a hinge (e.g., an upper, middle and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, the binding polypeptide used in the present invention may include a polypeptide chain containing 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, the polypeptide of the present invention includes a polypeptide chain containing a CH3 domain. In addition, the binding polypeptide used in the present invention may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As described above, it should be understood by those of ordinary skill in the art that these domains (e.g., heavy chain portions) may be modified to make their amino acid sequences different from naturally occurring immunoglobulin molecules.

[0212] 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 the same as the heavy chain portion of the second polypeptide chain of the multimer. Alternatively, the monomers containing the heavy chain portion of the invention are different. For example, each monomer can include a different target binding site, forming, for example, a bispecific antibody.

[0213] As used herein, the term "light chain portion" comprises an amino acid sequence derived from an immunoglobulin light chain, such as a kappa or lambda light chain. Preferably, the light chain portion includes at least one of a VL or CL domain.

[0214] "Antigen binding substances" as defined above can be described or specified with respect to the antigenic determinant or portion of the substance that it recognizes or specifically binds. The portion of the target polypeptide that specifically interacts with the antigen binding domain of an antibody is an "antigenic determinant" or "antigenic determinant". The target polypeptide can include a single antigenic determinant, but typically includes at least two antigenic determinants, and can include any number of antigenic determinants, which depends on the size, conformation and type in the antigen. In addition, it should be noted that the "antigenic determinant" on the target polypeptide can be or can include a non-polypeptide element, for example, the antigenic determinant can include a carbohydrate side chain.

[0215] The minimum size of the peptide or polypeptide epitope of antigen binding material is considered to be about four to five amino acids. Peptide or polypeptide epitope preferably contains at least seven, more preferably at least nine and most preferably at least about 15 to about 30 amino acids. Because CDR can identify the antigen peptide or polypeptide in tertiary form, it is not necessary to be continuous including the amino acid of epitope, and in some cases, even can not be on the same peptide chain. Peptide or polypeptide epitope identified by antigen binding molecules of the present invention can contain 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 continuous or non-continuous amino acid sequences.

[0216] "Antigen" means a substance that is capable of specifically or preferentially binding to an antibody-binding substance, such as an antibody or an antigen-binding fragment thereof.

[0217] In the case of antibodies (and similarly, other antigen-binding substances), "preferential binding" means that the antibody will more readily bind to an antigenic determinant than it will to a reference antigenic determinant (which may be a related, similar, homologous or similar antigenic determinant). Thus, an antibody that "preferentially binds" to a given antigenic determinant will more readily bind to that antigenic determinant than to a reference antigenic determinant, even though such an antibody may cross-react with the reference antigenic determinant.

[0218] In the case of antibodies (and similarly, other antigen-binding substances), "specific binding" means that the antibody binds to an antigenic determinant through its antigen binding domain, and that binding requires complementarity between the antigen binding domain and the antigenic determinant. According to this definition, an antibody is said to "specifically bind" to an antigenic determinant when it preferentially binds to that antigenic determinant through its antigen binding domain relative to a random, unrelated antigenic determinant.

[0219] As a non-limiting example, an antibody may be considered to bind a first antigenic determinant preferentially over a second antigenic determinant if, under the conditions of the experiment (e.g., the conditions used to perform the assay), the amount of binding to the first antigenic determinant relative to the amount of binding to the second antigenic determinant (expressed as a ratio) is greater than 1, 10, 100, 1,000, or 10,000. In another non-limiting example, an antibody may be considered to bind a first antigenic determinant preferentially over a second antigenic determinant if, relative to the equilibrium dissociation constant (K) of the antibody for the second antigenic determinant, the amount of binding to the first antigenic determinant (expressed as a ratio) is greater than 1, 10, 100, 1,000, or 10,000. D ), K binding to the first antigenic determinant D An antibody may be considered to bind a first antigenic determinant preferentially relative to a second antigenic determinant if the binding rate constant (also referred to as an association rate or k) for the antibody to the second antigenic determinant is less than 1, 0.1, 0.01, 0.001, or 0.0001. In another non-limiting example, an antibody may be considered to bind a first antigenic determinant preferentially relative to a second antigenic determinant if the binding rate constant (also referred to as an association rate or k) for the antibody to the second antigenic determinant is less than 1, 0.1, 0.01, 0.001, or 0.0001. on ), k binding to the first antigenic determinant on An antibody may be considered to bind a first antigenic determinant preferentially relative to a second antigenic determinant if the dissociation rate constant (also referred to as the dissociation rate or k) for the antibody against the second antigenic determinant is greater than 1, 10, 100, 1,000, or 10,000. In another non-limiting example, the antibody may be considered to bind a first antigenic determinant preferentially relative to a second antigenic determinant if the dissociation rate constant (also referred to as the dissociation rate or k) for the antibody against the second antigenic determinant is greater than 1, 10, 100, 1,000, or 10,000. off ), k dissociated from the first antigenic determinant off If the ratio of (expressed as a ratio) is less than 1, 0.1, 0.01, 0.001 or 0.0001, then the antibody can be considered to bind the first antigenic determinant preferentially relative to the second antigenic determinant.

[0220] When comparing the preference of two antibodies for a first antigenic determinant relative to a second antigenic determinant, the antibody with a stronger preference for the first antigenic determinant can be said to be more specific for the first antigenic determinant. The strength of the preference can be measured, for example, by the ratio of the amount of binding to the two antigenic determinants under the chosen experimental conditions, by the K D The ratio of values, through k on The ratio of values ​​and / or through k off The ratios of the values ​​are determined as described in the previous paragraph.

[0221] In general, by using high affinity and slow off-rate antibodies, assay sensitivity and robustness are improved. In embodiments, the antibodies or other antigen-binding substances disclosed herein have a K of less than or equal to 10 nM, 1 nM, 500 pM, 200 pM, 100 pM, 30 pM or 10 pM. D In embodiments, the antibodies or other antigen-binding substances disclosed herein bind to the target antigen with a K of less than or equal to 10 nM, 1 nM, 500 pM, 200 pM, 100 pM, 30 pM or 10 pM. DIn the examples, the antibodies or other antigen-binding substances disclosed herein are less than or equal to 5×10 -2 sec -1 , 10 -2 sec -1 , 5×10 -3 sec -1 or 10 -3 sec -1 K off In the examples, the antibodies or other antigen-binding substances disclosed herein are dissociated from the target antigen at a rate of less than or equal to 5×10 -4 sec -1 , 10 -4 sec -1 , 5×10 -5 sec -1 or 10 -5 sec -1 , 5×10 -6 sec -1 , 10 -6 sec -1 , 5×10 -7 sec -1 or 10 -7 sec -1 K off Dissociate from the target peptide.

[0222] The antigen binding substances of the present invention may have "multi-specificity", bi-specificity, tri-specificity or greater multi-specificity, meaning that they simultaneously recognize and bind to two or more different antigenic determinants present on one or more different antigens (e.g., proteins). Thus, an antigen binding substance is "mono-specific" or "multi-specific", for example "bi-specificity" refers to the number of different antigenic determinants that react with the binding polypeptide. Multi-specific antibodies may be specific for different antigenic determinants of a target, or may be specific for a target polypeptide as well as a heterologous antigenic determinant, such as a heterologous polypeptide.

[0223] As indicated previously, the subunit structures and three-dimensional configurations of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" comprises the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" comprises the first (most amino-terminal) constant region of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of an immunoglobulin heavy chain.

[0224] As used herein, the term "hinge region" comprises the portion of the heavy chain that joins the CH1 domain to the CH2 domain. This hinge region includes approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three different domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol. 161:4083 (1998)).

[0225] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​includes a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgGs, the CH1 and CL regions are linked by disulfide bonds and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).

[0226] As used herein, the terms "linked", "fused" and "fusion" are used interchangeably. These terms refer to joining two or more elements or components together by any means including chemical binding or recombinant means. "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 translation reading frame of the original ORF. Therefore, a recombinant fusion protein is a single protein that contains two or more segments corresponding to the polypeptides encoded by the original ORFs (the segments are not usually so joined in nature). Although the reading frame is thus continuous throughout the fused segment, the segments can be physically or spatially separated by, for example, in-frame linker sequences. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused in frame, but separated by polynucleotides encoding at least one immunoglobulin framework region or additional CDR region, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.

[0227] As used herein, the term "oligonucleotide" refers to a short polymer of a nucleic acid such as DNA or RNA. In an embodiment, the length of the oligonucleotide is about 5 to about 150 nucleotides. The oligonucleotide can be designed to hybridize specifically with a DNA or RNA sequence, for example as a probe for detecting a specific sequence complementary to the oligonucleotide. The oligonucleotide can be single-stranded or double-stranded. The oligonucleotides described herein can be produced by any means, including chemical synthesis.

[0228] The term "oligonucleotide" may include structural analogs including non-naturally occurring chemical structures. In an embodiment, an oligonucleotide includes a modification at its 5' end or 3' end, an internal modification, 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, connection modifications that can be used to connect an oligonucleotide to another substance or surface; fluorophores and fluorescence quenchers; modified bases; phosphorylation modifications, such as when an oligonucleotide is used as a ligase substrate; spacers, such as to create a distance between a nucleic acid sequence and a reactive functional group in an oligonucleotide; and phosphorothioate bonds, such as to increase the resistance of an oligonucleotide to nuclease degradation. Exemplary modifications are provided in Table A below.

[0229] Table A. Oligonucleotide modifications

[0230]

[0231] In embodiments, the modification comprises biotin, streptavidin, avidin, an amino group, a thiol group, an aldehyde group, a hydrazide group, an azide group, an alkyne group, a maleimide group, and / or an iodoacetamide group.

[0232] In one example, nucleotides and / or nucleic acids can include chemical modifications that connect them to another substance, such as a label, or provide reactive functional groups that can be connected to another substance, such as a label, such as by using amine or thiol-modified nucleotide bases, phosphates, or sugars. The term "reactive functional group" refers to an atom or a group of related atoms that can undergo another chemical reaction, such as to form a covalent bond with another functional group. Examples of reactive functional groups include, but are not limited to, amino, thiol, hydroxyl, and carbonyl groups. In one aspect, the reactive functional group includes a reactive thiol group. Labels that can be connected to nucleotides or nucleic acids by these chemical modifications include, but are not limited to, detectable moieties, such as biotin, haptens, fluorophores, and electrochemiluminescence (ECL) labels.

[0233] In another aspect, the nucleotide in the oligonucleotide can be modified to prevent the enzymatic or chemical extension of the nucleic acid chain incorporated therein, for example, by replacing ribose or deoxyribose groups with dideoxyribose. In another example, the backbone components connecting nucleotide bases (such as sugar and / or phosphate groups) together can be modified or replaced, for example, by using peptide nucleic acid (PNA) or by incorporating ribose analogs, such as those found in RNA, locked nucleic acid, bridged nucleic acid and morpholino nucleic acid substituted with 2'-O-methyl. These "backbone" analogs may be present in one, some or all of the backbones of nucleic acid and / or oligonucleotides, and some advantages may be provided, such as hybridization products with improved binding stability and / or connection stability with nuclease. In another example of nucleotide and nucleic acid structure analogs, non-natural nucleotide bases may be included. Non-natural (also referred to as "non-typical" bases) may be hybridized with natural (typical) bases or it may be hybridized with another non-natural base.

[0234] In the context of nucleic acids, a "probe" generally refers to an oligonucleotide (usually 5 to 50 bases) comprising a sequence that is complementary to another nucleic acid sequence. In some applications, a probe that hybridizes to a complementary region in a target sequence can enable the probe to be extended by a polymerase to serve as an origin of replication for an adjacent single-stranded region on the target sequence (in such cases, the probe may also be referred to as a "primer").

[0235] As used herein, "nucleic acid probes" include oligonucleotides that are (i) modified with one or more reactive moieties that can be used to react the oligonucleotide with another substance and thereby connect it, or (ii) connected to another substance (e.g., by a reaction as described in item (i)). In an embodiment, the nucleic acid probe is connected to a detection reagent. In an embodiment, the nucleic acid probe is connected to a polypeptide. In an embodiment, the nucleic acid probe is connected to an antibody or other antigen binding substance. In an embodiment, the reactive moiety is a reactive functional group. In an embodiment, the reactive functional group is an alkene, a strained alkene, an alkyne, a halide, an alcohol, a thiol, an amine, a phosphate, an aldehyde, a ketone, a carboxylic acid, a carboxylate, an amide, an ester, a thioester, an acyl phosphate, an acid halide, a nitrile, an anhydride, a hydrazine, a tetrazine, or an azide. In an embodiment, the reactive moiety is a member of a binding agent-binding partner pair, such as biotin or streptavidin. In an embodiment, the nucleic acid probe includes a sequence complementary to a template oligonucleotide for amplification. In an embodiment, the nucleic acid probe is combined with a circular template oligonucleotide for rolling circle amplification (RCA) of the circular template oligonucleotide. In an embodiment, RCA produces an extended sequence. In an embodiment, the nucleic acid probe is a primer for RCA.

[0236] "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 causing a chemical group or moiety to exhibit a detectable physical property, including, for example, an enzyme that catalyzes the conversion of a substrate into a detectable product. The label can be detected by spectroscopy, photochemistry, biochemistry, immunochemistry, 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 aspect, the label is a compound that is a member of a binding pair, wherein the first member of the binding pair (which may be referred to as a "primary binding agent") is attached to a substrate, such as an oligonucleotide, and the other member of the binding pair (which may be referred to as a "secondary binding agent") 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 an embodiment, a "labeled probe" includes an oligonucleotide and an electrochemiluminescent moiety. In embodiments, the oligonucleotide of the labeled probe comprises the complement of the extended sequence of the methods described herein. In embodiments, the labeled probe binds to the extended sequence, and the detectable label is used to enable measurement of the amount of the extended sequence. In embodiments of the methods described herein, measurement of the labeled probe (i.e., the amount of the extended sequence) determines the amount of analyte in the sample.

[0237] "Complementary" refers to nucleic acid molecules or nucleic acid molecule sequences that bind to each other (or "hybridize") by forming 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), between two RNA molecules (RNA-RNA hybridization), or between 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 with less than 100% nucleotide alignment based on a base pairing model such as the Watson-Crick base pairing model), although sequences with less sequence complementarity are less stable and less likely to hybridize than sequences with greater sequence complementarity. In one aspect, based on the Watson-Crick model, the nucleotides of the complement have 100% sequence complementarity. In another aspect, based on the Watson-Crick model, the nucleotides of the complement have at least about 90%, 95%, 97% or 99% sequence complementarity.

[0238] If two nucleic acids are able to hybridize or have hybridized, they are "hybridizable" or "hybridized", respectively. Whether two complements 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 (methods for producing stringent hybridization conditions are well known and exemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), especially Chapter 11 and Table 11.1 therein). The stringency of the hybridization conditions can be selected to selectively form or maintain the desired hybridization product of two complementary nucleic acid sequences in the presence of other potential cross-reacting or interfering sequences. Stringent conditions are sequence-dependent, and generally longer complements hybridize specifically at higher temperatures than shorter complements. In general, for a specific nucleotide sequence under defined ionic strength, chemical denaturant concentration, pH and hybridization partner concentration, stringent hybridization conditions are higher than the thermal melting point (T m ) (i.e., the temperature at which 50% of the sequence hybridizes to the substantial complement) is about 5°C to about 10°C lower. In general, nucleotide sequences having a higher percentage of G and C bases hybridize under more stringent conditions than nucleotide sequences having a lower percentage of G and C bases. In general, 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 (such as formamide, dimethylformamide, dimethyl sulfoxide, ethylene glycol, propylene glycol, and ethyl carbonate). Stringent hybridization conditions typically comprise a salt concentration or ionic strength of less than about 1 M, 500 mM, 200 mM, 100 mM, or 50 mM; a hybridization temperature greater than about 20°C, 30°C, 40°C, 60°C, or 80°C; and a chemical denaturant concentration greater than about 10%, 20%, 30%, 40%, or 50%. Because many factors can affect hybridization stringency, a combination of parameters may be more important than the absolute value of any single parameter.

[0239] Exemplary hybridization conditions include a buffer solution (e.g., a phosphate, tris or HEPES buffer solution having about 20 to 200 mM buffer component) at a pH of about 6.5 to 8.5 and an ionic strength of about 20 to 200 mM at a temperature of about 15 to 40° C. For example, the buffer may include a salt at a 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, (NH 4 ) 2 SO 4 、Na 2 SO4 and CH 3 COONH 4 One specific example is 50M Tris-HCl, pH 7.4 at room temperature (about 18 to 25°C). Another specific example is 66mM potassium acetate, 50mM potassium chloride, 10mM magnesium acetate, 33mM Tris buffer, pH 8.2 at 22°C to 37°C (or about 27°C).

[0240] In the case of nucleic acid sequences or amino acid sequences, the term "sequence identity" or "% identity" refers to the percentage of identical residues in the compared sequences when the sequences are aligned within a specified comparison window. In some embodiments, only specific portions of two or more sequences are aligned to determine sequence identity. In some embodiments, only specific domains of two or more sequences are aligned to determine sequence similarity. The comparison window can be a fragment of at least 10 to more than 1000 residues, at least 20 to about 1000 residues, or at least 50 to 500 residues, in which the 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. When referring to amino acid sequences, "percentage of identity" or "% identity" 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), as modified in Karlin and Altschul, Proceedings of the National Academy of Sciences USA 90:5873-5877 (1993). Such algorithms are incorporated into BLAST programs, such as BLAST+ or NBLAST and XBLAST programs, which are described in Altschul et al., Journal of Molecular Biology, 215:403-410 (1990). BLAST protein searches can be performed with a program such as the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. When there is a gap between two sequences, Gapped BLAST can be used 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 (e.g., XBLAST and NBLAST) can be used.

[0241] In some embodiments, the 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). In some embodiments, the 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).

[0242] Hybridization occurs when two nucleic acids contain complement, although depending on the stringency of hybridization, mispairing between bases is possible. In an embodiment, the sequence that can hybridize with the complement of the second sequence is substantially similar to the second sequence. In an embodiment, the sequence that can hybridize with the complement of the 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.

[0243] The present invention comprises an immunoassay method comprising (i) anchoring a detection complex formed between a target analyte used in the assay and one or more analyte binding reagents; and / or (ii) amplifying a signal from a labeled detection complex. Anchoring can be used to stabilize complexes involving low binding affinity interactions and / or high molecular weight markers or labeling sites. Signal amplification can be achieved by connecting an extension probe to a binding complex containing multiple markers or detection labeling sites, thereby amplifying a detectable signal for each individual detection complex. In a preferred embodiment, the method comprises connecting an extension probe containing multiple markers or detection labeling sites to a detection complex, and anchoring the complex to a surface to ensure that the complex is retained on the surface. This improved analytical method can be used to detect extremely low numbers of binding events, even individual analyte-binding reagent complexes. The basic method is not limited to immunoassays and can be used to perform binding analyses using other types of binding reagents.

[0244] In an embodiment, the present invention provides a binding analysis using a surface-bound anchoring agent to adhere a detection complex containing an analyte of interest to a surface and stabilize the detection complex. This method can be used to overcome the low binding affinity between reagents forming the detection complex and / or to prevent the complex from dissociating from the surface before subsequent processing. The use of an anchoring agent in a binding analysis is illustrated in FIG. 1( a). A surface (101) comprises a capture reagent (102) that binds analyte A, and an anchoring agent (103). In one or more steps, the analyte is bound to a capture reagent and also to a detection reagent (104) that binds the analyte, wherein the detection reagent is connected to a nucleic acid probe (105). The analyte can be bound to the capture reagent and the detection reagent simultaneously or substantially simultaneously, or the analyte can be bound to each of the capture reagent and the detection reagent sequentially (in any order). Therefore, a complex (106) comprising a capture reagent, an analyte, and a detection reagent is formed on the surface. The probe is extended to form an extension sequence (107), which comprises an anchoring region that binds the anchoring agent. The extension sequence is bound to the anchoring agent and the amount of extension sequence bound to the surface is measured.

[0245] Those skilled in the art of binding analysis will readily appreciate the range of capture reagents and concomitant binding partners that can be used in the methods of the present invention. A non-limiting list of such pairs includes (in either order) receptor / ligand pairs, antibody / antigen, natural or synthetic receptor / ligand pairs, hapten / antibody pairs, antigen / antibody pairs, antigenic determinant / antibody pairs, mimicking antigenic determinant / antibody pairs, aptamer / target molecule pairs, hybridization partners, and intercalator / target molecule pairs. In one embodiment, the binding analysis employs antibodies or other receptor proteins as capture reagents and / or detection reagents for the analyte of interest. The term "antibody" includes intact antibody molecules (including hybrid antibodies assembled by in vitro reassociation of antibody subunits), antibody fragments, and recombinant protein constructs comprising the antigen-binding domain of an antibody (as described, for example, in Porter, RR and Weir, RC, J. Cell Physiol., 67 (Suppl); 51-64 (1966) and Hochman, I. Inbar, D. and Givol, D., Biochemistry, 12: 1130 (1973)), as well as antibody constructs that have been chemically modified, for example, by the introduction of a detectable label.

[0246] Similarly, anchoring agents and corresponding anchoring members or regions can include any suitable binding pairs, such as receptor / ligand pairs, antibody / antigen, natural or synthetic receptor / ligand pairs, hapten / antibody pairs, antigen / antibody pairs, antigenic determinant / antibody pairs, simulated antigenic determinant / antibody pairs, aptamer / target molecule pairs, hybridization partners, intercalators / target molecule pairs, and use surfaces and anchoring agents bound by electrostatic charges. For example, anchoring agents can be oligonucleotide sequences, aptamers, aptamer ligands, antibodies, antigens, ligands, receptors, haptens, antigenic determinants or simulated antigenic determinants, and corresponding anchoring regions correspondingly include complementary oligonucleotide sequences, aptamer ligands, aptamers, antigens, antibodies, receptors, ligands or antibodies. In a specific embodiment, the anchoring region is an oligonucleotide sequence and the anchoring agent includes a DNA binding protein. Alternatively, if the anchoring region is a double-stranded oligonucleotide sequence, the anchoring agent can include an intercalator. In another embodiment, the anchor region may comprise one or more modified oligonucleotide bases and the corresponding anchor reagent comprises one or more moieties that bind to the modified bases on the anchor region. For example, the modified base may comprise a hapten or a ligand and the corresponding anchor reagent comprises one or more antibodies or ligands that are specific for the hapten or ligand, respectively. In addition, the anchor region may comprise a plurality of labeled nucleotide bases that can be used to detect the detection complex.

[0247] In the specific embodiment depicted in FIG. 1( b), the surface-bound anchoring reagent comprises an oligonucleotide for anchoring the detection complex to the surface. The anchoring oligonucleotide sequence is bound to a complementary oligonucleotide sequence that is attached to the detection complex. In this embodiment, the surface (108) comprises a capture reagent (109) that binds analyte A, and an anchoring reagent (110) that includes an anchoring oligonucleotide sequence (111). In one or more steps, the analyte is bound to the capture reagent and also to a detection reagent (112) that binds to the analyte, wherein the detection reagent is attached to a nucleic acid probe (113). As described above with reference to FIG. 1( a), the analyte may be bound to the capture reagent and the detection reagent simultaneously or substantially simultaneously, or the analyte may be bound to each of the capture reagent and the detection reagent sequentially (in either order). Thus, a complex (114) comprising a binding reagent, an analyte, and a detection reagent is formed on the surface. The probe is extended to form an extended sequence (115) that comprises an anchor sequence complement that is complementary to the anchor sequence. The anchor sequence hybridizes with the anchor sequence complement and measures the amount of the extended sequence bound to the surface. The extension sequence may also comprise a detection sequence, in which case a detection probe complementary to the detection sequence may be added and bind to the extension sequence, and the label measured to determine the amount of the extension sequence on the surface.

[0248] A specific embodiment of the method depicted in Figure 1(b) (wherein an anchoring agent is used to adhere the detection complex to a surface, and the probe attached to the detection complex is extended to produce an extended region that binds to the anchoring agent) further comprises binding the nucleic acid probe on the detection reagent to: (i) a circular oligonucleotide, which is then subjected to rolling circle amplification to produce an amplicon that binds to the anchoring agent, or (ii) a linear oligonucleotide, the ends of which are bound to the nucleic acid probe and are ligated to form a circular oligonucleotide that is then subjected to rolling circle amplification to produce an amplicon that binds to the anchoring agent. The surface comprises a capture reagent and an anchoring agent. In one or more steps, the analyte is bound to the capture reagent, the detection reagent comprising the nucleic acid probe, thereby forming a detection complex on the surface. The detection complex is contacted with: (i) a circular oligonucleotide comprising a sequence complementary to the nucleic acid probe, or (ii) a linear oligonucleotide having a first end sequence and a second end sequence that do not overlap with the nucleic acid probe. The circular or linear oligonucleotide is hybridized to the nucleic acid probe, and if a linear oligonucleotide is used, the end sequences of the linear oligonucleotide are ligated to form a circular target sequence that hybridizes to the nucleic acid probe. The nucleic acid probe is extended to produce an extended sequence comprising a binding agent combined with an anchoring agent by rolling circle hybridization, and the amount of the extended sequence combined with the surface is measured. The extended sequence can also include one or more detection sequences complementary to a labeled detection probe, which hybridizes with an amplicon and is used to measure the amount of the amplicon combined with the surface. In an alternative embodiment, the extension method incorporates the labeled nucleotide bases into the extended sequence for directly detecting the amplicon on the surface without adding one or more labeled probes complementary to the amplicon. The extended sequence that produces a sequence complementary to the anchoring oligonucleotide and / or the detection probe can be achieved by being incorporated into a circular or linear oligonucleotide sequence from an anchor and / or a detection probe.

[0249] The detection complex may include one or more detection reagents, for example, to enhance the specificity of the analysis for the analyte. If, for example, the analysis is designed to emit a detectable signal when each of the detection reagents is adjacent to the analyte, or if the signal from a single detection reagent bound to the analyte can be distinguished from the signal emitted by multiple detection reagents bound to the analyte, then the use of multiple detection reagents can enhance the specificity of the analysis. An embodiment of this type of analysis is shown in Figure 1 (c). The surface (116) includes a capture reagent (117) that binds to analyte A and an anchoring reagent (118) that includes an anchoring oligonucleotide sequence (119). In one or more steps, the analyte is bound to each of the two (or more) detection reagents (120 and 121, respectively) of the capture reagent and the analyte, wherein each of the first and second detection reagents is connected to a nucleic acid probe (122 and 123, respectively, the first and second nucleic acid probes). The analyte can be bound to the capture reagent and the detection reagent simultaneously or substantially simultaneously, or in a sequential, stepwise manner. Therefore, a complex (124) comprising a capture reagent, an analyte, and the first and second detection reagents is formed on the surface. Using an extension method that requires the first and second probes to be in proximity to each other, the first probe is extended to form an extended sequence that includes the anchor sequence complement that is complementary to the anchor sequence (125). In the penultimate step, the anchor sequence hybridizes to the anchor sequence complement and the amount of extended sequence bound to the surface is measured.

[0250] A specific embodiment of the method depicted in Fig. 1 (c) is shown in Fig. 2 (a), wherein an anchoring agent is used to adhere the detection complex to a surface, and the probe connected to the detection complex is extended to produce an extended region bound to the anchoring agent. In this embodiment, two detection agents bound to the neighboring probes are used to detect the complex. The method further includes joining the detection agent to a connection sequence, which is then connected to form a circular target sequence, and performing rolling circle amplification to produce an amplicon bound to the anchoring agent. The surface (201) comprises a capture agent (202) and an anchoring agent (203). In one or more steps, the analyte is bound to a capture agent, a first detection agent (204) comprising a first neighboring probe (205), and a second detection agent (206) comprising a second neighboring probe (207), thereby forming a detection complex (208) on the surface. The detection complex is contacted with two connection sequences (209a and 209b), each of which comprises an end sequence complementary to the non-overlapping region of the first neighboring probe and an end sequence complementary to the non-overlapping region of the second neighboring probe. The connection sequence is hybridized with the first and second adjacent probes, and the terminal sequence of the connection oligonucleotide is connected to form a circular target sequence (210) hybridized with the first and second adjacent probes. The second adjacent probe is extended to produce an amplicon including a binding agent combined with an anchoring agent by rolling circle hybridization, and the amount of the amplicon combined with the surface is measured. The first adjacent probe can be blocked or otherwise modified to prevent the first probe from extending. (In an alternative embodiment, the first adjacent probe is extended and the second adjacent probe can be blocked or otherwise modified to prevent extension). In the embodiment depicted in Fig. 2 (a), the amplicon also includes two or more detection sequences complementary to the labeled detection probe, and the labeled detection probe is hybridized with the amplicon and used to measure the amount of the amplicon combined with the surface. In an alternative embodiment (not depicted in Fig. 2 (a)), the extension method is incorporated into the amplicon by the labeled nucleotide base, for directly detecting the amplicon on the surface without adding one or more labeled probes complementary to the amplicon. FIG. 2( b ) is a schematic diagram showing the connection sequence components of the first and second connecting oligonucleotides (209 a and 209 b , respectively), wherein the first end of the first linker (C 1(E1) ) and the first end of the second linker (C 2(E1) ) are complementary to two non-overlapping regions of the first proximity probe, and the second end of the first linker (C 1(E2) ) and the second end of the second linker (C 2(E2) ) are complementary to two non-overlapping regions of the second proximity probe. The first and second linkers hybridize to the first and second proximity probes, and the first and second linkers are connected to form a circular target sequence that hybridizes to both the first and second proximity probes.

[0251] Figure 2(c) shows an alternative embodiment of a linker. The linker sequence 211 comprises an internal sequence (C IS ) and two terminal sequences complementary to the non-overlapping region of the first proximity probe (respectively C E1 and C E2 In this example, only one ligation event is required to form a circular target sequence for rolling circle amplification (i.e., the terminal C E1 and C E2 ligation), however, since the initiation / extension comes from the second proximity probe, the requirement for proximity of the two proximity probes is maintained. Preferably, the first proximity probe is capped or otherwise modified to prevent extension of the first probe.

[0252] Thereafter, the second proximity probe is extended by rolling circle amplification of the circular target sequence to generate an amplicon including a binding region bound to the anchoring agent, and the amount of amplicon bound to the surface is measured.

[0253] The first and second sequences of the neighboring probes can be designed by methods known to those skilled in the art. For example, the length of each in the probe is 20-50 bases, preferably 25-40 bases in length, and most preferably about 30-35 bases in length. The first and second neighboring probes also include sequences complementary to one or more tethered sequences or their parts used in the method as described herein. In one embodiment, the detection complex contacts with two tethered sequences (209a and 209b), and the two tethered sequences each include an end sequence complementary to the non-overlapping region of the first neighboring probe and an end sequence complementary to the non-overlapping region of the second neighboring probe. Therefore, in this embodiment, the first and second neighboring probes each include a non-overlapping region complementary to the end sequence of connexon. Alternatively, only one connexon can be used and the tethered sequence (211) includes an internal sequence complementary to the second neighboring probe (C IS ) and two terminal sequences complementary to the non-overlapping region of the first proximity probe (respectively C E1 and C E2 ). Therefore, in this embodiment, the first proximity probe comprises two terminal sequences (C E1 and C E2 ), and the second proximity probe comprises a non-overlapping region complementary to an internal sequence of the linker (C IS ) complementary sequence. The first neighboring probe may be capped or otherwise modified to prevent the first probe from extending. (In an alternative embodiment, the first neighboring probe is extended and the second neighboring probe may be capped or otherwise modified to prevent extension).

[0254] Therefore, the embodiments illustrated in Figures 1 to 2 show that the binding analysis can be modified to incorporate an anchoring agent and / or the signal from the detection complex can be amplified. In a preferred embodiment, an anchoring agent and a signal amplification method are used in the binding analysis. In an embodiment comprising the use of an anchoring agent, the concentration of the anchoring agent 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, only one or another method can be used to achieve enhanced binding analysis. Therefore, the present invention includes an analysis using a signal amplification method as described in Figures 1-2, wherein the anchoring agent is omitted.

[0255] In these embodiments where the anchoring agent comprises an anchor sequence that is directly or indirectly bound (e.g., by a binding reaction) to a surface, the anchoring agent can be produced using methods established in the art for fixing oligonucleotides, including covalent and non-covalent attachment methods. The anchoring agent can be directly fixed to the solid phase, or it can be indirectly fixed by a secondary binding agent, such as a targeting agent as described below. For example, the anchoring agent can be connected to or include a targeting agent that is bound to the fixed targeting agent complement on the solid phase. The binding of the targeting agent to its complement can be direct (e.g., the targeting agent can be streptavidin and the complement can be biotin) or indirect through a bridging agent (e.g., the targeting agent and the complement can be biotin, and the bridging agent can be a multivalent biotin binding receptor, such as streptavidin). In one embodiment, the targeting agent and its complement include a first oligonucleotide and a complementary oligonucleotide, a receptor-ligand pair, an antigen-antibody pair, a hapten-antibody pair, an antigenic determinant-antibody pair, a mimicking antigenic determinant-antibody pair, an aptamer-target molecule pair, a hybridization partner, or an intercalator-target molecule pair. The targeting agent and complement used in the multiple analysis of more than one analyte are selected so that the targeting agent and complement associated with the capture agent or detection agent for the analyte measured by the analysis are substantially not cross-reactive with the targeting agent and complement associated with the capture agent or detection agent for other analytes measured by the analysis. For example, the binding of the binding agent to its associated binding domain (through its associated targeting agent and targeting agent complement) should greatly exceed its binding to the binding domain associated with other analytes (and present different targeting agent complements). Preferably, the cross-reaction for binding the capture agent or detection agent for the analyte to the binding domain associated with other analytes is <1%, more preferably <0.1% and more preferably <0.01% relative to the binding with the appropriate binding domain. In a preferred embodiment, the targeting agent / targeting agent complement includes a pair of oligonucleotides comprising complement, and the targeting agent and its complement are contacted under conditions sufficient to hybridize the targeting agent with its complement.

[0256] When using targeting agent, there is some flexibility about when the anchoring agent used in the analytical method is fixed on the solid phase. In one embodiment, the anchoring agent is pre-fixed on the solid phase and provided to the user by the targeting agent-targeting agent complement interaction. In another embodiment, the anchoring agent connected with the targeting agent and the solid phase of the targeting agent complement fixed by support are provided as independent components. Therefore, the analytical method further includes the step of fixing the anchoring agent on the solid phase by combining the targeting agent with its complement (directly or by using a bridging agent). This step can be carried out before, simultaneously with or after the step related to forming the detection complex.

[0257] In one embodiment, the anchoring agent comprises a protein that is linked or otherwise bound to the anchor sequence. In this embodiment, any protein that can be fixed to a surface (covalently or non-covalently) and modified by an anchoring oligonucleotide can be used. Non-limiting examples include streptavidin, avidin, or bovine serum albumin (BSA). In a preferred embodiment, the anchoring agent comprises BSA. Proteins can be modified with anchoring oligonucleotides and used known methods (e.g., Figure 3 ), is linked to the surface using sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a well-established heterobifunctional crosslinker. The reaction of the N-hydroxysuccinimide (NHS) group of SMCC with bovine serum albumin (BSA) labels BSA with a thiol-reactive maleimide group. The maleimide group in turn reacts with a thiol-modified oligonucleotide to form a BSA-oligonucleotide conjugate linked by a stable thioether bond. In one embodiment, the array is formed by printing a series of BSA-oligonucleotide conjugates on 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 attached to the immobilized avidin or streptavidin through a biotin-avidin or biotin-streptavidin interaction.

[0258] The anchoring oligonucleotide connected to the anchoring agent can be any sequence that will hybridize with the extension sequence (or amplicon) generated during the extension method. The anchoring oligonucleotide can also include a non-complementary region (e.g., a poly (A) sequence) that is used as a connecting sequence between the surface and the complementary (hybridization) region to extend the complementary region away from the surface. In one embodiment, the hybridization sequence selection is not associated with the amplicon region ("inert" region) associated with the adjacent or detection probe. In a more specific embodiment, the hybridization sequence (preferably, a length of about 25 nucleotides) is included alone or in combination with, for example, a poly (A) arm having a length of up to 30 nucleotides to form a full-length complementary hybridization sequence to the inert region of the amplicon. Preferably, the anchoring oligonucleotide is selected from: (i) (full-length complement of the inert region of the amplicon, a length of 25 nucleotides)-(poly (A) arms of 20 nucleotides); or (ii) (complement of a part of the inert region of the amplicon, a length of 15 nucleotides)-(poly (A) arms of 30 nucleotides).

[0259] In one embodiment, the adjacent connection amplification (PLA) is carried out to extend the second adjacent probe. As described above with reference to Fig. 2 (a)-(c), the complex comprising two adjacent probes contacts with one or more connection oligonucleotides (209a-209b or 211), and the connection of the hybrid connection sequence forms a circular oligonucleotide that is then used to extend the second adjacent probe by the rolling circle amplification (RCA) of the ring. Suitable probe design and amplification conditions for adjacent connection amplification are fully established in the art. The unique aspect of the present invention is to include the same sequence used in the anchoring agent in one of the connexons. During the extension of the second adjacent probe, the extension region therefore includes the complement of the anchoring sequence, which hybridizes with the anchoring agent, thereby stabilizing the sandwich complex and preventing the second adjacent probe from dissociating. The second extended adjacent probe can contain a detectable label (for example, by including a labeled nucleotide during the RCA extension reaction), which can be measured to determine the amount of the analyte on the surface. Alternatively, multiple labeled probes including a detectable label are added and hybridized with the second extended adjacent probe, and the amount of the analyte combined with the surface is measured.

[0260] Any suitable amplification technique may be used to generate the extended sequence (or amplicon), including but not limited to PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), 3SR (self-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 has significant advantages in terms of sensitivity, multiplexing, dynamic range and scalability. Techniques for RCA are known in the art (see, e.g., Baner et al., Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al., Nature Genetics, 19:226, 1998; Schweitzer et al., Proc. Natl. Acad. Sci. USA, 97:10113-119, 2000; Faruqi et al., BMC Genomics, 2:4, 2000; Nallur et al., Nucl. Acids Res., 29:el18, 2001; Dean et al., Genome Res., 11:1095-1099, 2001; Schweitzer et al., Nature Biotech., 20:359-361). 365, 2002; U.S. Pat. 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 produces thousands of copies of a circular template, where the strands of the copies are linked to the original target DNA, allowing spatial resolution of the target and rapid amplification of the signal. RCA facilitates (i) detection of single target molecules; (ii) amplification of signals from proteins as well as DNA and RNA; (iii) identification of the location of molecules that have been amplified on a solid surface; (iv) simultaneous measurement of many different targets; and (v) analysis of one or more targets in solution or solid phase. Spatial localization of the RCA products with the detection complex is particularly advantageous when performing multiplex binding assays in an array or particle-based format.

[0261] A specific embodiment of the present invention is depicted in FIG4(a), wherein an anchoring agent and a signal amplification method are used. A complex is formed on a surface (401) between a capture agent (402), an analyte (403) and two detection agents (304 and 305), each of which comprises a first and a second proximity probe (406 and 407), respectively. A first and a second ligation oligonucleotide (Circ-1 (408) and Circ-2 (409) in FIG4(a)) are added, each of which hybridizes with and bridges the two proximity probes when both proximity probes are present in the complex. The bound ligation probes are connected 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, and thereby produces an amplicon comprising one or more detection sequences (413) and an anchoring oligonucleotide sequence complement (414) (comprising a partial anchor sequence complement (415)). The anchor oligonucleotide sequence (416) (connected to the capture portion (417)) and its complement are hybridized, multiple detection probes are hybridized 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 a first circular DNA template Circ-1 (408) (which is designed to hybridize to a first proximity probe (PP1)), a detection oligonucleotide sequence, an amplicon inert region (which may be used in whole or in part to bind to the anchor oligonucleotide sequence), and a portion PP2 (which is designed to hybridize to a second proximity probe). Another embodiment is depicted in Figure 4(c), in which the circular DNA template is amplified by rolling circle amplification to produce an amplicon including multiple detection sequences (418 and 419, respectively). In another embodiment, the anchor oligonucleotide sequence (416) connected to the capture portion 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).

[0262] In one embodiment, the assay format described herein utilizes a detection reagent coupled to a detection sequence at the 5' end, wherein the 3' end is exposed to facilitate ligation with a ligation 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 problem can be addressed by adding modified bases to prevent priming by the polymerase. Alternatively, the ligation template PP1 can be directly linked to the detection reagent via its 3' end, thereby preventing this oligonucleotide from participating as a primer for a DNA polymerase, even if it is degraded.

[0263] Another method of generating a target sequence amplified by RCA or any suitable amplification method is described in Figure 5In this embodiment, each of the proximity probes can be folded into a circular hairpin structure. The formation of these hairpin structures produces a single-stranded loop and a double-stranded portion containing a recombination signal. Adding a recombinase drives the recombination of the two hairpin structures to form a circular DNA template, which is then subjected to RCA as described above. The amplicon is labeled and optionally anchored to an anchoring reagent and the analyte is detected. The key element of this embodiment is the ability of the recombinase to catalyze site-specific recombination of DNA containing sequence-specific recombination sites. For example, the Cre recombinase from bacteriophage P1 catalyzes recombination at a site containing a loxP site, and other non-limiting examples include but are not limited to Flippase (flp, from yeast), Hin (Salmonella) and Tre (engineered (evolved) version of Cre). This alternative method does not require the addition of additional components, such as oligonucleotide templates, ATP and dNTPs. In this embodiment, the loxP (recombination) site is preferably modified to be asymmetric so that the normal equilibrium shifts toward the formation of the desired recombination product. In this embodiment, the loxP (recombination) site is preferably modified to be asymmetric so that the normal equilibrium shifts toward the formation of the desired recombination product. Figure 5 The recombination sites are illustrated by light / dark shading.

[0264] In addition, Fig. 6 (a) illustrates another method of producing a target sequence amplified by RCA or any suitable amplification method. Each of the neighboring probes connected to the detection reagent comprises a loxP site, which enables site-specific recombination between two oligonucleotides by Cre recombinase, so as to form a new oligonucleotide sequence consisting of a 5' part of a neighboring probe and a 3' part of another neighboring probe flanking the lox P site. The newly generated target sequence can be subsequently amplified, labeled as described above, optionally anchored and detected by any suitable method. Fig. 6 (a) illustrates this embodiment using the T7 RNA polymerase promoter as an operable element of amplification. It should also be understood that other RNA polymerase sites, such as T3 and SP6 connected at 3 or 5' parts of the neighboring probe, are equally applicable to this method. In this embodiment, the loxP (recombination) site is preferably modified to be asymmetric so that normal equilibrium is shifted toward forming a desired recombination product. As shown in Fig. 6 (b), the method can also be used to produce a circular DNA template that can be used in RCA.

[0265] The present invention includes a method for detecting an analyte, comprising combining an analyte with a capture reagent and two detection reagents on a surface to form a detection complex. The method includes measuring the detection complex, wherein the measurement method preferentially measures the complex comprising two detection reagents relative to a complex comprising 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 reagents. Any suitable cross-linking chemical method can be used to join the components of the detection complex. For example, the first and second detection reagents can include a reactive portion that reacts with a multifunctional cross-linking agent connected to the reactive portion and joins by adding the multifunctional cross-linking agent. In this embodiment, the reactive portion and the cross-linking agent can include amines, thiols, hydrazides, aldehydes, esters, iodoacetamide, maleimide, click chemistry reagents, and combinations thereof. In another embodiment, the first and second detection reagents can include a binding portion and the cross-linking agent is a multivalent binding partner of the binding portion. Several non-limiting examples of this embodiment include: (a) the first and second detection reagents are antibodies to an animal species and the cross-linker is a multivalent anti-species antibody that targets the antibodies to the animal species; (b) the first and second detection reagents include biotin and the cross-linker is streptavidin (or vice versa); (c) the first and second detection reagents are linked to streptavidin and the cross-linker is a polymer that includes multiple biotin molecules (or vice versa); or (d) the first and second detection reagents include first and second nucleic acid probes, respectively, and the cross-linker is an oligonucleotide that includes a sequence complementary to the first nucleic acid probe and an independent sequence complementary to the second nucleic acid probe.

[0266] In a specific embodiment, an analyte of interest in a sample can be detected by combining the analyte with an immobilized capture reagent, a first detection reagent, and a second detection reagent to form a complex, wherein the first detection reagent includes a first detectable label and a first nucleic acid probe, and the second detection reagent includes a second detectable label and a second nucleic acid probe. In this embodiment, the first and second detection reagents are cross-linked by (i) hybridizing the first probe to the second probe, (ii) hybridizing the first and second probes to a third nucleic acid having a region complementary to the first and second probes, or (iii) connecting the first and second probes.

[0267] Once the cross-linked product is bound to the surface, it can be detected, or optionally, the cross-linked product can be released from the surface into the eluent and detected. In this regard, only these individual cross-linked products containing both the first and second detectable labels in the eluent 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 is detected by single molecule fluorescence, such as fluorescence correlation spectroscopy and / or fluorescence cross-correlation spectroscopy to count the labeled cross-linked products present in the eluent. In this embodiment, single molecule fluorescence detection includes making the eluent flow through the capillary, focusing the light source on the volume in the capillary to produce an interrogation zone and observing the interrogation zone with a photodetector to detect the fluorescent molecule passing through the interrogation zone. The detection method may further include detecting a first fluorescent signal associated with the first label and a second fluorescent signal associated with the second label, and counting detection events when both signals are detected from the interrogation zone. Alternatively, one label is a fluorescence resonance energy transfer (FRET) donor and another label is a FRET acceptor, and the detection method may further include exciting the FRET donor in the interrogation zone and detecting the fluorescent signal from the FRET acceptor.

[0268] In a specific embodiment, the analyte in the sample can be detected as follows: by combining the analyte with an immobilized capture reagent, a first detection reagent, and a second detection reagent to form a complex, wherein the first detection reagent includes a first nucleic acid probe and the second detection reagent includes a second nucleic acid probe; extending the second nucleic acid probe to form an extended sequence including a detectable label, the extension depending on the co-localization of the first and second nucleic acid probes in the complex; releasing the extended sequence from the surface into an eluent; and counting each extended sequence 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 includes binding the first probe to the template nucleic acid sequence, forming a circular nucleic acid template, and extending the circular template by rolling circle amplification. The extension step can also include binding the first probe to the template nucleic acid sequence, binding the second probe to the template sequence, and connecting the first and second probes.

[0269] In the method of the present invention using a capture agent, the capture agent can be directly fixed to the solid phase, or it can be indirectly fixed by a secondary binding agent, such as a targeting agent as described below. For example, the capture agent can be connected to or include a targeting agent that binds to the fixed targeting agent complement on the solid phase. The binding of the targeting agent to its complement can be direct (e.g., the targeting agent can be streptavidin and the complement can be biotin) or indirect through a bridging agent (e.g., the targeting agent and the complement can be biotin, and the bridging agent can be a multivalent biotin binding receptor, such as streptavidin). In one embodiment, the targeting agent and its complement include a first oligonucleotide and a complementary oligonucleotide, a receptor-ligand pair, an antigen-antibody pair, a hapten-antibody pair, an antigenic determinant-antibody pair, a mimicking antigenic determinant-antibody pair, an aptamer-target molecule pair, a hybridization partner, or an intercalator-target molecule pair. The targeting agent and complement used in the analysis are selected so that the targeting agent and complement associated with the capture reagent or detection reagent for the analyte measured by the analysis are substantially not cross-reactive with the targeting agent and complement associated with the capture reagent or detection reagent for other analytes measured by the analysis. For example, the binding of the binding agent to its associated binding domain (through its associated targeting agent and targeting agent complement) should greatly exceed its binding to the binding domain associated with other analytes (and present different targeting agent complements). Preferably, the cross-reaction for binding the capture reagent or detection reagent for the analyte to the binding domain associated with other analytes is <1%, more preferably <0.1% and more preferably <0.01% relative to the binding with the appropriate binding domain. In a preferred embodiment, the targeting agent / targeting agent complement includes a pair of oligonucleotides comprising complement, and the targeting agent and its complement are contacted under conditions sufficient to hybridize the targeting agent with its complement.

[0270] When using targeting agent, there is some flexibility about when the capture reagent used in the analytical method is fixed on the solid phase. In one embodiment, the capture reagent is pre-fixed on the solid phase and provided to the user by the targeting agent-targeting agent complement interaction. In another embodiment, the capture reagent connected with the targeting agent and the solid phase of the targeting agent complement fixed by support are provided as independent components. Therefore, the analytical method further includes the step of fixing the capture reagent on the solid phase by combining the targeting agent with its complement (directly or by using a bridging agent). This step can be carried out before, simultaneously with or after the step related to the formation of the detection complex.

[0271] In a specific embodiment, the multifunctional targeting agent can be used in the analytical method and components described herein. The multifunctional targeting agent can include (a) a first segment designed to be bound to a capture agent by a first segment complement (i.e., a capture agent includes a targeting agent complement complementary to the first segment of a multifunctional targeting agent), and (b) a second segment designed to be bound to an amplicon (i.e., the second segment of the multifunctional targeting agent acts as an anchoring agent on the surface). Therefore, in this embodiment, the surface includes a multifunctional targeting agent, which is contacted with a capture agent bound to a targeting agent by a connection between the first segment and the first segment complement. 3-AB RCA / PLA analysis is performed as described herein, and the amplicon is combined with the anchoring segment of the multifunctional targeting agent before the measurement step. This method can be used to ensure that a capture agent and an anchoring agent with a 1:1 ratio are used in the analytical method.

[0272] A variety of surfaces are suitable for the methods of the present invention, including conventional surfaces from the field of binding analysis. The surface can be made of a variety of different materials, including polymers (e.g., polystyrene and polypropylene), ceramics, glass, composite materials (e.g., carbon-polymer composites, such as carbon-based inks). Suitable surfaces include surfaces of macroscopic objects, such as the inner surfaces of analytical containers (e.g., test tubes, colorimetric tubes, flow cells, microfluidic channels, capillaries (e.g., ELLA glass nanoreactors from BioTechne), FACS cell sorters, filter cartridges, wells in porous disks, etc.), slides, analytical chips (such as those used in gene or protein chip measurements), pins or probes, beads, filter media, lateral flow media (e.g., filter membranes for lateral flow test strips), etc.

[0273] Suitable surfaces also include particles (including but not limited to colloids or beads) commonly used in other types of particle-based analyses, 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 chromatographic applications, such as silica, alumina, polyacrylamide, polystyrene. The material can also be a fiber, such as carbon fibril. The micron particles can be inanimate, or can contain living biological entities, such as cells, viruses, bacteria, etc. The particles used in the method of the present invention can be composed of any material suitable for connection with one or more capture reagents or detection reagents, and can be collected by, for example, centrifugation, gravity, filtration or magnetic collection. A variety of different types of particles that can be connected to capture reagents or detection reagents are commercially available for binding analysis. These include non-magnetic particles and particles including magnetizable materials that allow particles to be collected by a magnetic field. In one embodiment, the particles are composed of conductive and / or semi-conductive materials, such as colloidal gold particles. The micron particles can have a variety of sizes and shapes. As an example and not limitation, the micron particles can be between 5 nanometers and 100 microns. Preferably, the size of the microparticles is between 20 nm and 10 microns. The particles may be spherical, rectangular, rod-shaped, etc., or they may be irregularly shaped.

[0274] Particle used in the inventive method can be encoded to allow to identify the particle subgroup in concrete particle or particle mixture.The use of this type of coded particle has been used to make the analysis of the solid support adopting particle as binding analysis can be multiplexed.In a method, make particle to comprise one or more fluorescent dyes and to identify concrete particle group based on the intensity and / or relative intensity of fluorescence emission under one or more wavelengths.This method has been used in Luminex XMAP system (see, for example, U.S. Patent No. 6,939,720) and BECTON DICKINSON Cytometric Bead Array system.Or, particle can be encoded by other physical properties, such as size, shape, embedding optical pattern etc.The one or more particles provided in particle mixture or set can be encoded to distinguish with other particles in mixture by means of particle optical properties, size, shape, embedding optical pattern etc.

[0275] In specific embodiments, the methods of the invention can be used in a multiplex format by combining a plurality of different analytes with a plurality of capture reagents for these analytes, the capture analytes being immobilized on coded beads such that the coding identifies the capture reagent (and analyte target) for a particular bead. The method can further comprise counting the number of beads with bound analytes (using the detection methods described herein).

[0276] Alternatively or additionally, the detection complex and / or capture reagent may be directly or indirectly bound to different discrete binding domains on one or more solid phases, such as in a binding array, where the binding domains are individual array elements, or in a collection of beads, where the binding domains are individual beads, such that discrete analytical signals are generated on each binding domain and measured from each binding domain. If capture reagents for different analytes are immobilized in different binding domains, then different analytes bound to these domains can be measured independently. In one example of such an embodiment, the binding domains are prepared by immobilizing discrete domains of capture reagents that bind to the analytes of interest on one or more surfaces. Optionally, the surface may partially define one or more boundaries of a container (e.g., a flow cell, a well, a colorimetric tube, etc.) through which a sample is held or passed. In a preferred embodiment, each binding domain is formed on an electrode for electrochemical or electrochemiluminescent analysis. Multiplexed measurements of analytes on a surface comprising multiple binding domains using electrochemiluminescence have been used in Meso Scale Diagnostics, LLC's and Imager product line (see, e.g., U.S. Patent Nos. 7,842,246 and 6,977,722, the disclosures of which are incorporated herein by reference in their entireties).

[0277] In addition, the detection complex and / or capture reagent may be directly or indirectly bound to the electrode surface, which optionally comprises different discrete binding domains, as described above. The electrode surface may be a component of a porous disk and / or a flow cell. The electrode may include a conductive material, such as a metal, such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, conductive allowing materials, etc. It may also include an oxide coated metal, such as aluminum coated with aluminum oxide. The electrode may include a working electrode and a relative electrode, which may be made of the same or different materials, such as a metal relative electrode and a carbon working electrode. In a specific embodiment, the electrode includes a carbon-based material, such as carbon, carbon black, graphite carbon, carbon nanotubes, carbon fibrils, graphite, graphene, carbon fibers, and mixtures thereof. In one embodiment, the electrode includes elemental carbon, such as graphite, carbon black, carbon nanotubes, etc. Advantageously, it may include conductive carbon-polymer composites, conductive particles and / or conductive polymers dispersed in a matrix (e.g., carbon ink, carbon paste, metal ink, graphene ink). One specific embodiment of the invention is an analytical module, preferably a porous disk, having electrodes (eg, working electrode and / or counter electrode) comprising carbon, such as a carbon layer and / or a screen-printed layer of carbon ink.

[0278] The present invention includes methods for detecting and counting each detection complex. In a specific embodiment, the surface can include a variety of capture reagents for one or more analyte molecules present in the sample and a variety of capture reagents distributed across multiple resolvable binding zones located on the surface. Under conditions for performing and analyzing measurements, a "resolvable binding zone" is the minimum surface area associated with each binding event, and the surface area can be distinguished and distinguished from another region where another individual binding event occurs. Therefore, the method consists of the following: combining one or more analyte molecules with one or more capture reagents on the surface, determining the presence or absence of analyte molecules in multiple resolvable binding zones on the surface and identifying the number of resolvable binding zones containing analyte molecules and / or the number of analyte domains that do not contain analyte molecules.

[0279] The resolvable binding region can be optically interrogated in whole or in part, i.e., each individual resolvable binding region can be optically interrogated separately, and / or the entire surface including multiple resolvable binding regions can be imaged and one or more pixels or pixel groups within the image can be mapped to each resolvable binding region. The resolvable binding region can also be a microparticle within a plurality of micron particles. The resolvable binding region that exhibits a change in optical signature can be identified by a conventional optical detection system. Depending on the type of detection (e.g., the type of fluorescent entity, etc.) and the operating wavelength, filters designed for specific wavelengths can be used for optical interrogation of the resolvable binding region. In embodiments using optical interrogation, the system can include more than one light source and / or multiple filters to adjust the wavelength and / or intensity of the light source. In some embodiments, a CCD camera is used to capture optical signals from multiple resolvable binding regions. Other non-limiting examples of camera imaging systems that can be used to capture images include charge injection devices (CIDs), complementary metal oxide semiconductor (CMOS) devices, scientific CMOS (sCMOS) devices, and time delay integration (TDI) devices, as will be known to those of ordinary skill in the art. In some embodiments, a scanning mirror system coupled with a photodiode or a photomultiplier tube (PMT) may be used for imaging.

[0280] The measuring step of the method can include imaging the optical signal from the surface (or its part) to produce an image consisting of multiple pixels, wherein each distinguishable binding zone is mapped to one or more pixels or pixel groups in the image. The image analysis of the pixel or pixel group with the signal indicating the binding event (detection complex) can be completed using a recognized method of the art, such as a rich image analysis algorithm and software that can be used to identify and count the labeled biological structures in the fluorescence microscopy image. In one embodiment, after filtering the image to remove the large-scale signal gradient, the image is converted into a binary image using a segmentation threshold. The distinguishable binding zone is found by identifying a continuous region higher than the threshold intensity. If the binding domain meets the size and intensity requirements, it is classified as a binding event.

[0281] In one embodiment, the distinguishable binding zone is an element of an array. In a preferred embodiment, the array is a micropore or nanopore, such as an array of individual depressions or holes of a single substrate. Preferably, the volume of the hole is less than 100nL, preferably less than 50nL. In one embodiment, the volume of the hole is between about 10aL-100pL. Optionally, the hole can be configured to accommodate microparticles.

[0282] In one embodiment, at least 50% of the resolvable binding zones located on the substrate and addressed during the analysis contain zero or one analyte molecule. Preferably, at least 80%, more preferably at least 95%, and most preferably at least 99% of the resolvable binding zones contain zero or more analyte molecules. The concentration of analyte molecules in the sample can be determined at least in part using a calibration curve, Poisson distribution analysis, and / or Gaussian step analysis of the number of binding zones containing at least one or one analyte molecule. In a specific embodiment, the surface includes a plurality of particles each containing a plurality of capture reagents for analyte molecules, and a plurality of particles distributed across a plurality of resolvable binding zones (e.g., micropore or nanopore arrays). Therefore, the method comprises: (i) binding one or more analyte molecules to one or more capture reagents on the surface, (ii) distributing a plurality of particles across an array of resolvable binding zones; and (iii) determining the presence or absence of analyte molecules in each resolvable binding zone to identify the number of binding domains containing analyte molecules and / or the number of binding domains without analyte molecules.

[0283] It may also be advantageous to detect analytes in a confined volume using one or more of the methods of the present invention. In these embodiments, analyte molecules in a sample are bound to a pair of detection reagents each carrying a distinguishable marker, and the analyte is distributed across multiple locations, such as holes or reaction vessels (referred to herein as "reaction vessels") on a substrate (e.g., a dish, a culture dish, a chip, an optical fiber, a grid, etc.) so that most reaction vessels contain one or less analytes. This method enables a user to detect analyte molecules by counting the number of reaction vessels containing each of the distinguishable markers attached to the analyte. In some cases, the addressed multiple reaction vessels are part or substantially all of the total number of reaction vessels that may contain at least one analyte molecule (e.g., associated with at least one analyte molecule or not associated with any analyte molecule). Reference is made to the following published U.S. patent applications: U.S. Patent Application No. 20070259448; U.S. Patent Application No. 20070259385; U.S. Patent Application No. 20070259381; and International Patent Application No. PCT / US07 / 019184; and International Patent Application No. PCT / US09 / 005428. The disclosure of each of these publications is incorporated herein by reference. At least a portion of the reaction vessel can be addressed and a measurement indicating the number / percentage of reaction vessels containing at least one analyte molecule or particle can be made. In some cases, based on the number / percentage, a measurement of the concentration of analyte molecules in the fluid sample can be determined.

[0284] In a specific embodiment enabling detection of analyte molecules in a confined volume, the analyte in the sample can be detected by combining the analyte with the first and second detection reagents to form a detection complex. Each detection complex comprises an analyte, a first detection reagent and a second detection reagent, and the first detection reagent and the second detection reagent have a first and a second detectable label, respectively. The detection complex can be formed simultaneously, substantially simultaneously or sequentially. The detection complex is distributed across multiple reaction vessels so that most reaction vessels contain one or less detection complexes, 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 complex is distributed across multiple reaction vessels so that the probability of detecting the unbound first detection reagent and the unbound second detection reagent in the same container is less than about 1 / 10, preferably less than about 1 / 100, more preferably less than about 1 / 1000, and most preferably less than about 1 / 10,000. Distributing the detection complex across multiple reaction vessels, i.e., dividing or separating the components or parts, is performed, for example manually by dividing a portion of the detection complex across multiple reaction vessels, and / or by flowing a solution including the detection complex across multiple reaction vessels so that the detection complex is separated into individual reaction vessels on a support.

[0285] In another embodiment, the analyte in the sample can be detected as follows: (a) the analyte is combined with a surface-bound capture reagent and a first and a second detection reagent to form a detection complex, wherein (i) each detection complex comprises a capture reagent, an analyte, a first detection reagent, and a second detection reagent, and (ii) the first detection reagent has a first detectable label and the second detection reagent has a second detectable label. The detection complex can be formed by adding components in any order, for example, by putting the components together simultaneously or substantially simultaneously, or by adding each component in sequence to construct the detection complex in a stepwise manner. The detection complex is distributed across multiple reaction vessels so that most reaction vessels contain one or less analyte, and the number of analyte molecules is detected by counting the number of reaction vessels containing the first and second detectable labels. The method can be performed with or without washing after each step and before the detection step.

[0286] 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 reagents are immobilized on a plurality of particles and the distribution of the analyte is achieved by allowing the analyte to bind to the capture reagent and distributing the particles to a plurality of reaction vessels; or (ii) the capture reagents are immobilized on a plurality of particles and the distribution of the analyte is achieved by distributing the particles to a plurality of reaction vessels, and then allowing the analyte to bind to the capture reagent.

[0287] The plurality of reaction vessels may also include water droplets dispersed in a water-in-oil emulsion. The emulsion may be made with droplets having a diameter of up to 100 μm and a volume approaching 1 nL. High capacity (i.e., greater than 10 10 The ease of preparing emulsions and their high stability under a wide range of conditions make them ideal for compartmentalizing biochemical analyses. Each droplet acts as an independent reaction vessel, and detection complexes, optionally attached to particles, can be distributed across multiple droplets.

[0288] Alternatively, the surface is a location within one of the reaction vessels, for example if the reaction vessel is a well of a dish, then the surface can be a domain or region within one of the wells of the dish. In this embodiment, the capture reagent can be immobilized on the domain or region of the plurality of reaction vessels, and the partitioning step is accomplished by binding the analyte molecules to the capture reagent. In another embodiment, the plurality of reaction vessels comprise an area immobilized with a targeting moiety, the capture reagent comprises a complement to the targeting moiety, and the partitioning step is accomplished by binding the complement to the targeting moiety to the target moiety disposed in the plurality of reaction vessels.

[0289] In additional or alternative embodiments, the binding assays described herein may also include a pre-concentration step to improve the performance of the assay, for example by increasing the concentration of the analyte in the sample and / or by reducing the concentration of extraneous materials that may be present in the sample that may hinder the performance of the assay. This may be performed as follows: (a) contacting a sample comprising the analyte of interest with a solid phase (e.g., a particle) attached to a first binding reagent that binds the analyte, thereby forming a complex comprising the analyte bound to the first binding reagent; (b) collecting the complex; (c) separating the unbound components of the sample from the complex; (d) releasing the complex. This method of concentrating the analyte may be performed prior to performing the binding assays described herein to remove impurities that may hinder the performance of the assay. In this regard, reference is made to U.S. Application Publication No. US2010 / 0261292, the disclosure of which is incorporated herein by reference.

[0290] Specifically, the concentration step involves treating a sample including an analyte under conditions sufficient to form an analyte complex, the analyte complex comprising the analyte bound to a first detection reagent, wherein the first detection reagent is linked to a first nucleic acid probe. Thereafter, the analyte complex formed at the end of the concentration step is bound to (i) a capture reagent on a surface comprising a capture reagent for the analyte and an anchor reagent comprising an anchor oligonucleotide sequence; and (ii) a second detection reagent for the analyte linked to a second nucleic acid probe; thereby forming a complex on the surface comprising the capture reagent, the analyte, and the first and second detection reagents. The surface-bound complex is subjected to an extension method requiring proximity of the first and second probes, extending the second probe to form an extended sequence comprising an anchor sequence complement that is complementary to the anchor sequence. The anchor sequence is then hybridized with the anchor sequence complement; and the amount of the extended sequence bound to the surface is measured. In a specific embodiment, the concentration step further comprises: (i) contacting the sample containing the analyte with a solid phase, wherein the solid phase is linked to a targeting agent complementary to at least a portion of the first nucleic acid probe, thereby forming a concentrated complex comprising the analyte bound to the solid phase through a binding reaction between the first nucleic acid probe and the targeting agent; (ii) collecting the concentrated complex; (iii) separating unbound components of the sample from the concentrated complex; and (iv) releasing the concentrated complex to separate the solid phase from the analyte to form an analyte complex.

[0291] As used herein, collection refers to the physical positioning of a material in a mixture. Collection includes positioning the material by a binding reaction or adsorption. For example, the material in the mixture can be collected on a solid phase by adsorbing the material on the solid phase or by binding the material to a binding agent on the solid phase. However, collection is not limited to positioning on a solid phase and can also include techniques in the art for positioning a material at a certain position / volume within a larger fluid volume, such as by using optical tweezers (which use light to manipulate microscopic objects as small as a single atom, where radiation pressure from a focused laser beam can capture small particles), electric or magnetic fields, focused flow, density gradient centrifugation, etc. to position the material.

[0292] Certain embodiments of the present invention include collecting microparticles or materials bound to microparticles. Suitable collection methods include many methods known in microparticle-based analytical techniques that achieve localization of microparticles from suspensions. These include filtration onto filter membranes or porous membranes under gravity or by centrifugal sedimentation, localization by applying a magnetic field (magnetizable particles), binding or adsorption of particles to a macroscopic solid phase, use of optical tweezers, etc.

[0293] As used herein, release refers to the delocalization of previously collected materials. Materials that are held in local positions by chemical bonds or by specific or nonspecific binding interactions can be allowed to delocalize by breaking bonds or interactions so that the material can diffuse or be mixed into the surrounding medium. There are many generally recognized cleavable chemical linking groups that can be used, which provide covalent bonds that can be cracked when harsh conditions are not required. For example, thiol or other reducing agents can be used to crack the linking groups containing disulfide bonds, periodate can be used to crack the linking groups containing cis-diols, and metal ligand interactions (such as nickel-histidine) can be cracked by changing pH or introducing competitive ligands. Similarly, there are many generally recognized reversible binding pairs that can be adopted (including those identified in affinity chromatography techniques). For example, the combination of many antibody-ligand pairs can be reversed by changing pH, adding protein denaturants or chaotropes, adding competitive ligands, etc. Other suitable reversible binding pairs include complementary nucleic acid sequences, and their hybridization can be reversed under a variety of conditions, including changing pH, reducing salt concentration, increasing temperature to a melting temperature higher than the pair and / or adding nucleic acid denaturants (such as formamide). Such reversible binding pairs can be used as targeting agents (as described above), for example, a first targeting agent can be attached to a first binding reagent that binds an analyte, a second targeting agent can be attached to a solid phase, and the binding interaction of the first and second targeting agents can be used to reversibly immobilize the first binding reagent on the solid phase.

[0294] Release also includes physical delocalization of materials by, for example, mixing, oscillation, vortexing, convective fluid flow, mixing by applying magnetic, electrical or optical forces, etc. When microparticles or materials bound to microparticles have been collected, such physical methods can be used to resuspend the particles in the surrounding matrix. Release can simply be the opposite of the previous collection step (e.g., by any of the mechanisms described above), or can be collected and released by two different mechanisms. In one such example, the collection of materials bound to particles (such as analytes or complexes comprising analytes) can be achieved by physical collection of particles. The material is then released by cleaving bonds or reversing the binding reaction that fixes the material on the particles. In a second such example, the material (such as an analyte of a complex comprising an analyte) is collected on the surface by a binding interaction with a binding reagent attached to the surface. The material is then released by a second binding interaction that destroys the bond or connects the binding reagent to the surface.

[0295] The collection is followed by release which can be used to concentrate and / or purify the analyte in the sample. By collecting in a first volume and releasing into a second smaller volume, the analyte in the sample can be concentrated. By concentrating, it is usually possible to significantly improve the sensitivity of subsequent measurement steps. By collecting and removing some or all of the uncollected samples from the sample, the potential analysis interferents in the sample can be reduced or eliminated. Optionally, removing the unbound sample can include washing the collected material with a defined liquid reagent (e.g., analysis or washing buffer) and releasing the collected material into the reagent to provide a uniform matrix for subsequent analysis steps.

[0296] As incorporated herein by reference in US 2010 / 0261292 Figure 3 As described in (a), the method includes contacting a sample including a target analyte with a particle attached to a first binding reagent that binds to the target analyte, wherein the first binding reagent is attached to a first targeting agent and the particle is attached to a second targeting agent, and the first binding reagent and the particle are attached by a binding reaction between the first and second targeting agents to form a complex including the target analyte bound to the first binding reagent. The complex is then collected and unbound components in the sample are separated from the complex. The complex is released and the released complex is contacted with a second binding reagent bound to a solid phase, wherein the second binding reagent binds to the complex. This specific embodiment is described in Fig.16. The particle (1601) is modified to include a capture oligonucleotide sequence 1602 that is at least partially complementary to the sequence of a proximity probe 1603 bound to a detection antibody 1604. The particle is mixed with the proximity probe to hybridize the capture sequence with the probe sequence to form a complex 1605. The complex is then mixed with a sample comprising an analyte 1606 and optionally one or more contaminants 1607-1608. The analyte is bound to the detection antibody (1609) and the contaminants are removed (1610). The particle is removed from the complex comprising the bound analyte under suitable conditions to form a concentrated solution of the analyte bound to the proximity probe (1611), which can be used in an immunoassay as described herein, wherein additional proximity probes are bound to the analyte and the 3-antibody complex is subjected to RCA-PLA to detect the presence of the analyte in the sample (1612), for example as described in FIG. 2 (a) and the accompanying instructions.

[0297] In another embodiment, an immunoassay complex between the detection antibody and the analyte is formed in solution, then amplified, and the amplified product is then attached to the 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 described in Fig.17 In. Analyte A (1701) is bound to the detection antibody (1702 and 1703 respectively) that is bound to each of the adjacent probes (1704 and 1705 respectively), and a detection complex (1706) comprising the analyte bound to each of the detection antibodies is formed. The detection complex is contacted with two connection sequences (1707a and 1707b), and the two connection sequences each comprise an end sequence complementary to the non-overlapping region of the first adjacent probe and an end sequence complementary to the non-overlapping region of the second adjacent probe. The connection sequence is hybridized with the first and second adjacent probes, and the end sequence of the connection oligonucleotide is connected to form a circular target sequence (1708) hybridized with both the first and second adjacent probes. The second adjacent probe is extended by rolling circle hybridization to produce an amplicon comprising a binding reagent complementary to an anchoring reagent. The amplicon is contacted with a surface (1709) comprising a capture reagent (1710) and an anchoring reagent (1711), and the amount of the amplicon bound to the surface is measured by marking with a plurality of labeled probes (1712).

[0298] Labeled probe

[0299] In an embodiment, the present invention provides a labeled probe comprising an oligonucleotide and at least one electrochemiluminescent portion. In an embodiment, the present invention provides a labeled probe comprising an oligonucleotide and at least two electrochemiluminescent portions. In an embodiment, the electroluminescent portion is an electrochemiluminescent label. In an embodiment, the labeled probe is used in the methods and analyses described herein to measure the amount of an extended sequence. In an embodiment, the extended sequence is an amplification product (or amplicon) of an RCA method. In an embodiment, the labeled probe comprises an oligonucleotide complementary to a detection sequence in the extended sequence. In an embodiment, in the methods described herein, a plurality of labeled probes are used to measure the amount of an extended sequence bound to a surface. In an embodiment, measuring the labeled probe determines the amount of an analyte in a sample.

[0300] Known detectable luminescent labels, such as fluorophores, have a self-quenching effect. Self-quenching is the reduction of the luminescent intensity of a label by another, usually increasing with high label concentrations or high label density. Therefore, multiple luminescent labels in close proximity are generally avoided to reduce the self-quenching effect. Therefore, the inventors unexpectedly found that the labeled probe of the present invention, although containing a structure that keeps multiple electrochemiluminescent labels in close proximity, still provides self-labeling to effectively generate ECL and improve signals relative to a probe with only one label. Therefore, in an embodiment, the labeled probe of the present invention includes more than one electrochemiluminescent label. In an embodiment, the labeled probe includes 2 to 10 electrochemiluminescent labels. In an embodiment, the labeled probe includes 2 to 5 electrochemiluminescent labels. In an embodiment, the labeled probe includes three electrochemiluminescent labels. In an embodiment, the labeled probe includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 electrochemiluminescent labels.

[0301] In an embodiment, the present invention provides a labeled probe comprising an oligonucleotide and a plurality of electrochemiluminescent labels. The probe may comprise (i) one or more (or two or more) labels attached to a modified nucleotide base of the oligonucleotide, (ii) a labeled portion having one or more (or two or more) labels, the portion being attached to the 5' end of the oligonucleotide, (iii) a labeled portion having one or more (or two or more) labels, the portion being attached to the 3' end of the oligonucleotide, or (iv) a combination of two or more of (i), (ii) and (iii).

[0302] In an embodiment, the present invention provides a labeled probe of Formula I:

[0303]

[0304] Where B is a nucleotide base, R is an electrochemiluminescent label, and L 1 is a linking group, L 2is a linking group, j is an integer between 0 and 11, k is an integer between 0 and 1, m is an integer between 0 and 11, and n is an integer between 0 and 5.

[0305] The nucleotide base B of formula I is any nucleotide base, as long as the nucleotide base does not interfere with the electrochemiluminescence ability of the labeled probe. In an embodiment, the nucleotide base is a naturally occurring nucleotide base. In an embodiment, the nucleotide base is a synthetic nucleotide base. In an embodiment, the nucleotide base is a purine or pyrimidine. In an embodiment, the nucleotide base is adenine, cytosine, guanine, thymine or uracil. In an embodiment, the nucleotide base is xanthine, hypoxanthine, 2,6-diaminopurine, 6,8-diaminopurine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, isoguanine or isocytosine.

[0306] R of 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. Patents 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

[0307]

[0308] In an embodiment, L 1 and L 2 L is independently alkyl, haloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl substituted cycloalkyl, heterosubstituted cycloalkyl, heteroalkyl, cyanoalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, phenyl or a combination thereof, having zero or one or more carbon chains optionally substituted with heteroatoms. In an embodiment, L 1 and L 2 is independently an alkyl linking group having zero or one or more carbon chains optionally substituted with heteroatoms. In embodiments, one or more heteroatoms are independently nitrogen, sulfur, phosphate or oxygen. In embodiments, L 1 The length of L is about 1 to about 20 carbon and / or heteroatoms. 1 The length of L is about 4 to about 15 carbon and / or heteroatoms. 1The length of L 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 carbons and / or heteroatoms. 2 The length of L is about 1 to about 30 carbon and / or heteroatoms. 2 The length of L is from about 7 to about 26 carbon and / or heteroatoms. 2 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 carbons and / or heteroatoms in length.

[0309] As used herein, "between" is a range that includes the ends of the range. For example, an integer between 0 and 11 specifically includes the integers 0 and 11, and any integer between 0 and 11, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. An integer between 0 and 5 specifically includes the integers 0 and 5, and any integer between 0 and 5, i.e., 1, 2, 3, and 4.

[0310] In an embodiment, R comprises a ruthenium complex RP 1 P 2 P 3 , where P 1 , P 2 and P 3 Each of is independently bipyridine, substituted bipyridine, phenanthroline or substituted phenanthroline. In an embodiment, the chemiluminescent label R is

[0311] In an embodiment, B is linked to L at the 5-position of uracil. 1 of uracil.

[0312] In an embodiment, L 1 include

[0313] or a combination thereof, wherein p is an integer between 1 and 12.

[0314] In an embodiment, L 2 include

[0315]

[0316] or a combination thereof, wherein q is an integer between 0 and 11.

[0317] In an embodiment, the present invention provides a labeled probe of Formula II:

[0318]

[0319] wherein j is an integer between 0 and 11, k is an integer between 0 and 1, m is an integer between 0 and 11, n is an integer between 0 and 5, and R is an electrochemiluminescent label:

[0320] In an embodiment, j is an integer between 0 and 5, k is 0, m is an integer between 0 and 5, and n is an integer between 2 and 7. In an embodiment, k is 0, j is 0, m is 1, and n is 5.

[0321] 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 an embodiment, the oligonucleotide of the labeled probe comprises a sequence having at least 88% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31). In an embodiment, the oligonucleotide of the labeled probe comprises a sequence having at least 90% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31). In an embodiment, the oligonucleotide of the labeled probe comprises a sequence having at least 95% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31). In an embodiment, the oligonucleotide of the labeled probe comprises a sequence having at least 98% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31). In an embodiment, the oligonucleotide of the labeled probe comprises a sequence having at least 99% sequence identity to 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31).

[0322] In an embodiment, the oligonucleotide of the labeled probe comprises 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31). In an embodiment, the oligonucleotide of the labeled probe comprises 5'-CAGTGAATGCGAGTCCGTCTAAG-3' (SEQ ID NO: 32). In an embodiment, the oligonucleotide of the labeled probe comprises one or more modifications described herein. In an embodiment, the labeled probe comprises an amino modifier. In an embodiment, the labeled probe comprises an internal amino-modified dT base (iAmMC6T). In an embodiment, the labeled probe comprises an internal spacer 18 (iSp18). In an embodiment, the labeled probe comprises a 3' amino modifier (3AmMO). In an embodiment, the oligonucleotide of the labeled probe comprises 5'-CAGTGAATGCGAGTCCGTCTAAG / iAmMC6T / iSp18 / iAmMC6T / iSp18 / 3AmMO / -3' (SEQ ID NO: 44 with modification).

[0323] In an embodiment, the present invention provides a method for measuring electrochemiluminescence, comprising: (a) applying a potential to an electrode under conditions where a complex proximate to the electrode will emit electrochemiluminescence, wherein the complex comprises a target oligonucleotide provided herein and a labeled probe, wherein the labeled probe comprises an oligonucleotide complementary to the target oligonucleotide; and (b) measuring the emitted electrochemiluminescence. Exemplary electrodes and methods for measuring electrochemiluminescence are described herein.

[0324] In an embodiment, the labeled probe is used in the measurement step of the analysis described herein, i.e., measuring the amount of the extended sequence bound to the surface. The labeled probe can be used in all methods described herein, such as methods using one detection reagent, two detection reagents, or two or more detection reagents. In an embodiment, the method of measuring electrochemiluminescence comprises: forming a composition comprising: (i) a target nucleic acid comprising a target sequence, and (ii) a labeled probe, wherein the labeled probe comprises an oligonucleotide complementary to the target sequence; cultivating the composition under conditions in which the labeled probe hybridizes with the target nucleic acid to form a complex; bringing the complex close to an electrode, applying a potential to the electrode under conditions in which the complex will emit electrochemiluminescence, and measuring the emitted electrochemiluminescence.

[0325] In an embodiment, the labeled probe is used in the detection step of the analysis described herein, i.e., measuring the amount of the extended sequence bound to the surface. The labeled probe can be used in all methods described herein, such as methods using one detection reagent, two detection reagents, or two or more detection reagents.

[0326] In an embodiment, the target nucleic acid is an extended sequence produced by an RCA reaction as described herein. In an embodiment, the target nucleic acid is fixed on an electrode. In an embodiment, the target nucleic acid is fixed on an electrode so that the formation of the complex brings the complex close to the electrode. In an embodiment, the target nucleic acid is directly fixed on an electrode, or it is indirectly fixed by a binding reagent as provided herein. In an embodiment, the complex further includes a binding reagent capable of binding to the target nucleic acid, wherein the binding reagent is fixed on an electrode. In an embodiment, the complex further includes a binding reagent capable of binding to the target nucleic acid, wherein the binding reagent is fixed on an electrode, and bringing the complex close to the electrode includes cultivating the composition together with the electrode under the condition that the target nucleic acid is bound to the binding reagent. In an embodiment, the binding reagent is an anchoring reagent. In an embodiment, the binding reagent includes a complement to the target nucleic acid.

[0327] In an embodiment, the target nucleic acid is fixed on a solid support. In an embodiment, the target nucleic acid is directly fixed on a solid support, or it is indirectly fixed by a binding reagent as provided herein. In an embodiment, a binding reagent capable of binding to a target nucleic acid is fixed on a solid support, wherein the solid support is fixed on an electrode. In an embodiment, the target nucleic acid is fixed on a solid support, and making the complex approach the electrode includes cultivating the composition with the electrode under the condition that the target nucleic acid is bound to the binding reagent. In an embodiment, the binding reagent is fixed on a solid support, and making the complex approach the electrode includes cultivating the composition with the solid support under the condition that the target nucleic acid is bound to the binding reagent, wherein the target nucleic acid is bound to the binding reagent, and collecting the solid support.

[0328] In embodiments, the cultivation lasts 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, the cultivation 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, the cultivation lasts about 10 minutes to about 8 hours at about 15°C to about 30°C. In embodiments, the cultivation lasts about 10 minutes to about 8 hours at about 15°C to about 30°C. In embodiments, the cultivation lasts about 20 minutes to about 6 hours at about 18°C ​​to about 29°C. In embodiments, the cultivation lasts about 20 minutes to about 6 hours at about 20°C to about 28°C. In embodiments, the incubation is at about 21° C. to about 26° C. for about 30 minutes to about 4 hours. In embodiments, the incubation is at about 22° C. to about 24° C. for about 40 minutes to about 2 hours. In embodiments, the incubation is at about 23° C. for about 1 hour.

[0329] Exemplary solid supports are described herein. In embodiments, the binding reagent comprises a complement to the target oligonucleotide. In embodiments, the solid support is a particle, and the particle is collected on an electrode using gravity, centrifugation, filtration, or application of a magnetic field.

[0330] In an embodiment, the present invention provides a kit for measuring electrochemiluminescence, which includes a labeled probe provided herein, and an electrode; an ECL reading buffer; a nucleic acid polymerase; a nucleic acid ligase; an analysis diluent; additional nucleic acid reagents; analysis consumables; or a combination thereof. Examples of additional nucleic acid reagents include buffers and reagents for dissolving, diluting and / or stabilizing nucleic acids. Examples of analysis consumables that may be included in the kit are analysis modules designed to contain samples and / or reagents during one or more steps of the analysis; pipette tips and other consumables for transferring liquid samples and reagents; covers and seals for analysis modules and other consumables used in the analysis; brackets for accommodating other analysis consumables; labels (including human-readable or machine-readable formats, such as barcodes, RFID, etc.) for identifying samples or other analysis consumables; and media (including paper and electronic media) for providing information about the analysis and / or instructions for performing the analysis.

[0331] In an embodiment, the test kit includes an electrode, and the electrode is a carbon-based electrode. In an embodiment, the test kit includes an analytical consumable, and the analytical consumable is a porous disk analytical consumable, and each hole of the disk includes a carbon ink electrode. In an embodiment, the test kit includes a porous analytical disk with a plurality of holes, and the analytical disk is used as a container for at least one binding reagent. In an embodiment, the binding reagent is fixed in the disk. A plurality of holes in the disk may have a binding reagent fixed therein. The binding reagent in each of these holes may be the same for all, some or none of these holes in these holes. In an embodiment, a plurality of binding reagents are fixed in each of these holes as a binding reagent array. Fixed binding reagents and / or arrays of fixed binding reagents may be fixed on electrodes in the holes (which may be carbon-based electrodes or more specifically, carbon ink electrodes).

[0332] In an embodiment, the kit includes an ECL read buffer, and the ECL read buffer includes tripropylamine. In an embodiment, the kit includes an ECL read buffer, and the ECL read buffer includes butyldiethanolamine. Exemplary ECL read buffers are described, for example, in US62 / 787,892 filed on January 3, 2019.

[0333] Method for producing labeled probes

[0334] The present invention includes methods for making labeled probes as 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 including an electrochemiluminescent label. The reaction mixture is then purified to isolate the product, wherein each of the amine moieties is coupled to an electrochemiluminescent label.

[0335] In an embodiment, the modified oligonucleotide having a reactive amino group has the structure shown in Formula VIII, and the product has the structure shown in Formula I, wherein R is an electrochemiluminescent label. In an embodiment, the modified oligonucleotide has the structure shown in Formula IX, and the product has the structure shown in Formula II, wherein R is an electrochemiluminescent label. The other components of the formula (L 1 , L 2 , k, m, etc.) are as described for Formulas I and II above.

[0336]

[0337] In embodiments, the amine-reactive labeling reagent comprises an active ester form of an electrochemiluminescent label and 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

[0338]

[0339] and the amine-reactive labeling reagent is

[0340]

[0341] In an embodiment, the modified oligonucleotide is prepared by solid phase synthesis. In an embodiment, the final product is purified by ion exchange chromatography. In an embodiment, the final product is purified by anion exchange chromatography. In an embodiment, the final product is purified by gel electrophoresis.

[0342] Nucleic acid probe

[0343] In an embodiment, the nucleic acid probe connected to the detection reagent is an oligonucleotide cross-linked or bound to the detection reagent. "Binding", "biobinding" or its variants 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 connected molecules may be referred to as a "conjugate" or "bioconjugate". In an embodiment, the conjugate includes a nucleic acid probe and a detection reagent.

[0344] In an embodiment, the nucleic acid probe includes one or more complementary regions to the template nucleic acid. In an embodiment, the template nucleic acid is a template for, for example, amplification by PCR. In an embodiment, the template nucleic acid is a circular nucleic acid template, connected to form one or more linear nucleic acid templates of a circular nucleic acid template (for example, for RCA). In an embodiment, the template nucleic acid sequence is a connection oligonucleotide used in the electrochemiluminescence measurement method as described herein. In an embodiment, the connection oligonucleotide is a linear oligonucleotide, and its 5' and 3' ends can be connected to produce a circular nucleic acid template. In an embodiment, the connection oligonucleotide includes a sequence at its 5' end complementary to the sequence at its 3' end, so that a ligase can connect 5' and 3' ends together to form a circular nucleic acid template. In an embodiment, the circular nucleic acid template is a template for rolling circle amplification (RCA). In an embodiment, the nucleic acid probe is a primer for RCA reaction, i.e., extends the circular nucleic acid template to form an extension sequence.

[0345] The inventors unexpectedly found that shorter nucleic acid probes improve the performance of the electrochemiluminescence measurement and analysis methods of the present invention. Conventional thinking is that appropriate performance will require relatively long oligonucleotides. Shorter nucleic acid probes provide the additional advantage of simplifying the binding scheme (and specifically, enabling the use of simpler, less labor-intensive and faster methods to separate protein-probe conjugates from unbound probes), and are also easier and cheaper to synthesize and purify. Therefore, in an embodiment, the nucleic acid probe of the present invention includes an oligonucleotide having a length of about 10 to about 30 nucleotides. In an embodiment, the nucleic acid probe includes an oligonucleotide having a length of about 12 to about 28 nucleotides. In an embodiment, the nucleic acid probe includes an oligonucleotide having a length of about 13 to about 26 nucleotides. In an embodiment, the nucleic acid probe includes an oligonucleotide having a length of about 14 to about 24 nucleotides. In an embodiment, the nucleic acid probe includes an oligonucleotide having a length of about 11 to about 22 nucleotides. In an embodiment, the nucleic acid probe includes an oligonucleotide having a length of about 12 to about 21 nucleotides. In an embodiment, the nucleic acid probe includes an oligonucleotide having a length of about 13 to about 20 nucleotides. In an embodiment, the nucleic acid probe comprises an oligonucleotide having a length of about 13 to about 18 nucleotides. In an embodiment, the nucleic acid probe comprises an oligonucleotide having a length of about 14 to about 19 nucleotides. In an embodiment, the nucleic acid probe comprises an oligonucleotide having a length of 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 an embodiment, the nucleic acid probe comprises an oligonucleotide having about 14 nucleotides. In an embodiment, the nucleic acid probe comprises an oligonucleotide having about 15 nucleotides. In an embodiment, the nucleic acid probe comprises an oligonucleotide having 5'-GACAGAACTAGACAC-3' (SEQ ID NO: 33). In an embodiment, the nucleic acid probe comprises an oligonucleotide having 5'-ACAGAACTAGACAC-3' (SEQ ID NO: 40). In an embodiment, the nucleic acid probe comprises an oligonucleotide comprising 5'-GACAGAACTAGACA-3' (SEQ ID NO: 41). In an embodiment, the nucleic acid probe comprises an oligonucleotide comprising 5'-TGCACAGCTCGACGC-3' (SEQ ID NO: 42). In an embodiment, the nucleic acid probe comprises an oligonucleotide, wherein the oligonucleotide is 14 to 24 nucleotides in length and comprises 14 or 15 consecutive nucleotides of 5'-GACAGAACTAGACAC-3' (SEQ ID NO: 33).

[0346] In an embodiment, the nucleic acid probe includes one or more nucleic acid modifications to allow binding to a detection agent. In an embodiment, a heterobifunctional cross-linking agent is used to complete the binding of the nucleic acid probe to a detection agent. In an embodiment, the nucleic acid probe includes a non-natural 5' modification containing a reactive functional group. Non-limiting examples of functional groups include, for example, olefins and strained olefins, alkynes, halides, alcohols, thiols, amines, phosphates, aldehydes, ketones, carboxylic acids, carboxylates, amides, esters, thioesters, acyl phosphates, acid halides, nitriles, anhydrides, hydrazines, tetrazines, azides, etc. In an embodiment, the reactive functional group is a thiol, amine, carboxylic acid, active ester, hydrazine, aldehyde, ketone, alkynes, strained olefins, azides or tetrazines. In an embodiment, the reactive functional group is a thiol. In an embodiment, the reactive functional group is a tetrazines. In an embodiment, the reactive functional group is a vinyl or strained olefin. In an embodimen...

Claims

1. A labeled probe of formula I, Where B is a nucleotide base, R is an electrochemiluminescent label, and L 1 is a linking group, L 2 is a linking group, j is an integer between 0 and 11, k is an integer between 0 and 1, m is an integer between 0 and 11, and n is an integer between 0 and 5.

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

3. The labeled probe according to claim 2, wherein the electrochemiluminescent label R is:

4. The labeled probe according to any one of claims 1 to 3, wherein B is uracil which is fused to L at position 5. 1 connect.

5. The labeled probe according to any one of claims 1 to 4, wherein L 1 include: or Its combination, Wherein p is an integer between 1 and 12.

6. The labeled probe according to any one of claims 1 to 5, wherein L 2 include: or Its combination, Wherein q is an integer between 0 and 11.

7. A labeled probe of formula II: wherein j is an integer between 0 and 11, k is an integer between 0 and 1, m is an integer between 0 and 11, n is an integer between 0 and 5, and R is an electrochemiluminescent label:

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

9. A labeled probe according to any one of claims 1 to 8, wherein the oligonucleotide comprises 5'-CAGTGAATGCGAGTCCGTCT-3' (SEQ ID NO: 31) has a sequence with at least 90% sequence identity.

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

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