Sandwich assay by ligation co-localization

Abstract

The present invention provides methods and systems for detecting and / or quantifying analytes, in particular methods and systems for simultaneously detecting and / or quantifying two or more analytes in a sample. In some embodiments, there is provided an assay co-localized by ligation (CLAMP) performed on a microparticle that includes two sets of conjugates pre-loaded on a carrier such that the two sets of conjugates are co-localized prior to contacting a sample.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of April 3, 2019, application number 201980038030.2, and invention name “Sandwich assay for co-localization by ligation” (the application date of the corresponding PCT application is April 3, 2019, and the application number is PCT / CA2019 / 050405).

[0002] Cross-application in related fields

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 651,943, filed April 3, 2018, entitled “Sandwich Assay for Colocalization by Ligation,” which is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention relates to the field of bioanalysis, and more particularly to methods and systems for detecting and / or quantifying biomolecules using sandwich assays with colocalization by ligation, and to multiplexed sandwich assays for simultaneously detecting and / or quantifying multiple biomolecules in a sample. Background Art

[0005] Rapid and specific detection of biological cells and biomolecules such as red blood cells, white blood cells, platelets, proteins, DNA and RNA has become increasingly important in diverse fields such as genomics, proteomics, diagnosis, treatment and pathology research. For example, rapid and accurate detection of specific antigens and viruses is essential for combating epidemic diseases such as AIDS, influenza and other infectious diseases. The maturation of genomic technologies and advances in personalized medicine will require faster and more sensitive assays for detecting and quantifying large numbers of cells and biomolecules. Advances in medical research will increasingly rely on accurate, timely and economical assessment of multiple proteins through proteomics. However, current automated, highly sensitive, low-cost assays cannot be efficiently multiplexed.

[0006] The sandwich assay is one of the most popular bioassay formats. In this format, capture probe molecules are immobilized on a surface. A biological sample containing target cells or biomolecules of interest is then applied to the surface. The target binds to the capture probe molecules immobilized on the surface in a concentration-dependent manner. In a subsequent step, the detection probe molecules are applied to the surface. The detection probe molecules bind to the target biomolecules, thereby "sandwiching" the target biomolecules between the capture probe and the detection probe molecules. In some assays, a second probe that binds to the detection probe molecules can also be applied to the surface. The second probe can be conjugated to a label such as a fluorophore, in which case a fluorescence scanner or fluorescence microscope can be used to detect the binding. In some cases, the second probe is conjugated to a radioactive element, in which case the radioactivity is detected to read the assay results. In some cases, the second probe is conjugated to an enzyme, in which case a solution containing the substrate is added to the surface and the conversion of the substrate by the enzyme is detected. In all cases, the intensity of the detected signal is proportional to the concentration of the target in the biological sample. The requirement for dual recognition in the sandwich assay provides a high-fidelity signal with low background noise, and therefore provides a highly sensitive detection.

[0007] Enzyme-linked immunosorbent assay (ELISA) is an example of a well-known sandwich assay. ELISA generally uses antibodies and a color change reaction to identify biomolecules in biological samples. For example, ELISA can use solid phase enzyme immunoassay (EIA) to detect the presence of biomolecules such as antigens in liquid or wet biological samples applied to the solid phase. ELISA is usually performed in a 96-well plate or 384-well polystyrene plate that passively binds antibodies and proteins. It is the binding and fixation of this reagent on the solid surface that makes the design and execution of ELISA so easy. Fixing the reagent on the microplate surface makes it easy to separate the bound target biomolecules from the unbound materials during the assay and wash away the non-specifically bound materials. In addition, the requirement for dual recognition by capture and detection probe molecules provides a high degree of specificity. Therefore, ELISA is a powerful tool for measuring specific target biomolecules in specific crude preparations.

[0008] Sandwich assays can be designed and constructed to measure or detect multiple analytes in parallel (also referred to as multiplexing). Multiple sandwich assays (MSA) can be implemented using microarrays such as DNA microarrays, protein microarrays, or antibody microarrays. A microarray is a collection of microdots containing biomolecules attached to a substrate surface such as glass, plastic, or silicon, thereby forming a "microscopic" array. Such microarrays can be used, for example, to simultaneously measure the expression levels of a large number of genes or proteins. Biomolecules such as DNA, proteins, or antibodies on a microarray chip are typically detected by optical readings of fluorescent labels that are attached to target molecules that specifically attach or hybridize to probe molecules. The labels used can be composed of, for example, enzymes, radioisotopes, or fluorophores.

[0009] MSA can also be performed on particles. In this case, particles suspended in a solution are attached with the necessary biomarkers to capture the target of interest, such as proteins or specific DNA molecules. In order to perform multiple assays, the particles must be encoded to distinguish between different assays in solution. A popular format is spectrally encoded microparticles, which are encoded with fluorescent or luminescent dyes. Particles can also be encoded graphically, so they are often called "barcoded particles." The size of the particles can range from nanometers (nanoparticles) to micrometers (microparticles). Among them, fluorescently encoded microparticles can be read out quickly and in high throughput on a cytometer.

[0010] However, current sandwich assays perform poorly when used to measure multiple biomolecules in a sample simultaneously (multiplexing). Multiplex ELISAs are limited by cross-reactions between reagents such as antibodies and proteins and are therefore susceptible to nonspecific signal transduction. In conventional multiplex sandwich assays in array and microbead formats, detection antibodies are usually applied as a mixture, but this approach can cause interactions between reagents, leading to cross-reactions. The application of a mixture of detection antibodies can therefore lead to false binding and, for example, to the capture and non-target analytes (e.g., Figure 1 False positive signals are generated from nonspecific binding events between the two targets (as shown), which are difficult to distinguish from the true target protein binding signal. This reagent-driven cross-reactivity is an inherent problem of MSA and scales quadratically with the number of targets, severely limiting the scale of multiplexing. Due to the problem of cross-reactivity, current MSAs are typically limited to 30-40 targets. Even so, lengthy and expensive optimization protocols are still required to discover and remove cross-reactive reagents (such as antibodies), which severely limits the applicability of these assays and increases their cost.

[0011] Cross-reactions therefore hinder other types of multiple determinations. For example, accurate protein phosphorylation analysis can be used to reveal cell signaling events where protein expression levels are not obvious. The methods and processes currently used to quantify the parts of specific protein post-translational modifications (PTMs) are severely limited in multiplexing because PTM-specific antibodies are usually not specific enough to the protein itself (i.e., phosphorus-specific antibodies are highly susceptible to reagent-driven cross-reaction problems). Therefore, conventional PTM plates cannot be multiplexed.

[0012] Conventional sandwich immunoassays are also not suitable for analyzing protein-protein interactions. Protein-protein interactions are a key part of cellular processes, and understanding the regulators of these interactions is extremely important for solving related diseases. However, the use of detection antibody mixtures allows undesired interactions and leads to false binding that can obscure the interaction signal. Current multiple sandwich assays are also expensive because expensive reagents such as antibodies are used inefficiently during manufacturing and performing the assay. For example, adding an antibody mixture to a solution requires high concentrations (nanomolar), however, the amount required to bind to proteins for quantitative analysis of microarrays or microbeads is 3 orders of magnitude less, corresponding to 99.9% antibody loss. Further, the sensitivity of a given sandwich immunoassay is severely affected by background signals, which are usually due to nonspecific binding of labeled detection antibodies and / or incomplete washing. Methods for reducing incomplete washing by increasing the washing cycle and including the addition of reagents have been used, however, these methods can lead to increased assay time and assay complexity.

[0013] U.S. Patent No. 9,481,945 describes an antibody colocalization microarray (ACM) that relies on a single detection antibody to address each capture antibody spot on the microarray, thereby avoiding the interactions between antibody reagents and reproducing assay conditions found in single-plex ELISA assays. Implementation of the method requires first spotting the capture antibody, removing the slide from the spotter, incubating it with the sample, washing and rinsing as needed, and then placing it back to spot the detection antibody, followed by binding and incubation. Therefore, the method relies on transferring n different reagents to n spots, each spot also using a different reagent, representing an n-to-n transfer. The need to perform spotting as part of the assay is cumbersome and slow, and has limited throughput.

[0014] U.S. Patent No. 7,306,904 describes a solution for detecting and / or quantifying one or more analytes using so-called proximity probes. The proximity probes include a binding moiety and a nucleic acid. The interaction of a nucleic acid from one proximity probe with a nucleic acid from another proximity probe is possible only when they are very close, i.e., when they are combined with an analyte that is specific to them. However, in general, multiplexed proximity-based assays need to be detected or read out in a single-plex format, and therefore require complex microfluidics to divide the sample into n parts for n-plex assays.

[0015] US Patent Application Publication No. US2016 / 0153973 describes a method and system for detecting analytes in immunoassays using a cleavable linker. However, due to high background signals and cross-reactions between reagents, this method and system are not suitable for multiplexing or simultaneously detecting multiple analytes in highly sensitive immunoassays. Summary of the invention

[0016] Methods and systems for detecting and / or quantifying biomolecules using biochemical assays are provided. It is an object of the present invention to improve at least certain disadvantages present in the prior art. Embodiments of the present technology were developed based on the inventors' recognition that there is a need for scalable, cost-effective, sensitive, rapid and / or simple multiplexed sandwich assays, for example, to replace ELISA for routine use. Therefore, in certain aspects, multiplexed sandwich assays are provided herein, including multiplexed sandwich immunoassays with minimal cross-reactions between reagents that are rapid, sensitive, cost-effective and / or scalable, allowing for simultaneous detection and / or quantification of multiple analytes in a sample.

[0017] The methods and systems provided herein are based at least in part on the design and construction of the connection between the reagent and the carrier, wherein these connections can achieve addressable and programmable topology and function. Without wishing to be limited by theory, it is believed that the systems and methods provided herein can reduce or eliminate the source of one or more background noise and / or false positives in multiple sandwich assays. In some embodiments, the cross-reaction between reagents in multiple assays is minimized or eliminated by minimizing or eliminating the interaction between non-homologous affinity binders. In some embodiments, the methods and systems provided herein can reduce or eliminate the background noise caused by incomplete cleaning and / or non-specific binding of the detection reagent. In some embodiments, the methods and systems provided herein can allow the multiple detection of post-translational modifications and / or the identification of protein-protein interactions to be achieved by assembling the combination of reagents on different assay carriers. In some embodiments, the surface structure, linker length and / or surface spacing of the reagent can be controlled to adjust the stringency and signal generation of the combination. In some embodiments, additional steps allow the assay signal to be stabilized by transducing the assay signal from a reversible reaction to a stable oligomer hybrid to minimize the unbound, thereby minimizing the signal loss after the assay is completed and thereby increasing the sensitivity.

[0018] In a first aspect, there is provided a biomolecule complex for detecting and / or quantifying an analyte in a sample, the biomolecule complex comprising:

[0019] an anchor chain attached to the carrier;

[0020] a capture agent attached to the carrier; and

[0021] a detection reagent releasably attached to the anchor, the detection reagent or anchor optionally attached to a first tag, the first tag being inactive or undetectable;

[0022] wherein: the capture reagent and the detection reagent can bind to the analyte (if present in the sample) simultaneously to form a triple complex; in the absence of the analyte, releasing the detection reagent from the anchor chain can cause the detection reagent to be released from the carrier; and when the detection reagent is released from the anchor chain, the first tag can be activated or detected. Thus, the presence of the analyte in the sample is determined by detecting the first tag on the carrier after the detection reagent has been released from the anchor chain, because the detection reagent only remains attached to the carrier when it binds to the analyte in the triple complex with the capture reagent. Thus, in some embodiments, the first tag is detected on the carrier only when the analyte is present.

[0023] In some embodiments, the amount of the first label detected on or from the support when the detection reagent is released from the anchor is proportional to the amount and / or concentration of the analyte in the sample.

[0024] In some embodiments, the detection reagent or anchor is optionally attached to the first tag. In some embodiments, the detection reagent is optionally attached to the first tag. In some embodiments, the anchor is optionally attached to the first tag.

[0025] In some embodiments, the detection reagent is releasably attached directly to the anchor by covalent bonds, biotin-streptavidin bonds, hydrogen bonding, hydrophobic interactions, affinity binding, or non-covalent interactions.

[0026] In other embodiments, the detection reagent is indirectly attached to the anchor chain via a hook chain, the detection reagent is connected to the hook chain, and the hook chain is releasably attached to the anchor chain, wherein in the absence of the analyte, releasing the hook chain from the anchor chain can release the detection reagent from the carrier, and the first tag can be activated or detected when the hook chain is released from the anchor chain. In some such embodiments, the amount of the first tag on the carrier when the hook chain is released from the anchor chain is proportional to the amount and / or concentration of the analyte in the sample.

[0027] In some embodiments, at least one of the detection agent and the tether is optionally attached to the first tag. For example, the first tag can be attached to the tether; the first tag can be attached to the detection agent; or the first tag can be attached to both the tether and the detection agent. In some embodiments, the first tag is not present, i.e., not attached to the first tether or the detection agent, for example, if the second tag is attached to a different component in the biomolecule complex.

[0028] In some embodiments, the capture agent is directly attached to the support, for example, by a covalent bond, a biotin-streptavidin bond, an oligonucleotide linker (such as a DNA oligonucleotide linker), or a polymer linker (such as a polyethylene glycol (PEG) linker). In other embodiments, the capture agent is indirectly attached to the support, for example, by connection to an anchor chain that is attached to the support, for example, via an oligonucleotide linker, a polymer linker, or a covalent bond. It should be understood that the capture agent can be attached to the support by any suitable means, including chemical interactions, affinity binding, etc.

[0029] In some embodiments, the anchor is a polymer such as polyethylene glycol (PEG), or an oligonucleotide such as a single-stranded DNA oligonucleotide, a single-stranded RNA oligonucleotide, or a double-stranded DNA or RNA oligonucleotide. It should be understood that the anchor can be attached to the carrier by any suitable means such as covalent bonds, chemical interactions, affinity binding, covalent bonds, biotin-streptavidin bonds, DNA oligonucleotide linkers, polymer linkers, etc.

[0030] The carrier is not particularly limited, and any suitable carrier can be used. Non-limiting examples of carriers include microparticles (such as microbeads), multi-well plate surfaces, slide surfaces, or hydrogel matrices. In some embodiments, the carrier is a microbead or microparticle, typically micrometer-sized, such as but not limited to polystyrene microbeads, magnetic microbeads, paramagnetic microbeads, plastic microbeads. In another embodiment, the carrier is a planar microarray. In some embodiments, the carrier is a barcoded microbead, for example, a microbead attached to a fluorescent or luminescent dye or a mixture thereof, or a spectral, graphical, or chemically encoded microbead.

[0031] Typically, the hook chain to which the detection reagent is attached is a linker of sufficient length and flexibility to allow the detection reagent and the capture reagent to bind to the analyte simultaneously to form a triple complex. Non-limiting examples of hook chains include polymers such as PEG and oligonucleotides such as single-stranded DNA oligonucleotides, single-stranded RNA oligonucleotides, or double-stranded DNA or RNA oligonucleotides.

[0032] In certain embodiments of the biomolecule complexes provided herein, the hook chain is absent and the detection reagent is releasably attached directly to the anchor chain, e.g., by covalent bonds, biotin-streptavidin bonds, affinity bonding, and the like.

[0033] The capture reagent can be any molecule that can specifically recognize and bind to the target analyte. Non-limiting examples of capture reagents include antibodies, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, aptamers, modified aptamers (such as modified aptamers or aptamers with low dissociation rates), and low molecular weight compounds. In certain embodiments, the capture reagent is an antibody, and the analyte is an antigen, protein, polypeptide, multi-protein complex, hormone, or exosome. In other embodiments, the capture reagent is an antigen, protein, polypeptide, multi-protein complex, or exosome, and the analyte is an antibody.

[0034] Similarly, the detection reagent can be any molecule that can specifically recognize and bind to the target analyte. Non-limiting examples of detection reagents include antibodies, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, and low molecular weight compounds. In certain embodiments, the detection reagent is an antibody and the analyte is an antigen, protein, polypeptide, multi-protein complex, or exosome. In other embodiments, the detection reagent is an antigen, protein, polypeptide, multi-protein complex, or exosome, and the analyte is an antibody.

[0035] It should be understood that if the capture reagent is an antibody and the analyte is an antigen, protein, polypeptide, multi-protein complex or exosome, then the detection reagent is also an antibody capable of binding to the analyte simultaneously with the capture reagent. Similarly, if the capture reagent is an antigen, protein, polypeptide, multi-protein complex or exosome and the analyte is an antibody, then the detection reagent will also be an antigen, protein, polypeptide, multi-protein complex or exosome capable of binding to the analyte simultaneously with the capture reagent.

[0036] The capture reagent and the detection reagent can be the same or different, as long as they both bind to the target analyte at the same time to form a triple complex. In some embodiments, the capture reagent and the detection reagent are both antibodies. They can be the same antibody or different antibodies. They can be different antibodies that bind to the same epitope on the analyte, or they can be different antibodies that bind to different epitopes on the analyte. In the case where the capture reagent and the detection reagent bind to the same epitope, they generally bind to different repeats of the epitope on the analyte, and the analyte has two or more repeats of the epitope.

[0037] The analyte is not intended to be particularly limited and can be any biological molecule or biological cell that needs to be detected and / or quantified in a sample. Non-limiting examples of analytes include antigens, antibodies, proteins, polypeptides, multi-protein complexes, hormones, exosomes, oligonucleotides, or low molecular weight compounds. The analyte can be detected in any sample of interest, and is not particularly limited to biological samples, such as but not limited to body fluids (e.g., urine, saliva, blood, serum, plasma, sweat), extracts (e.g., cell extracts), and solutions containing proteins and / or DNA (e.g., reaction mixtures).

[0038] In some embodiments, the detection reagent is attached to the first tag. In some embodiments, the shackle is attached to the first tag. In some embodiments, both the detection reagent and the shackle are attached to the first tag. In some embodiments where the first tag is not present, neither the detection reagent nor the shackle is attached to the first tag.

[0039] In some embodiments, the releasable joint between the hook chain and the anchor chain comprises a double-stranded DNA hybrid, and the hook chain and the anchor chain comprise complementary single-stranded DNA oligonucleotides, which are hybridized together to form a double-stranded DNA hybrid. In some such embodiments, the hook chain can be released from the anchor chain by raising the temperature of the anchor chain so that the DNA hybrid "melts" or is not combined. For example, in embodiments where the melting temperature of the double-stranded DNA hybrid is about 50 to about 80 degrees Celsius, the temperature can be raised to above the Tm so that the double-stranded DNA hybrid dissociates, thereby releasing the hook chain from the anchor chain.

[0040] In some embodiments, the biomolecule complex provided herein further comprises a displacing agent, which can release the hook chain from the anchor chain, thereby releasing the detection reagent from the carrier in the absence of an analyte. The displacing agent can be any reagent that can specifically break or release the connector between the hook chain and the anchor chain. For example, the displacing agent can be an enzyme or other reagent that can cut (or break) the releasable connector between the hook chain and the anchor chain. Non-limiting examples of displacing agents include enzymes, light and reducing agents such as DTT. The displacing agent can be capable of, for example, breaking the connector between the hook chain and the anchor chain by an enzymatic reaction or by photo-induced breaking.

[0041] In some embodiments, the displacer is an oligonucleotide. For example, when the releasable linker between the hook chain and the anchor chain comprises a double-stranded DNA hybrid, the displacer can be a single-stranded DNA or RNA oligonucleotide, which is hybridized with the hook chain or the anchor chain, thereby releasing the hook chain from the anchor chain by an oligonucleotide or DNA displacement reaction. In the embodiment where the displacer is hybridized with the hook chain hook chain, the displacer forms a double-stranded DNA or RNA hybrid with the hook chain. In some such embodiments, the displacer can be detectably labeled so that only when the detection reagent attached to the hook chain is combined with the analyte, the displacer will still remain on the carrier after cleaning, and the label on the displacer is detected to indicate the presence of analyte in the sample. In some such embodiments, the first label does not exist, and the label on the detection displacer is used to detect and / or quantify the analyte. In some such embodiments, the amount of the label on the displacer detected on the carrier is proportional to the quantity and / or concentration of the analyte in the sample. In the embodiment where the displacer is hybridized with the anchor chain, the displacer forms a double-stranded DNA or RNA hybrid with the anchor chain. It will be appreciated that in such embodiments, the displacing agent is unlabeled, but rather the tag is attached to the detection reagent and / or tether such that the tag is only detected on the support in the presence of analyte.

[0042] In some embodiments, if the first label is not present, and the label on the detection displacer is used to detect and / or quantify the analyte, and the displacer acts through a DNA displacement reaction, the displacer binds to the hook strand (e.g., hybridizes). In other embodiments, if the first label is present on the detection reagent or hook strand and the displacer is not labeled, and the displacer acts through a DNA displacement reaction, the displacer can bind to the hook strand or anchor strand.

[0043] In some embodiments, the biomolecule complex further comprises a stem chain complementary to the near-surface sequence of the anchor chain, the stem chain and the anchor chain are both single-stranded oligonucleotides, and the stem chain can be combined with the anchor chain to form a double-stranded oligonucleotide. In some embodiments, by forming a double-stranded oligonucleotide with the anchor chain, the stem chain can provide a structural carrier for the anchor chain, for example, to prevent the complex from collapsing onto the carrier surface, to provide rigidity, to produce a spacer between the carrier surface and the complex, or to provide structural stability. In some embodiments, the stem chain can be attached to a barcode, such as a fluorescent or luminescent dye, and is used to attach a barcode tag to a carrier. Generally, the stem chain is attached to the anchor chain, for example, by hybridization, and is directly bound to the carrier non-covalently.

[0044] In some embodiments where the biomolecule complex comprises a stem chain that binds to an anchor chain to form a DNA hybrid at a surface adjacent to the carrier, the anchor chain is attached to a tag hook chain (rather than a detection reagent, hook chain, or displacer, all of which are not labeled). In these embodiments, the anchor chain stem chain anchor chain is attached to a tag that is inactive or undetectable when the anchor chain hybridizes with the stem chain; the anchor chain is also directly connected to the detection reagent. After the DNA hybrid is cut at the site between the tag and the carrier, the tag is activated or becomes detectable. The detection reagent will be released from the carrier in the absence of an analyte so that a signal is detected only in the presence of an analyte and after cutting (e.g., after releasing the detection reagent).

[0045] In some embodiments, the relative density of the anchor strand and the capture agent on the support can be adjusted to control the effective affinity of the assay. In some embodiments, the length of the hook strand can be adjusted to control the effective affinity of the assay.

[0046] In some embodiments, the valence of the conjugation between the detection reagent and the hook chain is selected to minimize cross-reactions and optimize the performance of the multiplex assay. In one embodiment, the conjugation between the detection reagent and the hook chain is monovalent. In other embodiments, the conjugation between the detection reagent and the hook chain is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8, or less than 1:6, less than 1:8 or less than 1:10. In other embodiments, at least 90% of the detection reagents are connected to the carrier only by one hook chain.

[0047] In some embodiments where the capture reagent is attached to the anchor, the conjugation between the capture reagent and the linker of the anchor is monovalent. In some embodiments where the capture reagent is attached to the anchor, the conjugation between the capture reagent and the linker of the anchor is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8, or less than 1:6, less than 1:8, or less than 1:10. In some embodiments, at least 90% of the detection reagents are attached to the anchor via only one capture strand.

[0048] In some embodiments, the anchor chains and / or capture reagents are randomly distributed on the support.

[0049] In some embodiments, the length and / or flexibility of the hook chain can be selected to permit or optimize the binding of the detection reagent to the analyte in the presence of the capture reagent.

[0050] In some embodiments where the linker between the hook chain and the anchor chain is a double-stranded DNA hybrid, the melting temperature (Tm) of the double-stranded DNA hybrid is from about 50 to about 80 degrees Celsius.

[0051] In some embodiments, the concentration of the detection reagent after displacement is less than about 10 picomoles to avoid rebinding of the detection reagent to off-target reagents or analytes after displacement or release has occurred.

[0052] In further embodiments, the biomolecular complex comprises two detection reagents such that a stronger signal is generated upon binding of the analyte due to the presence of two copies of the tag. In these embodiments, the biomolecular complex further comprises a second anchor chain that is attached to the support; a second detection reagent that is attached to the second anchor chain, wherein a second hook chain is attached to the second anchor chain, and wherein at least one of the second detection reagent and the second hook chain is optionally attached to a third tag; wherein the capture reagent, the detection reagent, and the second detection reagent can simultaneously bind to the analyte (if present in the sample) to form a triple complex. Release of the second hook chain from the second anchor chain can release the second detection reagent from the support and can activate the third tag in the absence of the analyte.

[0053] In some embodiments, the second detection reagent is attached to the third tag. In some embodiments, the second hook chain is attached to the third tag. The third tag can be any suitable tag, such as but not limited to a fluorophore, a specific DNA sequence, or a biotin moiety.

[0054] In some embodiments where the third tag is attached to the second detection reagent and / or the second hook chain and is inactive or undetectable, the third tag is only activated or detected when the second hook chain is released from the second anchor chain and the analyte is present.

[0055] In some embodiments, the biomolecule complex further comprises a second displacer, which can release the second hook chain from the second anchor chain, so that the second detection reagent is released from the carrier when there is no analyte. The second displacer, like the displacer, can release the second hook chain from the second anchor chain by enzymatic reaction, by shearing or by oligonucleotide displacement reaction. The second displacer can also be detectably labeled like the displacer, in which case the second detection reagent and the second hook chain are generally not labeled (that is, the third label does not exist). The second displacer can be the same or different from the displacer. In some embodiments, the second displacer and the displacer are identical, so that a reagent can release the second hook chain and the hook chain from their corresponding anchor chains.

[0056] In one embodiment, a biomolecule complex for detecting an analyte in a sample is provided, the biomolecule complex comprising an anchor chain, wherein the anchor chain is connected to a carrier; a capture reagent, wherein the capture reagent is connected to the carrier; and a detection reagent, wherein the detection reagent is connected to the hook chain, wherein the hook chain is connected to the anchor chain and wherein the detection reagent is also labeled; wherein the capture reagent and the detection reagent can simultaneously bind to the analyte (if present in the sample) to form a triple complex, and the connection between the hook chain and the anchor chain can be broken, thereby releasing the detection reagent from the carrier in the absence of the analyte.

[0057] In another embodiment, a biomolecule complex for detecting an analyte in a sample is provided, the biomolecule complex comprising an anchor chain, wherein the anchor chain is connected to a carrier; a capture reagent, wherein the capture reagent is connected to the capture chain, wherein the capture chain is connected to the anchor chain; and a detection reagent, wherein the detection reagent is connected to the hook chain, wherein the hook chain is connected to the anchor chain and wherein the detection reagent is also labeled; wherein the capture reagent and the detection reagent can simultaneously bind to the analyte (if present in the sample) to form a triple complex; and the connection between the hook chain and the anchor chain can be broken, thereby releasing the detection reagent from the carrier in the absence of the analyte.

[0058] In another embodiment, a biomolecule complex for detecting an analyte in a sample is provided, the biomolecule complex comprising an anchor chain, wherein the anchor chain is connected to a carrier; a capture reagent, wherein the capture reagent is connected to a carrier; a detection reagent, wherein the detection reagent is connected to a hook chain, wherein the hook chain is connected to the anchor chain; and a displacer, wherein the displacer is capable of breaking the connection between the anchor chain and the hook chain by binding to the hook chain, resulting in the release of the detection reagent and the hook chain from the carrier in the absence of the analyte, and the displacer is labeled; wherein the capture reagent and the detection reagent can simultaneously bind to the analyte (if present in the sample) to form a triple complex.

[0059] In another embodiment, a biomolecule complex for detecting an analyte in a sample is provided, the biomolecule complex comprising an anchor chain connected to a carrier; a capture reagent connected to the capture chain, wherein the capture chain is connected to the anchor chain; a detection reagent connected to the hook chain, wherein the hook chain is connected to the anchor chain; and a displacer, the displacer is capable of breaking the connection between the anchor chain and the hook chain by binding to the hook chain, resulting in the release of the detection reagent and the hook chain from the carrier in the absence of the analyte, and the displacer is labeled; wherein the capture reagent and the detection reagent can simultaneously bind to the analyte (if present in the sample) to form a triple complex.

[0060] In another embodiment, a biomolecule complex for detecting an analyte in a sample is provided, the biomolecule complex comprising an anchor chain, the anchor chain being connected to a carrier; a capture reagent, the capture reagent being connected to the carrier and the anchor chain; a detection reagent, the detection reagent being connected to a hook chain, wherein the hook chain is connected to the anchor chain and comprises an inactivated label; and a displacement agent, the displacement agent being capable of breaking the connection between the anchor chain and the hook chain by the hook chain, resulting in the release of the detection reagent and the hook chain from the carrier in the absence of the analyte; wherein the capture reagent and the detection reagent can simultaneously bind to the analyte (if present in the sample) to form a triple complex, and wherein breaking the connection between the anchor chain and the hook chain activates the label on the hook chain.

[0061] In another embodiment, a biomolecule complex for detecting an analyte in a sample is provided, the biomolecule complex comprising an anchor chain connected to a carrier; a capture reagent connected to the capture chain, wherein the capture chain is connected to the anchor chain; a detection reagent connected to a hook chain, wherein the hook chain is connected to the anchor chain, and the hook chain comprises an inactivated label; and a displacer, the displacer is capable of breaking the connection between the anchor chain and the hook chain by binding to the hook chain, resulting in the release of the detection reagent and the hook chain from the carrier in the absence of the analyte; wherein the capture reagent and the detection reagent can simultaneously bind to the analyte (if present in the sample) to form a triple complex, and wherein breaking the connection between the anchor chain and the hook chain activates the label on the hook chain.

[0062] In another embodiment, a biomolecular complex for detecting an analyte in a sample is provided, the biomolecular complex comprising an anchor chain, the anchor chain being connected to a carrier; a capture reagent, the capture reagent being connected to a capture chain, wherein the capture chain is connected to the anchor chain; a detection reagent, the detection reagent being connected to a hook chain, wherein the hook chain is connected to the anchor chain, the hook chain comprising an inactivated tag; and a displacer, the displacer being capable of hooking and breaking the connection between the anchor chain and the hook chain, resulting in the release of the detection reagent and the hook chain from the carrier only in the absence of the analyte; wherein the capture reagent and the detection reagent are identical, and wherein the analyte has repeated epitopes, and the capture reagent and the detection reagent can simultaneously bind to the analyte (if present in the sample) to form a triple complex, wherein breaking the connection between the anchor chain and the hook chain activates the tag on the hook chain. Anchor Chain Hook Chain Hook Chain

[0063] In another embodiment, a biomolecule complex for detecting an analyte in a sample is provided, the biomolecule complex comprising an anchor chain, the anchor chain being connected to a carrier; a capture reagent, the capture reagent being connected to the capture chain, wherein the capture chain is connected to the anchor chain; a detection reagent, the detection reagent being connected to the hook chain, wherein the hook chain is connected to the anchor chain, and the hook chain comprises an inactivated label; and a displacer, the displacer being capable of breaking the connection between the anchor chain and the hook chain by binding to the hook chain, resulting in the release of the detection reagent and the hook chain from the carrier only in the absence of the analyte, wherein the displacer is labeled; wherein the capture reagent and the detection reagent are the same, and wherein the analyte has repeated epitopes, and the capture reagent and the detection reagent can simultaneously bind to the analyte (if present in the sample) to form a triple complex of anchor chain hook chain hook chain.

[0064] In another embodiment, a biomolecule complex for detecting an analyte in a sample is provided, the biomolecule complex comprising a first anchor chain, the first anchor chain being connected to a carrier; a second anchor chain, the second anchor chain being attached to a carrier; and a capture reagent, the capture reagent being connected to the carrier anchor chain; a first detection reagent, the first detection reagent being connected to a first hook chain, wherein the first hook chain is connected to the first anchor chain, the first hook chain comprising an inactivated first tag; a second detection reagent, the second detection reagent being connected to a second hook chain, wherein the second hook chain is connected to the second anchor chain, the second hook chain comprising an inactivated second tag; a first displacement agent, the first displacement agent being able to hook the first anchor chain to the second A connector between a hook chain is broken, resulting in the release of a first detection reagent and a first hook chain from the carrier in the absence of an analyte; and a second displacement agent, which is capable of breaking the connector between the second anchor chain and the second hook chain, resulting in the release of the second detection reagent and the second hook chain from the carrier only in the absence of an analyte; wherein the capture reagent, the first detection reagent and the second detection reagent can simultaneously bind to the analyte (if present in the sample) to form a quadruple complex, wherein breaking the connector between the anchor chain and the first hook chain activates the first tag on the first hook chain, and wherein breaking the connector between the anchor chain and the second hook chain activates the second tag on the second hook chain.

[0065] In another embodiment, a biomolecule complex for detecting an analyte in a sample is provided, the biomolecule complex comprising a first anchor chain, the first anchor chain being connected to a carrier; a second anchor chain, the second anchor chain being connected to a carrier, and a capture reagent, the capture reagent being connected to the carrier; a first detection reagent, the first detection reagent being connected to the first hook chain, wherein the first hook chain is connected to the first anchor chain; a second detection reagent, the second detection reagent being connected to the second hook chain, wherein the second hook chain is connected to the second anchor chain; a first A displacer, wherein the first displacer is capable of breaking the connection between the first anchor chain and the first hook chain by binding to the first hook chain, resulting in the release of the first detection reagent and the first hook chain from the carrier in the absence of an analyte, wherein the first displacer is labeled; and a second displacer, wherein the second displacer is capable of breaking the connection between the second anchor chain and the second hook chain by binding to the second hook chain, resulting in the release of the second detection reagent and the second hook chain from the carrier only in the absence of an analyte, wherein the first displacer is labeled; wherein the capture reagent, the first detection reagent and the second detection reagent can simultaneously bind to the analyte (if present in the sample) to form a quadruple complex.

[0066] In some embodiments, the carrier is a microparticle, a well plate surface, a glass slide surface, or a hydrogel matrix.

[0067] In some embodiments, the capture reagent and the detection reagent are antibodies. In some embodiments, the analyte is an antigen. In some embodiments, the analyte is a multiprotein complex. In some embodiments, the analyte is an exosome.

[0068] In other embodiments, the capture reagent and the detection reagent are antigens and the analyte is an antibody.

[0069] In some embodiments, the capture reagent is connected to the carrier by a covalent bond or by a biotin-streptavidin bond. In some embodiments, the capture reagent is connected to the carrier by a DNA oligonucleotide linker or by a polymer linker such as a PEG linker. In some embodiments, the detection reagent is connected to the carrier by a polymer linker such as a PEG linker or by a DNA oligonucleotide linker.

[0070] In one embodiment, the hook, anchor and displacer are DNA oligonucleotides.

[0071] In a further embodiment, the linker between the hook strand and the anchor strand is a double-stranded DNA hybrid.

[0072] In one embodiment, the linker between the anchor chain and the carrier is a covalent bond or a biotin-streptavidin bond. In another embodiment, the anchor chain is attached to the carrier by chemical interactions. It should be understood that the anchor chain can be attached to the carrier by any suitable means, such as but not limited to covalent bonds, biotin-streptavidin bonds, DNA oligonucleotide linkers, polymer linkers, or other chemical interactions such as hydrogen bonding, hydrophobic interactions, affinity binding, or non-covalent interactions.

[0073] In a further embodiment, the displacing agent breaks the connection between the hook strand and the anchor strand by a DNA strand displacement reaction.

[0074] In another embodiment, the displacing agent cleaves the connection between the hook and anchor strands by an enzymatic reaction.

[0075] In one embodiment, the tag is a fluorophore. In one embodiment, the tag is a specific DNA sequence. In one embodiment, the tag is a biotin moiety.

[0076] In another embodiment, the detection reagent recognizes the same antigen but a different epitope as the capture reagent.

[0077] In another embodiment, the detection reagent recognizes a different epitope bound on the same antigen to which the capture reagent is bound.

[0078] In another embodiment, the detection reagent recognizes the same epitope bound on the same antigen as the capture reagent.

[0079] In another embodiment, the biomolecule complex described herein further comprises a stem strand complementary to the surface-proximal sequence of the anchor strand, the stem strand making the anchor oligonucleotide double-stranded.

[0080] In one embodiment, the relative density of anchor and capture reagent is adjusted to control the effective affinity of the assay.

[0081] In another embodiment, the detection length of the analyte (eg, the length of the hook strand) is adjusted to control the effective affinity of the detection.

[0082] In another embodiment, the conjugation between the detection agent and the hook chain is monovalent.

[0083] In one embodiment, the anchor chains are randomly distributed. In another embodiment, the capture agents are randomly distributed.

[0084] Also provided is a method for detecting an analyte in a sample, the method comprising: providing a carrier, a capture reagent, an anchor chain, a hook chain and a detection reagent, wherein the capture reagent is connected to the carrier, the anchor chain is connected to the carrier and to the hook chain, wherein the hook chain is connected to the detection reagent, and wherein the detection reagent is labeled; incubating the sample with a carrier that allows the capture reagent and the detection reagent to bind to different epitopes on the analyte; breaking the bond between the hook chain and the anchor chain, and separating the detection reagent and the hook chain from the carrier in the absence of analyte bound to the capture reagent and the detection reagent; and quantifying the amount of bound analyte by analyzing the detection reagent label remaining on the carrier, wherein the concentration of the detection reagent label remaining on the carrier is proportional to the concentration of the bound analyte.

[0085] Also provided is a method for detecting an analyte in a sample, the method comprising providing a carrier, an anchor chain, a capture chain, a capture reagent, a hook chain and a detection reagent, wherein the anchor chain is connected to the carrier, to the capture chain and the hook chain, the capture chain is connected to the capture reagent and the hook chain, and wherein the hook chain is connected to the detection reagent; incubating the sample with a carrier that allows the capture reagent and the detection reagent to bind to different epitopes on the analyte; breaking the bond between the hook chain and the anchor chain by separating the detection reagent and the hook chain from the carrier in the absence of the analyte bound to the capture reagent and the detection reagent; and quantifying the amount of bound analyte by analyzing the detection reagent label remaining on the carrier, wherein the concentration of the detection reagent label remaining on the carrier is proportional to the concentration of the bound analyte.

[0086] Also provided is a method for detecting an analyte in a sample, the method comprising providing a carrier, a capture reagent, an anchor chain, a hook chain and a detection reagent, wherein the capture reagent is connected to the carrier, wherein the anchor chain is connected to the carrier and to the hook chain, wherein the hook chain is connected to the detection reagent; incubating the sample with a carrier that allows the capture reagent and the detection reagent to bind to different epitopes on the analyte; incubating with a displacer to break the bond between the hook chain and the anchor chain by binding to the hook chain, separating the detection reagent and the hook chain from the carrier in the absence of analyte bound to the capture reagent and the detection reagent, wherein the displacer is labeled; and quantifying the amount of bound analyte by analyzing the displacer label remaining on the carrier, wherein the concentration of the displacer label remaining on the carrier is proportional to the concentration of the bound analyte.

[0087] In another embodiment, a method for detecting an analyte in a sample is provided, the method comprising providing a carrier, an anchor chain, a capture chain, a capture reagent, a hook chain, and a detection reagent, wherein the anchor chain is connected to the carrier and to the capture chain, wherein the capture chain is connected to the capture reagent, the anchor chain is connected to the hook chain, and wherein the hook chain is connected to the detection reagent; incubating the sample with a carrier that allows the capture reagent and the detection reagent to bind to different epitopes on the analyte; incubating with a displacer to break the bond between the hook chain and the anchor chain by binding to the hook chain, separating the detection reagent and the hook chain from the carrier in the absence of analyte bound to the capture reagent and the detection reagent, wherein the displacer is labeled; and quantifying the amount of bound analyte by analyzing the displacer label remaining on the carrier, wherein the concentration of the displacer label remaining on the carrier is proportional to the concentration of the bound analyte.

[0088] Also provided is a method for detecting an analyte in a sample, the method comprising providing a carrier, a capture reagent, an anchor chain, a hook chain, and a detection reagent, wherein the capture reagent is connected to the carrier, wherein the anchor chain is connected to the carrier and to the hook chain, wherein the hook chain is connected to the detection reagent, wherein the hook chain comprises an inactivated tag; incubating the sample with a carrier that allows the capture reagent and the detection reagent to bind to different epitopes on the same analyte; incubating with a displacement agent to break the bond between the hook chain and the anchor chain, separating the detection reagent and the hook chain from the carrier in the absence of an analyte bound to the capture reagent and the detection reagent, wherein separating the hook chain from the anchor chain activates the tag on the hook chain; and quantifying the amount of bound analyte by analyzing the hook chain tag remaining on the carrier, wherein the concentration of the hook chain tag remaining on the carrier is proportional to the concentration of the bound analyte.

[0089] Also provided is a method for detecting an analyte in a sample, the method comprising providing a carrier, an anchor chain, a capture chain, a capture reagent, a hook chain and a detection reagent, wherein the anchor chain is connected to the carrier, the capture chain and the hook chain, wherein the capture chain is connected to the capture reagent, wherein the hook chain is connected to the detection reagent and comprises an inactivated tag; incubating the sample with a carrier that allows the capture reagent and the detection reagent to bind to different epitopes on the analyte; incubating with a displacement agent to break the bond between the hook chain and the anchor chain by binding to the hook chain, separating the detection reagent and the hook chain from the carrier in the absence of an analyte bound to the capture reagent and the detection reagent, wherein separating the hook chain from the anchor chain activates the tag on the hook chain; and quantifying the amount of bound analyte by analyzing the hook chain tag remaining on the carrier, wherein the concentration of the hook chain tag remaining on the carrier is proportional to the concentration of the bound analyte.

[0090] Also provided is a method for detecting an analyte in a sample, comprising providing a carrier, an anchor chain, a capture chain, a hook chain, a capture reagent and a detection reagent, wherein the anchor chain is connected to the carrier, to the capture chain and to the hook chain, wherein the capture chain is connected to the capture reagent, wherein the hook chain is connected to the detection reagent, wherein the hook chain comprises an inactivated tag and wherein the capture reagent and the detection reagent are structurally similar; incubating the sample with a carrier that allows the capture reagent and the detection reagent to bind to different epitopes on the analyte, wherein the epitopes are structurally similar; incubating with a displacement agent to break the bond between the hook chain and the anchor chain, separating the detection reagent and the hook chain from the carrier in the absence of an analyte bound to the capture reagent and the detection reagent, wherein separating the hook chain from the anchor chain activates the tag on the hook chain; and quantifying the amount of bound analyte by analyzing the hook chain tag remaining on the carrier, wherein the concentration of the hook chain tag remaining on the carrier is proportional to the concentration of the bound analyte.

[0091] Also provided is a method for detecting an analyte in a sample, the method comprising providing a carrier, an anchor chain, a capture chain, a hook chain, a capture reagent and a detection reagent, wherein the anchor chain is connected to the carrier, to the capture chain and the hook chain, wherein the capture chain is connected to the capture reagent, wherein the hook chain is connected to the detection reagent, and wherein the capture reagent and the detection reagent are structurally similar; incubating the sample with a carrier that allows the capture reagent and the detection reagent to bind to different epitopes on the analyte, wherein the epitopes are structurally similar; incubating with a displacer to break the bond between the hook chain and the anchor chain by binding to the hook chain, separating the detection reagent and the hook chain from the carrier in the absence of analyte bound to the capture reagent and the detection reagent, wherein the displacer is labeled; and quantifying the amount of bound analyte by analyzing the displacer chain label remaining on the carrier, wherein the concentration of the displacer chain label remaining on the carrier is proportional to the concentration of the bound analyte.

[0092] In one embodiment, the capture agent and the detection agent are peptides. In one embodiment, the capture agent is connected to the carrier via a DNA oligonucleotide linker. In another embodiment, the detection agent is connected to the carrier via a PEG linker.

[0093] In another embodiment, the hook chain, anchor chain and displacer are DNA oligonucleotides. In one embodiment, the connector between the hook chain and the anchor chain is a double-stranded DNA hybrid. In another embodiment, the connector between the anchor chain and the carrier is a biotin-streptavidin bond.

[0094] In one embodiment, the displacer breaks the connection between the hook chain and the anchor chain by a DNA strand displacement reaction. In one embodiment, the displacer breaks the connection between the hook chain and the anchor chain by an enzymatic reaction.

[0095] In another embodiment, the tag is a biotin moiety.

[0096] In yet another embodiment, the anchor is attached to the microparticle via a chemical interaction.

[0097] In another embodiment, the detection reagent recognizes the same antigen as the capture reagent but not the same epitope. In yet another embodiment, the detection reagent recognizes a different epitope bound to the same antigen to which the capture reagent binds. In one embodiment, the detection reagent recognizes the same epitope at a different position on the same antigen to which the capture reagent binds.

[0098] In one embodiment, the biomolecule complex described herein further comprises a stem strand complementary to the surface-proximal sequence of the anchor strand, thereby making the anchor oligonucleotide double-stranded.

[0099] In one embodiment, the relative densities of anchor and capture reagent are adjusted to control the effective affinity of the assay.

[0100] In one embodiment, the detection length of the analyte tether (eg, tether) is adjusted to control the effective affinity of the detection.

[0101] In one embodiment, the conjugation between the detection agent and the hook chain is monovalent.

[0102] In another embodiment, the anchor chains are randomly distributed.

[0103] In yet another embodiment, the capture agents are randomly distributed.

[0104] Also provided is a multiple complex detection system for detecting multiple analytes in a sample, the system comprising multiple carriers; multiple capture reagents, wherein each capture reagent is bound to its corresponding carrier; multiple detection reagents, wherein each detection reagent is bound to its corresponding carrier via a linker, wherein the detection reagent is labeled; wherein on each carrier, the capture reagent and the detection reagent can simultaneously bind to the carrier-specific analyte (if present in the sample) to form a triple complex; the linker between the detection reagent and its corresponding carrier can be broken, thereby releasing the detection reagent from the carrier in the absence of the analyte.

[0105] Also provided is a multiple complex detection system for detecting multiple analytes in a sample, the system comprising multiple carriers; multiple capture reagents, wherein each capture reagent is bound to its corresponding carrier; multiple detection reagents, wherein each detection reagent is bound to its corresponding carrier via a carrier-specific hook chain, wherein each hook chain comprises a carrier-specific inactivated label; a displacement agent, wherein the displacement agent is capable of breaking the bonds between the multiple hook chains and the multiple carriers to separate the detection reagents from their corresponding carriers, wherein on each carrier, the capture reagent and the detection reagent can simultaneously bind to the carrier-specific analyte (if present in the sample) to form a triple complex; wherein breaking the bond between the hook chain carrier and the hook chain activates the carrier-specific label on the hook chain.

[0106] Also provided is a multiple complex detection system for detecting multiple analytes in a sample, the system comprising multiple carriers; multiple capture reagents, wherein each capture reagent is bound to its corresponding carrier; multiple detection reagents, wherein each detection reagent is bound to its corresponding carrier through a carrier-specific hook chain; a displacer, wherein the displacer is capable of breaking the bonds between the multiple hook chains and the multiple carriers, wherein when the bonds between the hook chains and the carriers are broken, the displacer is bound to the hook chains, wherein the displacer is labeled; wherein on each carrier, the capture reagent and the detection reagent can simultaneously bind to the carrier-specific analyte (if present in the sample) to form a triple complex.

[0107] In one embodiment, a multiple complex detection system for detecting multiple analytes in a sample is also provided, the system comprising multiple carriers; multiple capture reagents, wherein each capture reagent is connected to its corresponding carrier, and multiple detection reagents, wherein each detection reagent comprises an inactivated label, wherein the connection between the detection reagent and its corresponding carrier can be broken; wherein on each carrier, the capture reagent and the detection reagent can simultaneously bind to the carrier-specific analyte (if present in the sample) to form a triple complex, wherein when the connection between the detection reagent and its corresponding carrier is broken, the detection reagent label is activated.

[0108] A method for detecting an analyte in a sample is also provided, the method comprising providing a carrier, an anchor chain, a capture chain, a hook chain, a capture reagent and a detection reagent, wherein the anchor chain is connected to the carrier, wherein the anchor chain is also connected to the capture chain, wherein the capture chain is connected to the capture reagent, wherein the anchor chain is also connected to the hook chain, wherein the hook chain is connected to the detection reagent; incubating the sample with the carrier while allowing the capture reagent and the detection reagent to bind to different epitopes on the same analyte; incubating with a displacer to break the bond between the hook chain and the anchor chain by binding to the hook chain, and separating the detection reagent and the hook chain from the carrier only in the absence of analyte bound to the capture reagent and the detection reagent, wherein the displacer is labeled; quantifying the amount of bound analyte by analyzing the displacer chain label remaining on the carrier, wherein the amount of the displacer chain label remaining on the carrier is proportional to the change in concentration of the bound analyte.

[0109] Also provided is a method for detecting an analyte in a sample, the method comprising providing a carrier, an anchor chain, a capture chain, a capture reagent, a hook chain and a detection reagent, wherein the anchor chain is connected to the carrier, wherein the capture reagent is connected to the carrier, wherein the anchor chain, wherein the hook chain is connected to the carrier, wherein the hook chain is connected to the detection reagent; incubating the sample with the carrier under conditions that allow the capture reagent and the detection reagent to bind to different epitopes on the same analyte; breaking the bond between the hook chain and the carrier, and separating the detection reagent and the hook chain from the carrier only in the absence of analytes bound to the capture reagent and the detection reagent; incubating with a bridge chain, wherein the bridge chain connects the anchor chain to the hook chain, wherein the bridge chain is labeled; and quantifying the amount of bound analyte by analyzing the bridge chain label remaining on the carrier, wherein the amount of the bridge chain label remaining on the carrier is proportional to the change in concentration of the bound analyte.

[0110] In some embodiments, the hook chain is labeled by including a tag sequence (i.e., a unique DNA sequence that can be detected). In some such embodiments, the hook chain also includes a rebinding sequence; after the hook chain is released from the anchor chain with a displacer oligonucleotide that binds to the anchor chain, a bridge chain is added, wherein the bridge chain can bind to the rebinding sequence on the hook chain anchor chain and the anchor chain, thereby indirectly reconnecting the hook chain to the anchor chain. In such embodiments, after the tag attached to the hook chain is activated or the anchor chain becomes detectable by release from the anchor chain, the hook chain with the active / detectable tag is reattached to the carrier.

[0111] In one embodiment, a biomolecule complex for detecting and / or quantifying an analyte in a sample is provided, the biomolecule complex comprising: a) an anchor chain attached to a carrier; b) a capture reagent attached to the carrier; c) a detection reagent connected to a hook chain, the hook chain is releasably attached to the anchor chain, the hook chain and the anchor chain are connected together by a double-stranded DNA hybrid, and d) a displacer, the displacer comprises a DNA oligonucleotide complementary to at least a portion of the hook chain and capable of hybridizing with the hook chain, thereby releasing the hook chain from the anchor chain through a DNA displacement reaction, and the displacer is detectably labeled; wherein the capture reagent and the detection reagent can simultaneously bind to the analyte (if present in the sample) to form a triple complex; and releasing the hook chain from the anchor chain by the displacer can release the detection reagent from the carrier in the absence of the analyte. In one embodiment, the capture reagent and the detection reagent are antibodies, the analyte is an antigen or protein, and the carrier is a barcoded microparticle.

[0112] In a second aspect, a multiplex sandwich assay system for simultaneously detecting and / or quantifying two or more analytes in a sample is provided, the system comprising two or more biomolecule complexes described herein, wherein each biomolecule complex is used to detect and / or quantify a different analyte in the sample.

[0113] In some embodiments, two or more biomolecule complexes are attached to the same carrier. For example, the carrier can be a flat surface, a multi-well plate surface, a slide surface, a hydrogel matrix, microparticles, etc. In such embodiments, each biomolecule complex is located at a different position on the carrier, thereby allowing each labeled complex (and each analyte) to be identified by its position.

[0114] In some embodiments, two or more biomolecule complexes are attached to different carriers, such as different barcoded microparticles. For example, a first biomolecule complex can be attached to a first barcoded microbead, such as spectrally, graphically, or chemically, such as with a first fluorescent or luminescent dye or dye mixture, and a second biomolecule complex can be attached to a second microbead that is also barcoded, such as spectrally, graphically, or chemically, such as with a second fluorescent or luminescent dye or dye mixture. After the first and second complexes have been assembled on their corresponding microbeads, they can be mixed and contacted together with a sample, allowing for simultaneous detection of two different analytes in the sample. Barcoding on the microbeads allows each labeled complex (and therefore each analyte) to be identified.

[0115] In some embodiments, one or more of the two or more biomolecule complexes comprises a second anchor, a second detection reagent linked to the second hook, etc., such that a quadruple complex is formed between the capture reagent, two detection reagents, and the analyte.

[0116] In some embodiments, two or more biomolecule complexes lack an optional first label on either the detection reagent or the hook chain, which is only provided on the displacer. In some embodiments, for each biomolecule complex, the corresponding hook chain is released from the corresponding anchor chain using the same labeled displacer, and each biomolecule complex (and its corresponding analyte) is identified by its position on the surface or by striping of the surface, particularly, where the surface is a microparticle. In other embodiments, different displacers with different labels can be used for each biomolecule complex.

[0117] In some embodiments, two or more biomolecule complexes can each detect and / or quantify the same analyte, wherein each biomolecule complex has a different effective affinity for the analyte. For example, the effective affinity of the biomolecule complex for the analyte can be selected by adjusting the length of the hook and / or anchor chain and / or adjusting the surface density of the capture reagent and / or detection reagent. In this way, the analyte can be determined over a large concentration range.

[0118] wherein the length of the hook and / or anchor chain can be adjusted to control the effective affinity of the assay; and / or wherein the surface density of the capture reagent and / or detection reagent can be adjusted to control the effective affinity of the assay.

[0119] It should be understood that the number of analytes that can be simultaneously detected and / or quantified in a multiplex sandwich assay system is not particularly limited. In some embodiments, the multiplex sandwich assay system can be used to simultaneously detect and / or quantify five or more analytes, ten or more analytes, 15 or more analytes, 20 or more analytes, 30 or more analytes, 40 or more analytes, 50 or more analytes, 75 or more analytes, or 100 or more analytes in a sample, the system comprising a corresponding biomolecular complex specific for each corresponding analyte. Therefore, the multiplex sandwich assay system is easily scalable for large-scale multiplexing.

[0120] In a third aspect, a method of detecting and / or quantifying an analyte in a sample using the biomolecular complex described herein is provided.

[0121] In some embodiments, methods are provided for simultaneously detecting and / or quantifying two or more analytes in a sample using two or more molecular complexes described herein, wherein each biomolecular complex is used to detect and / or quantify a different analyte in the sample. In some embodiments, methods are provided for simultaneously detecting and / or quantifying two or more analytes in a sample using a multiplex sandwich assay system described herein, the system comprising two or more biomolecular complexes described herein, wherein each biomolecular complex is used to detect and / or quantify a different analyte in the sample. It should be understood that the methods provided herein can be used to simultaneously detect and / or quantify a large number of analytes in a sample, which methods are extended to allow large-scale multiplexing.

[0122] In a practical embodiment, a method for detecting and / or quantifying an analyte in a sample is provided, the method comprising: a) providing a carrier, a capture reagent attached to the carrier, an anchor chain attached to the carrier, and a detection reagent optionally connected to the hook chain and the anchor chain, wherein the detection reagent or the hook chain is releasably connected to the anchor chain, and wherein at least one of the detection reagent and the hook chain is optionally attached to a first tag, and the first tag is inactive or undetectable; b) contacting the carrier with the sample under conditions that allow the capture reagent and the detection reagent to bind to the analyte simultaneously to form a triple complex; and c) adding a displacement agent optionally attached to a second tag, wherein the displacement agent releases the detection reagent or the hook chain from the anchor chain so that the detection reagent optionally connected to the hook chain is released from the carrier in the absence of the analyte, and wherein releasing the detection reagent or the hook chain from the anchor chain activates the first tag or makes the first tag detectable.

[0123] In some embodiments, the method further comprises the following steps: d) determining the presence and / or amount of the first label and / or the second label on the carrier, wherein the presence of the first label and / or the second label on the carrier indicates the presence of the analyte in the sample, and the amount of the first label and / or the second label is proportional to the amount and / or concentration of the analyte in the sample.

[0124] In some embodiments, the method further comprises the step of washing the support after step c) to remove any unbound reagent or material.

[0125] In some embodiments, the method further comprises the step of storing the vector after step c).

[0126] In some embodiments of the methods provided herein, the carrier further comprises a second anchor chain attached to the carrier, a second capture agent attached to the carrier, and a second detection agent optionally linked to the second hook chain, wherein the second detection agent or the second hook chain is linked to the second anchor chain, and wherein at least one of the second detection agent and the second hook chain is optionally attached to a third tag, and the third tag is inactive or undetectable, wherein the second capture agent and the second detection agent can bind to the second analyte in the sample simultaneously to form a second triple complex, wherein the displacer can also release the second detection agent or the second hook chain from the second anchor chain, so that the second detection agent optionally linked to the second hook chain is released from the carrier in the absence of the second analyte, and wherein releasing the second detection agent or the second hook chain from the second anchor chain can activate the third tag or make the third tag detectable; wherein the presence and / or amount of the third tag on the carrier indicates the presence of the second analyte in the sample, and the amount of the third tag on the carrier is proportional to the amount and / or concentration of the second analyte in the sample; so that the first analyte and the second analyte can be detected and / or quantified simultaneously in the sample.

[0127] In some embodiments, the second anchor chain and the second capture agent are positioned at a first position and a second position on the carrier, respectively. In other embodiments, the second anchor chain and the second capture agent are attached to the second carrier. The carrier and / or the second carrier can be, for example, microparticles, such as polystyrene microbeads. In one embodiment, the second carrier is a first barcoded microbead (i.e., a first microbead encoded with a first barcode, such as a first fluorescent or luminescent dye or a first dye mixture), and the second carrier is a second barcoded microbead (i.e., a second microbead encoded with a second barcode, such as a second fluorescent or luminescent dye or a second dye mixture), thereby allowing the corresponding microbead to be identified when the corresponding barcode is detected.

[0128] In some embodiments, the first carrier and the second carrier are mixed together before contacting with the sample. For example, the first carrier and the second carrier can be contacted with the sample at the same time. The sample can be a biological sample, such as but not limited to a body fluid, an extract, a solution containing protein and / or DNA, a cell extract, a cell lysate, or a tissue lysate. Non-limiting examples of body fluids include urine, saliva, blood, serum, plasma, cerebrospinal fluid, tears, semen, and sweat.

[0129] In some embodiments, the method uses a hook chain that is labeled by comprising a tag sequence (i.e., a unique DNA sequence that can be detected). In some such embodiments, the hook chain also comprises a rebinding sequence; after the hook chain is released from the anchor chain using a displacer oligonucleotide that binds to the anchor chain and an optional wash, a bridge chain is added, wherein the bridge chain binds to the rebinding sequence on the hook chain anchor chain and the anchor chain, thereby indirectly reconnecting the hook chain to the anchor chain. In such embodiments, after the tag attached to the hook chain is activated or becomes detectable by being released from the anchor chain, the hook chain with the active / detectable tag is reattached to the carrier.

[0130] In one embodiment, a method for detecting and / or quantifying an analyte in a sample is provided, the method comprising: a) providing a carrier, a capture reagent attached to the carrier, an anchor chain attached to the carrier, and a detection reagent connected to the hook chain, wherein the hook chain is releasably connected to the anchor chain through a double-stranded DNA hybrid; b) contacting the carrier with the sample under conditions that allow the capture reagent and the detection reagent to bind to the analyte simultaneously to form a triple complex; and c) adding a displacement agent attached to a detectable label, wherein the displacement agent is a DNA oligonucleotide that is complementary to at least a portion of the hook chain and capable of hybridizing with the hook chain, wherein the displacement agent releases the hook chain from the anchor chain through a DNA displacement reaction, so that the detection reagent is released from the carrier in the absence of the analyte; and d) optionally determining the presence and / or amount of the detectable label on the carrier, wherein the presence of the label on the carrier indicates the presence of the analyte in the sample, and the amount of the label is proportional to the amount and / or concentration of the analyte in the sample. In some embodiments, the capture reagent and the detection reagent are antibodies, the analyte is an antigen or protein, and the carrier is a barcoded microparticle. Barcoded microparticles may be barcoded, for example, spectrally, graphically or chemically.

[0131] In a fourth aspect, a method for preparing a multiplex sandwich assay system is provided, the method comprising: (a) providing a first container containing a first microparticle, the first microparticle being encoded with a first barcode; (b) attaching the first microparticle to a first capture reagent and a first detection reagent; (c) optionally, storing the first microparticle; (d) providing a second container containing a second microparticle, the second microparticle being encoded with a second barcode; (e) attaching the second microparticle to a second capture reagent and a second detection reagent; (f) optionally, storing the second microparticle; and (g) mixing the first microparticle and the second microparticle together for use in a multiplex sandwich assay system; wherein the first capture reagent and the first detection reagent are not mixed with the second capture reagent and the second detection reagent before being attached to their corresponding microparticles. The first and second barcodes can independently be spectral, graphic or chemical barcodes.

[0132] In some embodiments, the corresponding capture reagent and the corresponding detection reagent are attached to their corresponding microparticles simultaneously. In other embodiments, the corresponding capture reagent and the corresponding detection reagent are attached to their corresponding microparticles in a two-step reaction, wherein the capture reagent or the detection reagent is attached to the microparticles before the subsequent attachment of the other reagent is performed.

[0133] In some embodiments, the method further comprises washing the first microparticle and the second microparticle to remove unattached reagents before mixing them together in step (g). In some embodiments, the method further comprises one or more additional washing steps, performed before each step of attaching a capture reagent and / or a detection reagent is performed before performing a washing step to remove unattached and / or non-specifically attached reagents from the microparticles.

[0134] In some embodiments, the microparticles are microbeads, e.g., polystyrene microbeads. In some embodiments, the microparticles in step (a) are not barcoded, and the method further comprises a step of barcoding the microparticles (e.g., attaching a barcode such as a fluorescent or luminescent dye or a mixture thereof to the microparticles) before performing step (g), i.e., mixing the first microparticles and the second microparticles together.

[0135] In some embodiments, the first capture reagent, the first detection reagent, the second capture reagent, and the second detection reagent are antibodies.

[0136] In one embodiment, a method for preparing the multiplex sandwich assay system described herein is provided, the method comprising: (a) providing a carrier, which is a flat surface, a multi-well plate surface, a slide surface, or a hydrogel; (b) attaching a first capture reagent to the carrier at a first location; (c) washing the carrier to remove unattached first capture reagent; (d) attaching a second capture reagent to the carrier at a second location; (e) washing the carrier to remove unattached second capture reagent; (f) attaching a first detection reagent to the carrier at a first location via a first anchor chain attached to the carrier; (g) washing the carrier to remove unattached first detection reagent; (h) attaching a second detection reagent to the carrier at a second location via a second anchor chain attached to the carrier; and (i) washing the carrier to remove unattached second detection reagent; so that whenever the first capture reagent, the second capture reagent, the first detection reagent, and / or the second detection reagent are mixed together, no more than one reagent is not attached to the carrier at a time.

[0137] In some embodiments, the methods for preparing the multiple sandwich assay systems described herein are advantageous in minimizing cross-reactions because the different reagents are not mixed together in solution before being attached to a carrier and / or assembled in a biomolecule complex. In this way, non-specific binding of reagents to each other can be avoided or at least reduced to minimize cross-reactions. Unwanted background signals or "noise" can also be avoided or at least reduced. In some embodiments, the methods can also be expanded to allow for rapid and / or economical preparation of multiple sandwich assay systems. In some embodiments, much less capture and / or detection reagents are required compared to traditional assay systems, which can save a lot of cost for expensive antibody reagents, etc. In some embodiments, for example, less than one nanoliter of antibody reagent may be required to prepare a biomolecule complex.

[0138] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication and color drawings will be provided by the Office upon request and payment of the necessary fee.

[0140] In order to better understand the present invention and to more clearly show how it may be carried out, reference will now be made by way of example to the accompanying drawings which illustrate aspects and features according to embodiments of the present invention, in which:

[0141] Figure 1 is a schematic diagram illustrating (a) a typical ELISA reaction in which only one antibody pair is used (1-plex or monoplex assay); and (b) cross-reactions arising from nonspecific binding events between target biomolecules and mixed AB pairs (depicted as antibody pairs in the figure) in a multiplex assay.

[0142] Figure 2 Schematic diagrams illustrating certain embodiments of the present technology. (a) shows a CLAMP system in which cross-reactions are prevented by co-localizing antibody pairs on a single microbead using DNA linkage. In (b), each member of the CLAMP plate is made by a one-pot preparation of microbeads with a capture antibody (cAb) and a fluorescent barcode signal ratio. In (c), a collection of microbeads, each with a cAb and a pre-hybridized detection antibody (dAb), are mixed to form a CLAMP plate. In (d), sample addition and target protein binding are shown. In (e), non-specifically bound biomolecules are washed to remove. In (f), the sandwich complex on each microbead is labeled by fluorescent DNA chain displacement of the dAb.

[0143] Figure 3 A CLAMP assay according to an embodiment of the present technology is shown, wherein (a) an automated 4-color barcoding strategy is shown, which allows >580 microbead barcodes to be implemented on any multicolor cytometer; (b) a low-valent antibody-oligonucleotide conjugate is shown to significantly improve chain displacement efficiency (>99%) and minimize assay background signal; (c) a 1-complex CLAMP of uPA is shown, which is more sensitive than traditional (sequential) microbead immunoassays; (d) a low-valent conjugate is shown to further improve CLAMP sensitivity; (e) a 5-complex CLAMP is shown, assembled with antibody pairs that cross-react extensively in a traditional sandwich format, with no cross-reaction; and (f) a single standard curve for the 5-plex CLAMP is shown.

[0144] Figure 4 A schematic diagram is shown illustrating (a) a typical ELISA reaction using only one antibody pair (1-plex or monoplex assay); and (b) cross-reactivity resulting from nonspecific binding events between target biomolecules and mixed antibody pairs in multiplex analysis (depicted as antibody pairs in the figure). (c) A CLA system is shown in which cross-reactivity is prevented by co-localizing antibody pairs on a single microbead using DNA linkage.

[0145] Figure 5 Schematic diagrams illustrating exemplary embodiments of the CLA complex prior to contact and displacement with a biological sample, wherein the tag is absent or inactive / undetectable. (a) The detection AB is attached to a carrier using an anchor chain (i.e., directly attached to the anchor chain). (b) A stem oligomer is hybridized to the anchor chain, which is labeled and used to mask the label (e.g., a specific DNA sequence) so that it cannot be detected prior to release. (c) Both the capture antibody and the detection antibody are flexibly attached to the carrier. (d) The detection AB is attached to a hook chain, which is releasably attached to the anchor chain, optionally including an undetectable tag (the tag can be attached to the hook chain or the anchor chain). (e) The detection AB is attached to a hook chain, which is releasably attached to the anchor chain, and the hook chain is conjugated to a dye, which is quenched by a proximal quencher on the stem oligomer that is also bound to the anchor chain (before release). (f) Both the capture antibody and the detection antibody are attached to the anchor chain, the capture antibody is directly attached to the anchor chain, and the detection antibody is indirectly attached via a hook chain oligomer optionally attached to an undetectable tag (i.e., the tag is undetectable when the hook chain is attached to the anchor chain and is detectable after release). (g) The capture antibody and the detection antibody are indirectly attached to the anchor chain via their respective capture chains and hook chains.

[0146] Figure 6Schematic diagrams of exemplary embodiments for detecting CLA in the presence of an analyte after a successful displacement reaction are shown. (a) The label is a specific DNA sequence that can be detected by DNA hybridization methods. (b) The displacer is used to release the hook chain oligonucleotide from the anchor chain, thereby activating the label attached to the hook chain (in this case, the dye is not quenched after release). (c) The dye-labeled displacer preferentially binds to the hook chain oligonucleotide, releasing it from the anchor chain, and simultaneously labeling the hook chain and the bound triple complex. (d) The sequence-labeled displacer preferentially binds to the hook chain oligonucleotide, releasing it from the anchor chain, and simultaneously labeling the hook chain and the bound triple complex.

[0147] Figure 7 Schematics illustrating embodiments of displacement-dependent detection are shown. (a) A CLA embodiment is shown in which no label is present on the complex and in which the analyte is present and bound to the capture and detection AB in a triple complex. The dye-labeled displacer (an oligonucleotide in this embodiment) preferentially binds to the hook strand oligonucleotide, simultaneously labeling the hook strand and displacing it from the anchor strand. (b) AND (Boolean) logic gate representation of displacement-dependent detection, where detection at the complex level requires a bivalent capture target and successful hook-anchor displacement. Among the different potential outcomes considered for the presence of analyte and successful displacement, the scenario shown in (a) is the only scenario that results in a signal.

[0148] Figure 8 A schematic diagram of one embodiment of displacement-associated detection is shown, wherein the tag is a unique DNA sequence that is initially undetectable (i.e., cannot bind) because it is masked or hidden by hybridization with an anchor sequence. In the example shown in the figure, an unlabeled displacer hybridizes to a toe-hold retention sequence of the anchor to trigger displacement of the hook strand oligonucleotide, thereby enabling detection by secondary hybridization or other DNA detection and / or amplification methods, such as PCR.

[0149] Fig. 9 A schematic diagram illustrating a CLA embodiment with a displacement-dependent assay for sandwich antibody detection is shown. The same antigen (e.g., peptide) is used as both capture and detection AB, with one antigen attached to a hook that is releasably attached to an anchor. A labeled displacement agent (oligonucleotide in this embodiment) is used for bottom clamp-mediated displacement, performing the dual functions of displacement and labeling the hook. The labeled and released hook (as shown) remains attached to the carrier only in the presence of the target antibody.

[0150] Fig.10 Shown is a schematic diagram illustrating an embodiment of CLA for detecting multiplexed protein interactions, where an array (planar or microbeaded) is assembled with mismatched AB pairs, allowing for detection of protein-protein interactions based on the AB pairs.

[0151] Fig.11 A schematic diagram of a CLA embodiment for detecting multiplexed post-translational modifications (PTMs) is shown, where an array (planar or microbead) is assembled with AB pairs for total protein and PTM-specific proteins.

[0152] Fig.12 Shown is a schematic diagram illustrating a CLA embodiment, wherein the capture reagent is an antibody directly attached to a carrier and has two anchor chains. Each of the two anchor chains is attached to a hook chain attached to a detection reagent (i.e., an antibody) by DNA hybridization. In the presence of analyte, a quadruple complex is formed. The hook chains are released from their respective anchor chains using a labeled displacer oligonucleotide. The two tags may be the same or different.

[0153] Fig.13 A schematic diagram of an embodiment of a CLA is shown for stabilizing the signal using a rebinding mechanism after displacement and before detection. In this embodiment, the hook chain oligonucleotide includes a tag sequence and a rebinding sequence; after displacement and optional washing using a displacer oligonucleotide that binds to the anchor chain, the bridge chain binds to the rebinding sequence and the anchor chain, thereby indirectly reconnecting the hook chain to the anchor chain. In this way, the hook chain is reconnected to the carrier together with the active / detectable tag and the detection AB connected thereto.

[0154] Fig.14 Schematics of single-plex and multiplex sandwich assays and the CLA system on microparticles (“CLAMP”) are shown according to certain embodiments. (a) Single-plex sandwich immunoassays (also known as ELISAs) include a pair of matched antibodies. (b) MSAs of mixed antibodies are exposed to a large number of interactions between mismatched antibodies and proteins, often resulting in rCRs and false positives. (c) CLAMPs that pre-colocalize antibody pairs using DNA ligation allow sandwich binding while eliminating interactions between non-homologous antibodies. (d) dAbs are bound to a hook oligonucleotide (HO), which is tethered to the surface by partial hybridization with a capture oligonucleotide (CO) chain. Spacer oligonucleotides (SO) are used to control the density of CO and dAb-HO on the surface (see Fig.19 ). (e) Multiplexed CLAMP assays are performed by (i) mixing barcoded CLAMP microparticles with different targets, (ii) incubating the biological sample with the sandwich-bound microparticles only in the presence of the target analyte, (iii) washing, and (iv) displacing and labeling HO via bottom-clip-mediated displacement (inset) using a fluorescently labeled displacer oligonucleotide (DO), resulting in (v) labeling of the sandwich complex remaining on the surface. (f) An AND (Boolean) logic gate representation of detection via the labeled displacement step, where detection at the single-molecule level requires both capture of the target and successful release of HO.

[0155] Fig.15 Optimization of the displacement efficiency mediated by bottom clamping is shown. (a) Schematic representation of the displacement reaction, where Cy5-labeled HO is displaced using unlabeled DO. (b) Release efficiency (for n CO =10pmol, n CO = 100 pmol, blue and red, respectively). SO (i.e., n SO =0 pmol). The release efficiency was calculated as (I 0 –I f ) / (I 0 –I B ), where I 0 ,I f and I B The fluorescence before release, after release and background are shown respectively. The release efficiency increased significantly with the increase of ionic strength. The increase of CO concentration led to the increase of release efficiency, which may be due to the reduction of the proportion of non-specific binding oligonucleotide. (c) Release efficiency of CO concentration under high salt concentration (500mM NaCl)

[0156] Fig.16 Optimization of CLAMP by tuning conjugate valency and surface density is shown. (a) Normalized histogram comparing CLAMP background signal (i.e., residual signal after incubation with Cy5-labeled DO) of microparticles without HO (blue) and multivalent dAb-HO conjugates (yellow) (see Figure 6 ). (b) Illustration of how multivalent dAb-HO conjugates can increase background signal by labeling unsuccessfully displaced dAb-HO complexes despite the absence of sandwich binding of target analyte. (c) SDS-PAGE of mouse anti-goat IgGs conjugated to HOs of increasing valency and stained by silver amplification. (d) Assay background MFI plotted as a function of increasing conjugate valency (columns) and increasing CO density (rows). (e) SDS-PAGE of low-valent dAb-HO (mouse uPA monoclonal antibody) conjugates at different stages of the purification protocol, where (1) native dAb, (2) conjugated product dAb HO (unpurified), (3) recovered (unconjugated) dAb, and (4) purified dAb-HO. (f) MFI measurements from the x-uPACLAMP assay for standard dilutions of uPA antigen and different CO concentrations. Error bars are the standard deviation of the particle signal in the Cy5 channel. (g) MFI signals in the x-uPACLAMP assay with low (blue dots) and high (red dots) valency conjugates. Error bars are the standard deviation of the MFI signal across wells (n=3). The LOD shown on each curve was calculated as described below.

[0157] Fig.17 Elimination of cross-reactivity (CR) in CLAMP is shown. Schematic diagram of CR screening for (a) conventional MSAs and (d) CLAMP assays, where barcoded microparticles are mixed and incubated with one target at a time to display CR in a multiplexed format. SNRs quantify specific (diagonal) and nonspecific (off-diagonal) assay signals for conventional MSAs (b,c) and CLAMP (e,f) in response to the addition of (b,e) 1 ng / mL and (c,f) 100 ng / mL antigen, respectively. (g) Assay MFI of MCP-1 single-plex sandwich assay with MCP-1 (blue) and EGF (red) spike-in at specific concentrations (x-axis). (h) SNR signals for a 5-plex CLAMP dilution series. (Inset) SNRs were calculated using barcode-specific background and global standard deviation.

[0158] Fig.18 The sequences and melting temperatures of the oligonucleotides used in CLAMP according to one embodiment are shown. The capture oligonucleotide (CO, 42nt) is bound to the streptavidin surface via 5' biotin, and the 3' fluorescent labeling oligonucleotide (LO, 21nt) and the 5' antibody conjugated hook oligonucleotide (HO, 81nt) are attached to the particle surface. A displacer oligonucleotide (DO30nt) initially binds to the 9nt bottom clamp on the hook oligonucleotide to displace the HO-CO hybrid.

[0159] Fig.19 A schematic diagram of the synthesis of barcoded CLAMP microparticles according to one embodiment is shown. The schematic diagram depicts the main steps of CLAMP microparticle synthesis. (a) Oligonucleotide pre-annealing, antibody added to form a biotinylated reagent mixture. The mixture of biotinylated oligonucleotides includes a precisely controlled CO / SO ratio (90 pmol in total) and defines the CO (and later dAb-HO) surface density; the biotinylated oligonucleotides are annealed to a precisely controlled ratio of LO 0 / LO 1 / LO 2 (b) Thereafter, streptavidin MP is added to the biotinylated mixture to label them proportionally and randomly on the surface of the microparticles with reagents, where the oligonucleotide components (e.g., LO 1 / LO 2 ) remains constant on the surface. (c) dAb-HO is finally pulled onto the surface to complete the synthesis of CLAMP. The density of dAb on the surface is proportional to the density of CO and therefore to n CO Directly proportional.

[0160] Fig. 20Schematic diagram showing fine-tuning and precise control of surface density. (a) Detection of cAb using AF-647 labeled anti-goat antibody. (b) Detection of HO using Cy5 labeled complementary but non-displacing oligonucleotide. Fluorescence intensity of CLAMP microparticles with different coreacting amounts labeled with AF647 x-goat secondary antibody (red) and cy5 labeled oligonucleotide targeting HO (non-displacing, blue dots). (c) Detection of cAb and HO (in red and blue, respectively) when preparing CLAMP, increasing the starting amount of CO and decreasing SO to make n CO +n SO = 90 pmol. The linear fit of the data is shown by the dashed line, and the error bars represent the standard deviation of MP fluorescence.

[0161] Fig.21Schematic diagrams of several embodiments. (1) shows a CLAMP embodiment in which the capture and detection reagents are antibodies, the detection reagent is connected to the anchor chain by DNA hybridization, and the detection antibody is labeled; (2) shows an embodiment in which the capture and detection antibodies are attached to the anchor chain by oligonucleotide linkers and DNA hybrids, and the detection antibody is labeled. (3) Shows an embodiment in which the capture and detection reagents are antibodies, the detection reagent is connected to the anchor chain by DNA hybrids, and there is no label on the detection reagent or the hook chain. In the presence of analyte, a triple complex is formed, and the hook chain is displaced from the anchor chain by a labeled displacer oligonucleotide. (4) Shows an embodiment in which the capture antibody and the detection antibody are connected to the anchor chain by oligonucleotide linkers and DNA hybrids, and there is no label on the detection reagent or the hook chain. In the presence of analyte, a triple complex is formed, and the hook chain is displaced from the anchor chain by a labeled displacer oligonucleotide. (5) Shows an embodiment of (1), but in which the label is attached to the hook chain and is covered by the DNA hybrid that attaches the hook chain to the anchor chain. A triple complex is formed in the presence of an analyte, and the hook is displaced from the anchor by a displacer oligonucleotide that binds or hybridizes to the anchor. The tag on the hook is activated or de-masked after the displacement reaction. (6) An embodiment of (4) is shown, but as shown in (5), the tag is attached to the hook and is covered by a DNA hybrid that attaches the hook to the anchor. A triple complex is formed in the presence of an analyte, and the hook is displaced from the anchor by a displacer oligonucleotide that binds or hybridizes to the anchor. The tag on the anchor hook is activated or de-masked after the displacement reaction. (7) An embodiment is shown in which both the capture and detection reagents are antigens and are connected to the anchor by an oligonucleotide linker that hybridizes to the anchor. A complex is formed in the presence of an antibody that binds to two antigens (the analyte in this embodiment). The tag is attached to the hook and is covered by a DNA hybrid that attaches the hook to the anchor. The tag on the hook is activated or de-masked after separation from the anchor. (8) An embodiment is shown in which the capture reagent is an antibody directly linked to a support and has two anchor chains. Each of the two anchor chains is linked to a detection antibody via a DNA hybrid. In the presence of analyte, a quadruple complex is formed. Each of the two hook chains is labeled and the hook chain tags are unmasked by releasing the linker to the respective anchor chain. The two tags may be the same or different.

[0162] Fig. 22 Calculation of the displaced detection antibody concentration profile for a CLAMP assay according to one embodiment is shown, wherein the detection antibody concentration is plotted relative to the starting amount (y-axis) and the volume of solution (x-axis) during the displacement step.

[0163] Fig.23An AND (Boolean) logic gate representation of detection by the label displacement step is shown, where detection at the single-molecule level requires capture of the target and successful release of the hook oligonucleotide (HO).

[0164] Fig.24 Calibration curves obtained using two labeling methods are shown: direct detection of dAb using a BV421-labeled secondary antibody, and displacement-dependent detection using a Cy5-labeled displacer oligonucleotide. Calibration curves for IL-7, FN-γ, and MMP-9 were performed in buffer (PBST) by spiking in decreasing concentrations of protein standard solutions and analyzed as indicated.

[0165] Fig.25 CLAMP optimization achieved by adjusting the conjugate valency is shown. (a) SDS-PAGE of mouse anti-goat IgGs conjugated to HOs with increasing valency and stained by silver amplification. (b) Assay background MFI is plotted according to increasing conjugate valency (columns) and increasing CO density (rows). (c) MFI measurements of the x-uPACLAMP assay for standard dilutions of uPA antigen and different CO concentrations. The error bars are the standard deviation of the particle signal in the Cy5 channel. (g) MFI signals in the x-uPACLAMP assay with low (blue dots) and high (red dots) valency conjugates. The error bars are the standard deviation of the MFI signal across the wells (n=3). The LOD shown on each curve was calculated as described below.

[0166] Fig.26 A 40-plex specificity screen of a CLAMP assay for 40 proteins (cytokines and others) is shown. Antigens (recombinant) were spiked one by one into the buffer containing the multiplex CLAMP mix. Each well contained only one antigen. The signal-to-noise ratio of each well for each CLAMP was detected, read out, and plotted, indicating that the antigens had minimal interactions with off-target CLAMPs, as shown by the smallest off-diagonal signal in the heat map. DETAILED DESCRIPTION

[0167] As described herein, systems and methods are provided for detecting and / or quantifying one or more analytes using assays for colocalization by ligation. In particular, systems and methods are provided with sufficiently low background signals, sufficiently low cross-reactivity between reagents, and / or sufficiently high sensitivity to allow simultaneous detection and / or quantification of multiple biomolecules in a sample. Rapid, sensitive, economical, and / or scalable multiplex sandwich assays and methods for preparing the same are also provided.

[0168] It is to be understood that the present invention is not limited to specific apparatus, systems, methods or uses or process steps as such may vary.

[0169] For a clear and consistent understanding of the terms used in this specification, various definitions are provided below. Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which the present invention relates.

[0170] In the claims and / or the specification, the word "a" or "an" when used with the term "comprising" can mean "one", but is also consistent with the meaning of "one or more", "at least one" and "one or more". Similarly, the word "another" can mean at least a second or more.

[0171] As used in this specification and claims, the words "comprise" (and any form of inclusion, such as "including" and "consisting of..."), "have" (and any form of having, such as "having" or "containing"), "include" (and any form of inclusion, such as "including" and "covering") or "contains" (any form of inclusion, such as "containing" and "including") are inclusive or open-ended and do not exclude other unrecited elements or process steps.

[0172] As used herein, the term "about" in the context of a given value or range refers to a value or range that is within 20%, preferably 10%, and more preferably 5% of the given value or range.

[0173] As used herein, the term "and / or" is considered to be a specific disclosure of each of two specific features or components, whether or not there is another. For example, "A and / or B" is considered to be a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if individually listed herein.

[0174] As used herein, the term "carrier" refers to an immobilized structure, surface or matrix, such as but not limited to microparticles, nanoparticles, holes in a plate, porous polymers or hydrogels. It should be understood that carriers are not meant to be particularly limited, and any solid, semisolid, gel or gel-like structure can be used. For example, the carrier can be an array, microbeads (such as but not limited to polystyrene microbeads), the surface of a porous plate (such as a 96-well plate, a 384-well plate, etc.), the surface of a glass slide, a hydrogel matrix, a microfluidic chip, a lateral flow strip, a glass surface, a plastic surface, a silicon surface, a ceramic surface, etc. In one embodiment, the carrier is a microbead or microparticle or nanoparticle, typically micrometer-level or nanometer-level, such as but not limited to polystyrene microbeads, magnetic microbeads, paramagnetic microbeads, plastic microbeads, etc. In another embodiment, the carrier is a planar microarray. In one embodiment, the carrier is a nanoparticle. In one embodiment, the carrier is a microparticle.

[0175] As used herein, the term "analyte" refers to a biological molecule or biological cell of interest that is being identified, detected, measured and / or quantified. The analyte can be any biological molecule or biological cell that can be detected using the systems and methods provided herein, such as, but not limited to, proteins, nucleic acids (DNA, RNA, etc.), antibodies, antigens, proteins, cells, chemicals, biomarkers, enzymes, polypeptides, amino acids, polymers, carbohydrates, multi-protein complexes, exosomes, oligonucleotides, low molecular weight compounds, etc. Non-limiting examples of analytes include antibodies, antibody fragments (e.g., scFv, Fab, etc.), aptamers, modified aptamers, segments, aptamers, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, and low molecular weight compounds.

[0176] As used herein, "sample" refers to any liquid or liquid sample being analyzed for the purpose of detecting and / or quantifying an analyte. In some embodiments, the sample is a biological sample. Examples of samples include, but are not limited to, body fluids, extracts, solutions containing proteins and / or DNA, cell extracts, cell lysates, or tissue lysates. Non-limiting examples of body fluids include urine, saliva, blood, serum, plasma, cerebrospinal fluid, tears, semen, sweat, pleural effusions, liquefied feces, and lacrimal secretions.

[0177] As used herein, the term "encoded microparticles" refers to barcoded microparticles, for example, spectrally encoded according to the target analyte or the specific test to be performed in the assay. Barcoded (or encoded) microparticles are often used in multiple suspension assays because they can distinguish particles in a larger mixture. There is no particular limitation on the method of barcoding. For example, barcoding can be performed using spectral, graphical, or chemical methods. For example, spectral encoding of microparticles can be performed by labeling microparticles with precise ratios of multi-color dyes. This method allows for simple and high-throughput readout by flow cytometry. As another example, graphically coded microparticles are often engraved or otherwise engraved with visual patterns that can be characterized by a microscope. Microparticles can also be chemically barcoded, for example, using unique DNA sequences that can be detected later by DNA detection means.

[0178] As used herein, the term "non-specific binding" refers to unintended reactions between reagents and / or molecules within a sample, including but not limited to reactions between non-cognate antibodies and proteins attached by hydrophobic interactions.

[0179] As used herein, the terms "affinity binder" (AB), "binding substance" and "reactant" are used interchangeably to mean any molecule that can specifically recognize a target analyte, for example, through non-covalent interactions. Examples of affinity binders (ABs) include, but are not limited to, immunoglobulin G (IgG) antibodies (e.g., whole molecules or Fab fragments), aptamers, affimers, nanobodies, ankyrins, and single-chain variable fragments (scFv).

[0180] As used herein, the term "sandwich assay" refers to an analyte-targeted assay in which two ABs simultaneously bind to an analyte of interest and can be detected and / or quantified.

[0181] As used herein, the terms "multiplex sandwich assay," "multiplex sandwich analysis," and "MSA" are used interchangeably to refer to a sandwich assay targeting multiple (e.g., two or more) analytes from the same sample and / or assay volume simultaneously, using multiple AB pairs in the assay system.

[0182] As used herein, the term "cross-reaction" refers to instances of nonspecific binding or nonspecific reaction in multiplex assays where unintended complexes are formed involving non-cognate affinity binders, such as Figure 1 shown.

[0183] As used herein, the terms "capture affinity binder", "cAB", "capture AB", "capture binder", and "capture reagent" are used interchangeably and refer to an AB that is attached to a carrier in a biomolecule complex and is not released therefrom. The capture AB can be attached to the carrier (e.g., by a covalent bond, a biotin-streptavidin bond, a DNA oligonucleotide linker, or a polymer linker) directly or indirectly (e.g., by connection to an anchor chain, such as by conjugation or through a linker such as a capture chain). Non-limiting examples of capture reagents include antibodies, antibody fragments (e.g., scFv, Fab, etc.), aptamers, modified aptamers (e.g., slow dissociation rate modified aptamers or somamers), affibodies, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, and low molecular weight compounds.

[0184] The term "capture strand" refers to a linker (eg, oligonucleotide, polymer, etc.) that connects the capture agent to the anchor strand (and thereby the carrier to which the anchor strand is attached).

[0185] As used herein, the terms "detection affinity binder", "dAB", "detection antibody", "detection binder" and "detection reagent" are used interchangeably to refer to an AB in a biomolecular complex that is releasably attached to a carrier. dABs are generally used for signal transduction and measuring signal transduction. In some embodiments of the methods and systems provided herein, for example, the portion of the dAB that is not bound to the analyte is released from the carrier so that no signal is generated in the absence of bound analyte. In some embodiments, the dAB is bound to a label or device for signal transduction and measuring signal transduction. Non-limiting examples of detection reagents include antibodies, antibody fragments (e.g., scFv, Fab, etc.), aptamers, modified aptamers, aptamers, affibodies, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, and low molecular weight compounds.

[0186] As used herein, the term "anchor" refers to a linker attached to a fixed point on a carrier. Non-limiting examples of anchors include polymers such as polyethylene glycol (PEG), oligonucleotides (such as single-stranded DNA oligonucleotides, single-stranded RNA oligonucleotides, or double-stranded DNA or RNA oligonucleotides or DNA-RNA hybrids), and oligosaccharides.

[0187] As used herein, the term "hook" refers to a connector that connects the detection AB to the anchor and thus attaches it to the carrier. The hook is usually releasably attached to the anchor, for example, in a manner that the attachment can be released. Typically, when the attachment between the hook and the anchor is released, the hook-connected portion of the detection AB that is not bound to the target analyte will be released from the anchor, so that no signal of the AB is detected on the carrier in the absence of the target analyte. In this way, the signal on the carrier is only detected when the target analyte is present and the detection AB and the capture AB are bound.

[0188] In some embodiments, if the tag is on the hook and / or detection agent and is activated or detectable only after the hook and / or detection agent is released from the anchor, the signal is "release-dependent" because it is only detectable after the hook and / or detection agent is released from the anchor. Similarly, in some embodiments, if the tag is on a displacer that hybridizes to the hook, the signal is "displacement-dependent."

[0189] As used herein, the term "displacer" refers to an agent that directly or indirectly causes or initiates the release of a releasable connection between an anchor chain and a hook chain, thereby releasing the hook chain (and the detection AB connected thereto) from the carrier. The mechanism used by the displacer is not particularly limited. For example, the displacer may directly or indirectly cause or initiate the cutting, displacement or debinding of the connection between the anchor chain and the hook chain; other mechanisms are possible and may be expected. In some embodiments, a DNA oligonucleotide hybridized to the hook chain and / or the anchor chain is used to displace the hook chain from the anchor chain. Examples of displacers include, but are not limited to, displacing DNA oligonucleotides, monochromatic or polychromatic light sources, restriction enzymes, and reducing agents such as dithiothreitol (DTT). In some embodiments, if a photocleavable DNA fragment is used, the displacer may be light that affects the release through a photofragmentation reaction. In some embodiments, the displacer is labeled, for example, with a dye, a fluorophore, a specific DNA sequence, an enzyme, a biotin moiety, etc. When the displacer is labeled, it can simultaneously serve the dual functions of releasing the hook chain and labeling it.

[0190] As used herein, the term "tag" refers to any molecule or portion of a molecule that generates a signal, can be targeted with a signal generating molecule, or can be detected in other ways. Examples of tags include, but are not limited to, biotin, fluorophores, enzymes, enzyme substrates, and specific DNA sequences. An "inactive" or "undetectable" tag refers to a tag that is inactive, masked, or otherwise undetectable, e.g., incapable of generating a detectable signal, such as, but not limited to, a quenched fluorescent dye.

[0191] It should be understood that the systems and methods provided herein can be used for almost any type of sandwich assay in which two sets of ABs are used. However, for simplicity, whole molecule immunoglobulin G antibodies (IgG) are used as ABs to present specific embodiments of the present invention, which represent one of many possible embodiments. It should be understood that antibodies are not limited to whole molecule IgG, and many different antibodies, antibody fragments, etc. can be used. Further, AB is not limited to antibodies. Similarly, many different types of sandwich assays can be used in addition to the specific types described herein.

[0192] In some embodiments, a dual AB or sandwich assay is provided that avoids cross-reactivity by co-localizing two ABs (capture AB and detection AB) on a support prior to exposure to a biological sample containing an analyte of interest. Co-localization on the support does not allow mixing of different AB pairs prior to exposure to the analyte, thereby reducing or eliminating cross-reactivity between reagents and / or background (e.g., Figure 1 or Figure 4In one embodiment, a carrier attached to a mixture of capture and detection ABs is provided, wherein each set of capture and detection ABs is capable of binding to an analyte of interest, and the detection ABs are optionally attached to the carrier via a releasable linker. In one embodiment, a carrier attached to a mixture of capture and detection ABs is provided, wherein each analyte is capable of binding to both the capture AB and the detection AB at the same time, and wherein the detection ABs are optionally releasably attached to the carrier via a releasable hook chain. After releasing the detection reagent and / or hook chain, the corresponding detection antibody will only remain on the carrier when the capture AB is used to bind to the analyte in the triple complex.

[0193] It should be understood that the "linkers" and "chains" used in the methods and systems provided herein are not particularly limited. Non-limiting examples of linkers and chains include DNA oligonucleotides (also referred to as DNA oligonucleotides), polymers, polysaccharides, and the like. DNA connections can be covalent, such as conjugation between hook chain oligonucleotides and detection antibodies, or non-covalent, such as hybridization or base stacking between two complementary DNA sequences. In order to form a capture AB-antigen-detection AB triple complex, the hook chain is designed to have a flexible single-stranded portion. Several methods including but not limited to bottom clamping-mediated DNA displacement reactions, enzyme cleavage, and photosensitive cleavage can be used to displace DNA chains. Specific DNA sequences can also be used as tags, either by direct targeting using fluorescently labeled complementary sequences, or by hybridization chain reaction or polymerase chain reaction as amplification triggers or primers, or by sequencing readout.

[0194] It should be understood that the "oligonucleotides" (also referred to as "oligos") used in the methods and systems provided herein are not particularly limited. For example, oligonucleotides can be modified with fluorescent dyes at the 5' or 3' end, modified with photocleavable phosphodiester backbones, conjugated with proteins, biotin or enzymes, etc.

[0195] These embodiments may also be referred to herein as "co-localization assay by ligation" or "CLA". In some embodiments of CLA, the detection AB is labeled (i.e., attached to a tag). In some embodiments of CLA, the hook that connects the detection AB to the anchor is labeled (i.e., attached to a tag). Typically, the tag attached to the detection AB or the hook is inactive or undetectable so that the tag can be detected after the detection AB is released from the carrier (i.e., the hook is released from the anchor). Therefore, the signal detection from the tag is release-dependent (also referred to as "displacement-dependent" in some embodiments). In this way, only the detection AB bound to the analyte in the triple complex with the capture AB and released from the anchor AB is detected, because the unbound detection AB will be released from the carrier (and can be removed by washing, etc.). The background signal can also be reduced because the tag is inactive or undetectable before release, or if a given hook is not released (i.e., due to the release dependence or displacement dependence of the signal). Thus, in some embodiments, the methods and systems provided herein may be referred to as "release-dependent transduction" (or "RDT") or "displacement-dependent detection," to reflect release-dependent (or displacement-dependent) signaling.

[0196] Conventional sandwich assays generally rely on the presence of detection AB to transduce signals and detect the presence of analytes. Similarly, in some embodiments of the systems and methods described herein, detection AB and / or hook chains can be used as signal sensors. However, in contrast to conventional assays, in the systems and methods provided herein, only when the triple complex is formed with analyte and capture AB, the detection AB and / or hook chains optionally connected thereto are still on the carrier. It should be understood that if the detection reagent and / or hook chain cannot be successfully or completely released from the anchor chain, it is still on the carrier even in the absence of analyte. In this case, if the detection AB and / or hook chain are attached to a label that is active or detectable when attached to the anchor chain, any non-released, labeled detection AB and / or hook chain will transduce signals. In other words, in that case, any labeled and non-released detection reagent and / or hook chain will result in a signal independent of the presence of the analyte, which contributes to non-specific background signals and reduces assay performance and / or sensitivity. It should be understood that in that case, the background signal will be proportional to the portion of the non-released detection reagent and / or hook chain. It is also understood that it may be difficult to completely release the complex from the support due to steric hindrance, attachment, and / or incomplete washing. However, release-dependent transduction (RDT) can minimize or eliminate these problems because if the detection reagent and / or hook chain is not completely released from the anchor chain, signal transduction will not occur, such as Fig.24 shown.

[0197] In some embodiments, therefore, the systems and methods provided herein include additional redundancy levels to reduce background signals and / or increase sensitivity by using release-dependent transduction (RDT). In RDT, signal transduction occurs only when two of the following conditions are met: (i) formation of a triple capture AB-analyte-detection AB complex, and (ii) release of the corresponding detection AB and / or hook chain from the anchor chain. In this case, the non-released detection AB and / or hook chain will not contribute to the background signal. This signal transduction mechanism, which we refer to as "release-dependent transduction (RDT)" herein, can be achieved by a variety of means. For example, some embodiments may include a label on the hook chain, wherein the label is inactive or undetectable until it is released from the anchor chain, so that the non-released (e.g., undisplaced) hook chain and / or detection AB) is not conducive to or does not transduce the signal.

[0198] In some embodiments of RDT, the hook strand is labeled with a fluorescent dye that is quenched by the anchor strand or another proximal strand, such that release results in unquenching or activation of the fluorescent dye.

[0199] In some embodiments of RDT, the detection reagent and hook chain are not labeled, and instead, the displacer is labeled. In this case, the displacer hybridizes with the hook chain, thereby displacing it from the anchor chain and simultaneously labeling it. If the detection AB is not bound to the analyte and capture AB in the triple complex, the hook chain, displacer and label are washed off the carrier. Since the label is attached to the displacer, the label is only present on the carrier when the following two conditions are met: (i) it has been released or displaced from the anchor chain; and (ii) the analyte has bound to the capture and detection AB (e.g. Figure 7 ).

[0200] It should be understood that other implementations of the RDT are possible, and the mechanism of the RDT is not meant to be particularly limited.

[0201] In some embodiments of RDT, the detection AB or anchor is attached to a tag. In some embodiments, the hook linking the detection AB to the anchor is labeled (i.e., attached to a tag). Generally, the tag, anchor, or hook attached to the detection AB is inactive or undetectable, so that the tag can only be detected after the detection AB is released from the carrier (i.e., the hook is released from the anchor, such as Figure 6Only examples are shown). In this way, the only detection AB-hook oligonucleotide complex detected is the complex with the detection AB bound to the analyte in the triple complex, in which the capture AB and the hook chain are successfully released from the anchor chain. Otherwise, whether or not the analyte is bound, the unbound detection AB will be released from the carrier (and can be removed, for example, by washing), and all non-released chains are not detected. In this way, the background signal from the non-released detection AB and / or hook chain is alleviated, ensuring low background signal and / or high sensitivity detection.

[0202] In other embodiments of the RDT, the hook contains a label that remains inactive or undetectable until the hook is released from the anchor. For example, this can be achieved when the hook and anchor are bound together by hybridization to a DNA oligonucleotide, wherein the hook contains a DNA sequence tag that normally hybridizes with the anchor and thus binds or is undetectable. Releasing the hook oligonucleotide from the anchor oligonucleotide reveals a detectable label on the hook. This release can be achieved, for example, by enzymatic cleavage, DNA displacement, or photofragmentation using light.

[0203] In some such embodiments, a release or displacer is provided, which is an oligonucleotide that displaces the anchor-hook hybrid by binding to the anchor oligonucleotide via a bottom clamp displacement reaction. In one embodiment, neither the hook nor the detection AB is labeled, and the labeled displacer (e.g., a fluorescently labeled oligonucleotide) performs RDT by the dual functions of release (displacement) and labeling. In this way, by only labeling the displaced hook, a detectable signal / signal transduction occurs on the carrier only when two conditions are met (displacement of the hook and the presence of an analyte), similar to an "AND" logic gate (e.g., as Figure 7 B).

[0204] In some embodiments of the assays and systems provided herein, one or more groups of capture and detection ABs are attached to a carrier, each group being specific to the analyte of interest. In this way, the capture and detection ABs are preassembled and co-located on the carrier before being exposed to a biological sample containing an analyte. As described above, the detection AB is releasably attached to the carrier. In some embodiments, the detection AB is attached to the carrier via a releasable joint (hook chain) connected to an anchor chain attached to the carrier. The hook chain is usually flexible and allows the detection AB to be freely dispersed within the limits allowed by the length of the hook chain and / or anchor chain. The hook chain and the releasable chain are not particularly limited and their size, flexibility, structure, etc. can be varied as long as they allow the detection AB and the capture AB to bind to the analyte at the same time. The capture AB and the detection AB usually bind to different regions of the analyte, although they can bind to overlapping sites as long as they can bind to the analyte at the same time.

[0205] In some embodiments, a hook chain is used to link the detection AB to the carrier, and the hook chain is a DNA oligonucleotide that can specifically bind to the anchor chain attached to the carrier. After contacting and incubating with a biological sample (i.e., the target recognition step), the detection AB is separated from the anchor chain by breaking the connection between the hook chain and the anchor chain on the surface. This can release the portion of the detection AB that has not formed a triple-capture AB-analyte-detection AB complex. It should be understood that the connection between the hook chain and the anchor chain can be released or broken in various ways, such as, but not limited to, DNA strand displacement, enzymatic cleavage, photoexcitation cleavage, etc.

[0206] As included herein, multiple ABs targeting many different analytes can be mixed in the same assay volume (i.e., multiplexed); the interaction between different ABs on different carriers (different positions / different ABs at positions on the same carrier) is restricted by the connection to the carrier to avoid the interaction between ABs from different carriers / positions. This is contrary to traditional multiplexing techniques, where the interaction between ABs cannot be restricted when all ABs are mixed in solution. Further, the methods and systems described herein can be used to prepare different populations of microparticles in large quantities, with each population of microparticles containing different AB capture-detection pairs required for detecting a specific antigen.

[0207] In some embodiments, the multiplexed CLA method and system can thus avoid Figure 1 the cross-reaction scenarios shown. For example, as will be appreciated by those skilled in the art, the co-localization of homologous capture and detection ABs on their corresponding carriers (e.g., microparticles) will eliminate unwanted interactions, such as the binding between non-homologous detection and capture ABs. In addition to Figure 1 the scenarios shown, those skilled in the art will recognize that, contrary to traditional multiplexed sandwich assays, analytes bound or attached to off-target carriers cannot be detected by their homologous detection ABs in the methods and systems provided herein, and thus do not contribute to an increase in background signal.

[0208] In some embodiments, on each carrier, the local concentration of the capture and detection ABs can be relatively high, which can concentrate the analyte and increase the sensitivity. On the other hand, the total concentration of each capture and detection AB in the entire assay volume only depends on the concentration of the target-specific carriers (e.g., microparticles, microarray spots) and can be designed to obtain a relatively low total concentration of the detection ABs after release. For example, although the local concentration can be in the micromolar range, the use of a relatively small number of target-specific microparticles can result in an undetectably low (<pM) total concentration of the detection ABs to obtain any off-target binding, such as Fig. 22As shown. The total concentration can be further reduced by increasing the volume during the release step. Therefore, in certain embodiments, due to the lower concentration or amount of the detection AB used on the carrier, the methods and systems provided herein can further avoid cross-reactivity that occurs after the release of the detection AB.

[0209] In some embodiments, simultaneous binding of two co-localized binders (capture AB, detection AB) to two different epitopes of the same analyte (i.e., increased binding affinity) can result in a much lower effective off-rate (k) than traditional sandwich assays. off ), in the traditional sandwich assay, the capture and detection ABs are added sequentially. After the sample is introduced and incubated, the carrier in the methods and systems provided herein is rigorously cleaned because the analyte can be bound with high affinity. Therefore, in some embodiments of the methods and systems provided herein, rigorous cleaning can be used to reduce the assay background and / or improve sensitivity and / or specificity. In some embodiments, it may be necessary to quickly perform the assay step after the hook chain is released from the anchor chain until the assay signal is read, because the separation of the analyte can result in a reduction in the signal, which can contribute to a reduction in sensitivity, although this effect is generally reduced in CLA.

[0210] In one embodiment of the methods and systems provided herein, the carrier is encoded with microparticles, and both the capture reagent and the detection reagent are antibodies, wherein the capture reagent and its homologous detection reagent are co-located on the surface of the same carrier with a DNA chain (in other words, the hook chain and the anchor chain are single-stranded DNA oligonucleotides, connected together by double-stranded DNA hybrids). In some such embodiments, the detection reagent connected to the hook chain and the anchor chain is uniformly mixed and attached to the surface of the microparticle, wherein the anchor chain is connected to the hook chain by partial hybridization, and the hook chain is used for detection reagent conjugation, and the hook chain is flexible and releasable DNA joint. The hybrid between the anchor chain and the hook chain is generally stable during the conditions of sample incubation. In some embodiments, the capture reagent is also connected to the microparticle by a DNA joint. In some embodiments, the hook chain can be released from the anchor chain by a DNA displacement reaction mediated by the bottom clamp. In some embodiments, the displacer is an oligonucleotide, which is designed to bind to the bottom clamping sequence on the hook chain to drive the displacement reaction forward. In some such embodiments, the displacer is not used, for example, to release the hook chain from the anchor chain by heating, so that the DNA hybrid "melts" or debinds.

[0211] In one embodiment, the detection AB and / or hook chain are labeled, for example, with a dye, a biotin moiety that is detected using fluorescently labeled streptavidin in a subsequent step. In certain embodiments, the detection AB can be detected after the analyte is combined with a labeled conjugate, for example, a labeled species-specific second IgG can be used to target the IgG. In some embodiments, the detection AB and the hook chain are not labeled, instead, a displacement agent for releasing the hook chain from the anchor chain is labeled. In such embodiments, after the hook chain is released from the anchor chain, the labeled displacement agent is attached to the hook chain and / or the detection AB.

[0212] In some embodiments, the tag is a specific DNA sequence that can be detected or targeted in a subsequent step. For example, a specific DNA sequence can be targeted with a dye through a subsequent DNA hybridization step that marks it. In one embodiment, the specific DNA sequence is detected and amplified by polymerase chain reaction (PCR) or other enzymatic DNA amplification means. The specific DNA sequence can also be cut and detected by other means such as sequencing. The embodiment of using a DNA sequence as a tag is not limited and can include being part of a hook chain (thus initially inactive / undetectable) or present on a displacement agent (such as Figure 6 A. Figure 6 D).

[0213] In some embodiments, a detection AB is provided, which is attached to a hook chain and is indirectly attached to the microparticle through a releasable joint attached to the anchor chain. The hook chain is partially complementary to the anchor chain attached to the microparticle. The anchor chain can be attached to the microparticle by, for example, streptavidin / biotin interaction or chemical bonds. The detection AB is therefore attached to the microparticle. In this embodiment, a capture AB is further provided, which is attached to the surface of the microparticle, and wherein the detection AB recognizes the same antigen as the capture AB, and the two ABs can bind to the antigen at the same time. In addition, a displacement oligonucleotide (displacer) is provided, which has a sequence complementary to the hook chain and overlapping with the sequence of the anchor chain, so as to release the detection AB from the anchor chain and thus release it from the microparticle if the antigen is bound (i.e., if there is no triple complex between the capture AB-antigen-detection AB). In yet another embodiment, a fluorescently labeled second antibody is also provided, which binds to the detection AB remaining on the microparticle after the displacement reaction.

[0214] It should be noted that in embodiments where the capture and capture ABs are preassembled on a support and the detection AB is labeled with a detectable tag, any non-released hook chain-detection AB complex will result in the generation of an analyte-dependent signal that will contribute to background noise (e.g. Fig.24). Therefore, it will be appreciated that in order to avoid increasing the background signal, a nearly complete anchor-hook chain displacement reaction and washing of the hook chain-detection AB complex are required. It will be appreciated that such nearly complete release is not required even under optimized conditions ( Fig.15 ) is also difficult. In order to reduce such increased background signals caused by inefficient release of the anchor-hook link inversion, in some embodiments, the hook, anchor or detection AB is labeled with an inactive / undetectable label until the hook is displaced or released from the anchor. In another embodiment, the hook, anchor or detection AB is not labeled, and the displacement agent is labeled with a detectable label. In such embodiments, signal transduction only occurs at the carrier if two of the following conditions are met: (i) formation of a triple capture AB-analyte-detection AB complex, and (ii) displacement of the hook-anchor hybrid. It should be understood that, similarly, embodiments in which the label on the detection AB and / or hook is inactive or undetectable before release are beneficial, because non-released (e.g., undisplaced) hooks (or detection ABs) will be detrimental to signal generation.

[0215] In one embodiment, the labeled displacer (e.g., oligonucleotide) can perform both release (displacement) and labeling functions. In this way, by labeling only the displaced hook strand, detectable signal / signal transduction requires two conditions, similar to an "AND" logic gate ( Fig.23 One of the potential advantages of such embodiments is that they do not require changes to the design of the DNA sequence or the ligation properties of the detection complex including the hook and anchor strands.

[0216] In some embodiments, additional levels of redundancy can be achieved by using hook strands with inactive or undetectable tags that are activated or detectable only after displacement from the anchor strand. For example, in one embodiment, the hook strand is labeled with a dye that is quenched by a dye quencher that can be conjugated to the anchor strand. In another embodiment, similarly, displacement is achieved by using a restriction enzyme, followed by signal generation with a labeled oligonucleotide that targets the portion of the hook strand that was previously hybridized (and therefore not bound), thereby hybridizing only with the already displaced hook strand and labeling it.

[0217] In certain embodiments, a detection AB is provided which is connected to a microparticle via a hook chain, wherein the hook chain is an oligonucleotide which is connected to the detection AB. The hook chain oligonucleotide is partially complementary to the anchor chain, which is also connected to the microparticle by, for example, streptavidin / biotin interaction or chemical bonds, thereby attaching the detection AB to the microparticle. A capture AB is also provided, to which the remaining microparticle surfaces are connected, and wherein the detection AB recognizes the same antigen as the capture AB, and the two ABs can bind to the antigen simultaneously. In addition, a displacer can be provided which is an oligonucleotide containing a fluorescent tag or a DNA barcode sequence and has a sequence that is complementary to the hook chain oligonucleotide and overlaps with the sequence of the anchor chain oligonucleotide, so that the detection AB is released from the anchor chain and can therefore be released from the microparticle.

[0218] It should be understood that in the methods and systems provided herein, the use of co-localization and connection may require reasonable topological design to optimize the availability of two ABs (capture AB and detection AB) on the carrier. In some embodiments, when randomly distributed capture ABs and / or detection ABs are attached to the carrier, the proper binding of the analyte may require the optimization of two important design parameters: (i) the relative density of the capture and detection ABs, and (ii) the length of the hook chain. These two parameters control the time-averaged distance between the capture and detection ABs by taking into account the radius of gyration of the detection antibody. In some cases, the distance between the capture and detection ABs and the effective affinity at the final single molecule level may be random and difficult to control. Therefore, in some embodiments, it may be necessary to optimize the above two parameters to obtain optimal assay performance.

[0219] In another embodiment, both the capture AB and the detection AB are attached to anchors, allowing for simultaneous control of capture and detection AB density while maintaining nanoscale co-localization, potentially allowing for more precise control of assay performance (e.g. Figure 5 As shown in FG, Fig.21 Some embodiments in the present invention). In such embodiments, the capture and detection AB are co-localized, and their relative densities are the same and can be adjusted simultaneously. A potential advantage of this embodiment is that the average distance between the capture and detection AB for all line pairs on the carrier is uniform. A second potential advantage of this embodiment is that the structure of the capture and detection AB can be precisely controlled. For example, the stringency of binding can be controlled by reducing the length of the single-stranded portion of the anchor chain or hook chain, thereby providing a certain method to control the thermodynamics of the measurement system. Those skilled in the art will appreciate that increasing the stringency of binding can lead to a decrease in effective affinity. In some embodiments, this regulation of effective affinity can be used to control and expand the dynamic range of the measurement, among other applications.

[0220] In some such embodiments where the effective affinity can be tuned by varying the length of the hook or anchor (i.e., linker length) or by adjusting the surface density of the capture and detection ABs, multiplexed arrays (e.g., multiplexed microparticles) with different effective affinities can be made. This helps to expand the dynamic range of the assay for a particular analyte. For example, one skilled in the art will recognize that some proteins exist in blood at concentrations greater than 5 orders of magnitude; for such targets, several assays can be designed using different barcodes to be able to quantify such proteins over a larger dynamic range.

[0221] In one embodiment, the capture AB is conjugated to a capture oligonucleotide that hybridizes to one sequence domain of the anchor chain that is attached to the carrier. Another sequence domain of the anchor chain can hybridize to a hook chain that is attached to the detection AB. All of the above strategies for signal transduction and generation can also be utilized in this embodiment.

[0222] In one embodiment, two or more sets of distinguishable (i.e., multiplexed) complexes that detect the same target can be designed to increase the dynamic range of the multiplex assay, wherein the length of the hook link oligonucleotides of the two or more sets can be controlled to control the stringency of binding. For example, two or more sets of microparticles with different barcodes but targeting the same analyte can be prepared, wherein the first set of microparticles includes shorter hook link oligonucleotides to reduce flexibility and increase the stringency of binding, and wherein the second set of microparticles includes longer hook link oligonucleotides to increase flexibility and reduce the stringency of binding, etc. In this way, the first set of microparticles can be designed to quantify the analyte when the analyte is present at a higher concentration.

[0223] Those skilled in the art will recognize that another challenge of multiplexed assays is interference and matrix effects, which are difficult to control at the analyte level. In some embodiments, one of the advantages of the methods and systems provided herein is the ability to contact the same biological sample with multiple assay configurations within the same assay volume. This flexibility can provide the ability to individually control matrix effects on specific ABs and assay reagents. For example, certain samples may contain endogenous antibodies and other molecules that may positively or negatively affect the detection signal intensity of a specific analyte.

[0224] In another embodiment, different carriers or biomolecule complexes are provided, each analyte-specific carrier lacking one of the capture or detection ABs and serving as an analyte-specific internal standard that controls matrix effects and other potential failure modes of the assay. The assay signal of the fully formed biomolecule complex on the carrier can be compared to these individual AB controls. These internal controls can be used as markers for potential false positives.

[0225] Those skilled in the art will recognize that another challenge of the assay, particularly when using binders with non-zero or fast dissociation rates (k-off), is the dissociation of the analyte, thereby reducing the assay signal, which may occur in the time between washing of the biological sample and the assay signal readout. This debinding is particularly problematic for low concentration analytes, and the readout method cannot measure different assays (e.g., blood cell counts) in multiple assays. This problem may also exist in the CLA sensor program, so after release (e.g., after displacement), separation of the analyte from the capture AB or detection AB may result in loss of signal. Therefore, in yet another embodiment, the CLA methods and systems provided herein can be modified to alleviate this separation and time-dependent signal problem by entering a stable oligonucleotide hybrid from a reversible reaction (e.g., AB analyte) to stop further separation, and connect to a carrier that can be stored and read out later (e.g., Fig.13 As shown). A potential advantage of this embodiment is to minimize signal loss after the determination is completed, which helps to improve sensitivity. Another potential advantage of this embodiment is to normalize the signal drop between different determinations and samples read out within a non-negligible amount of time, thereby achieving better signal reproducibility and improved accuracy. In some such embodiments, the determination can be performed similarly to the previous embodiments, wherein the determination tag is a unique DNA sequence, wherein after cleaning the released detection AB, a replacement agent can be introduced to reconnect the hook chain to the anchor chain, thereby preserving the signal on the carrier. As appreciated by those skilled in the art, another potential advantage of this embodiment is the reproducibility of signal intensity, especially the elimination of any dependence of the determination signal on time-measurement and temperature.

[0226] In some such embodiments, a displacer is provided, which is an oligonucleotide that binds to the anchor chain oligonucleotide through a bottom clamp displacement reaction, followed by washing the released and unbound hook chain oligonucleotide-detection AB complex, and then adding a replacement oligonucleotide that can rebind the hook chain oligonucleotide to the anchor chain oligonucleotide by hybridizing with the two oligonucleotides.

[0227] Several applications will benefit significantly from the methods and systems provided herein, which are used in some embodiments to solve several sources of background noise and false positives in multiple determination sandwich assays. Specifically, in some embodiments, the multiplexing of protein analysis will be significantly achieved by the methods and systems provided herein. For example, due to the cross-reactivity of reagents, in traditional multiplexing, the analysis of factors such as cytokines and other soluble factors is limited. In some embodiments, the methods and systems provided herein can significantly improve multiple serological analysis. For example, multiple autoantibody assays for detecting many specific autoantibodies have been severely hindered by specificity. Typically, specific antibodies of people are captured by detecting autoantibodies or autoantibodies (e.g., antibodies). Therefore, the non-specific binding of any autoantibodies present in serum will be detected, and usually false positives will be caused, making this type of detection a single binder assay (in other words, limited to a single form). In contrast, the methods and systems provided herein can be used to perform double binder assays; that is, the analyte (here is an autoantibody) is recognized and detected by two specific ABs (here is a specific antigen). In such embodiments, the recombinant antigen or native antigen can be divided into two parts, representing the capture antibody and the detection antibody, which are conjugated to the capture chain and the hook chain, respectively, wherein the capture chain and the hook chain are both connected to the same anchor chain, wherein the anchor chain is connected to a carrier (e.g. Fig. 9 As previously described, the flexibility of the hook strand allows the analyte (here, an antibody) to bind to the capture antibody and the detection antibody (here, the same protein attached to different strands with different functions). After washing the unbound sample, signal transduction can be performed by displacement of the labeled strand, as described herein.

[0228] In some embodiments, the methods and systems provided herein can address the major challenges in multiplexing protein-protein interactions using ABs. To this end, AB pairs can be preassembled, each AB pair targeting a protein of interest, allowing CLA to detect interactions between the pairs, such as Fig.10 As shown. Since this multiplexed assay is completely isolated from another assay, cross-reactivity is significantly reduced, allowing for combined measurement of interactions between different protein-protein pairs. The modular approach of the manufacturing methods of the embodiments presented herein makes the implementation and manufacture of AB pairs for different proteins relatively simple. For example, large-scale preparation of CLA on microparticles allows for the overall functionalization of microparticles with capture ABs, followed by fractionation and addition of different detection ABs in each fractionated fraction.

[0229] In some embodiments, the methods and systems provided herein can address another major challenge in multiplexing post-translational modifications (PTMs) using ABs. For example, accurate protein phosphorylation assays can be used to reveal protein expression levels where cellular signaling events are not obvious. Current methods and workflows for quantifying the PTM portion of a particular protein are severely limited in multiplexing because PTM-specific ABs are not sufficiently specific for the protein itself (i.e., phosphor-specific ABs are highly susceptible to issues with reagent-driven cross-reactivity). Therefore, traditional PTM panels cannot be multiplexed. The multiplexed CLA assay methods and systems provided herein can address this problem by confining anti-PTM binders to analyte-specific supports (e.g., Fig.11 shown).

[0230] In some embodiments, the hook linker is a flexible and releasable linker and is an oligonucleotide that allows formation of a capture AB-analyte-detection AB triple complex such that a signal is generated only in response to recognition of the sandwich capture AB-analyte-detection AB after one of the unbound hook link oligonucleotides is released from the support.

[0231] In some embodiments, a detection AB is provided, which is an antibody attached to a carrier such as a microparticle by a hook chain, and the hook chain is an oligonucleotide connected to the detection AB. The hook chain oligonucleotide is partially complementary to the anchor chain oligonucleotide attached to the carrier (e.g., microparticle) by a streptavidin / biotin interaction, such as or a chemical bond, so that the detection AB is attached to the carrier. A capture AB is also provided, which is an antibody attached to the carrier, and wherein the detection AB recognizes the same antigen but does not recognize the same epitope as the capture AB. In some embodiments, a displacement agent is provided, which is an oligonucleotide containing a fluorescent tag or a DNA barcode sequence and having a sequence complementary to the hook chain oligonucleotide and overlapping with the sequence of the anchor chain oligonucleotide, so that the detection AB is released from the anchor chain oligonucleotide, and therefore can be released from the carrier in the absence of a target analyte. It should be understood that once the capture AB and the detection AB are bound to the analyte, a triple capture AB-analyte-detection AB is formed on the carrier (e.g., on the microparticle). After the formation of the triple complex, the unbound detection AB is removed from the carrier by washing, and the triple complex remains on the carrier. The presence of the ternary complex on the support is then detected and / or quantified.

[0232] In some embodiments, the methods and systems provided herein may be referred to as "co-localization assays on microparticles by ligation" or "CLAMP". The CLAMP methods and systems described herein may be highly usable and beneficial to users. For example, by providing microparticles with pre-assembled AB pairs (capture and detection AB pairs), users can quickly mix and match groups at will, quickly perform multiplexed assays, and read the assay results using, for example, any multi-color flow cytometer. The CLAMP assays provided herein are therefore suitable for existing biological experimental workflows, and in some embodiments can be read out using any multi-color flow cytometer.

[0233] It will be appreciated that the CLAMP embodiment is particularly suitable for large-scale, industrial-scale manufacturing of multiplexed panels that avoid cross-reactivity. Unlike planar arrays, CLAMPs can be manufactured individually in large batches and optionally stored and then mixed prior to assay. This manufacturing method allows CLAMPs to be manufactured independently without the need for interactions between non-homologous ABs, and therefore without cross-reactivity during the manufacturing steps, which is a key advantage over other CLA embodiments.

[0234] In some embodiments, to make multiple CLAMPs, AB pairs are attached to multiple groups of microparticles, where each target-specific AB pair is attached to its corresponding group of microparticles in different containers. The microparticles AB are barcoded before attachment, or are also barcoded during the process. The reaction can be performed in large quantities and the manufactured CLAMPs are stored. For assay, portions of the microbeads for each barcode / target are mixed together before contact with the biological sample. The microparticles are barcoded by any means, such as spectrally, graphically, or chemically.

[0235] In some embodiments, if the carrier is a microparticle (MP), certain advantages can be obtained. For example, in some embodiments, the ability to quickly read out a large number of MPs by a flow cytometer can increase accuracy and sample throughput. In addition, MP can be functionalized in large displacement, and then stored, used and read out in solution, which can reduce the difference between batches and realize quantitative analysis (Tighe, PJ et al., Proteomics-Clinical Applications 9,406 quantitative analysis (. Protein expression; Jani, IV et al., The Lancet 2,243al Applica; Krishhan, VV, Khan, IH & Luciw, Pa Multiplexed microbead immunoassays by flow cytometry for molecular profiling: Basic concepts; Tighe, P. et al., Utility, reliability and reproducibility of immunoassay multiplex kits. Methods (San Diego, Calif.) 1 Utilit; Fu, Q. et al., Clinical applications 4,271roducibil).

[0236] In some embodiments, the methods and systems provided herein can reduce or eliminate cross-reactivity of reagents. Figure 2 As shown in a, an embodiment of CLA is shown, using DNA oligonucleotides as flexible and addressable linkers to pre-colocate two sets of antibodies can eliminate the interaction between non-homologous antibodies. Further, after one of the flexible linkers is released from the surface, a signal is generated only in response to sandwich antibody-antigen-antibody recognition.

[0237] Figure 2 B to 2F show the nanoscale structure and working principle of CLAMP according to one embodiment. CLAMP populations are generated by one-step functionalization of microparticles with defined ratios of fluorescent oligonucleotides to antibodies ( Figure 2 B), followed by hybridization of the hook oligonucleotide-detection AB (dAB) complex to complete the construction of CLAMP ( Figure 2 B). In one embodiment, a stable biotin-streptavidin bond is used for reagent connection / attachment, and the microbead set is stored after manufacturing. The monovalent antibody oligonucleotides are then assembled in pairs on the barcode microbead set by hybridization (i.e., antibody pairs A1-A2 and B1-B2 are preassembled on microbeads A and B, respectively) ( Figure 2C), and then the beads are brought together. When a CLAMP plate is added to the sample, the target protein forms a sandwich complex, while non-specifically bound proteins do not form a complete sandwich ( Figure 2 D). After incubation, non-specifically bound proteins are removed by stringent washing ( Figure 2 E). Next, DNA strand displacement is used to simultaneously dehybridize and label one sandwich antibody on each bead population, which ensures that only sandwich binding events generate signal ( Figure 2 F) Finally, the CLAMP plate is automatically read out and the bead set decoded using any common multicolor flow cytometer.

[0238] In some embodiments, CLAMP panels have lower development costs than traditional immunoassays; not only do expensive re-optimization panels need to be avoided, but CLAMP also allows the use of significantly lower amounts of antibodies in each assay as new target analytes are added.

[0239] In some embodiments, in addition to overcoming reagent cross-reactivity, the surface-tethered antibody pairs in CLAMP can lead to binding affinity effects, giving CLAMP more advantages than traditional sandwich immunoassays. CLAMP has a higher affinity for the target because the dissociation rate (koff) of the antibody sandwich complex target in CLAMP is much lower than that of assays using sequential antibody addition. In some embodiments, CLMAP assay samples can be rigorously washed after incubation, reducing assay background and improving specificity. In addition, in some embodiments, CLAMP can reduce the disadvantage of false positives: erroneous binding events in CLAMP do not form a complete sandwich complex, so they do not result in false positive signals.

[0240] The present invention provides embodiments including but not limited to the following:

[0241] 1. A biomolecule complex for detecting and / or quantifying an analyte in a sample, the biomolecule complex comprising:

[0242] d) an anchor chain, said anchor chain being attached to the carrier;

[0243] e) a capture agent attached to the carrier; and

[0244] f) a detection agent, the detection agent being releasably attached to the anchor, the detection agent or the anchor being optionally attached to a first tag, the first tag being inactive or undetectable;

[0245] in:

[0246] The capture reagent and the detection reagent can bind to the analyte (if present in the sample) simultaneously to form a triple complex;

[0247] In the absence of the analyte, releasing the detection agent from the anchor may release the detection agent from the carrier; and

[0248] When the detection reagent is released from the anchor, the first tag can be activated or detected.

[0249] 2. The biomolecule complex according to embodiment 1, wherein the first label is detected on the carrier only when the analyte is present.

[0250] 3. The biomolecule complex according to embodiment 2, wherein the amount of the first label on the carrier when the detection reagent is released from the anchor chain is proportional to the amount and / or concentration of the analyte in the sample.

[0251] 4. The biomolecule complex according to any one of embodiments 1 to 3, wherein the detection reagent is directly and releasably attached to the anchor chain via a covalent bond, a biotin-streptavidin bond, hydrogen bonding, hydrophobic interaction, affinity binding or a non-covalent interaction.

[0252] 5. The biomolecule complex according to any one of embodiments 1 to 3, wherein the detection reagent is indirectly attached to the anchor chain via a hook chain, the detection reagent is connected to the hook chain and the hook chain is releasably attached to the anchor chain,

[0253] wherein, in the absence of the analyte, releasing the hook chain from the anchor chain releases the detection reagent from the carrier, and

[0254] The first tag may be activated or detected when the shackle is released from the anchor chain.

[0255] 6. The biomolecule complex according to embodiment 5, wherein the amount of the first label on the carrier when the hook is released from the anchor is proportional to the amount and / or concentration of the analyte in the sample.

[0256] 7. The biomolecule complex according to embodiment 5 or 6, wherein the first tag is optionally attached to the hook chain.

[0257] 8. The biomolecule complex according to embodiment 7, wherein at least one of the detection reagent and the hook chain is optionally attached to the first tag.

[0258] 9. A biomolecule complex according to embodiment 8, wherein the detection reagent is attached to the first tag and the hook is not attached to the first tag; wherein the hook is attached to the first tag and the detection reagent is not attached to the first tag; or wherein the detection reagent and the hook are both attached to the first tag.

[0259] 10. The biomolecule complex according to any one of embodiments 5 to 9, wherein the hook linker is a polymer, an oligonucleotide or a polysaccharide.

[0260] 11. The biomolecule complex according to embodiment 10, wherein the polymer is polyethylene glycol (PEG).

[0261] 12. The biomolecule complex according to embodiment 10 or 11, wherein the oligonucleotide is a single-stranded DNA oligonucleotide, a single-stranded RNA oligonucleotide, or a double-stranded DNA or RNA oligonucleotide.

[0262] 13. The biomolecule complex according to any one of embodiments 1 to 12, wherein the capture agent is directly attached to the carrier.

[0263] 14. The biomolecule complex according to any one of embodiments 1 to 13, wherein the capture agent is attached to the carrier via a covalent bond, a biotin-streptavidin bond, a DNA oligonucleotide linker or a polymer linker.

[0264] 15. The biomolecule complex according to any one of embodiments 1 to 14, wherein the capture agent is indirectly attached to the carrier by attaching to the anchor chain.

[0265] 16. The biomolecule complex according to embodiment 15, wherein the capture agent is releasably attached to the anchor chain by covalent bonds, biotin-streptavidin bonds, hydrogen bonding, hydrophobic interactions, affinity binding or non-covalent interactions.

[0266] 17. The biomolecule complex according to embodiment 15, wherein the capture agent is attached to the anchor chain via a capture chain connected to the capture agent and the anchor chain.

[0267] 18. The biomolecule complex according to embodiment 17, wherein the capture strand is an oligonucleotide, a polymer such as PEG or a polysaccharide.

[0268] 19. The biomolecule complex according to any one of embodiments 1 to 18, wherein the anchor chain is a polymer such as PEG, an oligonucleotide or an oligosaccharide.

[0269] 20. The biomolecule complex according to embodiment 19, wherein the oligonucleotide is a DNA oligonucleotide.

[0270] 21. The biomolecule complex according to embodiment 19 or 20, wherein the anchor is a single-stranded DNA oligonucleotide, a single-stranded RNA oligonucleotide, a double-stranded DNA or RNA oligonucleotide, or a DNA-RNA hybrid.

[0271] 22. The biomolecule complex according to any one of embodiments 1 to 21, wherein the carrier is a microparticle, a multi-well plate surface, a glass slide surface or a hydrogel matrix.

[0272] 23. The biomolecule complex according to embodiment 22, wherein the microparticles are polystyrene microbeads (optionally barcoded).

[0273] 24. A biomolecule complex according to any one of embodiments 1 to 23, wherein the anchor chain is attached to the carrier via a covalent bond, a biotin-streptavidin bond, an oligonucleotide linker, a DNA oligonucleotide linker, a polymer linker, a PEG linker or another chemical interaction, such as hydrogen bonding, hydrophobic interaction, affinity binding or a non-covalent interaction.

[0274] 25. A biomolecule complex according to any one of embodiments 1 to 24, wherein the capture agent is an antibody, an antibody fragment, an aptamer, a modified aptamer, a somamer, an affimer, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, an oligonucleotide or a low molecular weight compound.

[0275] 26. The biomolecule complex according to any one of embodiments 1 to 25, wherein the detection agent is an antibody, an antibody fragment, an aptamer, an affibody, a modified aptamer, an aptamer, an antigen, a protein, a polypeptide, a multiprotein complex, an exosome, an oligonucleotide or a low molecular weight compound.

[0276] 27. The biomolecule complex according to any one of embodiments 1 to 26, wherein the capture reagent and the detection reagent are different.

[0277] 28. The biomolecule complex according to any one of embodiments 1 to 26, wherein the capture reagent and the detection reagent are the same.

[0278] 29. The biomolecule complex according to any one of embodiments 1 to 28, wherein both the capture agent and the detection agent are antibodies.

[0279] 30. The biomolecule complex according to embodiment 28 or 29, wherein the capture reagent and the detection reagent are the same antibody.

[0280] 31. The biomolecule complex according to embodiment 27 or 29, wherein the capture reagent and the detection reagent are different antibodies and bind to different epitopes on the analyte.

[0281] 32. A biomolecule complex according to any one of embodiments 1 to 30, wherein the capture reagent and the detection reagent are linked to the same epitope on the analyte, the analyte has two or more repeats of the epitope, and the capture reagent and the detection reagent are linked to different repeats of the epitope on the analyte.

[0282] 33. The biomolecule complex according to any one of the preceding embodiments, wherein the capture reagent and the detection reagent are identical, structurally similar, or attached to a structurally similar part or epitope of the analyte.

[0283] 34. The biomolecule complex of any one of embodiments 1 to 33, wherein the analyte is an antigen, an antibody, an affibody, an aptamer, a modified aptamer, an aptamer, an antibody fragment, a protein, a polypeptide, a multiprotein complex, an exosome, an oligonucleotide, a hormone, a modified oligonucleotide or a low molecular weight compound.

[0284] 35. The biomolecule complex according to any one of the preceding embodiments, wherein the capture reagent and the detection reagent are both antigens, and the analyte is an antibody.

[0285] 36. The biomolecule complex according to any one of embodiments 5 to 35, wherein the releasable linker between the hook strand and the anchor strand comprises a double-stranded DNA hybrid.

[0286] 37. The biomolecule complex according to any one of embodiments 1 to 36, wherein the sample is a biological sample.

[0287] 38. The biomolecule complex according to any one of embodiments 1 to 37, wherein the sample is a body fluid, an extract, a solution containing protein and / or DNA, a cell extract, a cell lysate or a tissue lysate.

[0288] 39. The biomolecule complex of embodiment 38, wherein the body fluid is urine, saliva, blood, serum, plasma, cerebrospinal fluid, tears, semen or sweat.

[0289] 40. The biomolecule complex according to any one of embodiments 1 to 39, wherein the first tag is a fluorophore, a specific DNA sequence, an enzyme or a biotin moiety.

[0290] 41. The biomolecule complex according to any one of embodiments 1 to 40, wherein the detection reagent can be released from the anchor chain by an enzymatic reaction, by cleavage or by photo-fragmentation.

[0291] 42. The biomolecule complex according to any one of embodiments 5 to 40, wherein the hook chain can be released from the anchor chain by an enzymatic reaction, by cleavage, by photo-fragmentation or by oligonucleotide displacement.

[0292] 43. The biomolecule complex according to any one of embodiments 5 to 40 and embodiment 42 further comprises a displacing agent capable of releasing the hook chain from the anchor chain, thereby releasing the detection reagent from the carrier in the absence of the analyte.

[0293] 44. The biomolecule complex according to embodiment 43, wherein the hook chain and the anchor chain both comprise DNA oligonucleotides, and the releasable linker between the hook chain and the anchor chain is a double-stranded DNA hybrid.

[0294] 45. The biomolecule complex of embodiment 43 or 44, wherein the displacer is an enzyme, a DNA oligonucleotide, light or DTT.

[0295] 46. ​​The biomolecule complex according to any one of embodiments 43 to 45, wherein the displacer can release the hook chain from the anchor chain by an enzymatic reaction, by cleavage, by photo-fragmentation or by oligonucleotide displacement.

[0296] 47. A biomolecule complex according to any one of embodiments 43 to 46, wherein the releasable joint between the hook chain and the anchor chain is a double-stranded DNA hybrid; and the displacer is a DNA oligonucleotide, which is complementary to at least a portion of the hook chain and can hybridize with the hook chain, thereby releasing the hook chain from the anchor chain through a DNA displacement reaction.

[0297] 48. A biomolecule complex according to any one of embodiments 43 to 46, wherein the releasable joint between the hook chain and the anchor chain is a double-stranded DNA hybrid; and the displacer is a DNA oligonucleotide, which is complementary to at least a portion of the hook chain anchor chain and can hybridize with the anchor chain, thereby releasing the hook chain from the anchor chain through a DNA displacement reaction.

[0298] 49. The biomolecule complex according to any one of embodiments 43 to 48, wherein the displacing agent is attached to a second tag.

[0299] 50. The biomolecule complex according to embodiment 49, wherein the first tag is absent, and neither the detection reagent nor the hook chain is attached to the first tag.

[0300] 51. The biomolecule complex of embodiment 49 or 50, wherein the second tag is a fluorophore, a specific DNA sequence, a biotin moiety or an enzyme.

[0301] 52. The biomolecule complex according to any one of embodiments 43 to 51, wherein the displacing agent is an enzyme or light or DTT, and is capable of cleaving the connection between the hook chain and the anchor chain by an enzymatic reaction or by photo-cleavage.

[0302] 53. The biomolecule complex according to any one of embodiments 1 to 52, wherein the detection reagent and the capture reagent are both antibodies, the analyte is an antigen, and the detection reagent and the capture reagent can bind to different epitopes on the antigen.

[0303] 54. The biomolecule complex according to any one of embodiments 1 to 52, wherein the detection reagent and the capture reagent are both antibodies, the analyte is an antigen, and the detection reagent and the capture reagent can bind to the same epitope on the antigen.

[0304] 55. The biomolecule complex according to embodiment 54, wherein the antigen has two or more repeats of the epitope, and the detection reagent and the capture reagent can simultaneously bind to different repeats of the epitope on the antigen.

[0305] 56. The biomolecule complex according to any one of embodiments 1 to 55 further comprises a stem chain complementary to the near-surface sequence of the anchor chain, the anchor chain being a single-stranded oligonucleotide, wherein the stem chain is capable of binding to the anchor chain to form a double-stranded oligonucleotide anchor chain.

[0306] 57. The biomolecule complex according to any one of embodiments 1 to 56, wherein the relative density of the anchor chain and the capture agent on the support can be adjusted to control the effective affinity of the assay.

[0307] 58. A biomolecule complex according to any one of embodiments 5 to 57, wherein the length of the hook chain and / or the anchor chain can be adjusted to control the effective affinity of the assay; and / or wherein the surface density of the capture reagent and / or the detection reagent can be adjusted to control the effective affinity of the assay.

[0308] 59. The biomolecule complex according to any one of embodiments 5 to 58, wherein the conjugation between the detection agent and the hook chain is monovalent.

[0309] 60. The biomolecule complex according to any one of embodiments 5 to 59, wherein at least 90% of the detection reagents are connected to the carrier via only one hook chain.

[0310] 61. The biomolecule complex according to any one of embodiments 1 to 60, wherein the capture agent is connected to the anchor chain via a capture chain, and the conjugation between the capture agent and the hook chain and the capture chain is monovalent.

[0311] 62. The biomolecule complex according to any one of embodiments 1 to 53, wherein the capture reagent is connected to the anchor chain via a capture chain, and at least 90% of the detection reagents are connected to the hook chain anchor chain via only one of the capture chains.

[0312] 63. The biomolecule complex according to any one of embodiments 1 to 62, wherein the anchor chains are randomly distributed on the carrier.

[0313] 64. The biomolecule complex according to any one of embodiments 1 to 63, wherein the capture agents are randomly distributed on the carrier.

[0314] 65. The biomolecule complex of any one of embodiments 5 to 64, wherein the length and / or flexibility of the hook chain can be selected to be sufficient to allow the detection reagent to bind to the analyte in the presence of the capture reagent.

[0315] 66. The biomolecule complex according to any one of embodiments 5 to 65, wherein when the linker between the hook chain and the anchor chain is a double-stranded DNA hybrid, the melting temperature (Tm) of the double-stranded DNA hybrid is about 50 to about 80 degrees Celsius.

[0316] 67. The biomolecule complex of any one of embodiments 1 to 66, wherein the concentration of the detection reagent after release or displacement is less than about 10 picomolar.

[0317] 67a. A molecular biocomplex for detecting and / or quantifying an analyte in a sample, the molecular biocomplex comprising:

[0318] e) an anchor chain, said anchor chain being attached to the carrier;

[0319] f) a capture agent attached to the carrier; and

[0320] g) a detection agent connected to a hook chain, the hook chain being releasably attached to the anchor chain, the hook chain and the anchor chain being connected together by a double-stranded DNA hybrid;

[0321] h) a displacing agent comprising a DNA oligonucleotide that is complementary to at least a portion of the hook strand and capable of hybridizing to the hook strand, thereby releasing the hook strand from the anchor strand by a DNA displacement reaction, the displacing agent being detectably labeled;

[0322] in:

[0323] The capture reagent and the detection reagent can simultaneously bind to the analyte (if present in the sample) to form a triple complex; and

[0324] In the absence of the analyte, release of the hook chain from the anchor chain by the displacing agent may release the detection reagent from the carrier.

[0325] 68. The biomolecule complex according to any one of embodiments 1 to 67, further comprising:

[0326] a) a second anchor chain, the second anchor chain being attached to the carrier;

[0327] b) a second detection reagent releasably attached to the second anchor, wherein the second detection reagent is optionally attached to a third tag, wherein the third tag is inactive or undetectable;

[0328] in:

[0329] The capture reagent, the detection reagent and the second detection reagent can simultaneously bind to the analyte (such as present in the sample) to form a quadruple complex; and

[0330] In the absence of the analyte, releasing the second detection reagent from the second anchor may release the second detection reagent from the carrier; and

[0331] When the second detection reagent is released from the second anchor, the third tag can be activated or detected.

[0332] 69. The biomolecule complex of embodiment 68, wherein the second detection reagent is linked to a second tether, the second tether being releasably linked to the second anchor, wherein at least one of the second detection reagent and the second tether is optionally attached to the third tag, the third tag being inactive or undetectable;

[0333] wherein, in the absence of the analyte, releasing the second hook chain from the second anchor chain can release the second detection reagent from the carrier; and

[0334] The third tag may be activated or detected when the second shackle is released from the second anchor chain.

[0335] 70. The biomolecule complex of embodiment 68 or 69, wherein the second detection reagent is attached to the third tag; the second hook is attached to the third tag; or both the second detection reagent and the second hook are attached to the third tag.

[0336] 71. The biomolecule complex of any one of embodiments 68 to 70, wherein the third tag is a fluorophore, a specific DNA sequence, a biotin moiety or an enzyme.

[0337] 72. The biomolecule complex according to any one of embodiments 68 to 71, wherein the third tag is detected on the carrier only in the presence of the analyte.

[0338] 73. The biomolecule complex according to any one of embodiments 69 to 72, further comprising a second displacer, which is capable of releasing the second hook chain from the second anchor chain, thereby releasing the second detection reagent from the carrier in the absence of the analyte.

[0339] 74. The biomolecule complex according to embodiment 73, wherein the second hook chain and the second anchor chain are DNA oligonucleotides, and the connection between the second hook chain and the second anchor chain is a double-stranded DNA hybrid.

[0340] 75. The biomolecule complex according to embodiment 73 or 74, wherein the second displacer can release the second hook strand from the second anchor strand by an enzymatic reaction, by cleavage, by photo-fragmentation or by oligonucleotide displacement.

[0341] 76. A biomolecule complex according to any one of embodiments 73 to 75, wherein the connection between the second hook chain and the second anchor chain comprises a double-stranded DNA hybrid, and the second displacement agent is capable of breaking the connection between the second hook chain and the second anchor chain by a DNA chain displacement reaction.

[0342] 77. The biomolecule complex according to any one of embodiments 73 to 76, wherein the second displacer is detectably labeled.

[0343] 78. The biomolecule complex of embodiment 77, wherein the second displacer is labeled with a fluorophore, a specific DNA sequence, an enzyme or a biotin moiety.

[0344] 79. The biomolecule complex according to embodiment 77 or 78, wherein the third tag is absent, and neither the second detection reagent nor the second hook chain is attached to the third tag.

[0345] 80. A multiplex sandwich assay system for simultaneously detecting and / or quantifying two or more analytes in a sample, the system comprising two or more biomolecule complexes as described in any one of embodiments 1 to 79, wherein the two or more biomolecule complexes are attached to the same carrier, and wherein each biomolecule complex is used to detect and / or quantify a different analyte in the sample.

[0346] 81. The multiplex sandwich assay system of embodiment 80, wherein the carrier is a microparticle, such as a polystyrene microbead, a multi-well plate, a glass slide surface, or a hydrogel matrix.

[0347] 82. A multiplex sandwich assay system for simultaneously detecting and / or quantifying two or more analytes in a sample, the system comprising two or more biomolecule complexes as described in any one of embodiments 1 to 79, wherein the two or more biomolecule complexes are used to detect and / or quantify a corresponding first analyte and a corresponding second analyte in the sample,

[0348] The two or more biomolecule complexes are attached to a corresponding first carrier and a corresponding second carrier, and the sample contacts the first carrier and the second carrier simultaneously.

[0349] 83. A multiplex sandwich assay system according to embodiment 82, wherein the first carrier and the second carrier are the same carrier.

[0350] 84. A multiplex sandwich assay system according to embodiment 83, wherein the same carrier is a plane and the two or more biomolecule complexes are positioned at different positions on the plane.

[0351] 85. The multiplex sandwich assay system of embodiment 84, wherein the plane is a multi-well plate surface, a glass slide surface, or a hydrogel matrix.

[0352] 86. A multiplex sandwich assay system according to embodiment 82, wherein the first carrier and the second carrier are different carriers.

[0353] 87. A multiplex sandwich assay system according to embodiment 86, wherein the first carrier and the second carrier are both microparticles.

[0354] 88. A multiplex sandwich assay system according to embodiment 87, wherein the microparticles are polystyrene microbeads.

[0355] 89. The multiplex sandwich assay system of embodiment 87 or 88, wherein the microparticles are barcoded.

[0356] 90. The multiplex sandwich assay system of any one of embodiments 82 to 89, wherein the first carrier is a first microbead encoded with a first barcode and the second carrier is a second microbead encoded with a second barcode, thereby allowing identification of the corresponding microbeads.

[0357] 91. The multiplex sandwich assay system according to any one of embodiments 82 to 89, wherein the two or more biomolecule complexes comprise a corresponding first hook chain and a corresponding second hook chain and a corresponding first anchor chain and a corresponding second anchor chain, and the first hook chain, the second hook chain, the first anchor chain and the second anchor chain are all DNA oligonucleotides, and the connectors between the first hook chain and the first anchor chain and between the second hook chain and the second anchor chain are double-stranded DNA hybrids;

[0358] wherein the two or more bioanalytical complexes further comprise a corresponding first displacer and a corresponding second displacer, wherein the first displacer is capable of releasing the first hook chain from the first anchor chain and the second displacer is capable of releasing the second hook chain from the second anchor chain;

[0359] Wherein, the first displacer and the second displacer are the same.

[0360] 92. A multiple sandwich assay system according to embodiment 91, wherein the first displacer and the second displacer are displacer DNA oligonucleotides, which can bind to the first hook chain and the second hook chain, thereby displacing the first hook chain and the third hook chain from their corresponding anchor chains through a DNA displacement reaction.

[0361] 93. A multiplex sandwich assay system according to embodiment 92, wherein the displacer DNA oligonucleotide is detectably labeled.

[0362] 94. A multiplex sandwich assay system according to embodiment 93, wherein the label is a fluorophore, a specific DNA sequence, an enzyme or a biotin moiety.

[0363] 95. A multiple sandwich assay system according to any one of embodiments 82 to 94, wherein the first hook chain and the second hook chain are attached to a first tag and a third tag, respectively, the first tag and the third tag are inactive or undetectable, and the first tag and the third tag can be activated or detected when the first hook chain is released from the first anchor chain and the second hook chain is released from the second anchor chain.

[0364] 96. A multiplex sandwich assay system according to any one of embodiments 82 to 94, wherein the first tag and the third tag are not present and only the displacer DNA oligonucleotide is labeled.

[0365] 97. A multiplex sandwich assay system for simultaneously detecting and / or quantifying two or more analytes in a sample, the system comprising:

[0366] c) a first biomolecule complex for detecting and / or quantifying a first analyte in the sample, the first biomolecule complex comprising a first anchor, a first capture reagent and optionally a first detection reagent linked to the first hook, the first detection reagent or the first hook being releasably linked to the first anchor, at least one of the first detection reagent and the first hook being optionally attached to a first tag, wherein the first capture reagent and the first detection reagent can simultaneously bind to the first analyte (if present in the sample) to form a first triple complex; and

[0367] d) a second biomolecule complex for detecting and / or quantifying a second analyte in the sample, the second biomolecule complex comprising a second anchor, a second capture reagent and optionally a second detection reagent linked to the second hook, the second detection reagent or the second hook being releasably linked to the second anchor, at least one of the second detection reagent and the second hook being optionally attached to a second tag, wherein the second capture reagent and the second detection reagent can simultaneously form a second triple complex with the second analyte (if present in the sample);

[0368] wherein the first anchor chain, the first capture agent, the second anchor chain and the second capture agent are all attached to a carrier;

[0369] Wherein, the first tag and the second tag are inactive or undetectable, and can be activated or detected when their corresponding detection reagents or hooks are released from their corresponding anchors.

[0370] 98. The multiplex sandwich assay system of embodiment 97, wherein the first tag and the second tag are detected on the carrier only when the first analyte and the second analyte, respectively, are present.

[0371] 99. The multiplex sandwich assay system of embodiment 97 or 98, further comprising a displacer capable of releasing the first detection reagent or the first hook from the first anchor chain and releasing the second detection reagent or the second hook from the second anchor chain.

[0372] 100. A multiple sandwich assay system according to any one of embodiments 97 to 99, wherein the displacer is a DNA oligonucleotide; the first hook chain, the second hook chain, the first anchor chain and the second anchor chain comprise DNA oligonucleotides, and the connector between the hook chain and its corresponding anchor chain is a double-stranded DNA hybrid; and the displacer is capable of hybridizing with the first hook chain and the second hook chain and breaking the connector between the hook chain and its corresponding anchor chain through a DNA chain displacement reaction.

[0373] 101. The multiplex sandwich assay system of embodiment 99 or 100, wherein the displacer is detectably labeled.

[0374] 102. The multiplex sandwich assay system of embodiment 101, wherein the first tag and the second tag are absent and only the displacer is labeled.

[0375] 103. A multiplex sandwich assay system according to any one of embodiments 99 to 102, wherein the displacer is labeled with a fluorophore, a specific DNA sequence, an enzyme or a biotin moiety.

[0376] 104. A multiplex sandwich assay system according to any one of embodiments 97 to 103, wherein the carrier is a flat or multi-well plate surface, a glass slide surface or a hydrogel, and the first and second biomolecule complexes are positioned at a first position and a second position on the carrier, respectively.

[0377] 105. The multiplex sandwich assay system according to any one of embodiments 97 to 104, wherein the first biomolecule complex is attached to a first carrier and the second biomolecule complex is attached to a second carrier.

[0378] 106. A multiplex sandwich assay system according to embodiment 105, wherein the first carrier is a microparticle, such as a polystyrene microbead.

[0379] 107. The multiplex sandwich assay system of embodiment 105 or 106, wherein the second carrier is a microparticle, such as a polystyrene microbead.

[0380] 108. The multiplex sandwich assay system of any one of embodiments 105 to 107, wherein the first carrier and the second carrier are mixed together.

[0381] 109. The multiplex sandwich assay system of any one of embodiments 105 to 108, wherein the first vector and the second vector are barcoded.

[0382] 110. A multiplex sandwich assay system according to any one of embodiments 97 to 109, wherein the first molecular biocomplex system is the first molecular biocomplex system as described in any one of embodiments 1 to 79, and wherein the second molecular biocomplex system is the second biomolecule complex system as described in any one of embodiments 1 to 79.

[0383] 111. A method for detecting and / or quantifying an analyte in a sample, the method comprising:

[0384] d) providing a carrier, a capture agent attached to the carrier, an anchor attached to the carrier, and a detection agent optionally attached to the shackle, wherein the detection agent or the shackle is releasably attached to the anchor, and wherein at least one of the detection agent and the shackle is optionally attached to a first tag, the first tag being inactive or undetectable;

[0385] e) contacting the support with the sample under conditions that allow the capture reagent and the detection reagent to simultaneously bind to the analyte to form a triple complex; and

[0386] f) adding a displacing agent optionally attached to a second tag, wherein the displacing agent releases the detection reagent or the hook chain from the anchor chain to release the detection reagent optionally linked to the hook chain from the carrier in the absence of the analyte, and wherein the release of the detection chain or the hook chain from the anchor chain activates the first tag or makes the first tag detectable.

[0387] 112. The method according to embodiment 111, further comprising the following step (d):

[0388] g) determining the presence and / or amount of the first label and / or the second label on the carrier, wherein the presence of the first and / or the second label on the carrier indicates the presence of the analyte in the sample, and the amount of the first and / or the second label is proportional to the amount and / or concentration of the analyte in the sample.

[0389] 113. The method of embodiment 111 or 112, further comprising the step of washing the carrier after step (c) to remove any unattached reagents or materials.

[0390] 114. The method according to any one of embodiments 111 to 113, further comprising the step of storing the carrier after step (c).

[0391] 115. The method of any one of embodiments 111 to 114, wherein the first label is attached to the detection reagent.

[0392] 116. The method of any one of embodiments 111 to 115, wherein the first tag is attached to the shackle.

[0393] 117. The method of any one of embodiments 111 to 116, wherein the second tag is attached to the displacing agent.

[0394] 118. A method according to any one of embodiments 111 to 117, wherein the first tag and / or the second tag is a fluorophore, a specific DNA sequence, an enzyme or a biotin moiety.

[0395] 119. The method of any one of embodiments 111 to 118, wherein the first tag is absent and the displacing agent is attached to the second tag.

[0396] 120. The method of any one of embodiments 111 to 119, wherein the capture agent is directly attached to the carrier.

[0397] 121. A method according to any one of embodiments 111 to 120, wherein the capture reagent is attached to the carrier via a covalent bond, a biotin-streptavidin bond, a DNA oligonucleotide linker, or a polymer linker.

[0398] 122. The method of any one of embodiments 111 to 119, wherein the capture agent is indirectly attached to the carrier by attachment to the anchor chain.

[0399] 123. A method according to embodiment 122, wherein the capture reagent is attached to the anchor chain by a covalent bond, a biotin-streptavidin bond, hydrogen bonding, hydrophobic interaction, affinity binding or a non-covalent interaction.

[0400] 124. The method of embodiment 122, wherein the capture agent is attached to the anchor chain via a capture chain connected to the capture agent and the anchor chain.

[0401] 125. The method of embodiment 124, wherein the capture strand is an oligonucleotide, a polymer such as PEG, or a polysaccharide.

[0402] 126. A method according to any one of embodiments 111 to 125, wherein the anchor chain is a polymer such as PEG, an oligonucleotide or an oligosaccharide.

[0403] 127. A method according to embodiment 126, wherein the oligonucleotide is a single-stranded DNA oligonucleotide, a single-stranded RNA oligonucleotide, a double-stranded DNA or RNA oligonucleotide, or a DNA-RNA hybrid.

[0404] 128. A method according to any one of embodiments 111 to 127, wherein the carrier is a microparticle, a multi-well plate surface, a slide surface or a hydrogel matrix.

[0405] 129. The method of embodiment 128, wherein the microparticles are polystyrene microbeads.

[0406] 130. The method of embodiment 128 or 129, wherein the microparticles are barcoded.

[0407] 131. A method according to any one of embodiments 111 to 130, wherein the hook anchor is a polymer such as PEG, a DNA oligonucleotide such as a single-stranded DNA oligonucleotide, a single-stranded RNA oligonucleotide or a double-stranded DNA or RNA oligonucleotide or an oligosaccharide.

[0408] 132. A method according to any one of embodiments 111 to 131, wherein the hook chain is not present and the detection reagent is directly connected to the anchor chain via a covalent bond or a biotin-streptavidin bond.

[0409] 133. A method according to any one of embodiments 111 to 113, wherein the anchor chain is attached to the carrier by a covalent bond, a biotin-streptavidin bond, a DNA oligonucleotide linker, a polymer linker, or another chemical interaction such as hydrogen bonding, hydrophobic interaction, affinity interaction, or non-covalent interaction.

[0410] 134. A method according to any one of embodiments 111 to 133, wherein the capture reagent and / or the detection reagent is an antibody, an antibody fragment, an aptamer, a modified aptamer, an aptamer, an affibody, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, an oligonucleotide or a low molecular weight compound.

[0411] 135. A method according to any one of embodiments 111 to 134, wherein the capture reagent and the detection reagent are different.

[0412] 136. A method according to any one of embodiments 111 to 134, wherein the capture reagent and the detection reagent are the same, structurally similar, or bind to a structurally similar portion or epitope of the analyte.

[0413] 137. A method according to embodiment 136, wherein the analyte has two or more repeats of the portion to which the capture reagent and the detection reagent bind, and the capture reagent and the detection reagent bind to different repeats.

[0414] 138. A method according to embodiment 136, wherein the capture reagent and the detection reagent bind to the same epitope, the analyte has two or more repeats of the epitope, and the capture reagent and the detection reagent bind to different repeats of the epitope on the analyte.

[0415] 139. A method according to any one of embodiments 111 to 138, wherein the capture reagent and the detection reagent bind to different parts or epitopes of the analyte.

[0416] 140. A method according to any one of embodiments 111 to 139, wherein the capture reagent and the detection reagent are both antibodies.

[0417] 141. The method of embodiment 140, wherein the analyte is an antigen, a protein, a polypeptide, a multiprotein complex, a hormone, or an exosome.

[0418] 142. A method according to embodiment 141, wherein the analyte is an antigen.

[0419] 143. A method according to any one of embodiments 111 to 139, wherein the capture reagent and the detection reagent are antigens, proteins or peptides, and the analyte is an antibody.

[0420] 144. A method according to any one of embodiments 111 to 143, wherein the displacer releases the detection reagent or the hook chain from the anchor chain by an enzymatic reaction, by shearing, by photofragmentation, or by oligonucleotide displacement.

[0421] 145. A method according to any one of embodiments 111 to 144, wherein the hook chain and the anchor chain both comprise DNA oligonucleotides, and the connection between the hook chain and the anchor chain is a double-stranded DNA hybrid.

[0422] 146. A method according to any one of embodiments 111 to 145, wherein the displacing agent is an enzyme or light or DTT.

[0423] 147. A method according to any one of embodiments 111 to 145, wherein the displacer is a DNA oligonucleotide.

[0424] 148. The method of embodiment 147, wherein the displacer is complementary to at least a portion of the hook strand and hybridizes to the hook strand, thereby releasing the hook strand from the anchor strand via a DNA strand displacement reaction.

[0425] 149. The method of embodiment 147, wherein the displacer is complementary to at least a portion of the anchor strand and hybridizes to the anchor strand, thereby releasing the hook strand from the anchor strand by a DNA strand displacement reaction.

[0426] 150. The method of any one of embodiments 111 to 148, wherein the first tag is absent and the first displacing agent is attached to the second tag.

[0427] 151. A method according to embodiment 150, wherein the second tag is a fluorophore, a specific DNA sequence, an enzyme or a biotin moiety.

[0428] 152. A method according to any one of embodiments 111 to 151, wherein the carrier further comprises a stem chain, the stem chain is a single-stranded oligonucleotide complementary to the near-surface sequence of the anchor chain, and the anchor chain is a single-stranded oligonucleotide, wherein the stem chain is capable of binding to the anchor chain to form a double-stranded oligonucleotide anchor chain.

[0429] 153. A method according to any one of embodiments 111 to 152, wherein the relative density of the anchor chain and the capture agent on the support is adjusted to control the effective affinity of the assay.

[0430] 154. A method according to any one of embodiments 111 to 153, wherein the conjugation between the detection reagent and the hook chain is monovalent.

[0431] 155. A method according to any one of embodiments 111 to 154, wherein at least 90% of the detection reagents are connected to the carrier via only one of the hook chains.

[0432] 156. A method according to any one of embodiments 111 to 155, wherein the capture reagent is connected to the anchor chain through the capture chain, and the conjugation between the capture reagent and the capture chain is monovalent.

[0433] 157. The method according to any one of embodiments 111 to 155, wherein when the capture agents are attached to the anchor chain via the capture chains, at least 90% of the capture agents are attached to the anchor chain via only one of the capture chains.

[0434] 158. The method according to any one of embodiments 111 to 157, wherein the anchor chains are randomly distributed on the carrier.

[0435] 159. A method according to any one of embodiments 111 to 158, wherein the capture reagents are randomly distributed on the carrier.

[0436] 160. A method according to any one of embodiments 111 to 159, wherein the length and / or flexibility of the hook chain is selected to be sufficient to allow the detection reagent to bind to the analyte in the presence of the capture reagent.

[0437] 160a. A method according to any one of embodiments 111 to 160, wherein the length of the hook chain and / or the anchor chain is selected to control the effective affinity of the assay; and / or wherein the surface density of the capture reagent and / or the detection reagent is selected to control the effective affinity of the assay.

[0438] 161. A method according to any one of embodiments 111 to 160a, wherein the connection between the hook chain and the anchor chain is a double-stranded DNA hybrid with a melting temperature (Tm) of about 50 to about 80 degrees Celsius.

[0439] 162. A method according to any one of embodiments 111 to 161, wherein the capture reagent is connected to the anchor chain through an oligonucleotide linker so that the connector between the capture reagent and the anchor chain is a second double-stranded DNA hybrid, and the melting temperature (Tm) of the second double-stranded DNA hybrid is about 50 to about 80 degrees Celsius.

[0440] 163. A method according to any one of embodiments 111 to 162, wherein the concentration of the detection reagent after release or displacement is less than about 10 picomolar.

[0441] 163a. ​​A method for detecting and / or quantifying an analyte in a sample, the method comprising:

[0442] e) providing a carrier, a capture agent attached to the carrier, an anchor attached to the carrier, and a detection agent connected to the hook, wherein the hook is releasably connected to the anchor via a double-stranded DNA hybrid;

[0443] f) contacting the support with the sample while allowing the capture reagent and the detection reagent to bind to the analyte simultaneously to form a triple complex; and

[0444] g) adding a displacing agent attached to a detectable label, wherein the displacing agent is a DNA oligonucleotide complementary to at least a portion of the hook strand and capable of hybridizing to the hook strand, wherein the displacing agent releases the hook strand from the anchor strand by a DNA displacement reaction to release the detection reagent from the carrier in the absence of the analyte; and

[0445] h) optionally determining the presence and / or amount of the detectable label on the carrier, wherein the presence of the label on the carrier indicates the presence of the analyte in the sample and the amount of the label is proportional to the amount and / or concentration of the analyte in the sample.

[0446] 164. The method of any one of embodiments 111 to 163a, wherein:

[0447] - the carrier further comprises a second anchor chain attached to the carrier, a second capture reagent attached to the carrier, and a second detection reagent optionally connected to the second hook chain, wherein the second detection reagent or the second hook chain is connected to the second anchor chain, and wherein at least one of the second detection reagent and the second hook chain is optionally attached to a third tag, and the third tag is inactive or undetectable, wherein the second capture reagent and the second detection reagent can simultaneously bind to a second analyte in the sample to form a second triple complex;

[0448] - wherein the displacing agent also releases the second detection reagent or the second hook from the second anchor to allow the second detection reagent optionally attached to the second hook to be released from the carrier in the absence of the second analyte, and wherein the release of the second detection reagent or the second hook from the second anchor activates the third tag or renders the third tag detectable;

[0449] - wherein the presence and / or amount of the third label on the carrier indicates the presence of the second analyte in the sample, and the amount of the third label on the carrier is proportional to the amount and / or concentration of the second analyte in the sample;

[0450] So that the first analyte and the second analyte can be detected in the sample at the same time.

[0451] 165. The method of embodiment 164, wherein the second anchor chain and the second capture agent are positioned at a first position and a second position on the carrier, respectively.

[0452] 166. The method of embodiment 164, wherein the second anchor chain and the second capture agent are attached to a second carrier.

[0453] 167. The method of embodiment 166, wherein the second carrier is a microparticle, such as a polystyrene microbead.

[0454] 168. A method according to any one of embodiments 164 to 167, wherein the carrier is a microparticle, such as a polystyrene microbead.

[0455] 169. A method according to any one of embodiments 166 to 168, wherein the first carrier is a first microbead encoded with a first barcode, and the second carrier is a second microbead encoded with a second barcode, so that the corresponding microbead is identified when the corresponding barcode is detected.

[0456] 170. A method according to any one of embodiments 166 to 169, wherein the first carrier and the second carrier are mixed together before contacting with the sample.

[0457] 171. A method according to any one of embodiments 166 to 170, wherein the first carrier and the second carrier are contacted with the sample simultaneously.

[0458] 172. A method according to any one of embodiments 111 to 171, wherein the sample is a biological sample.

[0459] 173. A method according to any one of embodiments 111 to 172, wherein the sample is a body fluid, an extract, a solution containing protein and / or DNA, a cell extract, a cell lysate or a tissue lysate.

[0460] 174. The method of embodiment 173, wherein the body fluid is urine, saliva, blood, serum, plasma, cerebrospinal fluid, tears, semen or sweat.

[0461] 175. A method for preparing a multiplex sandwich assay system, the method comprising:

[0462] (h) providing a first container containing first microparticles, wherein the first microparticles are encoded with a first bar code;

[0463] (i) attaching the first microparticle to a first capture reagent and a first detection reagent, wherein the first capture reagent and the first detection reagent are capable of binding to a first analyte simultaneously;

[0464] (j) optionally, storing the first particles;

[0465] (k) providing a second container containing second microparticles, the second microparticles being encoded with a second bar code;

[0466] (1) attaching the second microparticle to a second capture reagent and a second detection reagent, wherein the second capture reagent and the second detection reagent are capable of binding to a second analyte simultaneously;

[0467] (m) optionally, storing the second particles;

[0468] (n) mixing the first microparticles and the second microparticles together for use in the multiplex sandwich assay system;

[0469] The first capture reagent and the first detection reagent are not mixed with the second capture reagent and the second detection reagent before being attached to their corresponding microparticles.

[0470] 175a. The method of embodiment 175, wherein the first barcode and the second barcode are independently spectral, graphic, or chemical barcodes.

[0471] 176. A method according to embodiment 175 or 175a, wherein the corresponding capture reagent and the corresponding detection reagent are simultaneously attached to their corresponding microparticles.

[0472] 177. A method according to embodiment 175 or 175a, wherein the corresponding capture reagent and the corresponding detection reagent are attached to their corresponding microparticles in a two-step reaction, wherein the capture reagent or the detection reagent is first attached to the microparticle, followed by the attachment of the other reagent.

[0473] 178. The method of any one of embodiments 175 to 177, further comprising washing the first microparticles and the second microparticles to remove unattached reagents before mixing them together in step (g).

[0474] 179. The method of any one of embodiments 175 to 178, wherein the first microparticles and the second microparticles are polystyrene microbeads.

[0475] 180. The method of any one of embodiments 175 to 179, wherein the first capture reagent, the first detection reagent, the second capture reagent, and the second detection reagent are antibodies.

[0476] 181. A method according to any one of embodiments 175 to 180, wherein the first detection reagent is connected to a first hook chain, the second detection reagent is connected to a second hook chain, and the first detection reagent and the second detection reagent are attached to their corresponding microparticles by connecting their corresponding hook chains to a first anchor chain and a second anchor chain respectively, and the first anchor chain and the second anchor chain have been attached to their corresponding microparticles in advance.

[0477] 182. A method according to any one of embodiments 175 to 181, wherein the method is used to prepare a multiplex sandwich assay system as described in any one of embodiments 80 to 110.

[0478] 183. A method for preparing a multiplex sandwich assay system, the method comprising:

[0479] (j) providing a carrier, wherein the carrier is a flat surface, a multi-well plate surface, a glass slide surface or a hydrogel;

[0480] (k) attaching a first capture reagent to the carrier at a first location;

[0481] (1) washing the support to remove unattached first capture reagent;

[0482] (m) attaching a second capture reagent to the support at a second location;

[0483] (n) washing the support to remove unattached second capture reagent;

[0484] (o) attaching a first detection reagent to the carrier via a first anchor chain attached to the carrier at the first position;

[0485] (p) washing the carrier to remove unattached first detection reagent;

[0486] (q) attaching a second detection reagent to the carrier via a second anchor attached to the carrier at the second location; and

[0487] (r) washing the carrier to remove unattached second detection reagent;

[0488] Such that whenever the first capture reagent, the second capture reagent, the first detection reagent, and / or the second detection reagent are mixed together, no more than one reagent is not attached to the carrier at a time.

[0489] 184. A method according to embodiment 183, wherein steps (b)-(c), (d)-(e), (f)-(g) and (h)-(i) can be performed in any order.

[0490] 185. A method according to embodiment 183 or 184, wherein the first capture reagent, the first detection reagent, the second capture reagent and the second detection reagent are antibodies.

[0491] Example

[0492] The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the invention and should not be construed as limiting the scope thereof in any way.

[0493] Unless otherwise defined or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0494] Example 1: One-pot microbead barcoding and CLAMP production

[0495] In some embodiments, a multiplexed assay system is implemented on spectrally encoded microbeads, where a one-pot microbead barcoding strategy and automated decoding method can be used in the methods and systems provided herein. Examples of such barcoding / decoding methods are described in US Patent Application No. 16 / 153,071 and Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925–932, 2018, the contents of each of which are incorporated herein by reference in their entirety. Such methods use an accurate model of fluorophore spectral overlap and multicolor Forster resonance energy transfer (FRET). For example, this strategy can use two lasers for barcoding and a third laser for analytical readout to process more than 580 barcodes (such as Figure 3 A). Cytometers with infrared lasers have the potential to expand this capacity to more than 5,000 barcodes.

[0496] The same manufacturing process is used to construct a version of the co-localization antibody assay described herein. That is, in the first step, streptavidin microbeads are co-coupled with biotinylated capture antibodies and biotinylated anchor or capture oligonucleotides modified with different dyes to produce distinguishable barcodes. Each barcode and target-specific antibody is made in a separate test tube. In the second step, the detection antibody (monoclonal) conjugated to the hook oligonucleotide is added to the corresponding functionalized microbeads in the first step. The hook oligonucleotide is complementary to the anchor oligonucleotide and hybridizes with it, resulting in the assembly and co-localization of matched antibody pairs. The microbeads can be stored separately for later use.

[0497] In some embodiments, low oligonucleotide:antibody conjugation ratios or valencies and / or two-step purification can be used to optimize (i.e., lower) background signal. For example, low-valency antibody-oligonucleotide conjugates have been shown to maximize CLAMP strand displacement efficiency and minimize background signal ( Figure 3B). After the first round of optimization (varying ionic strength, washing and incubation time, nanoscale design, and reagent concentrations), 1-plex CLAMP showed a 3-fold improvement in sensitivity to uPA compared to the conventional sandwich assay ( Figure 3 C). Implementation of monovalent rather than hypervalent conjugates resulted in a further 3-fold improvement ( Figure 3 D).

[0498] In one embodiment, the CLAMP system described herein comprises the following components:

[0499] 1) Microparticles, which hold all other components in place;

[0500] 2) a capture antibody (cAb) covalently coupled to the microparticle;

[0501] 3) a detection antibody (dAb) covalently linked to the hook oligonucleotide, wherein the detection antibody recognizes the same antigen as the cAb but not the same epitope;

[0502] 4) Anchor oligonucleotide (AO), which is attached to the microparticle, for example, via a streptavidin / biotin interaction;

[0503] 5) a stem oligonucleotide (SO) which is fully or partially complementary to AO, thereby making it at least partially double-stranded;

[0504] 6) a hook oligonucleotide (HO) that is covalently linked to the cAb and is partially complementary to the anchor oligonucleotide, thereby attaching the cAb to the particle; and

[0505] 7) Replacement oligonucleotide (DO), which has two functions:

[0506] a) Contains fluorescent tags and

[0507] b) have a sequence that is complementary to HO and overlaps with the sequence of AO, allowing for release of the dAb from AO and hence from the microparticles.

[0508] assembled a 5-plex CLAMP using antibodies that were highly cross-reactive in conventional sandwich immunoassays and confirmed that CLAMP completely avoided cross-reactivity ( Figure 3 E). Figure 3 F shows the standard curve for 5-plex ClAMP.

[0509] CLAMP was used to analyze human serum. The conjugated antibody and barcoded beads were stored independently for >1 month, and CLAMP obtained good recovery of PSA spike in serum (data not shown).

[0510] In one embodiment, the CLAMP system described herein is a 10-plex cytokine plate. The cytokines included herein are, for example, but not limited to, IL1 to IL17, MCP1 / 3, TNF, EGF / R, and / or VEGF / R. In another embodiment, the CLAMP system included herein is a 10-plex plate focused on breast cancer metastasis (for example, but not limited to HER2, CEA, p53, and / or CA15-3).

[0511] Example 2: CLAMP assay structure.

[0512] According to one embodiment, we prepared and tested a colocalization-ligation assay on microparticles (MPs) called "CLAMP". CLAMP is a versatile assay designed to eliminate reagent-driven cross-reactivity ("rCR") by colocalizing and confining each antibody pair on a set of barcoded MPs, thereby avoiding interactions between non-homologous antibodies ( Fig.14 C). Oligonucleotides (oligos) serve as programmable building blocks to implement the key molecular “operations” of CLAMP, including (i) flexible attachment of detection antibodies (dAbs), (ii) on-demand release of dAbs, (iii) transduction of assay signals, and (iv) fluorescent barcoding of MPs. Here, we detail the conceptual operation, experimental validation, and optimization of the CLAMP assay and demonstrate its efficacy in eliminating rCR using reagents that strongly cross-react in traditional MSAs.

[0513] exist Fig.14 The structure and working principle of one embodiment, referred to herein as the CLAMP assay, are schematically illustrated in panels d and e, respectively. To colocalize each pair of antibodies, an 82nt hook strand oligonucleotide (referred to as the hook oligonucleotide, or "HO") is covalently bound to a detection reagent that is an antibody (referred to as a detection antibody, or "dAb"), and partially hybridized to a capture strand oligonucleotide through a 21bp hybrid, referred to as a capture oligonucleotide, which is bound to the surface of a capture reagent that is an antibody, referred to as a capture antibody ("cAb"), coated microparticle ("MP"). However, the cAb is immobile on the surface, but the dAb is flexible due to the 61nt single-stranded domain of the HO; this flexibility allows the formation of a triple complex with the analyte ( Fig.14E). The restriction of antibody pairs excludes interactions between mismatched antibodies and restores a single-plex assay configuration on each MP, ensuring that single cross-reaction events (e.g., target analyte reacting with a non-cognate cAb) do not result in sandwich binding. A priori co-localization of antibodies allows rapid dual recognition of proteins, but requires a concomitant method for signal transduction and generation. One approach is to first break the HO-CO linkage. For example, by photoinduced or enzymatic cleavage, or bottom clamp-mediated displacement, and then, after washing the released dAb-HO complex, label the dAb remaining on the surface to indicate sandwich formation. However, continuous CO-HO linkage will result in labeling of the same dAb regardless of the presence of target analyte, thereby increasing the background signal. For example, 2% dAb coverage on a 3 μm MP is equivalent to 1000-5000 dAb, which, if labeled, may result in a large increase in background signal and severely hinder sensitive detection.

[0514] To mitigate this effect in the CLAMP assay, we designed a detection scheme to exclude only “successfully” released conjugates by using a fluorescently labeled displacer oligonucleotide (DO) that binds to the bottom-clamped domain on HO, simultaneously displacing and labeling it ( Fig.14 E. Figure 7 Importantly, this “displacement-detection” is a logic gate that requires dual capture by the protein and release by the dAb to detect a signal ( Fig.14 F). In this embodiment, CLAMP reagents are assembled on magnetic MPs in two steps, benefiting from the affinity of biotin-streptavidin bonds and Watson-Crick base pairing ( Fig.18 In the first step, a mixture of biotinylated oligonucleotides and antibodies are co-immobilized onto the surface of streptavidin-coated MPs. The one-step nature of the labeling provides precise control over the CO surface density ( Fig.19 ), while allowing MP encoding by one-pot labeling using multicolor sorting dyes, as described elsewhere (Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925-932, 2018). In the second step, the dAb-HO complex is pulled down by HO-CO hybridization to complete the assembly of CLAMP.

[0515] Example 3: CLAMP assay optimization.

[0516] We first optimized the efficiency of the bottom clamp-mediated displacement reaction by displacing unconjugated Cy5-labeled HO ( Fig.15). It was pulled down with MPs with different CO concentrations and then released with unlabeled DO. The increased ionic strength in the displacement buffer (MNaCl>500mM) facilitated the screening of negatively charged oligonucleotides and increased the efficiency of hybridization and release of DO from HO. With the increase of ionic strength and dissolved oxygen concentration (MNaCl~500mM and MDO=1μM), the displacement rate reached 98% over a wide range of CO density.

[0517] Next, we investigated the effect of antibody-oligonucleotide conjugates on the assay background by measuring the residual signal on the MP after a label displacement step in buffer (see Methods below). We first conjugated HO to immunoglobulin G (IgG) using a commercial kit (Solulink), yielding approximately 90% antibody conjugates with an average of 2 HO per IgG (i.e., λ ∼ 2). Using these conjugates, the assay background was an order of magnitude greater than that of the unbound HO ( Fig.16 A). The increase in background signal is due to the multivalent HO conjugate, which results in the non-release of the dAb-HO complex (due to the unbroken HO-CO linkage), which is labeled by hybridization of DO to at least one other HO chain, thus generating a fluorescent signal in the absence of a sandwich bound to a protein ( Fig.16 B). An effective way to minimize multivalent dAb-HO conjugation is to reduce its average conjugation valency, for example, targeting a lambda of 0.1, Poisson statistics indicate that <5% of dAbs will bind to multiple HOs. The cost of such a low conjugation valency is a decrease in antibody binding rate (10%), which leaves 90% of the antibody unreacted. To avoid wasting unreacted antibody, we developed a conjugation and purification workflow that preserves the unbound antibody in its native state and allows its recycling. The relative concentrations of dAb and HO were adjusted and adjusted from 1.25 to 0.1 ( Fig.16 C). On MP with different CO concentrations, different valence states of dAb-HO conjugates were obtained. As expected, the low valence state significantly reduced the residual assay background, which was consistent with 0.1<λ<0.2 ( Fig.16 D), resulting in less than 8% conjugates in the low-valent state for the multivalent conjugate. Consistent with the multivalency, increasing CO concentrations amplified the high background signal in the high-valent state of dAb-HO.

[0518] To optimize the assay performance, we adjusted the dAb-HO density. In CLAMP, the appropriate local dAb concentration is the key to sensitive and high-capacity sandwich binding, which at a certain HO length depends mainly on the surface density of dAb-HO and the surface density of CO capture by hybridization. CLAMP with different CO densities of anti-urokinase plasminogen activator (anti-uPACLAMP) was prepared using low-valent dAb-HO conjugates and multivalent conjugates with less than 8% valence. Fig.16 D; see Methods below). Anti-uPA CLAMPs were incubated with serial dilutions of recombinant uPA antigen, then washed and probed by label displacement. As expected, increasing CO concentration modulated the signal-to-noise ratio (SNR) of the assay, revealing a 10 14 m -2 The density of is necessary for sufficient SNR ( Fig.16 E) On the other hand, greater than 10 14 m -2 The density of the conjugates hardly improves the signal-to-noise ratio, as they also lead to an increase in background signal. Finally, to evaluate the importance of the conjugate valency on the assay performance, we compared high-valency (λ~2, Solulink) and low-valency conjugates (λ~=0.1, Fig.16 F) of anti-uPA. The low-cost conjugate significantly reduced the background signal (10-fold), and accordingly, the detection limit was improved by 3-fold ( Fig.16 F). On the other hand, since sandwich-bound dAb-HO conjugates are primarily labeled with a single dye, the fluorescence dynamic range of low-valent conjugates is reduced. In summary, these results emphasize the importance of conjugate valency on background signal and assay performance.

[0519] Example 4: Multiplex CLAMP Assay

[0520] To test the efficacy of CLAMP in eliminating reagent-driven cross-reactions ("rCR"), we screened the assay specificity of multiple CLAMP according to one embodiment. In addition, to challenge the CLAMP assay, we selected antibody pairs that showed different types of rCR when used together in a traditional multiple sandwich assay ("MSA"). To this end, antibody pairs for 6 targets (EpCAM, PSA, E-cadherin, EGF, uPA, and MCP) were screened from our previous 35 protein groups for specific and non-specific binding in traditional MSA (Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925–932, 2018). For traditional MSA, specificity screening involves incubating each individual antigen with a pool of cAb-coated barcoded MPs, and then adding a mixed dAb cocktail and a secondary antibody ("sAb") for detection and labeling, respectively ( Fig.17 A). The fluorescence of different barcodes was measured in response to antigen concentrations of 1 ng / mL and 100 ng / mL ( Fig.17 BC), two types of nonspecific binding that produce false positives were found, namely indiscriminate adhesion of antigens (observed for E-cadherin and uPA) and cross-reactivity between antigens and antibodies. On the other hand, specificity screening of CLAMP assays is performed by incubating a single antigen at a time with multiplex CLAMP and detecting by label displacement ( Fig.17 d- Fig.17 f; see Methods below). All but one of the nonspecific signals detected in the traditional MSA were completely eliminated using the CLAMP assay. For example, in the traditional MSA, general, nonspecific binding of E-cadherin (resulting in signal on all nontargeted beads) was undetectable in the CLAMP assay. In contrast, cross-reactivity was detectable between the MCP-1 antibody and the EGF antigen at 100 ng / mL, both in the traditional MSA and CLAMP. To investigate the source of the false-positive signals, we performed a singleplex assay using only the MCP-1 antibody and added MCP-1 or EGF at concentrations of 1 ng / mL or 100 ng / mL, respectively. Fig.17 G). Single MCP-1 antibody detection of EGF shows dCR. In fact, this dCR cannot be mitigated by CLAMP or ELISA and is a weak affinity binder. Overall, these results show the ability of CLAMP to eliminate rCR in multiplexed assays and to identify dCR in a multiplexed, combinatorial manner. Finally, dilution curves of the remaining 5 proteins were plotted and their SNRs are plotted, as shown in Figure 2. Fig.17 As shown in H.

[0521] In summary, we have successfully demonstrated the use of CLAMP, a homogeneous MSA that uses oligonucleotides to pre-colocalize antibody pairs on MPs. By confining each antibody pair to its corresponding MP during sample incubation, multiplexing can be achieved while maintaining a singleplex assay environment on each MP, which eliminates reagent-driven CRs. Notably, the pre-colocalization of antibodies in CLAMP represents a departure from traditional sandwich immunoassays where the matched antibodies are separate at the start of the assay. To detect correct sandwich binding, we have demonstrated that labeling with a displacer oligonucleotide can simultaneously release and label dAb complexes. We investigated and demonstrated the importance of using monovalent antibody-oligonucleotide conjugates to avoid labeling unreleased complexes and increasing background signal. We experimentally validated the approach, both in singleplex and multiplex, and screened for specificity of complex analysis using five pre-selected CR antibody pairs, demonstrating that CLAMP eliminates all rCRs in traditional MSAs.

[0522] CLAMP can offer several distinct advantages over currently available MSAs. First, CLAMP can be easily deployed as it does not require specialized equipment to read or introduce new workflows. Second, CLAMP can be a rapid assay as it can be completed in just over three hours. Finally, by eliminating the need to incubate detection antibodies in solution (which is typically performed at high concentrations), CLAMP can significantly reduce reagent consumption. Due to its high scalability and efficiency, CLAMP can be used to provide a truly scalable multiplex ELISA platform that can meet the growing needs of biomarker discovery and drug development.

[0523] Example 6: Low antibody concentration minimizes cross-reactivity in the CLAMP assay

[0524] Traditional multiplex sandwich immunoassays are usually performed in solution phase using a reagent mixture. Specifically, detection antibodies (dAbs) against different targets are mixed together and applied to the reaction. The application of such a dAb mixture leads to pseudo-binding and generates false positive signals from non-specific binding events (between cAbs or dAbs and non-targeted analytes) that are difficult to distinguish from true target protein binding signals. The risk of reagent-driven CR is ~4N 2 , the number of target analytes is N.

[0525] In contrast, in embodiments of CLAMP, reagents (e.g., antibodies) pairs can be pre-assembled and co-localized on barcoded microparticles to avoid reagent mixing. As described herein, the detection antibody (dAb) is released in solution only after the displacement reaction. In some embodiments, to avoid rebinding of off-target microbeads after the displacement reaction. The dAb released into solution is still optimally kept at a sufficiently low concentration. Fig.16 The dAb concentration profile is depicted relative to the starting amount (y-axis) and volume of solution during the displacement step. Typical dAb concentrations in traditional ELISAs are ~1 μg / mL (67 nM), and binding can still occur with dAbs as low as 1 nM (e.g. in the Simoa assay by Quanterix) given sufficiently long incubation times.

[0526] To ensure that unbinding is avoided after release, the amount of antibody for each target should ideally be kept at <10 pM. In a volume of 100 microliters, the amount of antibody is <1 molar. In the CLAMP assay, in some embodiments, the amount of Ab released from 1000 microparticles is estimated to be 0.1-1 femtomolar ( Fig. 22 ). These figures indicate that the CLAMP system releases dAb at significantly lower concentrations compared to other methods that require free diffusion-based reagent mixing.

[0527] Example 7: Displacement-dependent signaling minimizes background signal in CLAMP assays

[0528] In one embodiment of the CLAMP colocalization assay, both antibodies are pre-colocalized on a carrier, and signal transduction can be performed by detecting all dABs remaining on the surface after release and washing. However, any unreleased hook oligonucleotide-dAb complex may produce a signal unrelated to the analyte, which significantly contributes to background noise. Therefore, it will be appreciated that in order to avoid increasing background signals, in some embodiments, nearly complete anchor-hook displacement and cleaning of the hook oligonucleotide-dAB complex are required.

[0529] In some embodiments, the problem of increased background signal due to inefficient release can be solved by a displacement-dependent signal transduction mechanism. This mechanism will ensure that only the replaced hook-anchor strand is detectable, so that non-positional replaced strands that may occur due to ineffective displacement will not generate background signal. In such embodiments, molecular-level signal transduction will only occur when the following two conditions are met: (i) formation of the ternary complex and (ii) displacement of the hook-anchor strand.

[0530] Thus, in some embodiments, the detection Ab and hook strand are unlabeled, and displacement occurs using a labeled (e.g., fluorescently labeled) displacer oligonucleotide. In this embodiment, the displacer oligonucleotide can bind to the hook strand, which preferably (i) releases it from the anchor strand and (ii) labels it. On the other hand, the non-displacing hook oligonucleotide is unlabeled and does not contribute to the signal. This mechanism is equivalent to an AND gate, where the signal (output) depends on the presence (input 2) of the displaced (input 1) analyte, e.g. Fig.23 Shown

[0531] To demonstrate the effectiveness of displacement-dependent signaling, we performed calibration assays for IL-7, IFN-γ, and MMP-9. In the first experiment, the displacer oligonucleotide was unlabeled and the mouse dAbs were targeted with an anti-mouse BV421 secondary antibody. The BV421-labeled secondary antibody targets the dAb regardless of release, so labeling occurs regardless of displacement. In the second experiment, the displacer oligonucleotide was labeled with Cy5, which tested displacement-dependent signaling. As shown in the logic gate representation diagram ( Fig.23 ), BV421 signal will be introduced in conditions (i), (iii), and (iv), but Cy5 signal will only appear in condition (iv). Fig.24 Exemplary target calibration curves obtained by using these two labeling methods are shown. The signal background of BV421 is significantly increased compared to the Cy5 signal, while the assay performance in terms of sensitivity and dynamic range improves with increasing label displacement (Cy5).

[0532] Example 8: Low-valent antibody-oligonucleotides minimize background signals in CLAMP assays

[0533] In some embodiments, the hook and anchor chains are DNA oligonucleotides. Antibody-DNA conjugation can be performed, for example, by targeting lysine groups on IgG molecules. Heterobifunctional linkers such as sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfoSMCC) can be used to connect sulfhydryl-terminated DNA to IgG molecules. However, this reaction produces a heterogeneous conjugate in which the number of oligonucleotides per antibody depends on the DNA: antibody stoichiometry during the reaction. Multivalent conjugates (more than one oligonucleotide per antibody) can reduce displacement efficiency, thereby increasing background signal.

[0534] like Fig.25 As shown in AB, we adjusted the binding valency and measured it by SDS-page, and used the resulting conjugates to measure the displacement efficiency. High-valency conjugates lead to increased assay background. As expected, higher anchor oligonucleotide density leads to further increase in background signal. To determine the impact of high conjugate valency on signal background, we performed a CLA assay using a displacement-dependent detection mechanism ( Fig.25 C). uPA calibration curves were generated using high valency (λ, avg, number of oligonucleotides per antibody ~2) and low valency conjugates (λ ~0.1). The lower valency resulted in lower background and a 3-fold increase in sensitivity.

[0535] Example 9: Characterization of 40-plex cross-reactivity

[0536] To assess cross-reactivity in multiplexed assays with higher multiplexing, a panel of 40 targets was tested in which CLAMP cocktails against 40 targets (eg, Fig.26 ) were mixed together and incubated in buffer to which a high concentration (100 ng / mL) of target (protein standard, typically recombinant) was added to each well. Signals on the diagonal line indicate specific interactions between the correct antigen and its barcoded particle pair. Only a few off-target signals were measurable; however, these were not due to cross-reactivity of the reagents. Instead, the antigen is thought to cross-react with both antibodies and thus may also be evident in single-plex ELISAs, such as Fig.17 shown.

[0537] method

[0538] Materials and reagents. HPLC-purified oligonucleotides were purchased from IDT (Coralville, IA, USA); sequences and modifications were as follows Fig.11cAb, antigens and dAb were purchased from RnD Systems (Minneapolis, MN, USA) and stored at -20°C for up to 36 months. Streptavidin and protein G magnetic MP (M270) were purchased from Life Technologies (Carlsbad, CA, USA).

[0539] Synthesis of CLAMP. CLAMP was assembled in two steps on a magnetic MP with a diameter of 2.7 μm (M270 streptavidin). The first step included immobilizing a biotinylated mixture of antibodies and oligonucleotides to functionalize the MP and simultaneously encode it, as described elsewhere (Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925–932, 2018). Briefly, 90 pmol of biotinylated oligonucleotides (CO and SO) and a total of 90 pmol of LO (LO0-LO2) were mixed in 25 μL PBS + 0.05% Tween 20 + 300 mM NaCl (PBST0.05 + NaCl300). However, the ratio of each LO0: LO1: LO2 was designed to produce a unique collective fluorescence to define the barcode, while the ratio of CO: SO allowed the surface density of the pulled dAb-HO to be adjusted. The mixture was annealed by heating to 80 ° C and cooled to room temperature by removing the mixture from the heat source. Next, 5 μg of biotinylated cAb was added to 17 μL PBST0.05+NaCl300 and mixed with the annealed oligonucleotide mixture. The biotinylation reagent was then immobilized on the MP in a single step by adding 3.25M MP in 10 μL PBST0.05+NaCl300 and immediately mixing by pipette. The mixture was incubated at room temperature with end-to-end mixing for 90 minutes and then washed 3 times by magnetic accumulation in 150 μL PBST0.1. The barcoded and functionalized MPs were stored at 4$C until needed. In the second step, 100,000 copies of the prepared MPs were mixed with a HO solution (e.g., dAb-HO) diluted in PBST0.05+NaCl300 for 30 minutes. After pulling down HO, the fully assembled CLAMP was washed 3 times in PBST0.01 and stored at 4°C until the assay time for up to one week.

[0540] Characterization of CLAMP. To characterize CLAMP, the immobilization of antibodies and oligonucleotides was confirmed by labeling with anti-goat IgG conjugated with Alexa-Fluor647 (AF647) or by hybridization with oligonucleotides (LO) labeled with Cy5 targeting HO. As described elsewhere, the density of CO was estimated by fitting the overall fluorescence response of multicolor MP using a multicolor fluorescence model (Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925–932, 2018). In order to determine the expected measurement background signal of a specific CLAMP group, MP was incubated with 1 μM Cy5-labeled DO in PBST0.05+NaCl300 for 1 hour, followed by 3 magnetic washes in PBST0.05, and the residual signal was measured by cytometry.

[0541] Antibody Oligonucleotide Conjugation, Purification, and Characterization. Anti-uPA monoclonal antibodies were conjugated to amine-modified HO using hydrazone chemistry (Solulink) and then purified according to the manufacturer's protocol. Alternatively, monoclonal antibodies were conjugated to thiol-terminated HO using a heterobifunctional amine / thiol-reactive crosslinker. 40 μL of 30 μM thiol-modified HO was first reduced in 200 mM dithiothreitol (DTT) in PBST at 37°C for 1 hour. The reduced oligonucleotides were (i) buffer exchanged into PBS pH 7.0 using Zeba desalting spin columns (7K MWCO, Thermo), (ii) activated for 10 min using 8 μL of 9 mM sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfoSMCC), dissolved in 80% PBS pH 7.0 and 20% anhydrous dimethyl sulfoxide, (iii) buffer exchanged again into PBS pH 7.0 to remove excess sulfoSMCC, (iv) 1-10 μL portions (depending on the need) were reacted with 10 μL of 1 mg / mL antibody. The reaction was left at room temperature for 1 hour and then incubated overnight at 4 degrees Celsius. The conjugate was then purified in two separate steps, antibody and DNA purification.

[0542] Antibody oligonucleotide conjugation, purification and characterization. Anti-uPA monoclonal antibodies were conjugated to amine-modified HO using hydrazone chemistry (Solulink) and then purified according to the manufacturer's protocol. Alternatively, monoclonal antibodies were conjugated to sulfhydryl-terminated HO using a heterobifunctional amine / thiol-reactive crosslinker. 40 μL of 30 μM thiol-modified HO in PBST was first reduced in 200 mM DTT at 37 °C for 1 hour. The reduced oligonucleotides were (i) buffer exchanged into PBS pH 7.0 using Zeba desalting spin columns (7KMWCO, Thermo), (ii) activated for 10 min using 8 μL of 9 mM sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfoSMCC), dissolved in 80% PBS pH 7.0 and 20% anhydrous dimethyl sulfoxide, (iii) buffer exchanged again into PBS pH 7.0 to remove excess sulfoSMCC, (iv) 1-10 μL portions (depending on the desired valency) were reacted with 10 μL of 1 mg / mL antibody. The reaction was left at room temperature for 1 hour and then incubated overnight at 4°C. The conjugates were purified by two purification steps.

[0543] Single and multiplex CLAMP assays. Incubation was performed in a conical bottom 96-well plate at room temperature with horizontal shaking at 950 rpm. CLAMP was mixed at approximately 80 MP per barcode per μL and blocked with PBST0.05+NaCl150+0.5% BSA (PBST0.05+NaCl150+BSA0.5) for 30 minutes. A 25 μL aliquot of the blocked multiplex CLAMP mixture was added to each well and incubated with 25 μL of a solution containing the specific antigen at 2 times the concentration specified in PBST0.05+NaCl150+BSA0.25, incubated at 950 rpm for 3 hours. Magnetic aggregation and washing were performed with 150 μL of PBST0.1, repeated 4 times within 30 minutes. Finally, detection-displacement was performed by adding 1 μM DO-Cy5 in PBST0.05+NaCl300+BSA0.25 and incubating for 1 hour with shaking, followed by washing 3 times in PBST0.1.

[0544] Traditional MSA. To screen for specific and nonspecific binding in the traditional MSA format, MPs were barcoded and bound to their respective biotinylated cAbs during synthesis. For each assay, the MP mixture was combined to a final concentration of 2000 MPs per barcode. Incubation was performed in a conical bottom 96-well plate at room temperature with horizontal shaking at 950 rpm. Prior to incubation with the assay reagents, the MPs were first blocked in PBS for one hour with a 1% bovine serum albumin and 0.05% Tween 20 solution (PBST0.05). Incubate for 120 minutes at the specified antigen concentration. BMPs were incubated with the dAb mixture at 2 μg / mL for 60 minutes and then incubated with sAbs at a concentration of 4 μg / mL for 45 minutes. SNRAg was calculated by subtracting the cAb-specific mean assay background (n=6) from the MFI signal and normalizing to the global standard deviation of the assay background (i.e., across all barcodes, n=210).

[0545] Read out and perform data analysis. MP was read by BD FACS-CANTO-II cytometer with blue (488nm), red (633nm) and violet (405nm) lasers. In the blue laser flow cell, FAM and Cy3 used 530 / 30 and 585 / 42 bandpass filters, respectively. In the red laser flow cell, Cy5 / AF647 used 660 / 20 bandpass filters, respectively. MP was decoded using an automatic algorithm implemented on MATLAB (Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925–932, 2018). All data analysis was performed in MATLAB. Forward scattering intensity and side scattering intensity were used to distinguish single particles from microbead aggregates and other particles, and gating automation was achieved.

[0546] While the invention has been described in conjunction with specific embodiments thereof, it will be understood that the invention is capable of further modifications and this application is intended to cover any changes, uses or adaptations including departures therefrom that come within known or customary practice in the art and departures from the essential characteristics herein above described and which fall within the scope of the appended claims as follows.

[0547] The contents of all documents and references cited herein are incorporated by reference in their entirety.

Claims

1. A biomolecule complex for detecting and / or quantifying an analyte in a sample, the biomolecule complex comprising: a) an anchor chain, said anchor chain being attached to a carrier; b) a capture agent attached to the carrier ； and c) a detection reagent, the detection reagent being releasably attached to the anchor, the detection reagent or the anchor being optionally attached to a first tag, the first tag being inactive or undetectable; in: The capture reagent and the detection reagent can bind to the analyte (if present in the sample) simultaneously to form a triple complex; In the absence of the analyte, releasing the detection agent from the anchor may release the detection agent from the carrier; and When the detection reagent is released from the anchor, the first tag can be activated or detected. 2 . The biomolecule complex according to claim 1 , wherein the first label is detected on the carrier only when the analyte is present. 3 . The biomolecule complex according to claim 2 , wherein the amount of the first label on the carrier when the detection reagent is released from the anchor chain is proportional to the amount and / or concentration of the analyte in the sample.

4. The biomolecule complex according to any one of claims 1 to 3, wherein the detection reagent is directly releasably attached to the anchor chain by covalent bonds, biotin-streptavidin bonds, hydrogen bonding, hydrophobic interactions, affinity binding or non-covalent interactions.

5. A molecular biocomplex for detecting and / or quantifying an analyte in a sample, the molecular biocomplex comprising: a) an anchor chain, said anchor chain being attached to a carrier; b) a capture agent attached to the carrier; and c) a detection reagent connected to a hook chain, the hook chain being releasably attached to the anchor chain, the hook chain and the anchor chain being connected together by a double-stranded DNA hybrid; d) a displacing agent comprising a DNA oligonucleotide that is complementary to at least a portion of the hook strand and capable of hybridizing to the hook strand, thereby releasing the hook strand from the anchor strand by a DNA displacement reaction, the displacing agent being detectably labeled; in: The capture reagent and the detection reagent can bind to the analyte (if present in the sample) simultaneously to form a triple complex; and In the absence of the analyte, release of the hook chain from the anchor chain by the displacing agent may release the detection reagent from the carrier.

6. A multiplex sandwich assay system for simultaneously detecting and / or quantifying two or more analytes in a sample, the system comprising: a) a first biomolecule complex for detecting and / or quantifying a first analyte in the sample, the first biomolecule complex comprising a first anchor, a first capture reagent and a first detection reagent optionally linked to the first hook, the first detection reagent or the first hook being releasably linked to the first anchor, at least one of the first detection reagent and the first hook being optionally attached to a first tag, wherein the first capture reagent and the first detection reagent can simultaneously bind to the first analyte (if present in the sample) to form a first triple complex; and b) a second biomolecule complex for detecting and / or quantifying a second analyte in the sample, the second biomolecule complex comprising a second anchor, a second capture reagent and optionally a second detection reagent linked to the second hook, the second detection reagent or the second hook being releasably linked to the second anchor, at least one of the second detection reagent and the second hook being optionally attached to a second tag, wherein the second capture reagent and the second detection reagent can simultaneously form a second triple complex with the second analyte (if present in the sample); in, The first anchor chain, the first capture agent, the second anchor chain, and the second capture agent are all attached to a carrier; Wherein, the first tag and the second tag are inactive or undetectable, and can be activated or detected when their corresponding detection reagents or hooks are released from their corresponding anchors.

7. A method for detecting and / or quantifying an analyte in a sample, the method include: a) providing a carrier, a capture agent attached to the carrier, an anchor attached to the carrier, and a detection agent optionally attached to the shackle, wherein the detection agent or the shackle is releasably attached to the anchor, and wherein at least one of the detection agent and the shackle is optionally attached to a first tag, the first tag being inactive or undetectable; b) contacting the support with the sample under conditions that allow the capture reagent and the detection reagent to bind to the analyte simultaneously to form a triple complex; and c) adding a displacing agent optionally attached to a second tag, wherein the displacing agent releases the detection reagent or the hook chain from the anchor chain to release the detection reagent optionally linked to the hook chain from the carrier in the absence of the analyte, and wherein the release of the detection chain or the hook chain from the anchor chain activates the first tag or makes the first tag detectable.

8. A method for detecting and / or quantifying an analyte in a sample, the method comprising: include: a) providing a carrier, a capture agent attached to the carrier, an anchor chain attached to the carrier, and a detection agent connected to the hook chain, wherein the hook chain is releasably connected to the anchor chain through a double-stranded DNA hybrid; b) contacting the support with the sample while allowing the capture reagent and the detection reagent to bind to the analyte simultaneously to form a triple complex; and c) adding a displacing agent attached to a detectable label, wherein the displacing agent is a DNA oligonucleotide complementary to at least a portion of the hook strand and capable of hybridizing to the hook strand, wherein the displacing agent releases the hook strand from the anchor strand by a DNA displacement reaction to release the detection reagent from the carrier in the absence of the analyte; and d) optionally determining the presence and / or amount of the detectable label on the carrier, wherein the presence of the label on the carrier indicates the presence of the analyte in the sample and the amount of the label is proportional to the amount and / or concentration of the analyte in the sample.

9. A method for preparing a multiplex sandwich assay system, the method include: (a) providing a first container containing first microparticles, wherein the first microparticles are encoded with a first barcode; (b) attaching the first microparticle to a first capture reagent and a first detection reagent, wherein the first capture reagent and the first detection reagent are capable of binding to a first analyte simultaneously; (c) optionally, storing the first particles; (d) providing a second container containing second microparticles, wherein the second microparticles are encoded with a second bar code; (e) attaching the second microparticle to a second capture reagent and a second detection reagent, wherein the second capture reagent and the second detection reagent are capable of binding to a second analyte simultaneously; (f) optionally, storing the second particles; (g) mixing the first microparticles and the second microparticles together for use in the multiplex sandwich assay system; The first capture reagent and the first detection reagent are not mixed with the second capture reagent and the second detection reagent before being attached to their corresponding microparticles.

10. A method for preparing a multiplex sandwich assay system, the method include: (a) providing a carrier, wherein the carrier is a flat surface, a multi-well plate surface, a glass slide surface or a hydrogel; (b) attaching a first capture reagent to the support at a first location; (c) washing the support to remove unattached first capture reagent; (d) attaching a second capture reagent to the support at a second location; (e) washing the support to remove unattached second capture reagent; (f) attaching a first detection reagent to the carrier via a first anchor chain attached to the carrier at the first position; (g) washing the carrier to remove unattached first detection reagent; (h) attaching a second detection reagent to the carrier via a second anchor attached to the carrier at the second location; and (i) washing the carrier to remove unattached second detection reagent; Such that whenever the first capture reagent, the second capture reagent, the first detection reagent, and / or the second detection reagent are mixed together, no more than one reagent is not attached to the carrier at a time.

Images