Co-localization Sandwich Assay by Linkage

Abstract

Methods and systems for detecting and / or quantifying analytes are provided. In particular, methods and systems for simultaneous detection and / or quantification of two or more analytes in a sample are provided. In some embodiments, a colocalization assay (CLAMP) is provided that includes two sets of binding agents preassembled on a support such that the two sets of binding agents are colocalized before contacting the sample. [Selected Figure] Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 651,943, filed on Apr. 3, 2018, entitled “Colocalization - by - Linkage Sandwich Assays”, the entire content of which is incorporated herein by reference.

[0002] Field The present invention relates to the field of biological analysis, and more particularly, to systems and methods for detecting and / or quantifying biomolecules using a colocalization - by - linkage sandwich assay, and to multiplex sandwich assays for the simultaneous detection and / or quantification of multiple biomolecules in a sample.

Background Art

[0003] The 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 various fields such as genomics, proteomics, diagnostics, therapeutics, and pathological research. For example, the rapid and accurate detection of specific antigens and viruses is critically important for combating pandemic diseases such as AIDS, influenza, and other infectious diseases. The maturation of genomic technologies and the progress of personalized medicine require faster and more sensitive assays for detecting and quantifying multiple cells and biomolecules. The progress of medical research will increasingly rely on accurate, timely, and cost - effective assessment of multiple proteins via proteomics. However, current automated, high - sensitivity, and low - cost assays cannot be efficiently multiplexed.

[0004] The sandwich assay is one of the most common formats for biological assays. In this format, capture probe molecules are immobilized on a surface. Next, a biological sample containing the target cells or biomolecules of interest is applied to the surface. The target binds to the capture probe molecules immobilized on the surface in a concentration-dependent manner. In the next step, detection probe molecules are applied to the surface. The detection probe molecules bind to the target biomolecule, thereby "sandwiching" between the capture probe molecules and the detection probe molecules. In some assays, a secondary probe capable of binding to the detection probe molecules is also applied to the surface. The secondary probe can be conjugated to a label such as a fluorophore, in which case the binding can be detected using a fluorescence scanner or a fluorescence microscope. In some cases, the secondary probe is conjugated to a radioactive element, in which case the radioactivity is detected and read out as the assay result. In some cases, the secondary 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 of double recognition in the sandwich assay provides a high-fidelity signal with low background noise, and as a result, provides high-sensitivity detection.

[0005] Enzyme-linked immunosorbent assay (ELISA) is a well-known example of a sandwich assay. ELISA typically uses antibodies and color change reactions 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 a solid phase. ELISA is often performed in 96-well or 384-well polystyrene plates that passively bind antibodies and proteins. What makes the design and implementation of ELISA very easy is the binding and immobilization of reagents on this solid surface. By immobilizing reagents on the microplate surface, it becomes easy to separate the target biomolecules bound during the assay from unbound substances and wash away nonspecifically bound substances. Furthermore, the requirement for double recognition by both capture probe molecules and detection probe molecules provides high specificity. Thus, ELISA is a powerful tool for measuring specific target biomolecules in crude preparations.

[0006] Sandwich assays can be designed and assembled to measure or detect multiple analytes in parallel (also called multiplexing or multiplexed). Multiplexed sandwich assays (MSA) can be performed using microarrays such as DNA microarrays, protein microarrays, or antibody microarrays. A microarray is an aggregate of microscopic spots containing biomolecules attached to the surface of a substrate 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 multiple genes or proteins. Biomolecules such as DNA, proteins, or antibodies on the microarray chip are typically detected through the optical readout of fluorescent labels attached to probe molecules specifically attached or hybridized to target molecules. The labels used can consist of, for example, enzymes, radioisotopes, or fluorophores.

[0007] MSA can also be performed on particles. In this case, the particles suspended in solution are attached to the biomolecules necessary to capture the target of interest, such as proteins or specific DNA molecules. In order to perform assays in multiplexing, the particles must be encoded so that they can distinguish between the various assays in solution. A common format is spectrally encoded microparticles, which are encoded using fluorescent or luminescent dyes. The particles can also be encoded schematically and are thus often referred to as "barcoded particles". The particle size can range from nanometers (nanoparticles) to micrometers (microparticles). Among these, the fluorescently encoded microparticles can be read quickly and with high throughput on a cytometer.

[0008] However, current sandwich assays perform poorly when used to simultaneously measure multiple biomolecules in a sample (multiplexing). Multiplexed ELISA is limited by the cross-reactivity between reagents such as antibodies and proteins, and as a result, non-specific signal transduction is likely to occur. In conventional multiplexed sandwich assays in both array and bead formats, the detection antibodies are typically applied as a mixture, but this method causes interactions between reagents that constitute the negative element of cross-reactivity. Therefore, the application of the detection antibody mixture results in false binding and, for example, generates false positive signals from non-specific binding events between the capture and non-target analytes (shown here in Figure 1), which can be difficult to distinguish from the actual target protein binding signal. Such reagent-driven cross-reactivity is an inherent problem in MSA, scales quadratically with the number of targets, and severely limits the scale of multiplexing. Due to the problems associated with cross-reactivity, current MSA is generally limited to 30-40 targets. Nevertheless, long and costly optimization protocols are required to discover and remove cross-reactive reagents (e.g., antibodies), which severely limits the applicability of these assays and increases their costs.

[0009] Cross-reactivity also hinders other types of multiplex assays. For example, accurate protein phosphorylation analysis can be used to reveal cellular signaling events that are not apparent from protein expression levels. Current methods and workflows for quantifying the fraction of post-translational modifications (PTMs) of a specific protein are severely limited in multiplexing because PTM-specific antibodies often have insufficient specificity for the protein itself (i.e., fluorophore-specific antibodies are very sensitive to cross-reactivity problems driven by reagents). As a result, conventional PTM panels are not multiplexed.

[0010] Conventional sandwich immunoassays are also not suitable for analyzing protein-protein interactions. Protein-protein interactions are key parts of cellular processes, and understanding the modulators of these interactions is extremely important for addressing the associated diseases. However, the use of detection antibody mixtures can allow unwanted interactions and lead to false bindings that can obfuscate the interaction signals. Current multiplex sandwich assays are also costly because expensive reagents such as antibodies are inefficiently used during assay production and implementation. For example, the addition of an antibody mixture in solution requires high concentrations (nanomolar concentrations), while the amount required to bind to the protein for quantification on a microarray or microbead is three orders of magnitude less, which corresponds to a 99.9% loss of the antibody. Furthermore, the sensitivity of a given sandwich immunoassay is greatly affected by background signals often caused by non-specific binding of labeled detection antibodies and / or incomplete washing. Methods have been used to reduce incomplete washing by increasing the wash cycles and including additive reagents, but these methods result in increased assay time and assay complexity.

[0011] U.S. Patent No. 9,481,945 describes an Antibody Co - localization Microarray (ACM) that relies on the addressing of each capture antibody spot on a microarray by a single detection antibody, thereby avoiding interactions between antibody reagents and reproducing the assay conditions found in single - plex ELISA assays. The implementation of this method requires first spotting capture antibodies, removing the slide from the spotter, incubating it with a sample, washing and rinsing it as needed, and returning it for spotting of the detection antibody, followed by binding and incubation. Thus, this method relies on the transfer of n different reagents to n spots, each having a different reagent as well, representing an n - to - n transfer. The need to perform spotting as part of the assay is cumbersome and slow, and throughput is limited.

[0012] U.S. Patent No. 7,306,904 describes an assay for the detection and / or quantification of one or more analytes that is a solution using so - called proximity probes. Proximity probes contain a binding moiety and a nucleic acid. The nucleic acid from one proximity probe can interact with the nucleic acid from another proximity probe only when they are in proximity, i.e., when they are bound to the analyte to which they are specific. However, generally, multiplexed proximity - based assays require detection or readout in a single - plex format and thus require complex microfluidics to fractionate a sample into n fractions for an n - plex assay.

[0013] U.S. Patent Application Publication No. US 2016 / 0153973 describes a method and system for using a cleavable linker to detect analytes in immunoassays. However, this method and system are not suitable for the multiplexing or simultaneous detection of multiple analytes in highly sensitive immunoassays due to high background signals and cross - reactions between reagents. SUMMARY OF THE INVENTION

[0014] Methods and systems are provided for the detection and / or quantification of biomolecules using biochemical assays. The object of the present invention is to improve at least some of the deficiencies existing in the prior art. Embodiments of the present technology have been developed based on the understanding of the inventors that, for example, there is a need for a scalable, cost-effective, sensitive, rapid and / or simple multiplex sandwich assay to replace ELISA for everyday use. Therefore, in some aspects, provided herein is a multiplex sandwich assay that includes a multiplex sandwich immunoassay with minimal cross-reactivity between reagents, which is rapid, sensitive, cost-effective, and / or scalable, and enables the simultaneous detection and / or quantification of multiple analytes in a sample.

[0015] The methods and systems provided herein are based, at least in part, on the design and construction of linkages between reagents and supports, where the linkages enable addressable and programmable topologies and functions. Without wishing to be bound by theory, it is believed that the systems and methods provided herein can reduce or eliminate one or more causes of background noise and / or false positives in multiplex sandwich assays. In some embodiments, cross-reactivity between reagents in a multiplex assay is minimized or eliminated by minimizing or eliminating interactions between non-cognate affinity binders. In some embodiments, the methods and systems provided herein can reduce or eliminate background noise caused by incomplete washing and / or non-specific binding of detection reagents. In some embodiments, the methods and systems provided herein can enable multiplex detection of post-translational modifications and / or identification of protein-protein interactions through the assembly of combinatorial reagent pairs on individual assay supports. In some embodiments, the surface structure, linker length, and / or surface spacing of the reagents can be controlled to modulate the stringency of binding and signal generation. In some embodiments, an additional step enables stabilization of the assay signal by converting a reversible reaction to a stable oligohybrid to minimize non-binding, and thus minimize signal loss after assay completion, thereby increasing sensitivity.

[0016] In a first aspect, there is provided a biomolecular complex for the detection and / or quantification of an analyte in a sample, which comprises an anchor chain attached to a support, a capture reagent attached to the support, and a detection reagent releasably attached to the anchor chain, wherein the detection reagent or the anchor chain is optionally attached to a first label, and the first label is inert or undetectable, the detection reagent comprising Here, when the capture reagent and the detection reagent are present in the sample, they can simultaneously bind to the analyte to form a three-component complex. The release of the detection reagent from the anchor chain can release the detection reagent from the support in the absence of the analyte, and the first label can be activated or detected when the detection reagent is released from the anchor chain. Thereby, the presence of the analyte in the sample is determined through the detection of the first label on the support after the detection reagent is released from the anchor chain. This is because the detection reagent remains attached to the support only when it binds to the capture reagent and the analyte in the three-component complex. Therefore, in some embodiments, the first label is detected only on the support when the analyte is present.

[0017] In some embodiments, the amount of the first label on or detected on the support when the detection reagent is released from the anchor chain is proportional to the amount and / or concentration of the analyte in the sample.

[0018] In some embodiments, the detection reagent or the anchor chain is optionally attached to the first label. In some embodiments, the detection reagent is optionally attached to the first label. In some embodiments, the anchor chain is optionally attached to the first label.

[0019] In some embodiments, the detection reagent is directly releasably attached to the anchor chain via a covalent bond, a biotin-streptavidin bond, a hydrogen bond, a hydrophobic interaction, an affinity bond, or a non-covalent interaction.

[0020] In other embodiments, the detection reagent is indirectly attached to the anchor chain via a hook chain, the detection reagent is linked to the hook chain, and the hook chain is releasably attached to the anchor chain, where release of the hook chain from the anchor chain can release the detection reagent from the support in the absence of the analyte, and a first label can be activated or detected when the hook chain is released from the anchor chain. In some such embodiments, the amount of the first label on the support when the hook chain is released from the anchor chain is proportional to the amount and / or concentration of the analyte in the sample.

[0021] In some embodiments, at least one of the detection reagent and the hook chain is optionally attached to a first label. For example, the first label may be attached to the hook chain, the first label may be attached to the detection reagent, or the first label may be attached to both the hook chain and the detection reagent. In some embodiments, the first label is absent, i.e., not attached to either the first chain or the detection reagent. For example, a second label is attached to different components in the biomolecular complex.

[0022] In some embodiments, the capture reagent is directly attached to the support via, for example, 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 aspects, the capture reagent is indirectly attached to the support via, for example, linkage to an anchor chain attached to the support, via, for example, an oligonucleotide linker, a polymer linker, or a covalent bond. It should be understood that the capture reagent may be attached to the support using any suitable means including chemical interactions, affinity binding, etc.

[0023] In some embodiments, the anchor strand is a polymer such as PEG, or an oligonucleotide such as single-stranded DNA oligonucleotide, single-stranded RNA oligonucleotide, or double-stranded DNA oligonucleotide or double-stranded RNA oligonucleotide. It should be understood that the anchor strand may be attached to the support by any suitable means such as covalent bond, chemical interaction, affinity bond, covalent bond, biotin-streptavidin bond, DNA oligonucleotide linker, polymer linker, etc.

[0024] The support is not particularly limited, and any suitable support may be used. Non-limiting examples of the support include microparticles (such as beads), the surface of a multi-well plate, the surface of a slide glass, or a hydrogel matrix. In some embodiments, the support is beads or microparticles, typically of micron size, such as, but not limited to, polystyrene beads, magnetic beads, paramagnetic beads, plastic beads, etc. In another embodiment, the support is a flat microarray. In some embodiments, the support is barcoded beads, such as beads attached with fluorescent dyes or luminescent dyes or mixtures thereof, or beads encoded spectrally, graphically, or chemically.

[0025] The hook strand attached to the detection reagent is generally a linker of sufficient length and flexibility to allow the detection reagent and the capture reagent to simultaneously bind to the analyte to form a three-component complex. Non-limiting examples of the hook strand include polymers such as PEG, and oligonucleotides such as single-stranded DNA oligonucleotide, single-stranded RNA oligonucleotide, or double-stranded DNA oligonucleotide or double-stranded RNA oligonucleotide.

[0026] In certain embodiments of the biomolecular complexes provided herein, there is no hook strand, and the detection reagent is directly releasably bound to the anchor strand, for example, via covalent bonding, biotin-streptavidin binding, affinity binding, and the like.

[0027] The capture reagent can be any molecule capable of specifically recognizing and binding to a target analyte. Non-limiting examples of capture reagents include antibodies, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, aptamers, modified aptamers (such as slow-off-rate modified aptamers or somamers), 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.

[0028] Similarly, the detection reagent can be any molecule capable of specifically recognizing and binding to a 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.

[0029] It should be understood that when the capture reagent is an antibody and the analyte is an antigen, protein, polypeptide, multi-protein complex, or exosome, the detection reagent is also an antibody that can bind to the analyte simultaneously with the capture reagent. Similarly, when the capture reagent is an antigen, protein, polypeptide, multi-protein complex, or exosome and the analyte is an antibody, the detection reagent is also an antigen, protein, polypeptide, multi-protein complex, or exosome that can bind to the analyte simultaneously with the capture reagent.

[0030] The capture reagent and the detection reagent may be the same or different as long as they can both bind to the target analyte simultaneously to form a third complex. In some embodiments, both the capture reagent and the detection reagent are antibodies. These may be the same antibody or different antibodies. They may be different antibodies that bind to the same epitope on the analyte, or they may be different antibodies that bind to different epitopes on the analyte. When 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 this analyte has two or more repeats of the epitope.

[0031] The analyte is not meant to be particularly limited and may be any biomolecule or biological cell for which detection and / or quantification in a sample is desired. Non-limiting examples of analytes include antigens, antibodies, proteins, polypeptides, multi-protein complexes, hormones, exosomes, oligonucleotides, or low molecular weight compounds. The analyte may be detected in any sample of interest, particularly biological samples such as, but not limited to, body fluids (e.g., urine, saliva, blood, serum, plasma, sweat), extracts (e.g., cell extracts), and proteins and / or DNA (e.g., reaction mixtures).

[0032] In some embodiments, the detection reagent is attached to the first label. In some embodiments, the hook strand is attached to the first label. In some embodiments, both the detection reagent and the hook strand are attached to the first label. The first label is absent. In some embodiments, neither the detection reagent nor the hook strand is attached to the first label.

[0033] In some embodiments, the releasable linkage between the hook strand and the anchor strand comprises a double-stranded DNA hybrid, the hook strand, and the anchor strand, which together hybridize to form a double-stranded DNA hybrid comprising complementary single-stranded DNA oligonucleotides. In some such embodiments, release of the hook strand from the anchor strand can be effected by raising the temperature such that the DNA hybrid "melts" or becomes unbound. For example, if the melting temperature (Tm) of the double-stranded DNA hybrid is from about 50 to about 80 °C, this temperature may be raised above Tm so that the double-stranded DNA hybrid dissociates, thereby releasing the hook strand from the anchor strand.

[0034] In some embodiments, the biomolecular complex provided herein further comprises a displacer agent capable of releasing the hook strand from the anchor strand, thereby releasing the detection agent from the support in the absence of the analyte. The displacer agent may be any agent capable of specifically disrupting or releasing the linkage between the hook strand and the anchor strand. For example, the displacer agent may be an enzyme or other agent that cleaves (or otherwise disrupts) the releasable linkage between the hook strand and the anchor strand. Non-limiting examples of displacer agents include enzymes, light, and reducing agents such as DTT. The displacer agent may be capable of disrupting the linkage between the hook strand and the anchor strand, for example, via an enzymatic reaction or by photocleavage.

[0035] In some embodiments, the displacing agent is an oligonucleotide. For example, if the releasable linkage between the hook strand and the anchor strand comprises a double-stranded DNA hybrid, the displacing agent can be a single-stranded DNA or RNA oligonucleotide that hybridizes to the hook strand or the anchor strand, thereby releasing the hook strand from the anchor strand via an oligonucleotide or DNA displacement reaction. In embodiments where the displacing agent hybridizes to the hook strand, the displacing agent forms a double-stranded DNA or RNA hybrid with the hook strand. In some such embodiments, the displacing agent can be detectably labeled such that, if the detection reagent to which the hook strand is attached is bound to the analyte, after washing, only the displacing agent is retained on the support, and detection of the label on the displacing agent thereby indicates the presence of the analyte in the sample. In some such embodiments, there is no first label, and detection of the label on the displacing agent is used to detect and / or quantify the analyte. In some such embodiments, the amount of label on the displacing agent detected on the support is proportional to the amount and / or concentration of the analyte in the sample. In embodiments where the displacing agent hybridizes to the anchor strand, the displacing agent forms a double-stranded DNA or RNA hybrid with the anchor strand. In such embodiments, the displacing agent is not labeled; instead, the label is attached to the detection reagent and / or the hook strand, such that it is understood that the label will only be detected on the support in the presence of the analyte.

[0036] In some embodiments, there is no first label, detection of the label on the displacing agent is used to detect and / or quantify the analyte, and when the displacing agent acts via a DNA displacement reaction, the displacing agent binds to (e.g., hybridizes to) the hook strand. In other embodiments, there is a first label on the detection reagent or the hook strand, the displacing agent is not labeled, and when the displacing agent acts via a DNA displacement reaction, the displacing agent may bind to either the hook strand or the anchor strand.

[0037] In some embodiments, the biomolecular complex further comprises a stem strand that is complementary to the surface proximal sequence of the anchor strand, wherein both the stem strand and the anchor strand are single-stranded oligonucleotides, and the stem strand can bind to the anchor strand to form a double-stranded oligonucleotide. In some embodiments, by forming a double-stranded oligonucleotide with the anchor strand, the stem strand can, for example, prevent the complex from collapsing onto the surface of the support, provide rigidity, form a spacer between the surface of the support and the complex, or provide structural stability by providing structural support to the anchor strand. In some embodiments, the stem strand can also be attached to, for example, a barcode, such as a fluorescent or luminescent dye, and can be used to attach a barcode label to the support. Generally, the stem strand is attached to the anchor strand, for example, by hybridization and does not directly covalently bind to the support.

[0038] In some embodiments where the biomolecular complex comprises a stem strand bound to an anchor and forms a DNA hybrid in proximity to the surface of the support, the anchor strand is attached to a label (instead of a detection reagent, hook strand, or displacer agent, all of which are unlabeled). In these embodiments, the anchor strand is attached to a label that is inactive or undetectable when the anchor strand hybridizes to the stem strand, and the anchor strand is also directly attached to the detection reagent. When the DNA hybrid at the site between the label and the support is cleaved, the label is activated or becomes detectable. The detection reagent is released from the support in the absence of the analyte, such that the signal is detected only in the presence of the analyte and after cleavage (i.e., after release of the detection reagent).

[0039] In some embodiments, the relative densities of the anchor strand and the capture reagent 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.

[0040] In some embodiments, the valency of the conjugation between the detection reagent and the hook chain is selected to minimize cross-reactivity and optimize performance in a multiplex assay. In one embodiment, the conjugation between the detection reagent and the hook chain is monovalent. In other aspects, the binding 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 reagent is linked to the support via only one hook chain.

[0041] In some embodiments where the capture reagent is linked to the anchor chain, the conjugation between the capture reagent and the linker to the anchor chain is monovalent. In some embodiments where the capture reagent is linked to the anchor chain, the conjugation between the capture reagent and the linker to the anchor 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 some embodiments, at least 90% of the capture reagent is linked to the anchor chain via only one capture chain.

[0042] In some embodiments, the anchor chain and / or the capture reagent are randomly dispersed on the support.

[0043] In some embodiments, the length and / or flexibility of the hook chain can be selected to allow or optimize the binding of the detection reagent to the analyte in the presence of the capture reagent.

[0044] In some embodiments where the linkage 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 °C.

[0045] In some embodiments, the concentration of the detection reagent after substitution is less than about 10 picomolar to avoid rebinding of the detection reagent to the off-target reagent or analyte after substitution or release has occurred.

[0046] In further embodiments, the biomolecule complex includes two detection reagents, such that when an analyte is attached, two copies of the label are present, enabling a stronger signal to be generated. In these embodiments, the biomolecule complex further includes a second anchor chain linked to a support and a second detection reagent linked to a second hook chain, the second hook chain being linked to the second anchor chain, and at least one of the second detection reagent and the second hook chain being optionally attached to a third label, the capture reagent, the detection reagent, and the second detection reagent being able to simultaneously bind to the analyte, when present in a sample, to form a four-component complex. Release of the second hook chain from the second anchor chain can release the second detection reagent from the support in the absence of the analyte and can activate the third label.

[0047] In some embodiments, the second detection reagent is attached to the third label. In some embodiments, the second hook chain is attached to the third label. The third label may be any suitable label, such as, but not limited to, a fluorophore, a specific DNA sequence, or a biotin moiety.

[0048] In some embodiments where the third label is attached to the second detection reagent and / or the second hook chain and is inactive or undetectable, the second hook chain is released from the second anchor chain and the third label can be activated or detected only in the presence of the analyte.

[0049] In some embodiments, the biomolecular complex further comprises a second displacer agent capable of releasing the second hook strand from the second anchor strand, such that the second detection agent is released from the support in the absence of the analyte. The second displacer agent, like the displacer agent, can release the second hook strand from the second anchor strand by an enzymatic reaction, cleavage, or oligonucleotide substitution reaction. The second displacer agent can also be detectably labeled, like the displacer agent, in which case the second detection reagent and the second hook strand are generally unlabeled (i.e., no third label is present). The second displacer agent may be the same as or different from the displacer agent. In some embodiments, the second displacer agent and the displacer agent are the same, such that one agent can release both the second hook strand and the hook strand from their respective anchor strands.

[0050] In one embodiment, there is provided a biomolecular complex for detecting an analyte in a sample, comprising an anchor strand linked to a carrier, a capture reagent linked to a support, and a detection reagent linked to a hook strand, wherein the hook strand is linked to the anchor strand and the detection reagent is also labeled, wherein, when present in the sample, the capture reagent and the detection reagent can bind to the analyte simultaneously to form a three-component complex, the linkage between the hook strand and the anchor strand can be disrupted, and the detection reagent is released from the carrier in the absence of the analyte.

[0051] In another embodiment, provided is a biomolecular complex for detecting an analyte in a sample, comprising an anchor chain linked to a support, a capture reagent linked to a capture chain, wherein the capture chain is linked to the anchor chain, and a detection reagent linked to a hook chain, wherein the hook chain is linked to the anchor chain and the detection reagent is also labeled, wherein, when present in the sample, the capture reagent and the detection reagent can simultaneously bind to the analyte to form a three-component complex, the linkage between the hook chain and the anchor chain can be disrupted, and in the absence of the analyte, the detection reagent is released from the support.

[0052] In a further embodiment, provided is a biomolecular complex for detecting an analyte in a sample, comprising an anchor chain linked to a carrier, a capture reagent linked to a support, a detection reagent linked to a hook chain, wherein the hook chain is linked to the anchor chain, and a displacer agent capable of disrupting the linkage 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 support in the absence of the analyte, wherein the displacer is labeled, and wherein, when present in the sample, the capture reagent and the detection reagent can simultaneously bind to the analyte to form a third complex.

[0053] In a further embodiment, provided is a biomolecular complex for detecting an analyte in a sample, comprising an anchor chain linked to a carrier, a capture reagent linked to a capture chain, wherein the capture chain is linked to the anchor chain, a detection reagent linked to a hook chain, wherein the hook chain is linked to the anchor chain, and a displacer agent capable of disrupting the linkage 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 support in the absence of the analyte, wherein the displacer agent is labeled, and wherein, when present in the sample, the capture reagent and the detection reagent can simultaneously bind to the analyte to form a three-component complex.

[0054] In another embodiment, there is provided a biomolecular complex for detecting an analyte in a sample, comprising an anchor chain linked to a support, a capture reagent linked to the support, and a detection reagent linked to a hook chain, wherein the hook chain is linked to the anchor chain and contains an inactivated label, and a displacing agent capable of disrupting the linkage between the anchor chain and the hook chain to effect release of the detection reagent and the hook chain from the support in the absence of the analyte. The capture reagent and the detection reagent, when present in the sample, are capable of simultaneously binding to the analyte to form a three-component complex and disrupting the linkage between the anchor chain and the hook chain.

[0055] In another embodiment, there is provided a biomolecular complex for detecting an analyte in a sample, comprising an anchor chain linked to a support, a capture reagent linked to a capture chain which is linked to the anchor chain, and a detection reagent linked to a hook chain which is linked to the anchor chain, wherein the hook chain contains a detection reagent with an inactivated label, and a displacing agent capable of disrupting the linkage between the anchor chain and the hook chain upon binding to the anchor chain to effect release of the detection reagent and the hook chain from the support in the absence of the analyte. The capture reagent and the detection reagent, when present in the sample, are capable of simultaneously binding to the analyte to form a three-component complex and disrupting the linkage between the anchor chain and the hook chain, and disrupting the linkage between the anchor chain and the hook chain activates the label on the hook chain.

[0056] In another embodiment, provided is a biomolecular complex for the detection of an analyte in a sample, comprising an anchor chain linked to a support, a capture reagent linked to a capture chain linked to the anchor chain, a detection reagent linked to a hook chain linked to the anchor chain and comprising an inactivated label, and a displacer agent capable of disrupting the linkage between the anchor chain and the hook chain to effect release of the detection reagent and the hook chain from the support only in the absence of the analyte, wherein the capture reagent and the detection reagent are the same, the analyte has a repetitive epitope, and when present in the sample, the capture reagent and the detection reagent bind simultaneously to the analyte to form a three-component complex, and disrupting the linkage between the anchor chain and the hook chain activates the label on the hook chain.

[0057] In a further embodiment, provided is a biomolecular complex for the detection of an analyte in a sample, comprising an anchor chain linked to a support, a capture reagent linked to a capture chain linked to the anchor chain, a detection reagent linked to a hook chain linked to the anchor chain, and a displacer agent capable of disrupting the linkage between the anchor chain and the hook chain to effect release of the detection reagent and the hook chain from the support only in the absence of the analyte, the displacer agent being labeled, the capture reagent and the detection reagent being the same, the analyte having a repetitive epitope, and when present in the sample, the capture reagent and the detection reagent being able to bind simultaneously to the analyte to form a three-component complex.

[0058] In another embodiment, a first anchor chain coupled to a support, a second anchor chain coupled to the support, a capture reagent coupled to the support, a first detection reagent coupled to the first anchor chain and coupled to a first hook chain comprising an inactivated first label, a second detection reagent coupled to the second anchor chain and coupled to a second hook chain comprising an inactivated second label, a first displacer agent capable of breaking the linkage between the first anchor chain and the first hook chain to effect release of the first detection reagent and the first hook chain from the support in the absence of an analyte, and a second displacer agent capable of breaking the linkage between the second anchor chain and the second hook chain to effect release of the second detection reagent and the second hook chain from the support in the absence of an analyte, wherein the capture reagent, the first detection reagent, and the second detection reagent, when present in the sample, are capable of simultaneously binding to an analyte to form a four-component complex, and breaking the linkage between the anchor chain and the first hook chain activates the first label on the first hook chain, and breaking the linkage between the anchor chain and the second hook chain activates the second label on the second hook chain, are provided for the detection of an analyte in a sample.

[0059] In another embodiment, a first anchor chain coupled to a support, a second anchor chain coupled to the support, a capture reagent coupled to the support, a first detection reagent coupled to a first hook chain coupled to the first anchor chain, a second detection reagent coupled to a second hook chain coupled to the second anchor chain, a labeled first displacer agent that can disrupt the linkage between the first anchor chain and the first hook chain by binding to the first hook chain and cause the release of the first detection reagent and the first hook chain from the support in the absence of an analyte, and a labeled second displacer agent that can disrupt the linkage between the second anchor chain and the second hook chain by binding to the second hook chain and cause the release of the second detection reagent and the second hook chain from the support in the absence of an analyte are provided for the detection of an analyte in a sample, wherein the capture reagent, the first detection reagent, and the second detection reagent, when present in the sample, can simultaneously bind to the analyte to form a four-component complex.

[0060] In some embodiments, the support is a microparticle, the surface of a well plate, the surface of a glass slide, or a hydrogel matrix.

[0061] 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 multi-protein complex. In some embodiments, the analyte is an exosome.

[0062] In other embodiments, the capture reagent and the detection reagent are antigens and the analyte is an antibody.

[0063] In some embodiments, the capture reagent is attached to the carrier via a covalent bond or via a biotin-streptavidin bond. In some embodiments, the capture reagent is linked to the support via a DNA oligonucleotide linker or via a polymer linker such as a PEG linker. In some embodiments, the detection reagent is attached to the support via a polymer linker such as a PEG linker or via a DNA oligonucleotide linker.

[0064] In one embodiment, the hook strand, the anchor strand, and the displacer agent are DNA oligonucleotides.

[0065] In a further embodiment, the linkage between the hook strand and the anchor strand is a double-stranded DNA hybrid.

[0066] In one embodiment, the linkage between the anchor strand and the support is a covalent bond or a biotin-streptavidin bond. In another embodiment, it should be understood that the anchor strand may be attached to the support using 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 bonds, hydrophobic interactions, affinity bonds, or non-covalent interactions.

[0067] In a further embodiment, the displacer agent disrupts the linkage between the hook strand and the anchor strand via a DNA strand displacement reaction.

[0068] In another embodiment, the displacer agent disrupts the linkage between the hook strand and the anchor strand via an enzymatic reaction.

[0069] In one embodiment, the label is a fluorophore. In one embodiment, the label is a specific DNA sequence. In one embodiment, the label is a biotin moiety.

[0070] In another embodiment, the detection reagent recognizes the same antigen as the capture reagent but does not recognize the same epitope.

[0071] In another embodiment, the detection reagent recognizes a different epitope bound on the same antigen bound by the capture reagent.

[0072] In another embodiment, the detection reagent recognizes the same epitope on the same antigen bound by the capture reagent.

[0073] In another embodiment, the biomolecular complex described herein further comprises a stem strand complementary to the surface proximity sequence of the anchor strand, and the stem strand renders an anchor oligonucleotide duplex.

[0074] In one embodiment, the relative densities of the anchor strand and the capture reagent are adjusted to control the effective affinity of the assay.

[0075] In another embodiment, the length of the analyte detection (e.g., the length of the hook strand) is adjusted to control the effective affinity of the detection.

[0076] In another embodiment, the conjugation between the detection reagent and the hook strand is monovalent.

[0077] In one embodiment, the anchor strands are randomly dispersed. In another embodiment, the capture reagents are randomly dispersed.

[0078] A method for detecting an analyte from a sample, comprising the steps of providing a support, a capture reagent, an anchor chain, a hook chain, and a detection reagent, wherein the capture reagent is linked to the support, the anchor chain is linked to the support and the hook chain, the hook chain is linked to the detection reagent, and the detection reagent is labeled; incubating the sample with the support to enable binding of the capture reagent and the detection reagent to different epitopes on the analyte; disrupting the bond between the hook chain and the anchor chain; separating the detection reagent and the hook chain from the support 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 support. The labeled concentration of the detection reagent remaining on the support is proportional to the concentration of the bound analyte.

[0079] A method for detecting an analyte from a sample, comprising the steps of providing a support, an anchor chain, a capture chain, a capture reagent, a hook chain, and a detection reagent, wherein the anchor chain is linked to the support, the capture chain, and the hook chain, the capture chain is linked to the capture reagent, and the hook chain is linked to the detection reagent; incubating the sample with the support to enable binding of the capture reagent and the detection reagent to different epitopes on the analyte; disrupting the bond between the hook chain and the anchor chain by separating the detection reagent and the hook chain from the support 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 support. The labeled concentration of the detection reagent remaining on the support is proportional to the concentration of the bound analyte.

[0080] A method for detecting an analyte from a sample, the method comprising the steps of providing a support, a capture reagent, an anchor chain, a hook chain, and a detection reagent, wherein the capture reagent is linked to the support, the anchor chain is linked to the support and the hook chain, and the hook chain is linked to the detection reagent; incubating the sample with the support to enable the capture reagent and the detection reagent to bind to different epitopes on the analyte; separating the detection reagent and the hook chain from the support in the absence of analyte bound to both the capture reagent and the detection reagent, wherein the displacer agent is labeled; and quantifying the amount of bound analyte by analyzing the displacer agent remaining on the support, wherein the labeled concentration of the displacer agent remaining on the support is proportional to the concentration of the bound analyte.

[0081] In another embodiment, a method for detecting an analyte from a sample, the method comprising the steps of providing a support, an anchor chain, a capture chain, a capture reagent, a hook chain, and a detection reagent, wherein the anchor chain is linked to the support and the capture chain, the capture chain is linked to the capture reagent, the anchor chain is linked to the hook chain, and the hook chain is linked to the detection reagent; incubating the sample with the support to enable the capture reagent and the detection reagent to bind to different epitopes on the analyte; incubating with a displacer 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 support in the absence of analyte bound to both the capture reagent and the detection reagent, wherein the displacer agent is labeled; and quantifying the amount of bound analyte by analyzing the displacer agent remaining on the support, wherein the labeled concentration of the displacer agent remaining on the support is proportional to the concentration of the bound analyte.

[0082] A method for detecting an analyte from a sample, comprising the steps of providing a support, a capture reagent, an anchor chain, a hook chain, and a detection reagent, wherein the capture reagent is linked to the support, the anchor chain is linked to the support and the hook chain, the hook chain is linked to the detection reagent, and the hook chain contains an inactivated label; incubating the sample with the support to enable binding of the capture reagent and the detection reagent to different epitopes on the same analyte; incubating with a displacing agent to break the bond between the hook chain and the anchor chain; separating the detection reagent and the hook chain from the support in the absence of analyte bound to both the capture reagent and the detection reagent, wherein separating the hook chain from the anchor chain activates the label on the hook chain; and analyzing the hook chain label remaining on the support to quantify the amount of bound analyte. The label concentration of the hook chain remaining on the support is proportional to the concentration of the bound analyte.

[0083] A method for detecting an analyte from a sample, comprising the steps of providing a support, an anchor chain, a capture chain, a capture reagent, a hook chain, and a detection reagent, wherein the anchor chain is linked to the support, the capture chain, and the hook chain, the capture chain is linked to the capture reagent, the hook chain is linked to the detection reagent, and the hook chain contains an inactivated label; incubating the sample with the support to enable binding of the capture reagent and the detection reagent to different epitopes on the analyte; incubating with a displacing agent to break the bond between the hook chain and the anchor chain by binding to the anchor chain; separating the detection reagent and the hook chain from the support only in the absence of analyte bound to both the capture reagent and the detection reagent, wherein separating the hook chain from the anchor chain activates the label on the hook chain; and analyzing the hook chain label remaining on the support to quantify the amount of bound analyte. The label concentration of the hook chain remaining on the support is proportional to the concentration of the bound analyte.

[0084] A method for detecting an analyte from a sample, comprising the steps of providing a support, an anchor chain, a capture chain, a hook chain, a capture reagent, and a detection reagent, wherein the anchor chain is linked to the support, the capture chain, and the hook chain, the capture chain is linked to the capture reagent, the hook chain is linked to the detection reagent and contains an inactivated label, and the capture reagent and the detection reagent are structurally similar; incubating the sample with the support to allow binding of the capture reagent and the detection reagent to different epitopes on the analyte, wherein these epitopes are structurally similar; incubating with a displacing agent to break the bond between the hook chain and the anchor chain; separating the detection reagent and the hook chain from the support in the absence of the analyte bound to both the capture reagent and the detection reagent, wherein separating the hook chain from the anchor chain activates the label on the hook chain; and quantifying the amount of bound analyte by analyzing the hook chain label remaining on the support. Also provided is a method wherein the label concentration of the hook chain remaining on the support is proportional to the concentration of the bound analyte.

[0085] A method for detecting an analyte from a sample, the method comprising the steps of providing a support, an anchor chain, a capture chain, a hook chain, a capture reagent, and a detection reagent, wherein the anchor chain is linked to the support, the capture chain, and the hook chain, the capture chain is linked to the capture reagent, and the capture reagent and the detection reagent are structurally similar; incubating the sample with the support to enable binding of the capture reagent and the detection reagent to different epitopes on the analyte, wherein these epitopes are structurally similar; incubating with a displacer agent to disrupt 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 support in the absence of the analyte bound to both the capture reagent and the detection reagent, wherein the displacer agent is labeled; and quantifying the amount of the bound analyte by analyzing the label of the displacer chain remaining on the support. The labeled concentration of the displacer chain remaining on the support is proportional to the concentration of the bound analyte.

[0086] In one embodiment, the capture reagent and the detection reagent are peptides. In one embodiment, the capture reagent is attached to the support via a DNA oligonucleotide linker. In another embodiment, the detection reagent is attached to the support via a PEG linker.

[0087] In a further embodiment, the hook chain, the anchor chain, and the displacer agent are DNA oligonucleotides. In one embodiment, the linkage between the hook chain and the anchor chain is a double-stranded DNA hybrid. In a further embodiment, the linkage between the anchor chain and the support is a covalent bond. In one embodiment, the linkage between the anchor chain and the support is a biotin-streptavidin bond.

[0088] In one embodiment, the displacer agent disrupts the bond between the hook chain and the anchor chain via a DNA strand displacement reaction. In one embodiment, the displacer agent disrupts the bond between the hook chain and the anchor chain via an enzymatic reaction.

[0089] In another embodiment, the label is a biotin moiety.

[0090] In a further embodiment, the anchor chain is attached to the microparticle via a chemical interaction.

[0091] In another embodiment, the detection reagent recognizes the same antigen as the capture reagent but does not recognize the same epitope. In a further embodiment, the detection reagent recognizes a different antigen bound to the same antigen as that bound by the capture reagent. In one embodiment, the detection reagent recognizes the same epitope at a different position of the same antigen as that bound by the capture reagent.

[0092] In one embodiment, the biomolecular complex described herein further comprises a stem strand complementary to the surface proximal sequence of the anchor chain, rendering an anchor oligonucleotide duplex.

[0093] In one embodiment, the relative densities of the anchor chain and the capture reagent are adjusted to control the effective affinity of the assay.

[0094] In one embodiment, the length of detection of an analyte (e.g., hook strand) is adjusted to control the effective affinity of the detection.

[0095] In one embodiment, the binding between the detection reagent and the hook strand is monovalent.

[0096] In another embodiment, the anchor chains are randomly dispersed.

[0097] In a further embodiment, the capture reagents are randomly dispersed.

[0098] Also provided are multiple complex detection systems for detecting multiple analytes in a sample, which include a plurality of supports, a plurality of capture reagents, each of which is bound to its respective support, and a plurality of detection reagents, each of which is bound to its respective support via a linker and is labeled. On each support, each detection reagent is attached to its respective support via a linker, the detection reagent is labeled, and on each support the detection reagent is labeled. The capture and detection reagents, when present in the sample, can simultaneously bind to a support-specific analyte to form a three-component complex. The linker between the detection reagent and its respective support is cleaved, and the detection reagent can be released from the support in the absence of the analyte.

[0099] Further provided are multiple complex detection systems for detecting multiple analytes in a sample, which include a plurality of supports, a plurality of capture reagents, each of which is bound to its respective support, and a plurality of detection reagents, each of which is bound to its respective support via a support-specific hook chain, each hook chain including a support-specific inactivation label, and a displacing agent capable of breaking the bond between the plurality of hook chains and the plurality of supports and separating the detection reagents from their respective supports. Here, on each support, the capture and detection reagents, when present in the sample, can simultaneously bind to a support-specific analyte to form a three-component complex, and breaking the bond between the support and the hook chain activates the support-specific label on the hook chain.

[0100] Also provided are multiple complex detection systems for detecting multiple analytes in a sample, which include a plurality of supports, a plurality of capture reagents, each capture reagent being coupled to its respective support, a plurality of detection reagents, each detection reagent being coupled to its respective support via a support-specific hook chain, a plurality of hook chains and a displacer agent that disrupts the binding between the hook chains and the supports and that is labeled and binds to the hook chains upon disruption of the binding between the hook chains and the supports. Here, on each support, the capture reagent and the detection reagent, if present in the sample, can simultaneously bind to a support-specific analyte to form a three-component complex.

[0101] In one embodiment, also provided are multiple complex detection systems for detecting multiple analytes in a sample, which include a plurality of supports, a plurality of capture reagents, each capture reagent being coupled to its respective support, a plurality of detection reagents, all detection reagents being coupled to their respective supports, each detection reagent including an inactivated label, the linkage between the detection reagents and their respective supports being disruptable, where on each support, the capture reagent and the detection reagent, if present in the sample, can simultaneously bind to a support-specific analyte to form a three-component complex, and where the detection reagent label is activated upon disruption of the linkage between the detection reagents and their respective supports.

[0102] There is further provided a method for detecting a plurality of analytes in a sample, which comprises the steps of providing a support, an anchor chain, a capture chain, a hook chain, a capture reagent and a detection reagent, wherein the anchor chain is linked to the support, the anchor chain is also linked to the capture chain, the capture chain is also linked to the capture reagent, the anchor chain is also linked to the hook chain, and the hook chain is also linked to the detection reagent; incubating the sample together with the support under conditions that allow the capture reagent and the detection reagent to bind to different epitopes on the same analyte; incubating with a displacer agent to disrupt 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 support only in the absence of analyte bound to both the capture reagent and the detection reagent, wherein the displacer agent is labeled; and quantifying the amount of bound analyte by analyzing the displacer chain label remaining on the support, wherein the concentration of the displacer chain label remaining on the support is proportional to the change in the concentration of the bound analyte.

[0103] There is further provided a method for detecting a plurality of analytes in a sample, which comprises the steps of providing a support, an anchor chain, a capture chain, a hook chain, a capture reagent and a detection reagent, wherein the anchor chain is linked to the support, the capture chain is linked to the support, there is an anchor chain, the hook chain is linked to the support, and the hook chain is linked to the detection reagent; incubating the sample together with the support under conditions that allow the capture reagent and the detection reagent to bind to different epitopes on the same analyte; disrupting the bond between the hook chain and the support; separating the detection reagent and the hook chain from the support only in the absence of analyte bound to both the capture reagent and the detection reagent; incubating with a cross-linking chain that links the anchor chain to the hook chain and that is labeled; and quantifying the amount of bound analyte by analyzing the cross-linking chain label remaining on the support, wherein the concentration of the cross-linking chain label remaining on the support is proportional to the change in the concentration of the bound analyte.

[0104] In some embodiments, the hook strand is labeled by including a tag sequence, i.e., a unique DNA sequence that can be detected. In some such embodiments, the hook strand further includes a recombination sequence, and after releasing the hook strand from the anchor strand using a displacer agent oligonucleotide that binds to the anchor strand, a cross-linking strand is added, where the cross-linking strand can bind to the recombination sequences of both the hook strand and the anchor strand, thereby indirectly reconnecting the hook strand to the anchor strand. In such embodiments, after the label attached to the hook strand has been activated or made detectable by release from the anchor strand, the hook strand with the active / detectable label is reattached to the support.

[0105] In one embodiment, there is further provided a biomolecular complex for the detection and / or quantification of an analyte in a sample, which comprises: a) an anchor strand attached to a support; b) a capture reagent attached to the support; c) a detection reagent linked to a hook strand, wherein the hook strand is releasably attached to the anchor strand, and the hook strand and the anchor strand are linked to each other by a double-stranded DNA hybrid; and d) 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 via a DNA displacement reaction, and the displacer agent is detectably labeled, where the capture reagent and the detection reagent, when present in the sample, simultaneously bind to the analyte to form a three-component complex, and the release of the hook strand from the anchor strand by the displacer agent can release the detection reagent from the support in the absence of the analyte. In one embodiment, the capture reagent and the detection reagent are antibodies, the analyte is an antigen or a protein, and the support is a barcoded microparticle.

[0106] In a second aspect, a multiplex sandwich assay system is provided for the simultaneous detection and / or quantification of two or more analytes in a sample, the system comprising two or more biomolecular complexes as described herein, each biomolecular complex for the detection and / or quantification of a different analyte in the sample.

[0107] In some embodiments, two or more biomolecular complexes are attached to the same support. For example, the support may be a flat surface, the surface of a multiwell plate, the surface of a glass slide, a hydrogel matrix, microparticles, etc. In such embodiments, each biomolecular complex is disposed at a distinct location on the support, enabling each labeled complex (and thus each analyte) to be identified by its position.

[0108] In some embodiments, two or more biomolecular complexes are attached to different carriers, such as different barcoded microparticles. For example, a first biomolecular complex may be attached to barcoded first beads, such as first fluorescent or luminescent dyes or mixtures of dyes, such as first fluorochromes or luminescent dyes or mixtures of dyes, such as first beads spectrally, graphically, or chemically attached, and a second biomolecular complex may also be attached to barcoded second beads, such as second fluorescent or luminescent dyes or mixtures of dyes, such as second fluorochromes or luminescent dyes or mixtures of dyes, such as second beads spectrally, graphically, or chemically attached. After the first and second complexes are assembled on their respective beads, they can be mixed and contacted with the sample, enabling the simultaneous detection of two different analytes in the sample. The barcoding on the beads enables each labeled complex (and thus each analyte) to be identified.

[0109] In some embodiments, one or more of the two or more biomolecular complexes include a second anchor chain, a second detection reagent linked to the second hook chain, etc., such that a four-component complex is formed between the capture reagent, the two detection reagents, and the analyte.

[0110] In some embodiments, all of the two or more biomolecular complexes lack any first label on the detection reagent or the hook chain, and the label is provided only on the displacer agent. In some aspects, the same labeled displacer agent is used to release each hook chain from its respective anchor chain of each biomolecular complex, and each biomolecular complex (and its respective analyte) is identified by its position on the surface or, particularly when the surface is a microparticle, by barcoding of the surface. In other embodiments, different displacer agents having different labels may be used for each biomolecular complex.

[0111] In some embodiments, two or more biomolecular complexes can each detect and / or quantify the same analyte, and each biomolecular complex has a different effective affinity for the analyte. For example, the effective affinity of a biomolecular complex for an analyte can be selected by adjusting the length of the hook chain and / or the anchor chain, and / or by adjusting the surface density of the capture reagent and / or the detection reagent. In this way, an analyte can be assayed over a wide range of concentrations.

[0112] The length of the hook chain and / or the anchor chain can be adjusted to control the effective affinity of the assay, and / or the surface density of the capture reagent and / or the detection reagent can be adjusted to control the effective affinity of the assay.

[0113] 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 for the simultaneous detection and / or quantification of 5 or more analytes, 10 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, and this system includes respective biomolecular complexes specific to each analyte. Thus, the multiplex sandwich assay system can be easily extended for large-scale multiplexing.

[0114] In a third aspect, a method for detecting and / or quantifying an analyte in a sample is provided using the biomolecular complex described herein.

[0115] In some embodiments, a method for the simultaneous detection and / or quantification of two or more analytes in a sample is provided using two or more of the biomolecular complexes described herein, where each biomolecular complex is for the detection and / or quantification of a different analyte in the sample. In some embodiments, a method for the simultaneous detection and / or quantification of two or more analytes in a sample is provided using the multiplex sandwich assay system described herein, which system includes two or more biomolecular complexes as described herein, and each biomolecular complex is for the detection and / or quantification of a different analyte in the sample. It should be understood that the methods provided herein can be used for the simultaneous detection and / or quantification of a large number of analytes in a sample, and this method can be extended to enable large-scale multiplexing.

[0116] In one embodiment, a method for detecting and / or quantifying an analyte in a sample is provided, the method comprising: a) providing a support, a capture reagent bound to the support, an anchor chain attached to the support, and a detection reagent optionally linked to a hook chain, wherein the detection reagent or the hook chain is releasably linked to the anchor chain, and at least one of the detection reagent and the hook chain is optionally attached to a first label, the first label being inactive or undetectable; b) contacting the support with the sample under conditions that allow simultaneous binding of the capture reagent and the detection reagent to the analyte to form a three-component complex; and c) adding a displacer agent optionally attached to a second label, the displacer agent releasing the detection reagent or the hook chain from the anchor chain, such that the detection reagent optionally linked to the hook chain is released from the support in the absence of the analyte, and the release of the detection chain or the hook chain from the anchor chain activates the first label or makes the first label detectable.

[0117] In some embodiments, the method further comprises determining the presence and / or amount of the first label and / or the second label on the support, the presence of the first and / or second label on the support indicating the presence of the analyte in the sample, and the amount of the first label and / or the second label being proportional to the amount and / or concentration of the analyte in the sample.

[0118] In some embodiments, the method further comprises washing the support to remove unbound reagents or substances after step (c).

[0119] In some embodiments, the method further comprises storing the support after step (c).

[0120] In some embodiments of the methods provided herein, the support further comprises a second anchor chain attached to the support, a second capture reagent attached to the support, and a second detection reagent attached to the second hook chain, wherein the second detection reagent or the second hook chain is attached to the second anchor chain, and at least one of the second detection reagent and the second hook chain is optionally attached to a third label, the third label being inert or undetectable, and the second capture reagent and the second detection reagent are capable of binding simultaneously to a second analyte in the sample to form a second three-component complex, and the displacing agent also releases the second detection reagent or the second hook chain from the second anchor chain, such that in the absence of the second analyte, the second detection reagent optionally linked to the second hook chain is released from the support, and the release of the second detection reagent or the second hook chain from the second anchor chain activates or makes detectable the third label, and the presence and / or amount of the third label on the support indicates the presence of the second analyte in the sample, and the amount of the third label on the support is proportional to the amount and / or concentration of the second analyte in the sample, such that the first analyte and the second analyte can be detected and / or quantified simultaneously in the sample.

[0121] In some embodiments, the second anchor chain and the second capture reagent are disposed at a first location and a second location on the support, respectively. In other embodiments, the second anchor chain and the second capture reagent are attached to a second support. The support and / or the second support may be, for example, microparticles such as polystyrene beads. In one embodiment, the first support is a first barcoded bead (i.e., a first bead encoded with a first barcode, such as a first fluorescent dye or a luminescent dye or a mixture of first dyes), and the second support is a second barcoded bead (i.e., a second bead encoded with a second barcode, such as a second fluorescent dye or a luminescent dye or a mixture of second dyes), enabling the identification of each bead when its respective barcode is detected.

[0122] In some embodiments, the first support and the second support are mixed together before contacting the sample. For example, the first support and the second support can be contacted with the sample simultaneously. The sample can be a biological sample, such as, but not limited to, a body fluid, an extract, a solution containing proteins 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.

[0123] In some embodiments, the method uses a hook strand that is labeled by including a labeled sequence, i.e., a unique DNA sequence that can be detected. In some such embodiments, the hook strand further includes a recombination sequence and uses a displacer agent oligonucleotide that binds to the anchor strand to release the hook strand from the anchor strand, and after any washing, the cross-linking strand binds to both the recombination sequence on the hook strand and the anchor strand, thereby indirectly recombining the hook strand to the anchor strand. In such a manner, after the label attached to the hook strand is activated or made detectable by release from the anchor strand, the hook strand having the active / detectable label is reattached to the support.

[0124] In one embodiment, a method for the detection and / or quantification of an analyte in a sample is provided, the method comprising: a) providing a support, a capture reagent attached to the support, an anchor chain attached to the support, and a detection reagent bound to a hook chain, the hook chain being releasably linked to the anchor chain by a double-stranded DNA hybrid; b) contacting the support with the analyte under conditions that allow simultaneous binding of the capture reagent and the detection reagent to the analyte to form a three-component complex; and c) adding a displacer agent attached to a detectable label, the displacer agent being a DNA oligonucleotide complementary to at least a portion of the hook chain and capable of hybridizing to the hook chain, the displacer agent releasing the hook chain from the anchor chain via a DNA displacement reaction such that the detection reagent is released from the support in the absence of the analyte; and d) optionally determining the presence and / or amount of the detectable label on the support, the presence of the label on the support indicating the presence of the analyte in the sample and the amount of the label being 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 a protein, and the support is barcoded microparticles. The barcoded microparticles may be barcoded, for example, spectrally, graphically, or chemically.

[0125] In a fourth aspect, a method for preparing a multiplex sandwich assay system is provided, the method comprising: (a) providing a first container comprising first microparticles encoded with a first barcode; (b) attaching the first microparticles to a first capture reagent and a first detection reagent; (c) optionally, storing the first microparticles; (d) providing a second container comprising second microparticles encoded with a second barcode; (e) attaching the second microparticles to a second capture reagent and a second detection reagent; (f) optionally, storing the second microparticles; and (g) mixing the first microparticles and the second microparticles 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 prior to their respective attachment to their microparticles. The first barcode and the second barcode may independently be a spectral, a pattern, or a chemical barcode.

[0126] In some embodiments, each capture reagent and each detection reagent are attached to their respective microparticles simultaneously. In other embodiments, each capture reagent and each detection reagent are attached to their respective microparticles in a two-step reaction, where either the capture reagent or the detection reagent is first attached to the microparticle, followed by subsequent attachment of the other reagent.

[0127] In some embodiments, the method of the invention further comprises washing the first microparticles and the second microparticles to remove unattached reagents prior to mixing them together in step (g). In some embodiments, the method of the invention further comprises additional washing steps, one or more of each step of attaching the capture reagent and / or the detection reagent, followed by a washing step to remove unattached reagents and / or non-specifically attached reagents from the microparticles.

[0128] In some embodiments, the microparticles are beads, e.g., polystyrene beads. In some embodiments, the microparticles in step (a) are not barcoded, and the method further includes barcoding the microparticles (e.g., attaching a barcode such as a fluorescent dye or a luminescent dye or a mixture thereof to the microparticles) before step (g), i.e., before mixing the first microparticles and the second microparticles together.

[0129] In some embodiments, the first capture reagent, the first detection reagent, the second capture reagent, and the second detection reagent are antibodies.

[0130] In one embodiment, a method of preparing the multiplex sandwich assay system described herein is provided, the method including: (a) providing a support that is a flat surface, the surface of a multiwell plate, the surface of a glass plate, 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 support via a first anchor chain attached to the support at the first location; (g) washing the support to remove unattached first detection reagent; (h) attaching a second detection reagent to the support via a second anchor chain attached to the support at the second location; and (i) washing the support to remove unattached second detection reagent, such that no more than one reagent attaches to the support at a time whenever the first capture reagent, the second capture reagent, the first detection reagent, and / or the second detection reagent are mixed together.

[0131] In some embodiments, the method of preparing the multiplex sandwich assay system described herein is advantageous in minimizing cross-reactivity because different reagents (capture reagents, detection reagents) are not mixed together in solution before being attached to a carrier and / or assembled into a biomolecule complex. In this way, nonspecific binding of the reagents to each other is avoided or at least reduced in order to minimize cross-reactivity. Unwanted background signal or "noise" may be avoided or at least reduced. In some embodiments, the method is also scalable, enabling rapid and / or cost-effective preparation of multiplex sandwich assay systems. In some embodiments, far fewer capture and / or detection reagents are required than in conventional assay systems, which can lead to substantial cost savings for expensive antibody reagents and the like. In some embodiments, for example, less than a nanoliter of antibody reagent may be required to prepare a biomolecule complex.

[0132] Other aspects and features of the present invention will become apparent to those skilled in the art by considering the following description of specific embodiments of the present invention in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0133] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. For a better understanding of the present invention, and to more clearly show how it may be carried out and what effects it may bring, reference is made by way of example to the accompanying drawings that illustrate aspects and features according to embodiments of the present invention.

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DETAILED DESCRIPTION OF THE INVENTION

[0134] Detailed Description As described herein, systems and methods are provided for detecting and / or quantifying one or more analytes using a co-localization assay by ligation. In particular, systems and methods are provided that have a sufficiently low background signal, sufficiently low cross-reactivity between reagents, and / or a sufficiently high sensitivity to simultaneously detect and / or quantify multiple biomolecules in a sample. Also provided are multiplex sandwich assays and methods for their preparation that are rapid, sensitive, cost-effective, and / or scalable.

[0135] The present disclosure is not limited to a particular apparatus, system, method, or use or process step and is to be understood to be capable of variation in such a manner.

[0136] To provide a clear and consistent understanding of the terms used herein, a number of definitions are provided below. Further, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0137] In the claims and / or specification, when used in connection with the term "comprising", the use of the word "a" or "an" may mean "one", but this also conforms to the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" may mean at least a second or more.

[0138] As used herein and in the claims, the terms "comprising" (and any form of comprising, such as "comprises" and "comprising"), "having" (and any form of having, such as "has" and "having"), "including" (and any form of including, such as "includes" and "including"), "containing" (and any form of containing, such as "contains" and "containing") are inclusive or non-limiting and do not exclude additional, unrecited elements or process steps.

[0139] As used herein, the term "about" in the context of a given value or range refers to a value or range within 20%, preferably within 10%, more preferably within 5% of the given value or range.

[0140] As used herein, the term "and / or" should be taken as specific disclosure of each of two specific features or components, with or without others. For example, A and / or B should be taken as (i) A, (ii) B, and (iii) specific disclosure of each of A and B, as if each were individually set forth herein.

[0141] As used herein, the term "support" refers to an immobilization structure, surface or substrate, such as, but not limited to, microparticles, nanoparticles, wells of plates, porous polymers, or hydrogels. It is meant that the support is not particularly limited, and it should be understood that any solid, semi-solid, gel or gel-like structure may be used. For example, the support may be an array, beads (e.g., without limitation, polystyrene beads), the surface of a multi-well plate (e.g., 96-well plate, 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 support is beads or microparticles or nanoparticles, typically of micron size or nano size, e.g., without limitation, polystyrene beads, magnetic beads, paramagnetic beads, plastic beads, etc. In another embodiment, the support is a planar microarray. In one embodiment, the support is nanoparticles. In one embodiment, the support is microparticles.

[0142] As used herein, the term "analyte" refers to a target biomolecule or biological cell of interest that is being identified, detected, measured, and / or quantified. An analyte can be any biomolecule or biological cell that can be detected using the systems and methods provided herein, for example, without limitation, proteins, nucleic acids (such as DNA, RNA), antibodies, antigens, proteins, cells, chemicals, biomarkers, enzymes, polypeptides, amino acids, polymers, carbohydrates, multi-protein complexes, exosomes, oligonucleotides, low molecular weight compounds, and the like. Non-limiting examples of analytes include antibodies, antibody fragments (such as scFv, Fab, etc.), aptamers, modified aptamers, somamers, affimers, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, and low molecular weight compounds.

[0143] As used herein, "sample" refers to any fluid or liquid sample being analyzed for the detection and / or quantification of 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 effusion, liquefied fecal matter, and lacrimal gland secretions.

[0144] As used herein, the term "encoded microparticle" refers to a barcoded microparticle, e.g., spectrally encoded by either a barcode or by a particular test being performed in an assay, such as a target analyte. Barcoded (or encoded) microparticles are often used in multiplexed suspension assays so that particles in a large mixture can be distinguished. The method of barcoding is not limited. Barcoding can be performed using, for example, spectral, graphical, or chemical means. For example, spectral encoding of microparticles can be done by labeling the microparticles with an exact ratio of multicolor dyes. This approach enables simple and high-throughput readout by flow cytometry. As another example, graphically barcoded microparticles are typically etched or otherwise patterned using a visual pattern that can be characterized via a microscope. Microparticles can also be chemically barcoded using, for example, unique DNA sequences that can later be detected via DNA detection means.

[0145] As used herein, the term "non-specific binding" means an unintended reaction between reagents and / or molecules in a sample, including but not limited to, reactions between non-cognate antibodies and proteins that adhere via hydrophobic interactions.

[0146] As used herein, the terms "affinity binding agent" (AB), "binding agent", and "reactant" are used interchangeably to mean any molecule capable of specifically recognizing a target analyte via, for example, non-covalent interactions. Examples of affinity binding agents (AB) include, without limitation, immunoglobulin-G (IgG) antibodies (e.g., whole molecules or Fab fragments), aptamers, affimers, nanobodies, ankyrins, and single-chain variable fragments (scFv).

[0147] As used herein, the term "sandwich assay" is used to mean an analyte-targeted assay in which two ABs can bind simultaneously to a target analyte of interest and be used to detect and / or quantify it.

[0148] As used herein, the terms "multiplex sandwich assay", "multiplexed sandwich assay", and "MSA" are used interchangeably to mean a sandwich assay that targets multiple (e.g., two or more) analytes simultaneously from the same sample and / or assay volume, and multiple AB pairs are used simultaneously in the assay system.

[0149] As used herein, the term "cross-reactivity" is used to mean a particular case of non-specific binding or non-specific reaction in a multiplex sandwich assay, where, for example, as shown in FIG. 1, an unintended complex containing a non-cognate affinity binder is formed.

[0150] As used herein, the terms "capture affinity binder", "cAB", "capture AB", "capture binder", and "capture reagent" are used interchangeably to refer to an AB that is attached to a support in a biomolecular complex and not released therefrom. The capture AB can be attached directly to the support (e.g., via a covalent bond, biotin-streptavidin binding, DNA oligonucleotide linker, or polymer linker) or indirectly (e.g., via linkage to an anchor chain, e.g., through a linker such as a conjugate or capture chain). Non-limiting examples of capture reagents include antibodies, antibody fragments (e.g., scFv, Fab, etc.), aptamers, modified aptamers (such as slow-off-rate modified aptamers or somamers), affimers, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, and low molecular weight compounds.

[0151] The term "capture linker" refers to a linker (e.g., an oligonucleotide, a polymer, etc.) that attaches a capture reagent to an anchor chain (and thus the support to which the anchor chain is attached).

[0152] As used herein, the terms "detection affinity binder", "dAB", "detection AB", "detection binder", and "detection reagent" are used interchangeably to refer to the AB in a biomolecular complex releasably attached to a support. dAB is generally used for signal transduction and assay signaling. In some embodiments of the methods and systems provided herein, for example, the fraction of dAB not bound to an analyte is released from the support such that no signal is generated in the absence of the bound analyte. In some embodiments, the dAB is bound to a label or means for signal transduction and assay signaling. Non-limiting examples of detection reagents include antibodies, antibody fragments (e.g., scFv, Fab, etc.), aptamers, modified aptamers, somamers, affimers, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, and low molecular weight compounds.

[0153] As used herein, the term "anchor chain" refers to a linker that attaches to a fixed point on a support. Non-limiting examples of anchor chains include polymers such as polyethylene glycol (PEG), oligonucleotides (e.g., single-stranded DNA oligonucleotides, single-stranded RNA oligonucleotides, or double-stranded DNA or double-stranded RNA oligonucleotides, or DNA-RNA hybrids), and oligosaccharides.

[0154] As used herein, the term "hook chain" refers to a linker that couples the detection AB to the anchor chain, i.e., attaches it to a support. The hook chain is typically releasably attached to the anchor chain, for example, in such a way that the attachment can be released. Generally, when the attachment between the hook chain and the anchor chain is released, a fraction of the detection AB that is coupled to a hook chain not bound to the target analyte is released from the anchor chain and thus also from the support so that in the absence of the target analyte, the signal from the detection AB cannot be detected on the support. In this way, the signal on the support is detected only when the target analyte is present and is bound by the detection AB and the capture AB.

[0155] In some embodiments where the label is on the hook chain and / or the detection reagent and is only activatable or detectable after the hook chain and / or the detection reagent have been released from the anchor chain, the signal is release-dependent since it is only detectable after the release of the hook chain and / or the detection reagent from the anchor chain. Similarly, in some embodiments, if the label is present on a displacer agent that hybridizes to the hook chain, the signal is "displacement-dependent".

[0156] As used herein, the term "displacer agent" refers to an agent that directly or indirectly effects the release of a releasable bond between an anchor chain and a hook chain, thereby releasing the hook chain (and any detection AB linked thereto) from the support. The mechanism by which the displacer agent functions is not particularly limited. For example, the displacer agent can directly or indirectly cause or initiate cleavage, displacement, or unlinking of the bond between the anchor chain and the hook chain, and other mechanisms are possible and contemplated. In some embodiments, the hook chain is displaced from the anchor chain using a DNA oligonucleotide that hybridizes to the hook chain and / or the anchor chain. Examples of displacer agents include, but are not limited to, substituted DNA oligonucleotides, light sources of single or multiple colors, restriction enzymes, and reducing agents such as dithiothreitol (DTT). In some embodiments where a photocleavable DNA segment is used, the displacer agent may be light that effects release via a photocleavage reaction. In some embodiments, the displacer agent is labeled, for example, with a dye, fluorophore, specific DNA sequence, enzyme, biotin moiety, etc. When the displacer agent is labeled, it can serve a dual function of releasing the hook chain and simultaneously labeling it.

[0157] As used herein, the term "label" refers to a molecule or any part of a molecule that can generate a signal, can target a signal generating molecule, or is otherwise detectable. Examples of labels include, but are not limited to, biotin, fluorophores, enzymes, enzyme substrates, and specific DNA sequences. An "inactive" or "undetectable" label refers to a label that is not active, is masked, or is otherwise undetectable, e.g., a quenched fluorescent dye, non-limitingly.

[0158] It should be understood that the systems and methods provided herein can be used in virtually any type of sandwich assay that uses two sets of A and B. However, for simplicity, specific embodiments of the present invention are presented herein using whole molecule immunoglobulin G antibody (IgG) as A and B, which represents one of many possible embodiments. It should be understood that the antibody is not limited to whole molecule IgG and that many different antibodies, antibody fragments, etc. can be used. Further, A and B are not limited to antibodies. Similarly, many different types of sandwich assays other than those specifically described herein can be used.

[0159] In some embodiments, a dual A and B or sandwich assay is provided that can avoid cross-reactivity by co-localizing two A and B (capture A and B) on a support prior to exposure of a biological sample containing the analyte of interest to the support. Co-localization on the support does not allow any mixing of different A and B pairs prior to exposure to the analyte, so cross-reactivity between reagents and / or background can be reduced or eliminated (e.g., as shown in FIG. 1 or FIG. 4). In one embodiment, a support attached to a mixture of capture A and B is provided, where each set of capture A and B can bind to the analyte of interest and the detection A is optionally attached to the support via a releasable linker. In one embodiment, a support attached to a mixture of capture A and B is provided, where each analyte can bind simultaneously to both capture A and B and the detection A is optionally releasably attached to the support via a releasable hook chain. When the detection reagent and / or hook chain is released, the corresponding detection A remains on the support only when bound to the analyte in a three-component complex with the capture A.

[0160] 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 oligos), polymers, polysaccharides, and the like. DNA binding can be a covalent bond such as conjugation between a hook chain oligo and a detection AB, or a non-covalent bond such as hybridization or base stacking between two complementary DNA sequences. To enable the formation of a three-component complex of capture AB - antigen - detection AB, the hook chain is designed to have a flexible single-stranded portion. Substitution of DNA ligation includes, but is not limited to, footprint retention DNA substitution reactions, enzymatic cleavage, and photoactivated cleavage. Specific DNA sequences can also be used as labels that can be directly targeted using a fluorescently labeled complementary sequence, used as amplification triggers or primers via hybridization chain reaction or polymerase chain reaction, and read via sequencing.

[0161] 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, oligos can be modified using fluorescent dyes at the 5' or 3' termini and can be modified with a photocleavable phosphodiester backbone conjugated to a protein, biotin, or enzyme, etc.

[0162] These embodiments may 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 label). In some embodiments of CLA, the hook chain that links the detection AB to the anchor chain is labeled (i.e., attached to a label). Generally, the label attached to the detection AB or the hook chain is inactive or undetectable, such that the label can be detected after release of the detection AB from the support (i.e., after the hook chain has been released from the anchor chain). Thus, signal detection from the label is release-dependent (in some embodiments, also referred to as “substitution-dependent”). In such a manner, the analyte in the three-component complex having the capture AB is bound, and only the detection AB released from the anchor chain is detected. Unbound detection AB is released from the support (e.g., can be removed by washing). Also, since the label is inactive or undetectable prior to release, or if a given hook chain is not released (i.e., due to the release-dependency or substitution-dependency of the signal), the background signal can be reduced. Thus, in some embodiments, the methods and systems provided herein may be referred to as release-dependent (or substitution-dependent) transduction or “RDT” or “substitution-dependent detection” to reflect the release-dependent (or substitution-dependent) signal transduction.

[0163] Conventional sandwich assays generally rely on the presence of a detection AB to convert a signal and detect the presence of an analyte. Similarly, in certain embodiments of the systems and methods presented herein, the detection AB and / or hook strand can act as signal transduction factors. However, in contrast to conventional assays, in the systems and methods provided herein, the detection AB and / or hook strand optionally linked thereto can only remain on the support when a three-component complex is formed with the analyte and capture AB. It is understood that if the detection reagent and / or hook strand is not successfully or completely released from the anchor strand, this can remain on the support even in the absence of the analyte. In this case, the detection AB and / or hook strand, whether attached to the anchor strand or attached to an active or detectable label, any non-released, labeled detection AB, and / or hook strand transduces a signal. In other words, in that case, any label and non-released detection reagent and / or hook strand can result in a signal that does not depend on the presence of the analyte, contributes to non-specific background signal, and reduces assay performance and / or sensitivity. It is understood that the background signal in that case is proportional to the fraction of non-released detection reagent and / or hook strand. It should also be understood that near-complete release of the complex from the support may be difficult to achieve due to steric hindrance, immobilization, and / or incomplete washing. However, release-dependent transduction (RDT) can minimize or eliminate these problems because, as demonstrated in FIG. 24, signal transduction does not occur if the release of the detection reagent and / or hook strand from the anchor strand is not complete.

[0164] In some embodiments, thus, the systems and methods provided herein include an additional level of redundancy in order to reduce background signal and / or increase sensitivity through the use of release-dependent conversion (RDT). In RDT, signaling occurs only if both of the following conditions are met: (i) formation of a three-component capture AB-analyte-detection AB complex, and (ii) release of the corresponding detection AB and / or hook strand from the anchor strand. In such cases, non-released detection AB and / or hook strands do not contribute to the background signal. As used herein, this signaling mechanism, which the inventors refer to as “release-dependent conversion (RDT)”, can be achieved via a variety of means. For example, some embodiments can include a label on the hook strand, where the label is inactive or undetectable until after release from the anchor strand so that non-released (e.g., unsubstituted) hook strands and / or detection ABs do not contribute to the signal or are not converted to that signal.

[0165] In some embodiments of RDT, the hook strand is labeled with a fluorophore quenched by a quencher on the anchor or another proximal strand such that release results in quenching or activation of the fluorophore.

[0166] In some embodiments of RDT, the detection reagent and the hook strand are unlabeled, and instead, a displacer agent is labeled. In this case, the displacer agent hybridizes to the hook strand, displacing it from the anchor strand and simultaneously labeling it. If the detection AB does not bind to the analyte and the capture AB in the three-component complex, the hook strand, the displacer agent, and the label are washed from the support. Since the label is attached to the displacer agent, the label is present only on the support when both conditions are met: (i) release or displacement from the anchor strand occurs, and (ii) the analyte is bound to both the capture AB and the detection AB (e.g., as shown in FIG. 7).

[0167] Other embodiments of the RDT are possible, and it is understood that the mechanism of the RDT is not particularly limited.

[0168] In some embodiments of the RDT, the detection AB or the anchor chain is attached to a label. In some embodiments, the hook chain that links the detection AB to the anchor chain is labeled (i.e., attached to a label). Generally, the label attached to the detection AB, the anchor chain, or the hook chain is inactive or undetectable, such that the label can only be detected after the release of the detection AB from the support (i.e., after the hook chain has been released from the anchor chain, e.g., as shown in FIG. 6). In this way, the only detection AB-hook oligo complex that is detected is the one that involves the detection AB bound to the analyte in the three-component complex having the capture AB and the hook chain successfully released from the anchor chain. Otherwise, the unbound detection AB is released from the support (and can be removed, e.g., by washing), and all non-released chains are not detected, regardless of whether the analyte is bound or not. In this way, the background signal from non-released detection AB and / or hook chains is reduced, ensuring a low background signal and / or high-sensitivity detection.

[0169] In other embodiments of the RDT, the hook chain contains a label that remains inactive or undetectable until the hook chain is released from the anchor chain. For example, this can be achieved if the hook chain and the anchor chain are DNA oligonucleotides bound together via hybridization, where the hook chain contains a DNA sequence label that normally hybridizes to the anchor chain and thus is not available for binding or is undetectable, or contains a DNA sequence. Release of the hook chain oligo from the anchor chain oligo reveals a detectable label on the hook chain. Such release can be achieved, for example, via enzymatic cleavage, DNA displacement, or photocleavage using light.

[0170] In some such embodiments, a release or displacer agent, which is an oligonucleotide that replaces the anchor - hook chain hybrid by binding to the anchor chain oligo via a footprint replacement reaction, is provided. In one embodiment, neither the hook chain nor the detection AB is labeled, and a labeled displacer agent (e.g., a fluorescently labeled oligonucleotide) performs the RDT via the dual functions of release (replacement) and labeling. In this way, detectable signal / signal transduction is homologous to an "AND" logic gate only by labeling only the replaced hook chain, when two conditions (replacement of the hook chain and presence of the analyte) are met (e.g., as shown in FIG. 7B).

[0171] In some embodiments of the assays and systems provided herein, one or more sets of capture ABs and detection ABs are attached to a support, with each set being specific for the analyte of interest. In this way, the capture ABs and detection ABs are pre - assembled and co - localized on the support prior to exposure to a biological sample containing the analyte of interest. As described above, the detection AB is removably attached to the support. In some embodiments, the detection AB is attached to the support by a releasable linker (hook chain) linked to an anchor chain attached to the support. The hook chain is generally flexible, allowing the detection AB to diffuse freely within the limits permitted by the lengths of the hook chain and / or the anchor chain. The hook chain and the releasable linker are not particularly limited as long as they allow for simultaneous binding of the analyte by the detection AB and the capture AB, and can be varied in size, flexibility, structure, etc. The capture AB and the detection AB can bind to overlapping sites as long as they can bind the analyte simultaneously, but generally bind to separate regions of the analyte.

[0172] In some embodiments, the detection AB is attached to the support using a hook strand that is a DNA oligonucleotide capable of specifically binding to an anchor strand attached to the support. After contact and incubation with the biological sample (i.e., the target recognition step), the detection AB is separated from the anchor strand by disrupting the bond between the hook strand and the anchor strand on the surface. This releases the fraction of the detection AB that was not part of the three-component capture AB - analyte - detection AB complex. It should be understood that the linkage between the hook strand and the anchor strand can be released or disrupted in several ways, such as, but not limited to, DNA strand displacement, enzymatic cleavage, photoactivated cleavage, etc.

[0173] As included herein, many different ABs targeting many different analytes can be mixed (i.e., multiplexed) in the same assay volume, and the interactions between different ABs on different supports (or between different ABs at different positions / places on the same support) are restricted by binding to the support(s), so that interactions between ABs from different supports / places are avoided. This is in contrast to conventional multiplexing techniques that cannot limit the interactions between ABs when all ABs are mixed in solution. Further, in the methods and systems described herein, different microparticle populations can be manufactured separately in large batches, each containing a different AB capture - detection pair required to detect a specific antigen, ensuring that no cross - reactivity occurs during manufacture.

[0174] In some embodiments, the multiplexed CLA methods and systems can thus avoid cross-reactive scenarios such as those shown in FIG. 1, for example. As will be appreciated by those skilled in the art, co-localization on each support (e.g., microparticles) of cognate capture ABs and detection ABs eliminates unwanted interactions such as binding between non-cognate detection ABs and capture ABs. In addition to these scenarios shown in FIG. 1, those skilled in the art will recognize that, unlike conventional multiplexed sandwich assays, analytes that bind or adhere non-specifically to off-target supports cannot be detected by their cognate detection ABs in the methods and systems provided herein, and thus do not contribute to an increase in background signal.

[0175] In some embodiments, on each support, the local concentrations of the capture AB and the detection AB can be increased, which can help to concentrate the analyte and increase sensitivity. On the other hand, the total concentration of each capture AB and detection AB in the total assay volume depends only on the concentration of the target-specific support (e.g., microparticles, microarray spots) and can be designed to result in a low bulk density of the detection AB upon release. For example, the local concentration can be within the micromolar range, but the use of a small number of target-specific microparticles can result in a bulk detection AB concentration that is too low (<pM) to cause any off-target binding, as shown, for example, in FIG. 22. This bulk concentration can be further decreased by increasing the volume during the release step. Thus, in certain embodiments, the methods and systems provided herein can further avoid cross-reactions that occur after the release of the detection AB because the concentration or amount of the detection AB used on the support is low.

[0176] In some embodiments, the simultaneous binding of two copolymerized binders (capture AB, detection AB) to different epitopes of the same analyte (i.e., increased binding avidity) can result in a much lower effective off-rate (koff) compared to conventional sandwich assays where the capture AB and detection AB are added sequentially. After sample introduction and incubation, the support in the methods and systems provided herein can be washed rigorously because the analyte is attached with high binding avidity. Thus, in some embodiments of the methods and systems provided herein, stringent washing can be used to reduce assay background and / or improve sensitivity and / or specificity. In some embodiments, it may be desirable to perform the assay steps rapidly after release of the hook chain from the anchor chain and until readout of the assay signal. Because off-binding of the analyte can result in a reduced signal that can contribute to a reduction in sensitivity, and such effects are generally reduced in CLA.

[0177] In one embodiment of the methods and systems provided herein, the support is an encoded micron-sized particle, both the capture reagent and the detection reagent are antibodies, and the capture reagent and its cognate detection reagent are co-localized on the surface of the same support using DNA ligation (in other words, the hook strand and the anchor strand are single-stranded DNA oligonucleotides linked to each other via a double-stranded DNA hybrid). In some such embodiments, the detection reagents linked to the hook strand and the anchor strand are uniformly mixed and attached to the surface of the particle, where the anchor strand is linked to the hook strand through partial hybridization, the hook strand is conjugated to the detection reagent, and the hook strand is a flexible and releasable DNA linker. The hybrid between the anchor and the hook strand is generally stable under the conditions of sample incubation. In some embodiments, the capture reagent is also linked to the particle in a similar manner via a DNA linker. In some such embodiments, the release of the hook strand from the anchor strand can be effected via a footprint-mediated DNA displacement reaction. In such embodiments, the displacer agent is an oligonucleotide designed to bind to the footprint sequence on the hook strand and drive the displacement reaction forward. In some such embodiments, the release of the hook strand from the anchor strand can be effected without a displacer agent, for example, by raising the temperature such that the DNA hybrid complex "melts" or unbinds, by raising the temperature.

[0178] In one embodiment, the detection AB and / or hook chain are labeled with a biotin moiety that can be detected, for example, using a dye, fluorescently labeled streptavidin, in subsequent steps such as. In certain embodiments, the detection AB can be detected after binding the analyte to the label binder, for example, IgG can be targeted using a labeled species-specific secondary-lgg. In some embodiments, the detection AB and hook chain are not labeled, and instead, the displacer agent used to release the hook chain from the anchor chain is labeled. In such embodiments, the labeled displacer agent attaches to the hook chain and / or detection AB after release of the hook chain from the anchor chain.

[0179] In some embodiments, the label is a specific DNA sequence that can be detected or targeted in subsequent step(s). For example, the specific DNA sequence can be targeted using a subsequent DNA hybridization step that labels it with a dye. In one embodiment, the specific DNA sequence is amplified through polymerase chain reaction (PCR) or other enzymatic DNA amplification means in which the specific DNA sequence is detected and amplified. The specific DNA sequence can also be cleaved and detected by other means such as sequencing. Embodiments using a DNA sequence as a label are not limited and can include a sequence that is part of the hook chain (and thus initially inactive / undetectable) or present on the displacer agent (e.g., as shown in FIGS. 6A, D).

[0180] In some embodiments, a detectable AB is provided that is indirectly attached to the microparticle via a releasable linkage to an anchor strand that is linked to and attached to the hook strand. The hook strand is partially complementary to the anchor strand attached to the microparticle. The anchor strand can be attached to the microparticle, for example, via streptavidin / biotin interactions or chemical bonds. In this way, the detectable AB is attached to the microparticle. In this embodiment, a capture AB that is attached to the microparticle surface is further provided, the detectable AB recognizes the same antigen as the capture AB, and both ABs can bind the antigen simultaneously. Further, a substituted oligonucleotide (displacer agent) having a sequence complementary to the hook strand that overlaps with the sequence of the anchor strand is provided, such that the detectable AB is released from the anchor strand and thus released from the microparticle if the antigen is not bound (i.e., if no ternary complex exists between the capture AB - antigen - detectable AB). In a further embodiment, a fluorescently labeled secondary antibody that binds to the detectable AB remaining on the microparticle after the displacement reaction is also provided.

[0181] In embodiments where the capture AB and the detection AB are pre - assembled on a support and the detection AB is labeled with a detectable label, it should be noted that any non - released hook - chain detection AB complex gives a signal that is independent of the analyte, which can contribute to background noise (as shown in FIG. 24). Thus, it is understood that in order to avoid an increase in the background signal, an almost complete anchor - chain - hook - chain substitution reaction and washing of the hook - chain - detection AB complex are required. Also, it is understood that such almost complete release can be difficult even when using optimized conditions (FIG. 15). To reduce such increased background signal resulting from inefficient release of the anchor - chain - hook - chain linkage, in some embodiments, the hook - chain, the anchor - chain, or the detection AB is labeled with a label that remains inactive / undetectable until substitution or release of the hook - chain from the anchor - chain. In another embodiment, the hook - chain, the anchor - chain, or the detection AB is not labeled and the displacer agent is labeled with a detectable label. In such embodiments, signal transduction on the support occurs only when both (i) the formation of the three - component capture AB - analyte - detectable AB complex and (ii) the conditions for substitution of the hook - chain - anchor - chain hybrid are met. In these embodiments, the non - substituted hook - chain does not contribute to the signal. Similarly, it should be understood that embodiments where the label on the detection AB and / or the hook - chain is inactive or undetectable until after release may be advantageous since non - released (e.g., non - substituted) hook - chains (or detection ABs) do not contribute to the signal.

[0182] In one embodiment, a labeled displacer agent (e.g., an oligonucleotide) can perform a dual function of release (substitution) and labeling similar to an “AND” logic gate. In this way, detection - capable signal / signal transduction requires two conditions by simply labeling the substituted hook - chain (FIG. 23). A potential advantage of such embodiments is that they do not require changes in the DNA sequence or ligation properties of the detection complex that includes the hook - chain and the anchor - chain.

[0183] In some embodiments, an additional level of redundancy can be achieved by using a hook strand having an inactive or undetectable label that is only activated or detectable upon substitution 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 conjugate to the anchor strand. In another embodiment, substitution can be similarly achieved using a restriction enzyme, followed by signal generation using a labeled oligo that targets a portion of the previously hybridized (and thus not available for binding) hook strand, thereby only being able to hybridize to and label the already substituted hook strand.

[0184] In certain embodiments, a detection AB linked to microparticles via a hook strand is provided, where the hook strand is an oligo attached to the detection AB. The hook strand oligo is partially complementary to an anchor strand that is also an oligo linked to the microparticles via, for example, a streptavidin / biotin interaction or a chemical bond, thus attaching the detection AB to the microparticles. Further, a capture AB linked to the microparticle surface is provided, where the detection AB recognizes the same antigen as the capture AB and both ABs can bind to the antigen simultaneously. Also, an oligonucleotide containing a fluorescent label or a DNA barcode sequence and having a sequence complementary to the hook strand oligonucleotide that overlaps with the sequence of the anchor strand oligonucleotide can be provided as a substitute, such that the detection Ab is released from the anchor strand and thus can be released from the microparticle.

[0185] In the methods and systems provided herein, it should be understood that the use of co-localization and conjugation can require a reasonable topology design in order to optimize the availability of both ABs (capture AB and detection AB) across the support. In some embodiments, proper binding of the analyte using stochastically distributed capture ABs and / or detection ABs attached to the support can require optimization of two important design parameters: (i) the relative density of the capture AB and the detection AB, and (ii) the length of the hook strand. These two parameters help control the time-averaged distance between the capture AB and the detection AB by considering the radius of rotation of the detection AB. In some cases, the distance between the capture AB and the detection AB, and ultimately the effective affinity at the single-molecule level, can be stochastic and difficult to control. Thus, in some embodiments, it may be desirable to optimize the two aforementioned parameters for optimal assay performance.

[0186] In another embodiment, both the capture AB and the detection AB are linked to an anchor strand, allowing for simultaneous control of the capture AB and detection AB densities while maintaining co-localization at the nanoscale, potentially allowing for more precise control of assay performance (e.g., as shown in some embodiments of FIGS. 5F-G and 21). In such embodiments, the capture AB and the detection AB are co-localized, their relative densities are identical, and can be adjusted simultaneously. One potential advantage of this embodiment is the uniform average distance between the capture AB and the detection AB for all pairs on the support. A second potential advantage of this embodiment is that the architecture of the capture AB and the detection AB can be precisely controlled. For example, by decreasing the length of the single-stranded portion of the anchor strand or the hook strand, the stringency of binding can be controlled, providing a decisive means of controlling the thermodynamics of the assay system. It is understood by those skilled in the art that increasing the stringency of binding can result in a decrease in effective affinity. In some embodiments, such adjustment of effective affinity can be used, among other applications, to control and extend the dynamic range of the assay.

[0187] In some such embodiments, where the effective affinity can be adjusted by varying the length of the hook or anchor chain (i.e., the linker length), or by modulating the surface density of the capture AB and the detection AB, multiplexed arrays (such as multiplexed microparticles) designed with different effective affinities can be fabricated. This can be useful for extending the dynamic range of a particular assay for a particular analyte. For example, one of ordinary skill in the art will understand that some proteins are present in blood at concentrations ranging over five orders of magnitude, and that for such targets, several assays can be designed using different barcodes, and that such proteins can be quantified over a larger dynamic range.

[0188] In one embodiment, the capture AB is conjugated to a capture oligonucleotide that hybridizes to one array domain of a support-linked anchor chain. Another array domain of the anchor chain can be hybridized to a hook chain linked to the detection AB. In this embodiment, all of the aforementioned strategies for signal transduction and generation can also be utilized.

[0189] 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 multiplexed assay, where the length of the hook chain oligos for the two or more sets, and thus the stringency of binding, can be controlled. For example, two or more sets of microparticles having different barcodes but targeting the same analyte can be fabricated, where the first set of microparticles includes shorter hook chain oligos to reduce flexibility and increase the stringency of binding, and the second set of microparticles includes longer hook chain oligos to increase flexibility and decrease the stringency of binding. In this way, the first set of microparticles can be designed to quantify the analyte when present at higher concentrations.

[0190] One skilled in the art recognizes that another challenge in multiplex assays is interference and matrix effects, which can be difficult to control at the analyte level. One advantage of the methods and systems provided herein is the ability, in some embodiments, 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, a particular sample can contain endogenous antibodies and other molecules that can have a positive or negative impact on the intensity of the assay signal for a particular analyte.

[0191] In another embodiment, a separate support or biomolecule complex is provided, which is accompanied by all analyte-specific supports lacking either the capture AB or the detection AB, and acts as an analyte-specific internal standard to control matrix effects and other potential modes of assay failure. The assay signal of the fully formed biomolecule complex on the support can then be compared to these single-AB controls. These internal controls can be used as flags for potential false positives.

[0192] One further problem for assays, particularly when using binding agents having a non-zero or high off-rate (k-off), is the unbinding of the analyte that can occur, particularly during the time from the washing of the biological sample to the readout of the assay signal, and thus the decrease of the assay signal. This unbinding is particularly problematic for low concentrations of analyte and readout methods that cannot measure different assays in multiplexing (e.g., cytometry). This problem can also exist in the CLA sensor procedure, whereby, after release (e.g., after displacement), unbinding of the analyte to either the capture AB or the detection AB can result in signal loss. In yet another embodiment, therefore, the CLA methods and systems provided herein can be modified to mitigate this problem of unbinding and time-dependent signals by converting the assay signal (e.g., AB analyte) from a reversible reaction into a stable oligo-hybrid to stop further unbinding, and is coupled to a support (e.g., as shown in FIG. 13) that allows storage and readout at a later time point. A potential advantage of this embodiment is to minimize signal loss after assay completion that can be useful for increasing sensitivity. Another potential advantage of this embodiment is the standardization of signal decrease across different assays and samples, which can be read out over a non-negligible amount of time, allowing for better signal reproducibility and improved accuracy. In some such embodiments, the assay can be performed as in previous embodiments, the assay label is a unique DNA sequence, where, after washing of the released detection AB, a displacing agent is introduced to rebind the hook strand onto the anchor strand, thereby preserving the signal on the support. As will be appreciated by those skilled in the art, another potential advantage of this embodiment is the reproducibility of signal intensity, particularly in removing the time-dependence of the assay signal with respect to measurement and temperature.

[0193] In some such embodiments, a displacer agent is provided that is an oligo that binds to the anchor - chain oligo via a footprint replacement reaction and then replaces the anchor - chain - hook - chain hybrid, by washing the released, unbound hook - chain oligo - detection AB complex and then adding a replacement oligo that hybridizes to both oligos to enable re - binding of the hook - chain oligo to the anchor - chain oligo, thereby replacing the anchor - chain - hook - chain hybrid.

[0194] Some applications would benefit significantly from the methods and systems provided herein, which, in some embodiments, serve to address some of the causes of background noise and false positives in multiplex sandwich assays. In particular, in some embodiments, multiplexing of protein analysis is made significantly possible by the methods and systems provided herein. For example, profiling of proteins such as cytokines and other soluble factors has been limited in conventional multiplexing due to reagent cross-reactivity. In some embodiments, the methods and systems provided herein can significantly improve multiplexed serological assays. For example, multiplexed autoantibody assays used to detect many specific autoantibodies have been significantly hampered by specificity. Autoantibodies are typically captured by specific recombinant or native antigens on a solid support and then detected by species-specific detection antibodies (e.g., anti-human Fc IgG). As a result, any non-specific binding of autoantibodies present in the serum is detected and often results in false positives, making this type of assay a single-binding assay (i.e., limited to singleplex form). In contrast, the methods and systems provided herein can be utilized to perform a double-binding assay, i.e., an analyte (here, an autoantibody) is recognized and detected by two specific ABs (here, specific antigens). In such an embodiment, the recombinant or native antigen can be split into two fractions representing a capture AB and a detection AB conjugated to a capture chain and a hook chain, respectively, where both the capture chain and the hook chain are linked to the same anchor chain, and the anchor chain is attached to a support (as shown in FIG. 9). As discussed above, the flexibility of the hook chain can allow for simultaneous binding of the analyte (here, an antibody) to the capture AB and the detection AB (here, the same protein conjugated to separate chains with different functionalities). Following washing of the unbound sample, signal transduction can proceed via label strand displacement as described herein.

[0195] In some embodiments, the methods and systems provided herein can address a major challenge in multiplexed analysis of protein-protein interactions using ABs. For that purpose, as shown in FIG. 10, the AB pairs can be pre-assembled, each AB pair targeting one protein of interest and enabling the CLA to detect the interaction between the pairs in question. To completely isolate such a multiplexed assay from others, cross-reactivity is significantly reduced, enabling combinatorial measurements of interactions across different protein-protein pairs. The modular approach of the manufacturing methods of the embodiments presented herein facilitates the realization and production of AB pairs targeting different proteins. For example, large batch production of CLA on microparticles enables bulk functionalization of the microparticles with capture ABs, followed by fractionating and adding different detection ABs to all fractions.

[0196] In some embodiments, the methods and systems provided herein can address another major challenge in multiplexed analysis of post-translational modifications (PTMs) using ABs. For example, accurate protein phosphorylation analysis can be used to reveal cellular signaling events not apparent from protein expression levels. Current methods and workflows for quantifying the fraction of PTMs of a particular protein are severely limited in multiplexing because PTM-specific ABs lack sufficient specificity for the protein itself (i.e., fluorophore-specific ABs are very sensitive to reagent-driven cross-reactivity issues). As a result, conventional PTM panels are not multiplexed. The multiplexed CLA assay methods and systems provided herein can address this problem by restricting the anti-PTM binding agents to analyte-specific supports (as in FIG. 11).

[0197] In some embodiments, the hook strand is a flexible releasable linker and is an oligonucleotide that enables the formation of a three-component complex of capture AB - analyte - detection AB, such that a signal is generated only in response to the recognition of the sandwich capture AB - analyte - detection AB when one of the unbound hook strand oligos is released from the support.

[0198] In some embodiments, a detection AB, which is an antibody attached to a support such as a microparticle, is provided via a hook strand that is an oligonucleotide linked to the detection AB. The hook strand oligonucleotide is partially complementary to an anchor strand oligonucleotide attached to the support (e.g., a microparticle) via a streptavidin / biotin interaction, e.g., a chemical bond, and thus attaches the detection AB to the support. A capture AB, which is an antibody attached to the support, is further provided, where the detection AB recognizes the same antigen as the capture AB but does not recognize the same epitope. In some embodiments, a displacer agent is provided that is an oligonucleotide having a sequence complementary to the hook strand oligonucleotide, including a fluorescent label or a DNA barcode sequence, and overlapping with the sequence of the anchor strand oligonucleotide, such that the detection AB is released from the anchor strand and can thus be released from the support in the absence of the target analyte. It should be understood that once the capture AB and the detection AB bind to the analyte, a three-component capture AB - analyte - detection AB complex is formed on the support (e.g., on the microparticle). After the formation of the three-component complex, the unbound detection AB is removed from the support by washing, while the three-component complex is retained on the support. Thereafter, the presence of the three-component complex on the support can be detected and / or quantified.

[0199] In some embodiments, the methods and systems provided herein can be referred to as "Colocalization Assay by Ligation on Particles" or "CLAMP". The CLAMP methods and systems described herein can be very user-friendly and advantageous to the user. For example, by providing particles having pre-assembled AB pairs (pairs of capture AB and detection AB), the user can arbitrarily and rapidly mix and match panels, perform multiplexed assays, and, for example, read assay results using any multicolor flow cytometer. Thus, the CLAMP assays provided herein can be adapted to existing experimental workflows in biology and, in some embodiments, can be read using any multicolor flow cytometer.

[0200] It is understood that embodiments of CLAMP are uniquely adaptable for the large-scale industrial-scale manufacture of multiplexed panels that avoid cross-reactivity. In contrast to planar arrays, CLAMP can be manufactured separately in large batches that are stored as needed and then mixed prior to the assay. This manufacturing method enables the independent manufacture of CLAMP without interactions between non-cognate ABs and thus without cross-reactivity during the manufacturing process, which is a significant advantage over other CLA embodiments.

[0201] In some embodiments, to manufacture multiplexed CLAMP, the AB pairs are attached to a set of particles, where each target-specific AB pair is attached to a respective set of particles in a separate container. The particles can be barcoded before AB binding or can be barcoded during this process. This reaction can be carried out in large batches and the manufactured CLAMP can be stored. To perform the assay, fractions of beads for each barcode / target are mixed together before contacting with the biological sample. The particles can be barcoded by any means, e.g., spectrally, graphically, or chemically.

[0202] In some embodiments, certain advantages can be obtained when the support is microparticles (MPs). For example, in some embodiments, the ability to rapidly read out a large number of MPs by flow cytometry can provide increased accuracy and sample throughput. In addition, MPs can be functionalized in large batches and then stored, used, and read out in solution, which can reduce lot-to-lot variability and enable quantitative analysis (Tighe, P.J., et al., Proteomics-Clinical Applications 9, 406-422, 2015; Jani, I.V., et al., The Lancet 2, 243-250, 2002; Krishhan, V.V., Khan, I.H. & Luciw, P.a. 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.) 1-7 2013; Fu, Q., et al., Clinical applications 4, 271-84, 2010).

[0203] In some embodiments, the methods and systems provided herein can reduce or eliminate reagent cross-reactivity. As shown in FIG. 2a, which illustrates one aspect of CLA, the pre-colocalization of two sets of antibodies on a surface using a DNA oligonucleotide as a flexible and addressable linker can eliminate interactions between non-cognate antibodies. Further, upon release of one of the flexible linkers from the surface, a signal is generated only in response to sandwich antibody-antigen-antibody recognition.

[0204] Figures 2B-2F show the nanostructure and the operating principle of CLAMP according to one embodiment. Through one-pot functionalization of microparticles with defined ratios of fluorescent oligonucleotides and antibodies, a CLAMP population was created (Figure 2B), followed by hybridization of hook oligo-detection AB (dAB) complexes to complete the construction of CLAMP (Figure 2B). In one embodiment, stable biotin-streptavidin binding is used for reagent ligation / attachment along with bead sets preserved after manufacture. Next, monovalent antibody-oligo conjugates are assembled as pairs on barcode bead sets via hybridization (i.e., antibody pairs A1-A2 and B1-B2 are pre-assembled on beads A and B, respectively) (Figure 2C), and then the bead sets are pooled together. When the CLAMP panel is added to a sample, target proteins generate sandwich complexes, while non-specifically bound proteins do not form complete sandwiches (Figure 2D). After incubation, stringent washing removes non-specifically bound proteins (Figure 2E). Next, DNA strand displacement is used to simultaneously dehybridize and label one antibody of the sandwich on each bead population, ensuring that only sandwich binding events generate signals (Figure 2F). Finally, the CLAMP panel is automatically read using any commonly available multi-color flow cytometer, and the bead sets are decoded.

[0205] In some embodiments, the CLAMP panel can have a lower development cost than conventional immunoassays, and not only avoid the costly re-optimization of the panel when new target analytes are added, but CLAMP can also use significantly lower amounts of antibody per assay.

[0206] In some embodiments, in addition to overcoming the cross-reactivity of the reagents, the pair of surface-linked antibodies in CLAMP can provide a binding activity effect, thereby providing additional advantages over conventional sandwich immunoassays. The off-rate (koff) of the target from the antibody sandwich complex in CLAMP can be made much lower than that of assays using continuous antibody addition, so CLAMP can exhibit higher affinity for the target. In some embodiments, the CLAMP assay can be washed rigorously after incubation to reduce assay background and improve specificity. Further, in some embodiments, CLAMP can have a reduced disadvantage due to false positives, and since incorrect binding events in CLAMP do not form a complete sandwich complex, they do not lead to false positive signals.

Example

[0207] The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the invention and should in no way be construed as limiting the scope of the invention.

[0208] Unless otherwise defined or the context clearly indicates 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 this invention belongs. It is to 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.

[0209] Example 1. One-Pot Bead Barcoding and Production of CLAMP In some embodiments, the multiplex assay system is performed on spectrally encoded beads, and in the methods and systems provided herein, a one-pot bead barcode strategy and an automated decoding method can be used. Examples of such barcoding / decoding methods are described in U.S. Patent Application No. 16 / 153,071 and Dagher, M. et al, Nature Nanotechnology, vol. 13, pp. 925-932, 2018, the contents of which are incorporated herein by reference in their entirety. Such methods use accurate models of fluorophore spectral overlap and multicolor Förster resonance energy transfer (FRET). For example, such a strategy can have the ability to barcode more than 580 barcodes using two lasers for barcoding and a third laser for assay readout (shown in FIG. 3A). A cytometer using an infrared laser can potentially extend performance up to more than 5,000 barcodes.

[0210] Using the same manufacturing workflow, a version of the co-localization antibody assay as described herein was constructed. That is, in the first step, streptavidin beads were co-coupled with biotinylated capture antibodies and capture oligosaccharides modified with biotinylated anchors or different dyes to obtain distinguishable barcodes. Each barcode and target-specific antibody was manufactured in a separate tube. In the second step, a detection antibody (monoclonal) conjugated to a hook oligo was added to the corresponding functionalized beads from the first step. The hook strand oligo was complementary to the anchor strand oligo and hybridized to it, thereby resulting in the assembly and co-localization of the matching antibody pair. The beads can be stored separately for later use.

[0211] In some embodiments, background signal can be optimized (i.e., made lower) using a low oligo:antibody binding ratio or valence and / or two-step purification. For example, low valence antibody-oligo conjugates have been shown to maximize CLAMP strand displacement efficiency and minimize background signal (Figure 3B). After a first round of optimization (changes in ionic strength, wash and incubation times, nanoscale design, and reagent concentrations), the sensitivity of 1-plex CLAMP for uPA was improved 3-fold over the conventional sandwich assay (Figure 3C). By performing monovalent instead of multivalent binding, an additional 3-fold improvement was achieved (Figure 3D).

[0212] In one embodiment, a CLAMP system as described herein includes the following components. 1) Particles that hold all other components in place 2) One type of capture antibody (cAb) covalently attached to the particles 3) One type of detection antibody (dAb) covalently linked to a hook oligonucleotide, where the detection antibody recognizes the same antigen as the cAb but does not recognize the same epitope as the cAB 4) An anchor oligonucleotide (AO) linked to the particles, for example, via a streptavidin / biotin interaction 5) A stem oligonucleotide (SO) that is fully or partially complementary to the AO and thereby forms at least a partial duplex 6) A hook oligonucleotide (HO) covalently linked to the cAb and partially complementary to the anchor oligonucleotide, thus attaching the cAb to the particles 7) A displacement oligonucleotide (DO) having two functions a) Contains a fluorescent label, and b) Is complementary to the HO, has a sequence that overlaps with the sequence of the AO, such that the dAb is released from the AO and thus from the particles.

[0213] A 5-plex CLAMP was assembled using antibodies, where the antibodies were highly cross-reactive in a conventional sandwich immunoassay and it was confirmed that the CLAMP completely avoided cross-reactivity (Figure 3E). The standard curve of the 5-plex CLAMP is shown in Figure 3F.

[0214] The CLAMP was used to profile human sera. The conjugated antibodies and bar-coded beads were stored independently for >1 month and the CLAMP produced a good spike in the recovery of PSA in sera (data not shown).

[0215] In one embodiment, the CLAMP system described herein is a 10-plex cytokine panel. The cytokines included herein are, for example, but not limited to, IL1-IL17, MCP1 / 3, TNF, EGF / R, and / or VEGF / R. In another embodiment, the CLAMP system included herein is a 10-plex panel focused on breast cancer metastasis that targets HER2, CEA, p53 and / or CA15-3.

[0216] Example 2. CLAMP assay architecture The inventors prepared and tested a co-localization assay by ligation on microparticles (MPs) called "CLAMP" according to one embodiment. CLAMP is a multiplexed assay designed to eliminate reagent-driven cross-reactivity ("rCR") by co-localizing and confining each antibody pair on a set of barcoded MPs, thereby avoiding interactions between non-cognate antibodies (Figure 14C). Oligonucleotides (oligos) are used as programmable building blocks to perform the key molecular "operations" of CLAMP, including (i) flexible ligation of detection antibodies (dAbs), (ii) on-demand release of dAbs, (iii) introduction of assay signals, and (iv) fluorescent barcoding of MPs. Here, the inventors use reagents that strongly cross-react in conventional MSAs to eliminate rCR and describe in detail the conceptual operation, experimental validation, and optimization of the CLAMP assay and showcase.

[0217] In this specification, the architecture and operating principle of one embodiment, referred to as the CLAMP assay, are illustrated schematically in panels d and e of FIG. 14, respectively. To co-localize each pair of antibodies, an 82 nt hook-strand oligonucleotide (referred to as the hook oligo, or "HO") is covalently attached to a detection reagent that is an antibody called the detection antibody or "dAb", and is partially hybridized via a 21 bp hybrid to a capture strand oligonucleotide called the capture oligo (CO) that is bound to the surface of a capture reagent that is an antibody called the capture antibody (cAb) coated microparticle (MP). The cAB is immobile on the surface, but the dAb is flexible due to the 61 nt single-stranded domain of the HO. This flexibility allows the formation of a three-component complex with the analyte (FIG. 14E). The confinement of the antibody pair eliminates interactions between non-matching antibodies, restores a single plex assay configuration on all MPs, and ensures that a single cross-reaction event (e.g., a target analyte that reacts with a non-cognate cAb) does not result in a sandwich binding. Empirical co-localization of antibodies allows for rapid dual recognition of proteins, but requires an accompanying method for signal transduction and generation. One approach is to first disrupt the HO-CO linkage. For example, via photoinduced or enzymatic cleavage or linker-mediated displacement, and then, after washing the released dAb-HO complex, label the dAb remaining on the surface to signal sandwich formation. However, an unbroken CO-HO bond results in labeling of the corresponding dAb regardless of the presence of the target analyte, and as a result, increases the background signal. For example, a 2% dAb coating on 3 μm MPs corresponds to 1000 - 5000 dAbs, which, when labeled, results in a large increase in the background signal and can significantly interfere with sensitivity detection.

[0218] To mitigate this effect in the CLAMP assay, the inventors designed a detection scheme to exclusively label the “successfully” released conjugate through the use of a fluorescently labeled displacer oligo (DO) that binds to and simultaneously replaces and labels the footprint holding domain on HO (FIGS. 14E, 7). Importantly, this “detection by displacement” operates as an AND logic gate and requires both protein double capture and dAb release for a detectable signal (FIG. 14F). In this embodiment, the CLAMP reagent is assembled onto magnetic MPs in two steps, benefiting from biotin-streptavidin binding and the affinity of Watson-Crick base pairing (FIG. 18). In the first step, a mixture of biotinylated oligosaccharide and antibody is co-immobilized onto the surface of streptavidin-coated MPs. The one-pot nature of the labeling enables precise control over the CO surface density (FIG. 19), while at the same time enabling MP encoding via one-pot labeling using multicolor barcoding 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 via HO-CO hybridization to complete the assembly of CLAMP.

[0219] Example 3. Optimization of the CLAMP assay The inventors first optimized the efficiency of the footprint-mediated displacement reaction by replacing unbound Cy5-labeled HO (FIG. 15). HO was pulled down on MPs with different CO concentrations and then released using unlabeled DO. The increased ionic strength in the displacement buffer (MNaCl > 500 mM) served to screen the negatively charged oligos and improved the efficacy of DO hybridization to HO and the release of HO. 98% displacement was achieved over a wide range of CO concentrations with increased ionic strength and DO concentration (MNaCl ~ 500 mM and MDO = 1 μM, respectively).

[0220] Next, the inventors studied the effect of the antibody-oligonucleotide conjugate on the assay background by measuring the residual signal on the MPs after the labeling substitution step in buffer (see the method below). The inventors first conjugated HO to immunoglobulin-G (IgG) using a commercial kit (Solulink) that gave an antibody binding yield of approximately 90% and an average of 2 HO per IgG (i.e., λ~2). Using these conjugates, the assay background was several orders of magnitude higher than the assay background of un-conjugated HO (Figure 16A). The increase in the background signal was due to multivalent HO conjugates, which resulted in non-released dAb-HO complexes (due to intact HO-CO bonds) labeled by hybridization of the DO with at least one of the other HO strands, thereby generating a fluorescent signal in the absence of sandwich binding to the protein (Figure 16B). An effective way to minimize multivalent dAb-HO conjugates is to reduce the average conjugation valence, e.g., aiming for a λ of 0.1, for which the Poisson statistic indicates that <5% of the dAbs are bound to multiple HOs. The trade-off for such a low conjugation valence number is a decrease in the conjugation yield of the antibody (10%), which leaves 90% of the unreacted antibody. To avoid wasting the unreacted antibody, the inventors developed a binding and purification workflow that maintains the native state of the un-conjugated antibody and allows their recycling. The relative concentrations of dAb and HO were adjusted, varying from 1.25 to 0.1 (Figure 16C). dAb-HO conjugates with varying valences were pulled down on MPs with varying CO concentrations. As expected, the lower valence resulted in a significant decrease in the residual assay background, yielding lower valence conjugates with less than 8% multivalent conjugates that matched the background signal shown by un-conjugated HO at 0.1 < λ < 0.2 (Figure 16D). Consistent with the multivalent scenario, increasing the CO density amplified the higher background signal for the less numerous higher valence dAb-HOs.

[0221] To optimize assay performance, the dAb-HO concentration was adjusted. In CLAMP, the appropriate local dAb concentration is key for sensitive and high-capacity sandwich binding, which depends mainly on the surface density of dAb-HO for a given HO length and, through hybridization capture, on the CO. CLAMPs against urokinase plasminogen activator (anti-uPA CLAMP) with various CO concentrations were prepared using low-cost dAb-HO conjugates with less than 8% multivalent conjugates (see Figure 16D and the method below). The anti-uPA CLAMP was incubated with serial dilutions of recombinant upa antigen, followed by detection by washing and label displacement. As expected, increasing the CO concentration adjusted the signal-to-noise ratio (SNR) of the assay, revealing that a density greater than 10 14 m -2 was required for sufficient SNR (Figure 16E). On the other hand, a density exceeding 10 14 m -2 also resulted in an increased background signal and thus provided little improvement in SNR. Finally, to evaluate the importance of conjugate valency on assay performance, we compared high-valency (λ~2, Solulink) anti-uPA to low-valency conjugates (λ~0.1, Figure 16F). The low-valency conjugate resulted in a significantly lower background signal (10-fold), corresponding to a 3-fold improvement in the limit of detection (Figure 16F). On the other hand, the low-cost conjugate showed a decrease in the fluorescence dynamic range since the sandwich-binding dAb-HO complex was mainly labeled with a single dye. Taken together, these results generally highlight the importance of conjugate valency in background signal and assay performance.

[0222] Example 4. Multiplexed CLAMP assay To test the effectiveness of CLAMP in eliminating reagent-driven cross-reactivity (“rCR”), the inventors screened the assay specificity of multiplexed CLAMP according to one embodiment. Further, to challenge the CLAMP assay, the inventors selected antibody pairs that have been shown to exhibit different types of rCR when used together in a conventional multiplexed sandwich assay (“MSA”). For this purpose, antibodies against six targets (EpCAM, PSA, E-cadherin, EGF, uPA and MCP) were selected from a 35-protein panel (Dagher, M, et al, Nature Nanotechnology, vol, 13, pp. 925-932, 2018) that the inventors had previously characterized for specific and non-specific binding in a conventional MSA. For the conventional MSA, the specificity screen consisted of incubating each individual antigen with a pool of cAB-coated barcoded MPs, followed by adding a mixed dAb cocktail and a secondary antibody (“sAb”) for detection and labeling, respectively (Figure 17A). Measuring fluorescence across different barcodes in response to antigen concentrations of 1 and 100 ng / ml (Figure 17B-C) discriminates two types of non-specific binding that generate false positives, namely, antigen attachment (observed for E-cadherin and uPA) and cross-reactivity between the antigen and the antibody. On the other hand, the specificity screening for the CLAMP assay was performed by incubating a single antigen with multiplexed CLAMP for -a hours and performing detection by label-displacement (Figure 17d-f, see methods below). All but one of the non-specific signals detected in the conventional MSA were completely removed using the CLAMP assay. For example, the spreading non-specific binding of E-cadherin that resulted in signals on all off-target beads in the conventional MSA could not be detected in the CLAMP assay. In contrast, cross-reactivity was detectable between the 100 ng / ml MCP-1 antibody and the EGF antigen in both the conventional MSA and CLAMP.To investigate the source of this false positive signal, the inventors performed a single plex assay using only the MCP-1 antibody and spiked MCP-1 or EGF separately at 1 or 100 ng / ml (Figure 17G). Detection of EGF by the MCP-1 antibody in the single plex showed dCR. In fact, this dCR could not be reduced by CLAMP or ELISA and is a binder with poor affinity. Overall, these results are the strength of CLAMP in removing rCR in multiplexed assays and identifying dCR in a multiplexed, combinatorial manner. Finally, dilution curves for the remaining 5 proteins were generated and their SNR was plotted as shown in Figure 17H.

[0223] In summary, the inventors have successfully demonstrated a homogeneous MSA using CLAMP and oligonucleotides on MPs pre-co-localized with antibody pairs. By restricting each antibody pair to its respective MP during sample incubation, CLAMP can be multiplexed while maintaining a single plex assay environment on each MP, thereby eliminating reagent-driven CR. In particular, pre-co-localization of antibodies in CLAMP represents a departure from traditional sandwich immunoassays where the matching antibodies are separated at the start of the assay. To detect accurate sandwich binding, the inventors showed that labeled displacer oligos can be used to simultaneously release and label dAb-oligo complexes. The inventors studied and demonstrated the importance of using monovalent antibody-oligo conjugates to avoid labeling un-released complexes and increasing background signal. The inventors experimentally verified the assay in both single plex and multiplexed formats and screened the specificity of the assay in multiplexing using 5 pre-selected antibody pairs for CR, thereby demonstrating that CLAMP eliminates all rCR experienced in traditional MSAs.

[0224] CLAMP can offer several different advantages over currently available MSAs. First, CLAMP can be easily deployed as it does not require dedicated 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 (typically done at high concentrations), CLAMP can significantly reduce reagent consumption. Due to its highly scalable and very efficient nature, CLAMP can be used to provide a truly scalable multiplexed ELISA platform that meets the increasing demands in biomarker discovery and drug development.

[0225] Example 6. Low antibody concentrations minimize cross-reactivity in the CLAMP assay Conventional multiplexed sandwich immunoassays are generally performed using a mixture of reagents in solution phase. In particular, detection antibodies (dAbs) against different targets are mixed and applied to the reaction. Application of such dAb cocktails results in false binding and generates false positive signals from non-specific binding events (between cAb or dAb and non-target analytes) that are difficult to distinguish from true target protein binding signals. The risk of reagent-driven CR scales with the number of target analytes N at ~4N 2 scale.

[0226] In contrast, in embodiments of CLAMP, pairs of reagents (e.g., antibodies) can be pre-assembled and co-localized on barcoded microparticles to avoid reagent mixing. The detection antibody (dAb) is only released in solution after the substitution reaction, as described herein. In some embodiments, the dAb released into solution should be optimally maintained at a sufficiently low concentration to avoid rebinding on off-target beads after the substitution reaction. FIG. 16 plots the dAb concentration profile against the starting amount (y-axis) and volume of the solution during the substitution process. A typical dAb concentration in a conventional ELISA is ~1 μg / mL (67 nM), and with a sufficiently long incubation, binding can still occur if the dAb is low (such as in the Simoa assay by Quanterix) to about 1 nM.

[0227] To ensure that off-binding is avoided after release, the amount of antibody per target should ideally be kept below <10 pm. At a volume of 100 μl, the amount of antibody is <1 fmol. In the CLAMP assay, in some embodiments, the amount of Ab released from 1000 microparticles was estimated to be 0.1 - 1 fmol (FIG. 22). The numbers indicate that the dAb concentration released from the CLAMP system is significantly lower compared to other methods that require free diffusion-based reagent mixing.

[0228] Example 7. Substitution-dependent signaling minimizes background signal in the CLAMP assay In embodiments of the CLAMP co-localization assay where both antibodies are pre-co-localized on the support, signaling can be performed by detecting all dAB remaining on the surface after release and washing. However, any non-released hook oligo-dAb complex can give rise to an analyte-independent signal and significantly contribute to background noise. Thus, in some embodiments, it is understood that nearly complete anchor-hook substitution and washing of the hook oligo-dAB complex are required to avoid an increase in background signal.

[0229] In some embodiments, the problem of increased background signal due to inefficient release can be addressed through a substitution-dependent signal transduction mechanism. Such a mechanism ensures that only the substituted hook anchor strand is detectable, and as such, unsubstituted strands that may result from inefficient substitution do not generate a background signal. In such embodiments, signal transduction at the molecular level occurs only when both (i) the formation of the three-component complex and (ii) the conditions for substitution of the hook anchor strand are met.

[0230] In some embodiments, thus, the detection Ab and the hook strand are unlabeled, and substitution occurs using a labeled (e.g., fluorescently labeled) displacer oligo. In this mode, the displacer oligo can preferentially (i) release it from the anchor strand and (ii) bind to the hook strand that labels it. On the other hand, the unsubstituted hook oligo is unlabeled and does not contribute to the signal. This mechanism is equivalent to an AND gate where the signal (output) depends on both substitution (input 1) and analyte presence (input 2), as shown in FIG. 23.

[0231] To demonstrate the effectiveness of displacement-dependent signal transduction, the inventors conducted calibration assays for IL-7, IFN-γ, and MMP-9. In the first test, the displacer oligo was unlabeled and the mouse-dAb was targeted using an anti-mouse BV421 secondary antibody. The BV421-labeled secondary antibody was targeted with dAb regardless of whether it was released, and thus, labeling occurred regardless of substitution. In the second test, the displacement oligo was labeled with Cy5, which was used to test displacement-dependent signal transduction. As shown in the logic gate display chart (Figure 23), the BV421 signal was introduced in conditions (i), (iii), (iv), while the Cy5 signal appeared only in condition (iv). An example of the calibration curve from the target obtained by using the two labeling methods is shown in Figure 24. The signal background from BV421 was significantly higher compared to the Cy5 signal, but the assay performance regarding sensitivity and dynamic range was improved using the labeled substitution (Cy5).

[0232] Example 8. Low-valency antibody-oligos minimize background signal in the CLAMP assay In some embodiments, the hook strand and the anchor strand are DNA oligonucleotides. Antibody-DNA conjugation can be performed, for example, by targeting lysine groups on the IgG molecule. A heterobifunctional linker such as sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) can be used to bind the thiol-terminated DNA to the IgG molecule. However, this reaction results in a heterogeneous conjugate, where the number of oligos per antibody depends on the DNA:antibody stoichiometry during the reaction. Multivalent conjugates (more than one oligo per antibody) can reduce the substitution efficiency and thus increase the background signal.

[0233] As shown in FIGS. 25A - B, the inventors adjusted the valency, measured it by SDS - PAGE, and determined the displacement efficiency using the resulting conjugates. High - valency conjugates led to an increase in assay background. As expected, higher anchor - chain oligo density led to a further increase in background signal. To determine the effect of high - valency conjugates on signal background, the inventors performed a CLA assay using a displacement - dependent detection mechanism (FIG. 25C). Calibration curves for uPA were generated using high - valency (λ, average, number of oligos per antibody ~ 2) and low - valency conjugates (λ ~ 0.1). Low valency resulted in a decrease in background and a three - fold improvement in sensitivity.

[0234] Example 9.40 - Cross - reaction characteristics in a 40 - plex To evaluate cross - reactivity in a multiplex assay with higher multiplexing, a panel of 40 targets was tested, and a mixture of CLAMPs for the 40 targets (shown in FIG. 26) was mixed together and incubated in buffer spiked with one of the high - concentration (100 ng / mL) targets (protein standard, typically recombinant) in each well. The signals seen on the diagonal indicate specific interactions between the correct antigen and its barcoded microparticle pair. Only a few off - target signals were measurable, but these were not due to reagent cross - reactivity. Instead, the antigen was considered to cross - react with both antibodies and thus was as significant for single - plex ELISA as shown in FIG. 17.

[0235] Methods Materials and reagents. HPLC - purified oligonucleotides were purchased from IDT (Coralville, IA, USA). Sequences and modifications are shown in FIG. 11. cAB, antigens, and dAbs were purchased from Rnd Systems (Minneapolis, MN, USA) and stored at - 20 °C for up to 36 months. Streptavidin - and protein G magnetic MPs (M270) were purchased from Life technologies (Carlsbad, CA, USA).

[0236] Synthesis of CLAMP. CLAMP was assembled on streptavidin-coated magnetic MPs (M270-streptavidin) with a diameter of 2.7 μm in two steps. The first step consisted of immobilizing a biotinylated mixture of antibodies and oligos, functionalizing the MPs, and simultaneously encoding them as described in detail elsewhere (Dagher, M. et al, Nature Nanotechnology, vol. 13, pp. 925-932, 2018). Briefly, 90 picomoles of biotinylated oligos (CO, SO) and a total of 90 picomoles of LO (LO0-LO2) were mixed together in 25 μL of PBS + 0.05% Tween20 + 300 mM NaCl (PBST0.05 + NaCl300). The ratio of each LO0:LO1:L02 was designed to generate a unique ensemble fluorescence that defines the barcode, while the ratio of CO:SO allows for adjustment of the surface density of the pulled dAb-HO. The mixture was annealed by heating the mixture to 80 °C and then cooling it back to room temperature by removing the mixture from the heat source. Next, 5 μg of biotinylated cAB in 17 μl of PBST0.05 + NaCl300 was added to the annealed oligonucleotide mixture and mixed. Subsequently, the biotinylated reagents were co-immobilized onto the MPs in a single step by adding 3.25 M MP in 10 μl of PBST0.05 + NaCl300 and immediately mixing by pipetting. The mixture was incubated for 90 minutes at room temperature using end-over-end mixing, followed by three washes by magnetic aggregation in 150 μl of PBST0.1. The barcoded and functionalized MPs were stored at 4 °C until needed. In the second step, 100,000 of the prepared MPs were mixed with an HO-containing solution (e.g., dAb-HO) diluted with PBST0.05 + NaCl300 for 30 minutes. After the pull-down of HO, the fully assembled CLAMP was washed three times in PBST0.01 and stored at 4 °C until the assay time up to one week.

[0237] Characterization of CLAMP. To characterize CLAMP, immobilization of antibodies and oligos was confirmed by labeling with anti-goat IgG conjugated to Alexa-Fluor647 (AF647) or hybridization of Cy5-labeled oligos (LO) targeting HO. As described elsewhere (Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925 - 932, 2018), the density of CO was estimated by fitting the ensemble fluorescence responses of multi-color MPs using a multi-color fluorescence model. To determine the expected assay background signal for a particular set of CLAMP, MPs were incubated with 1 μM Cy5-labeled DO in PBST0.05 + NaCl300 for 1 h, followed by three magnetic washes in PBST0.05, and the remaining signal was measured by cytometry.

[0238] Antibody-oligo conjugation, purification, and characterization. Anti-uPA monoclonal antibodies were conjugated to amine-modified HPs using hydrazone chemistry (Solulink) and subsequently 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 h. The reduced oligo was (i) buffer-exchanged to PBS pH 7.0 using a Zeba desalting spin column (7K MWCO, Thermo), (ii) activated for 10 min using 80% PBS pH 7.0 and 8 μL of dissolved 9 mM sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), (iii) buffer-exchanged back to PBS pH 7.0 to remove excess sulfo-SMCC, and (iv) reacted with 1 - 10 μl fractions (as desired) with 10 μL of 1 mg / ml antibody. The reaction was left at room temperature for 1 h and then incubated overnight at 4 °C. The conjugates were then purified in two purification steps, in an antibody and a DNA purification step, respectively.

[0239] Antibody-Oligonucleotide Conjugation, Purification, and Characterization. Anti-uPA monoclonal antibody was conjugated to amine-modified HP using hydrazone chemistry (Solulink) and subsequently purified according to the manufacturer's protocol. Alternatively, the monoclonal antibody was 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 oligo was (i) buffer-exchanged to PBS pH 7.0 using a Zeba desalting spin column (7K MWCO, Thermo), (ii) activated for 10 minutes using 80% PBS pH 7.0 and 8 μL of dissolved 9 mM sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), (iii) buffer-exchanged back to PBS pH 7.0 to remove excess sulfo-SMCC, and (iv) 1 - 10 μl fractions (as desired) 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 conjugate was then purified in two purification steps.

[0240] Singleplex and multiplex CLAMP assays. Incubations were performed at room temperature in conical bottom 96-well plates with horizontal shaking at 950 rpm. CLAMPs were mixed at approximately 80 MP / barcode / 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 and multiplexed CLAMP mixture was added to each well and incubated with 25 μl containing a specific antigen(s) at 2× the specified concentration in PBST0.05 + NaCl150 + BSA0.25, and the incubation was carried out with shaking at 950 rpm for 3 hours. Magnetic aggregation and washing with 150 μL of PBST0.1 were repeated 4 times over a total of 30 minutes. Finally, detection by displacement was performed through the addition of 1 μM DO-Cy5 in PBST0.05 + NaCl300 + BSA0.25, incubated for 1 hour with shaking, and then washed 3 times with PBST0.1.

[0241] Conventional MSA. To screen for specificity and non-specific binding in the conventional MSA format, as described above, MPs were barcoded and coupled to their respective biotinylated cAbs during synthesis. The MP mixture was adjusted to a final concentration of 2000 MPs per barcode per assay. Incubations were performed at room temperature in conical bottom 96-well plates with horizontal shaking at 950 rpm. Prior to incubation with assay reagents, MPs were first blocked with 1% bovine serum albumin in 0.05% Tween-20 in PBS (PBST0.05) for 1 hour. Incubation with antigen was performed for 120 minutes at the specified concentration. BMP was incubated with the dAb cocktail at 2 μg / ml for 60 minutes, followed by incubation with sAb at 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).

[0242] Reading and data analysis. MPs were read out using a FACS CANTOII cytometer by BD using blue (488 nm), red (633 nm), and violet (405 nm) lasers. In the blue laser flow cell, 530 / 30 and 585 / 42 bandpass filters were used for FAM and Cy3, respectively. In the red laser flow cell, a 660 / 20 bandpass filter was used for Cy5 / AF647. MPs were decoded using an automated algorithm implemented on MATLAB (Dagher, M. et al, naturenanotechnology, vol. 13, pp. 925 - 932, 2018). All data analysis was performed in MATLAB. Single beads were distinguished from bead aggregates and other microparticles using forward scatter intensity and side scatter intensity, and gating was automated.

[0243] Although the present disclosure has been described in connection with its particular embodiments, it is intended to embrace any modifications, uses, or adaptations including such departures from the present disclosure, within the scope of known or customary practice in the art, and applicable to the essential features herein described, as will be understood from the following in the appended claims.

[0244] The content of all documents and references cited herein are hereby incorporated by reference in their entirety.

Claims

Claim 1 A composition for the detection and / or quantification of an analyte, comprising: a) a support; b) an anchor chain attached to the support; c) a capture reagent attached to the support; d) a detection reagent linked to a hook chain, wherein the hook chain is releasably attached to the anchor chain, and the hook chain and the anchor chain are linked to each other by a double-stranded DNA hybrid; and e) a displacer agent that is complementary to at least a portion of the hook chain and is hybridizable to the hook chain, thereby releasing the hook chain from the anchor chain via a DNA displacement reaction, and is detectably labeled, wherein when the analyte is present, the capture reagent and the detection reagent can simultaneously bind to the analyte to form a complex, the complex comprising: (i) the analyte bound to the capture reagent attached to the support; (ii) the detection reagent bound to the analyte and linked to the hook chain; and (iii) the displacer agent hybridized to the hook chain, and after release of the hook chain from the anchor chain by the displacer agent, the detection reagent and the displacer agent are retained on the support via the complex; wherein when the analyte is absent, after release of the hook chain from the anchor chain by the displacer agent, the detection reagent and the displacer agent are not retained on the support. Claim 2 The composition according to claim 1, wherein the support is a microparticle, the surface of a multi-well plate, the surface of a slide glass, or a hydrogel matrix. Claim 3 The composition according to claim 1 or 2, wherein the analyte is from a liquid sample, and the liquid sample is a body fluid, an extract, a solution containing protein and / or DNA, a cell extract, a cell lysate, or a tissue lysate. Claim 4 The composition according to any one of claims 1 to 3, wherein the capture reagent is an antibody, an antibody fragment, an aptamer, a modified aptamer, an affimer, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, an oligonucleotide, or a low molecular weight compound. Claim 5 The composition according to any one of claims 1-4, wherein the detection reagent is an antibody, antibody fragment, aptamer, affimer, modified aptamer, antigen, protein, polypeptide, multi-protein complex, exosome, oligonucleotide, or low molecular weight compound.

6. The composition according to any one of claims 1-5, wherein the capture reagent and the detection reagent are different.

7. The composition according to any one of claims 1-6, wherein both the capture reagent and the detection reagent are antibodies.

8. The composition according to claim 7, wherein the capture reagent and the detection reagent are different antibodies that bind to different epitopes on the analyte.

9. The composition according to any one of claims 1-8, wherein the analyte is an antigen, antibody, affimer, aptamer, modified aptamer, antibody fragment, protein, polypeptide, multi-protein complex, exosome, oligonucleotide, hormone, modified oligonucleotide, or low molecular weight compound.

10. a) A second anchor chain attached to the support; b) The composition according to any one of claims 1-9, further comprising a second detection reagent releasably attached to the second anchor chain.

11. The composition according to claim 10, wherein the second detection reagent is linked to a second hook chain, and the second hook chain is releasably linked to the second anchor chain.

12. The composition according to claim 10 or 11, further comprising a second displacer agent that is hybridizable to the second hook chain and thereby releases the second hook chain from the second anchor chain via a DNA displacement reaction, and the second displacer agent is detectably labeled.

13. A multiplex sandwich assay system for simultaneous detection and / or quantification of two or more analytes, the system comprising two or more supports as defined in any one of claims 1-12, each support being for detection and / or quantification of a different analyte.

14. The multiplex sandwich assay system according to claim 13, wherein the support is a microparticle, multi-well plate, surface of a slide glass, or hydrogel matrix.

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