Compounds, compositions, and methods for improving assays

Abstract

To provide a kit for detecting a target in a sample, and to provide a method for detecting a target in a sample.SOLUTION: Provided is a kit comprising: (i) a solid support (e.g., a bead) comprising: an encoding agent (e.g., a dye); a first nucleic acid sequence (e.g., an oligonucleotide and / or primer); and a first molecular recognition element (e.g., an antibody); and (ii) a detection reagent comprising a second molecular recognition element (e.g., an antibody) and a second nucleic acid sequence, at least a portion of the first nucleic acid sequence and at least a portion of the second nucleic acid sequence being configured to participate in a nucleic acid amplification process.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] (Federal Government Assistance Statement) This invention was made with Government support under Grant No. HR0011-12-2-0001 awarded by the Department of Defense (DARPA). The United States Government has certain rights in this invention.

[0002] The present invention relates to compounds, compositions, and methods for improving assays, such as, for example, multiplexed immuno-PCR assays. [Background technology]

[0003] Immunoassays for target molecules such as proteins are generally based on pairs of molecular recognition elements (such as antibodies or aptamers) that first capture the target molecule and then label it so that it can be quantified. Typically, the capture antibody is bound to a solid support such as the wall of a microwell plate or the surface of a microbead. The target molecule is captured by incubating the sample with the antibody-coated surface. After a washing step to remove the sample matrix, the captured target molecule is labeled with a detection recognition element (such as an antibody or aptamer) that can either be directly conjugated to a reporter molecule (tag) or conjugated to a linking reagent (such as biotin) that can then be coupled to the tag. Alternatively, the detection recognition element can be detected using a secondary antibody that is further detected by the tag. The tag can include nanoparticles, dyes, enzymes, or nucleic acids (NAs) that can be detected by direct observation or by chemical or biochemical assays.

[0004] For applications that do not require high sensitivity, the tag can be a gold nanoparticle (common in lateral flow type assays) or a fluorescent dye that can be directly detected. One common immunoassay that uses direct labeling is commercialized by Luminex Corp. In this assay, a multiplexed immunoassay is performed using coded microbead sets. Each bead set is coded by staining the beads with a unique combination of dyes. In addition, each bead set is functionalized with a different affinity reagent (e.g., antibody, antigen, etc.). These beads can be mixed together to form a multiplexed bead library. During the assay, these beads are incubated with the sample, which captures the target molecule if present. After washing the beads are incubated with a detection antibody that is labeled with a reporter dye or a detection antibody that is biotinylated so that it can later be labeled with a streptavidin-conjugated reporter dye. Flow cytometry or fluorescence microscopy is used to measure both the coding dye combination (to identify the type of bead) and the assay signal for each bead (i.e., the signal from the reporter dye). The concentration of target molecules in a sample is determined by comparing the average assay signal measured for each bead type to a standard curve. In this approach, captured target molecules are labeled with approximately one to five fluorophores, depending on the number of fluorophores attached to the detection antibody or its degree of biotinylation.

[0005] Enzyme-linked immunosorbent assay (ELISA) is a well-known immunoassay that uses signal amplification to improve assay sensitivity. ELISA uses an affinity reagent pair to capture and label target molecules with an enzyme, such as horseradish peroxidase, alkaline phosphatase, glucuronidase, or galactosidase. The enzyme then converts a substrate into a detectable product in a concentration-dependent manner, allowing the concentration of the target molecule to be determined (Figure 1). This approach is generally more sensitive than direct labeling approaches, since one enzyme label can rapidly convert many substrate molecules into detectable product molecules.

[0006] In ELISA, the capture antibody is bound to a solid phase, such as the surface of a microwell plate, and an analog signal is measured using a reader, such as a fluorescent plate reader. Multiplexing can be performed in space where the sample is separated between different wells of a microwell plate that contain different capture antibodies and to which different detection antibodies are added. Single-vessel multiplexing can also be performed, but is generally not practical due to the additional complexity of needing a different enzyme-substrate pair for each target. Alternatively, the capture antibody can be linked to microbeads or magnetic microbeads to make them easier to handle or improve the workflow (Figure 1).

[0007] In addition to ELISA, proteins and other molecules can be quantified using immunoPCR (iPCR). This approach uses affinity reagents similar to ELISA, but unlike ELISA, the detection reagent (e.g., antibody) is labeled with a nucleic acid (NA) tag rather than an enzyme. The NA tag can be covalently linked to the antibody (forming a conjugate) or linked by an interaction such as biotin-streptavidin binding. The tag is typically amplified by quantitative PCR (qPCR) to determine the concentration of the target molecule. Similar to ELISA, iPCR can be performed in an analog signal format using microwell plates coated with capture antibodies. The immune complexes can be left intact or separated to release the NA tags before PCR. The released tags can be detected using digital PCR in a droplet or microwell array format, resulting in more accurate quantification. Multiplexing can be achieved by splitting the sample between singleplex reactions or by using different colored probe sets with multiple unique tag sequences in a single reaction.

[0008] Similar to ELISA, iPCR can also be performed using capture antibodies bound to beads (Figure 2). Differently coded bead sets are functionalized with different capture antibodies, so that the identity of the capture antibody bound to each bead can be decoded from the optical signal (e.g., fluorescence from different concentrations of multiple dyes attached or embedded in the beads). Different bead sets can be mixed together to form bead libraries or assay panels that can quantify multiple analytes in a single sample (Figure 3).

[0009] In a typical bead-based iPCR assay, beads are incubated with the sample to capture the target molecules, washed to remove the sample matrix, and then labeled with a detection antibody (Figure 4). If the detection antibody is covalently linked to a nucleic acid tag (i.e., detection antibody-NA conjugate or "detection conjugate"), the immune complex is complete. However, if the detection antibody is biotinylated, these beads are subsequently incubated with streptavidin and biotinylated NA tags. Quantitative PCR is used to determine the number of NA tags and therefore the concentration of the original sample. Multiplex assays are more complex than singleplex assays because each target molecule requires a different pair of capture and detection antibodies.

[0010] Similar to the digital ELISA described above, in a digital iPCR assay, PCR reactions are separated from each other by loading beads into individual reaction wells. Most wells contain only one bead and a small amount of PCR master mix containing primers to amplify the tag sequence. PCR amplification is performed after sealing the wells, for example with immiscible oil. The identity of the beads is determined by examining their optical properties (e.g., fluorescence intensity at one or more wavelengths). PCR-positive beads can be identified by several methods, such as measuring the fluorescent signal of dyes or hydrolysis probes (e.g., TaqMan probes) that intercalate into double-stranded DNA (dsDNA).

[0011] At low target molecule concentrations, most beads have zero or one detection conjugate, and the percentage of PCR positive beads (i.e., digital signal) can be used with Poisson statistics to determine the average number of detection conjugates per bead and thus the concentration of target molecules in the sample. At high target concentrations, most beads may have more than one detection conjugate, and an analog signal (e.g., real-time PCR or another quantitative measure of target molecule population) can be used to determine the number of detection conjugates on each bead. The combination of digital and analog PCR signals allows for a very large dynamic range (up to about 10 under certain conditions). 10 ), while maintaining the accurate quantification capability of digital PCR at low target concentrations. Summary of the Invention [Problem to be solved by the invention]

[0012] In all immunoassays detailed above, the concentration of the detection affinity reagent (e.g., antibody) must be optimized for each target molecule. For example, if the detection antibody concentration is too low, the captured target molecules will not be labeled efficiently and the assay will have insufficient sensitivity. Increasing the detection antibody concentration will improve the sensitivity as more captured target molecules will be labeled, but this can also increase the background signal of the assay. The increase in background signal is often caused by nonspecific binding (NSB) of the detection reagent to the microwell or microbead surface, interaction with the capture antibody, or interaction with the coding dye of the beads in bead-based immunoassays. Unfortunately, it is not possible to distinguish a positive signal derived from a properly formed immune complex (Figure 5, complex A) from nonspecific interactions such as the interaction between the capture antibody and the detection antibody (Figure 5, complex B), the interaction of the capture antibody with the NA tag (Figure 5, complex C), or the interaction of the NA tag with the bead surface (Figure 5, complex D). It should be noted that the antibody portion of the detection antibody conjugate may also interact with the bead surface (Figure 5, complex E), and NSB may occur between the detection antibody and other proteins in the sample matrix that may be specifically or nonspecifically bound to the beads or capture antibody.

[0013] The above NSB interactions become more severe when combining singleplex assays into a multiplex panel, often requiring reoptimization of detection antibody concentrations and other assay parameters. The main reason for increased NSB in multiplex assays is that as more target molecules are added to the assay, the total detection antibody concentration increases, generally increasing the signal observed from NSB. Furthermore, cross-reactivity between antibodies and off-target molecules can become a bigger problem in multiplex assay formats. There are many forms of cross-reactivity that can affect the assay signal. For example, an antigen may bind to the wrong (off-target) capture antibody and then be detected by its own detection antibody, resulting in a false positive signal. In a similar example, an antigen may bind to the wrong (off-target) capture antibody but not be detected by the detection antibody. In this case, the wrongly captured antigen may block (i.e., competitively inhibit) the detection of the actual target following its binding, resulting in a false negative signal.

[0014] Although signal due to cross-reactivity and NSB can sometimes be mitigated in less sensitive direct label or bulk analog assays, this signal is a significant issue for single molecule assays, as even a single NSB event per bead can be detrimental to digital assay performance. Highly sensitive multiplexed assays are usually limited to about 5-10 different targets, as NSB and cross-reactivity between detection chemistries greatly reduce the signal-to-noise ratio and raise the detection limit.

[0015] In addition to the problems highlighted above, multiplexed PCR often suffers from off-sequence amplification and competition between primer and probe sets. To achieve highly sensitive iPCR assays at higher levels of multiplexing, alternative approaches are required. [Means for solving the problem]

[0016] Aspects of the invention relate to compounds, compositions, kits, systems, devices and methods for improving assays, such as multiplexed PCR assays (eg, multiplexed immuno-PCR assays).

[0017] One aspect of the present invention relates to a kit comprising (i) a solid support (e.g., a bead) comprising a coding agent (e.g., a dye), a first nucleic acid sequence (e.g., an oligonucleotide and / or primer) and a first molecular recognition element (e.g., an antibody), and (ii) a detection reagent comprising a second molecular recognition element (e.g., an antibody) and a second nucleic acid sequence, wherein at least a portion of the first nucleic acid sequence and at least a portion of the second nucleic acid sequence participate in a nucleic acid amplification process. In some embodiments, the solid support and the detection reagent are stored separately. In some embodiments, the solid support and the detection reagent are present in the same composition and / or stored and / or provided together.

[0018] Another aspect of the invention relates to a solid support (eg, a bead) comprising a coding agent (eg, a dye), a nucleic acid sequence, and a molecular recognition element (eg, an antibody).

[0019] A further aspect of the invention relates to a plurality of solid supports comprising: (i) a first plurality of solid supports, each solid support comprising a first coding agent (e.g., a dye), a first nucleic acid sequence, and a first molecular recognition element (e.g., an antibody) that provides a first coding signal; and (ii) a second plurality of solid supports, each solid support comprising a second coding agent (e.g., a dye), a second nucleic acid sequence, and a second molecular recognition element (e.g., an antibody) that provides a second coding signal, wherein the first coding signal is different from the second coding signal, the first nucleic acid sequence is different from the second nucleic acid sequence, and the first molecular recognition element is different from the second molecular recognition element.

[0020] Another aspect of the invention relates to a plurality of detection reagents comprising: (i) a first plurality of detection reagents, each detection reagent comprising a first molecular recognition element (e.g., an antibody) and a first nucleic acid sequence; and (ii) a second plurality of detection reagents, each detection reagent comprising a second molecular recognition element (e.g., an antibody) and a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence comprise a first portion and a second portion, respectively, and the nucleic acid sequence of the first portion for the first nucleic acid sequence is different from the nucleic acid sequence of the first portion for the second nucleic acid sequence. In some embodiments, the nucleic acid sequences of the first nucleic acid sequence and the second portion for the second nucleic acid sequence are the same.

[0021] A further aspect of the invention relates to a method of detecting a target in a sample, comprising combining a solid support as described herein with a sample comprising a target to form a first composition comprising a solid support bound to a target, optionally washing (e.g., with an aqueous solution) the solid support bound to the target, combining a detection reagent with the solid support bound to the target to form a complex comprising the solid support, the target, and a detection reagent, wherein the detection reagent comprises a second molecular recognition element and a second nucleic acid sequence, and at least a portion of the first nucleic acid sequence and at least a portion of the second nucleic acid sequence are configured to participate in a nucleic acid amplification process, optionally washing (e.g., with an aqueous solution) the complex, optionally combining an amplification agent (e.g., a reverse primer, a ssDNA splint, a ligase, etc.) with the complex, optionally releasing the nucleic acid sequence from the solid support, and amplifying at least a section of the second nucleic acid sequence, thereby detecting the target in the sample.

[0022] Another aspect of the invention relates to a method of improving the specificity and / or reducing background signal of an assay, comprising providing a solid support as described herein or a plurality of solid supports as described herein, and performing an assay in the presence of said solid support or said plurality of solid supports, thereby improving the specificity and / or reducing background signal of the assay.

[0023] It should be noted that aspects of the invention described with respect to one embodiment may be incorporated in a different embodiment even if not specifically described in that context. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination. The applicants reserve the right to modify any originally filed claims and / or to file any new claims, including the right to amend any originally filed claims to depend on and / or incorporate any features of any other claim or claims even if not originally so claimed. These and other objects and / or aspects of the invention are described in detail in the specification set forth below. Further features, advantages and details of the invention will be apparent to those skilled in the art from a reading of the drawings and the following detailed description of the preferred embodiments, such description being merely illustrative of the invention. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic diagram of a complex formed from an exemplary affinity reagent pair for use in an ELISA. [Diagram 2] FIG. 1 is a schematic diagram of a complex formed from an exemplary affinity reagent pair for use in iPCR. [Diagram 3] FIG. 1 is a schematic diagram of three different coded bead sets functionalized with separate capture antibodies, with each bead set corresponding to a different detection antibody. [Figure 4] FIG. 1 is a schematic diagram of exemplary method steps in a bead-based iPCR assay. [Diagram 5]FIG. 1 is a schematic diagram of interactions that can result in a true or false positive in an immunoassay. [Figure 6] FIG. 1 is a schematic diagram of an exemplary composite formed in accordance with some embodiments of the present invention. [Figure 7] FIG. 1 is a schematic diagram of bead sets prepared by encoding each set with a unique dye or combination of dyes, and the corresponding detection reagents for each set contain different nucleic acid tags according to some embodiments of the present invention. [Figure 8] FIG. 1 is a schematic diagram of an exemplary complex that is correctly formed (i.e., the capture antibody on the bead binds to the appropriate target and the corresponding detection reagent is also bound to the target) resulting in a fluorescent signal, and a complex that is incorrectly formed (i.e., the capture antibody on the bead binds to a detection reagent that is not the corresponding detection reagent of the bead) resulting in no fluorescent signal, according to some embodiments of the present invention. [Figure 9A] Graph showing the percentage of positive beads obtained in singleplex assays for various targets. [Figure 9B] Graph showing the percentage of positive beads obtained in a multiplex assay for different targets. [Figure 9C] 9A and 9B are graphs showing background signals for the singleplex and multiplex assays referenced in FIGS. 9A and 9B, respectively. [Figure 10] FIG. 1 is a schematic diagram of a method for preparing a solid support according to some embodiments of the present invention. [Figure 11] 1 is a plot of the fluorescence intensity of coding dyes bound to representative beads from various bead sets. [Figure 12] Figure 1 shows a graph comparing the percentage of positive beads of conventional iPCR beads with the percentage of positive beads prepared according to some embodiments of the present invention. For all assays, iPCR dAb conjugates were used with one set of primers contained in the PCR master mix. [Figure 13]1 shows a graph comparing the activity of a conventional iPCR detection reagent conjugate with the activity of a detection reagent prepared according to some embodiments of the present invention. For all assays, iPCR beads were used with one set of primers contained in a PCR master mix. [Figure 14] 1 shows a graph comparing the percentage of positive beads for various targets in a baseline singleplex assay using a conventional iPCR system with the percentage of positive beads for the same targets using a system according to some embodiments of the present invention. [Figure 15] Graphs showing the signal obtained for each bead type in a 3-plex iPCR assay and comparing different assay conditions are shown. [Figure 16] 1 shows graphs illustrating the signal obtained for each bead type in a 3-plex assay according to some embodiments of the present invention, as well as graphs comparing different assay conditions. [Figure 17] A comparative graph is shown for a 3-plex iPCR assay in FIG. 15 and a comparative graph is shown for a 3-plex assay according to some embodiments of the present invention in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0026] The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0027] Unless otherwise specified, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Furthermore, terms as defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of this application and related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. The terms used in the description of the invention herein are intended only to describe specific embodiments and are not intended to limit the invention. The entire contents of all publications, patent applications, patents, and other references cited herein are incorporated herein by reference. In the event of conflict, the present specification shall control.

[0028] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0029] Unless the context clearly indicates otherwise, it is specifically contemplated that the various features of the invention described herein can be used in any combination. Moreover, the invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein can be excluded or omitted. By way of example, if a composite is described herein as comprising components A, B, and C, it is specifically contemplated that any of A, B, or C, or any combination thereof, can be omitted or discarded.

[0030] As used herein, the transitional phrase "consisting essentially of" (and grammatical variants) should be construed to include the recited materials or steps of the claimed invention "as well as materials or steps which do not materially affect one or more of the basic and novel characteristics." See In re Herz, 537 F.2d 549,551-52,190 USPQ461,463 (CCPA 1976) (emphasis in original); see also MPEP section 2111.03. Thus, the term "consisting essentially of," as used herein, should not be construed as equivalent to "comprising."

[0031] It is also understood that, as used herein, the terms "example," "exemplary," and grammatical variations thereof are intended to refer to non-limiting example and / or alternative embodiments discussed herein, and are not intended to indicate a preference for one or more of the embodiments discussed herein over one or more other embodiments.

[0032] The term "about" as used herein when referring to a measurable value, such as an amount or concentration, is meant to encompass the specified value as well as a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value. For example, "about X," where X is a measurable value, means to encompass X as well as a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. Ranges given herein for measurable values ​​may include any other ranges and / or individual values ​​therein.

[0033] As used herein, the terms "increase," "increases," "increased," "increasing," "enhance," and similar terms refer to at least about a 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more increase in a specified parameter.

[0034] As used herein, the terms "reduce", "reduces", "reduced", "reduction", "inhibit" and similar terms refer to at least about a 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% decrease in a specified parameter.

[0035] When an element is referred to as being "on," "attached," "connected," "coupled," "contacting," or the like, the element may be directly on, attached to, connected to, coupled with, or in contact with the other element, or intervening elements may be present. In contrast, when an element is referred to as being, for example, "directly on," "directly attached," "directly connected," "directly coupled," or "directly contacting" another element, no intervening elements are present. It will also be understood by those skilled in the art that a reference to a structure or feature being located "adjacent" to another feature may have portions that overlap the adjacent feature or portions that underlie the adjacent feature.

[0036] Spatially relative terms such as "under," "below," "lower," "over," "upper," and the like may be used herein for ease of description to describe the relationship of an element or feature shown in the figures to one or more other elements or features. It is understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation shown in the figures. For example, if the device in the figures is inverted, an element described as "under" or "beneath" the other element or feature would be oriented "over" the other element or feature. Thus, the exemplary term "under" can encompass both an orientation of "over" and "under." The device may be otherwise oriented (rotated 90 degrees or in other orientations) and the spatially relative descriptors used herein may be interpreted accordingly. Similarly, terms such as "upwardly," "downwardly," "vertical," "horizontal," etc. are used herein for descriptive purposes only, unless specifically indicated otherwise.

[0037] In this specification, various elements may be described using terms such as "first" and "second", but it is understood that these elements should not be limited by these terms. These terms are used only to distinguish one element from another. Thus, a "first" element discussed below could also be referred to as a "second" element without departing from the teachings of the present invention. The order of operations (or steps) is not limited to the order shown in the claims or figures unless specifically indicated otherwise.

[0038] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleotide sequence," "nucleic acid sequence," "oligonucleotide," and "polynucleotide" refer to linear or branched, single-stranded or double-stranded RNA or DNA or hybrids thereof. These terms also encompass RNA / DNA hybrids. In some embodiments, the RNA and / or DNA can contain chemically modified bases, e.g., not commonly found in nature, which improve the specificity of the assay (e.g., PCR reaction) and / or reduce non-specific binding between other RNA and / or DNA. When dsRNA is made synthetically, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc. can be used for pairing antisense, dsRNA, and ribozymes. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense inhibitors of gene expression. Other modifications, such as modifications to the phosphodiester backbone or to the 2'-hydroxy of the ribose sugar group of RNA, can also be made.

[0039] As used herein, the terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably to refer to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' end to the 3' end of a nucleic acid molecule, including DNA or RNA molecules, including cDNA, DNA fragments or portions, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, any of which may be single-stranded and / or double-stranded. The terms "nucleotide sequence," "nucleic acid," "nucleic acid molecule," "oligonucleotide," and "polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides. Nucleic acid molecules and / or nucleotide sequences presented herein are presented in a 5' to 3' orientation from left to right and are represented using the standard symbols for representing nucleotide symbols as set forth in the United States Sequencing Code, 37 CFR 1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25. As used herein, the "5' region" refers to the region of a polynucleotide closest to the 5' end. Thus, for example, an element in a 5' region of a polynucleotide may be located anywhere from a first nucleotide located at the 5' end of the polynucleotide to a nucleotide located midway through the polynucleotide. As used herein, a "3' region" refers to the region of a polynucleotide closest to the 3' end. Thus, for example, an element in a 3' region of a polynucleotide may be located anywhere from a first nucleotide located at the 3' end of the polynucleotide to a nucleotide located midway through the polynucleotide.

[0040] The term "complementary" or "complementarity" as used herein refers to the natural binding of polynucleotides by base pairing under permissive salt and temperature conditions. For example, the sequence "AGT" (5' to 3') binds to the complementary sequence "TCA" (3' to 5'). Complementarity between two single-stranded molecules can be "partial," where only a portion of the nucleotides bind, or complete, where there is overall complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

[0041] "Complementarity," as used herein, may mean 100% complementarity with a comparison nucleotide sequence, or it may mean less than 100% complementarity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc. complementarity).

[0042] A "portion" or "section" of a nucleic acid sequence of the invention may be truncated (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250 or more) relative to a reference nucleic acid sequence. "Nucleic acid moiety" is understood to mean a nucleic acid sequence of length (reduced by 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) of consecutive nucleotides to a reference nucleic acid sequence. Such a nucleic acid moiety according to the invention may be suitably comprised in any portion of the larger polynucleotide of which it is a constituent.

[0043] "Fragments" of the amino acid sequences of the invention are truncated (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250 or more amino acids) relative to the reference amino acid sequence. "A" is understood to mean an amino acid sequence of length (shortened by 1 amino acid) that comprises an amino acid sequence of consecutive amino acids that is identical or nearly identical (e.g. 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a reference amino acid sequence. Such amino acid fragments according to the invention may be comprised, as appropriate, in a larger constituent protein or peptide.

[0044] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or peptide sequences are invariant over the entire window of alignment of components, e.g., nucleotides or amino acids. "Identity" may be used in a variety of applications, including, but not limited to, Computational Molecular Biology (Lesk, AM, ed.), Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects (Smith, DW, ed.), Academic Press, New York (1993), Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.), Humana Press, New Jersey (1994), Sequence Analysis in Molecular Biology (von Heinje, G., eds.), Academic Press (1987), and Sequence Analysis Introduction to Molecular Biology (von Heinje, G., eds.), Academic Press (1989). This can be readily calculated by known methods, including those described in "Theoretical and Experimental Primer" (Gribskov, M. and Devereux, J., eds.), Stockton Press, New York (1991).

[0045] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, "percent identity" may refer to the percentage of identical amino acids in an amino acid sequence.

[0046] As used herein, the phrase "substantially identical" or "substantial identity" in the context of two nucleic acid molecules, nucleotide sequences or protein sequences refers to two or more sequences or subsequences that have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and / or 100% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the invention, the substantial identity exists over a region of contiguous nucleotides of the nucleotide sequences of the invention that is about 4 nucleotides to about 30 nucleotides, about 5 nucleotides to about 20 nucleotides, about 16 nucleotides to about 30 nucleotides, about 18 nucleotides to about 25 nucleotides, about 30 nucleotides to about 40 nucleotides, about 50 nucleotides to about 60 nucleotides, about 70 nucleotides to about 80 nucleotides, about 90 nucleotides to about 100 nucleotides or more nucleotides in length, and over any range therein up to the full length of the sequence. In some embodiments, the nucleotide sequences can be substantially identical over at least about 5, 10, 15, 20 or 25 contiguous nucleotides. In some embodiments, the nucleotide sequences can be substantially identical over at least about 5 contiguous nucleotides. In some embodiments, the substantially identical nucleotide or protein sequences perform substantially the same function as the substantially identical nucleotide or protein sequences.

[0047] In sequence comparison, generally, one sequence serves as a reference sequence, and this sequence is compared with test sequence.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the percent sequence identity of one or more test sequences to reference sequence based on designated program parameters.

[0048] Optimal alignment of sequences to align a comparison window is well known to those skilled in the art and can be performed by tools such as the Smith and Waterman local homology algorithm, the Needleman and Wunsch homology alignment algorithm, the Pearson and Lipman search for similarity method, or by computer implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA, optionally available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, Calif.). The "percent identity" of an aligned interval between a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence interval, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. Percent sequence identity is expressed as the percentage of identity multiplied by 100. The comparison of one or more polynucleotide sequences can be to the full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of the present invention, "percent identity" can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0049] Two nucleotide sequences can also be considered to be substantially complementary if they hybridize to each other under stringent conditions. In some representative embodiments, two nucleotide sequences that are considered to be substantially complementary hybridize to each other under highly stringent conditions.

[0050] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence dependent and vary under different environmental parameters. An extensive guide to nucleic acid hybridization can be found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York (1993). In general, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.

[0051] The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences having more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide containing 1 mg heparin at 42°C, where hybridization is performed overnight. An example of very stringent washing conditions is 0.15 M NaCl at 72°C for about 15 minutes. An example of stringent washing conditions is a 0.2x saline-sodium citrate (SSC) wash at 65°C for 15 minutes (see Sambrook below for a description of SSC buffer). Often, a low stringency wash precedes a high stringency wash to remove background probe signal. An example of a medium stringency wash, for example for duplexes of more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An example of a low stringency wash, for example for duplexes of more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes (e.g., about 10-50 nucleotides), stringent conditions typically include a salt concentration of less than about 1.0 M Na ion at pH 7.0-8.3, typically about 0.01 M to 1.0 M Na ion (or other salt), and a temperature of typically at least about 30° C. Destabilizing agents such as formamide can also be added to achieve stringent conditions. In general, a signal-to-noise ratio of 2x (or more) than that observed with an unrelated probe in a particular hybridization assay indicates detection of specific hybridization.

[0052] The following are examples of sets of hybridization / wash conditions that may be used to clone homologous nucleotide sequences that are substantially identical to a reference nucleotide sequence of the present invention. In one embodiment, the reference nucleotide sequence is washed in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO at 50° C. 4 In another embodiment, the reference nucleotide sequence is hybridized to the "test" nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO at 50° C. with a wash in 2×SSC, 0.1% SDS in 1 mM EDTA. 4 , washed in 1×SSC, 0.1% SDS at 50° C. with 1 mM EDTA, or hybridized to a “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO at 50° C. 4 In a further embodiment, the reference nucleotide sequence is hybridized to the "test" nucleotide sequence in 0.5xSSC, 0.1% SDS at 50°C with 1 mM EDTA and a wash in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , washed in 0.1×SSC, 0.1% SDS at 50° C. with 1 mM EDTA, or hybridized to a “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO at 50° C. 4 , washed in 0.1×SSC, 0.1% SDS at 65° C. with 1 mM EDTA, and hybridized to a “test” nucleotide sequence.

[0053] The term "target" as used herein refers to any analyte of interest that may be present in a sample. Examples of targets include, but are not limited to, synthetic or natural chemical and / or biological macromolecules, nanoparticles, small molecule compounds, DNA, nucleic acids / polynucleic acids, peptides, proteins, and / or the like. A target may be one or more analyte molecules. In some embodiments, a target may be present in a sample, optionally derived from a source (e.g., a subject, a water supply, a food supply, etc.). A target and / or sample may include one or more polar metabolites, such as, for example, amino acids and / or charged molecules, peptides, and / or proteins. A target and / or sample may include one or more chemical and / or biological compounds extracted from a biological fluid, tissue, blood, serum, urine, cell growth medium, lysed cells, beverages, and / or foods, etc. In some embodiments, a target may be derived from and / or present in an environmental sample, such as, for example, a water, air, and / or soil sample. In some embodiments, a target may be derived from and / or present in a forensic sample. In some embodiments, the target and / or sample may include a pooled sample that includes a mixture of two or more samples.

[0054] As used herein, "contact," "contacting," "contacted," and grammatical variations thereof, refer to placing two or more components together or in sufficient proximity such that a desired reaction can occur under appropriate conditions (e.g., nucleic acid binding, transcriptional regulation, genome editing, nicking, cleavage, and / or amplification).

[0055] Embodiments of the invention may address and / or reduce one or more problems in assays (e.g., multiplexed iPCR assays), such as, for example, background signal problems, such as problems caused by non-specific binding (NSB) and / or antibody cross-reactivity problems.Embodiments of the invention include and / or utilize unique combinations of nucleic acid tags and provide solid support (e.g., bead)-based delivery for the formation of spatially multiplexed PCR reactions, which may avoid the usual complications associated with single-reaction multiplexed PCR.

[0056] According to some embodiments of the present invention, a solid support is provided. As used herein, the term "solid support" includes one or more objects, such as, for example, microbeads, chips, plates, slides, pins, plate wells, beads, microspheres, nanoparticles, microwells, or other members that can be used to obtain nucleic acid sequences and can be coupled to or labeled with coding agents (e.g., fluorescent compounds, chemiluminescent compounds, radioactive elements, and / or enzymes, etc.) and / or dynamic elements. The nucleic acid sequences, molecular recognition elements, coding agents, and / or dynamic elements can be directly or indirectly attached or embedded (partially or completely) within the solid support, such as by covalent attachment, non-covalent attachment, and / or physical incorporation. In some embodiments, the solid support can be an array printed on a solid surface. In some embodiments, the solid support can be a microsphere. In some embodiments, the solid support may be a solid surface that is contacted with a sample, such as, for example, a chemical and / or biological component (e.g., a tissue sample), and a molecular recognition element (optionally including one or more coding agents) on the solid support may be attached to a portion of the sample and / or a target therein. The molecular recognition element may or may be an antibody or fragment thereof, an aptamer, a DNA hybridization probe, an oligonucleotide, a peptide, a protein, and / or a combination thereof. In some embodiments, the solid support may include a labile reagent and / or a support bond, each of which may enable cleavage of a component (e.g., a nucleic acid sequence) from the solid support.

[0057] In some embodiments, one or more coding agents can be attached to a solid support (e.g., a bead or a microsphere) to provide a coded solid support (e.g., a coded bead or a coded microsphere). In some embodiments, one or more coding agents can be attached to a molecular recognition element (e.g., an antibody) to provide a coded molecular recognition element (e.g., a coded antibody) that can be attached to a solid support to provide a coded solid support. In some embodiments, the coded molecular recognition element can be attached to a particle, e.g., a nanoparticle. In some embodiments, the method can include providing or attaching one or more coding agents and / or coded molecular recognition elements to a solid support. In some embodiments, the solid support and / or coded solid support can be as described in WO 2007 / 112025, the entire contents of which are incorporated herein by reference.

[0058] The term "bead" or "beads" as used herein refers to a solid phase member, such as, for example, particles, granules, or microspheres, typically magnetic microspheres, which may be one or more materials such as polymers, plastics, glass, silicon dioxide, metal oxides or semi-metal oxides (including but not limited to aluminum oxide, titanium oxide, zirconium oxide, or other oxides), quantum dots, metal particles, and / or the like, which may be porous, superficially porous, or porous, e.g., suitable for use in a reaction well. In some embodiments, a multiplexed bead set may be provided that includes a plurality of beads (e.g., microspheres) that are uniquely coded to distinguish one population of beads from another population.

[0059] In some embodiments, the solid support is a bead, e.g., a magnetic bead or a superparamagnetic bead. In some embodiments, the solid support is not a magnetic bead or a superparamagnetic bead. In some embodiments, the solid support is a bead that is porous and / or has a coating that increases the surface area. In some embodiments, the solid support is a bead made by forming aggregates of microparticles and / or nanoparticles, optionally with some or all of the microparticles and / or nanoparticles functionalized such that they can be used to retain reagents, e.g., capture affinity reagents, primers, probes, blocking agents, polymerases, reverse transcriptases, and / or other enzymes.

[0060] In some embodiments, a solid support (e.g., a bead) comprises a coding agent (e.g., a dye), a nucleic acid sequence, and a molecular recognition element (e.g., an antibody). In some embodiments, the coding agent is directly or indirectly attached to or at least partially embedded in the solid support. In some embodiments, the nucleic acid sequence and / or the molecular recognition element of the solid support comprises the coding agent. The molecular recognition element may include or be an antibody or fragment thereof, an aptamer, a modified aptamer, a DNA hybridization probe, an oligonucleotide, a peptide, a protein, a carbohydrate, and / or a combination thereof.

[0061] The term "coding agent" as used herein refers to one or more agents (e.g., chemicals, proteins, etc.) that provide and / or generate a coding signal on each solid support (which may be all or a portion of it) associated with (e.g., applied to, attached to, bound to, formulated with, used to manufacture or create) a solid support and / or material of the solid support and / or material in contact with the solid support. In some embodiments, the one or more coding agents provide and / or generate a detectable coding signal that allows for identification of a population or subpopulation of solid supports (e.g., beads). For example, in some embodiments, the one or more coding agents may provide and / or generate a detectable coding signal on an individual solid support (e.g., bead) with which the one or more coding agents are associated (e.g., attached) and / or the one or more coding agents may provide and / or generate a detectable coding signal on a particular portion of the solid support to which the one or more coding agents are attached (e.g., the portion of the solid support to which a molecular recognition element comprising one or more coding agents is bound).

[0062] The coded signal may be provided and / or generated by one or more coding agents associated with the solid support and / or by the solid support itself and / or materials (e.g., compounds) associated with the solid support. In some embodiments, the coded signal may be the size and / or shape of the solid support. In some embodiments, the coded signal is a signal (e.g., optical and / or electrical signal) generated by one or more coding agents (e.g., chemicals, proteins, etc.) associated with (e.g., applied to, attached to, bound to, compounded with, used to manufacture or produce, etc.) the solid support. The detectable coded signal may be optically and / or electronically detectable. The detectable coded signal may be visually perceptible by the human eye and / or electronically read, detected, and / or obtained. The detectable coded signal may include a change in intensity, color (e.g., hue, color intensity, and / or color value), color and intensity, and / or size, shape, and / or radioactivity, typically above a defined threshold. In some embodiments, the detectable coded signal includes luminescence intensity, which may include fluorescence, phosphorescence, and / or chemiluminescence intensity.

[0063] The coding signal of each solid support (which may be all or a part of it) may be detectable (e.g., optically, electronically, electrochemically, electrostatically, magnetically, etc. detectable) or may not be detectable. In some embodiments, the coding signal of each solid support (which may be all or a part of it) may change. For example, the coding signal for each solid support may change from detectable to undetectable, from undetectable to detectable, from a higher value to a lower value (e.g., from a higher signal amplitude, fluorescence intensity value, diameter, etc.), and / or from a lower value to a higher value. The change in the coding signal for each solid support may occur over time and / or may occur due to at least one chemical and / or physical change associated with the solid support. In some embodiments, the coding signal for the solid substrate may change and / or may be provided and / or generated by a change in the environment in which the solid support is present (e.g., a change in the solution in which the solid support and / or the coding agent are in contact). For example, the coding signal for a solid support may change and / or may be provided and / or generated by changes in magnetic behavior, pH, light scattering, physical size (increase or decrease), shape, refractive index, solubility, absorbance and / or emission intensity or maximum wavelength shift, conductivity, dielectric constant, viscosity, and radio emission from isotope decay, and combinations of the above.

[0064] The term "decoding" refers to the (typically electronic / programmatic) identification of distinct populations of each sample (e.g., including samples of solid supports and / or samples containing multiple coded molecular recognition elements) based, at least in part, on a coded signal from one or more coded solid supports and / or coded molecular recognition elements. Detection and / or identification of one or more coded signals allows for the decoding of the sample to determine the identity of a particular population of solid supports and / or to detect a target.

[0065] As used herein, a "dynamic element" refers to a chemical and / or biological compound that provides or exhibits a physical and / or chemical change in response to a defined event. The physical and / or chemical change can alter the coding signal (which can be all or a portion thereof) for each solid support at a time point compared to the coding signal at a different (e.g., earlier) time point and can be detectable (e.g., optically and / or electrically detectable). A "dynamic element" can include a coding agent (e.g., a dye, a protein, etc.) and / or a solid support (e.g., a microsphere). The physical and / or chemical change can be a physical and / or chemical change that affects the solid support (e.g., the change can be a change in charge and / or color of the solid support and / or a change in size and / or shape of the solid support). Thus, the dynamic element can be a solid support and / or a compound attached to and / or associated with the solid support. In some embodiments, the physical and / or chemical change is a physical and / or chemical change that affects the coding agent associated with the solid support (e.g., the change can be a change in the stability of a linker that connects the coding agent to the solid support, a change in the solvent accessibility of the coding agent, and / or the presence and / or absence of a quencher). Thus, the dynamic element can be the coding agent and / or a compound (e.g., a quencher) that is attached to and / or associated with the coding agent. The physical and / or chemical change can be reversible or irreversible. In some embodiments, the dynamic element can be the coding agent. In some embodiments, the dynamic element and / or the coding agent are those described in WO 2007 / 112025, the entire contents of which are incorporated herein by reference.

[0066] Coding of the solid support can be accomplished by any method known to one of skill in the art (e.g., using varying characteristics such as color, fluorescent color, size, shape, thermolabile or photolabile dyes, and / or any combination thereof). In some embodiments, the solid support can include a coding agent that is covalently or non-covalently attached to the solid support. In some embodiments, the dyes are embedded in the solid support (e.g., beads), for example, via solvent swelling and entrapment.

[0067] In some embodiments, the solid support includes one or more linkers, which may be the same or different. The one or more linkers can attach and / or bind (e.g., covalently or non-covalently) the molecular recognition element and / or the nucleic acid sequence to the solid support. For example, the linker can be used to attach or bind one or more molecular recognition elements to the solid support, and / or the same or different linkers can be used to attach or bind one or more nucleic acid sequences to the solid support. As used herein, "linker" refers to any molecule, moiety, and / or functional group that can be used to attach or bind one component (e.g., a solid support) to another component (e.g., a molecular recognition element). Examples of linkers include, but are not limited to, chemical moieties and / or compounds such as, for example, amines (e.g., amine derivatives, isocyanates, isothiocyanates, iodoacetamides, azides, etc.), acids (e.g., acid derivatives, N-hydroxysuccinimide esters, acid hydrazides, etc.), aldehydes, sulfonyl chlorides, sulfonyl hydrazides, epoxides, hydroxyl groups, thiol groups, and / or maleimides, and / or biomolecules such as, for example, proteins, peptides, DNA, and / or RNA. In some embodiments, the linker is a biomolecule that is a member of a specific binding pair. "Specific binding pair" and "ligand-receptor binding pair" are used interchangeably herein and refer to two different molecules where both molecules have an affinity for each other because one of the molecules has an area on the surface or within a cavity of the molecule that specifically attracts or binds to a particular spatial or polar organization of the other molecule. The members of a specific binding pair may be referred to as a ligand and a receptor (antiligand). The terms ligand and receptor are intended to include the entire ligand or receptor or a sufficient portion thereof for binding to occur between the ligand and receptor.Examples of ligand-receptor binding pairs include, but are not limited to, hormones and hormone receptors, e.g., epidermal growth factor and epidermal growth factor receptor, tumor necrosis factor alpha and tumor necrosis factor receptor, and interferon and interferon receptor, avidin and biotin or anti-biotin, antibody and antigen pairs, enzymes and substrates, drugs and drug receptors, cell surface antigens and lectins, two complementary nucleic acid strands, nucleic acid strands and complementary oligonucleotides, interleukins and interleukin receptors, and stimulatory factors and their receptors, such as granulocyte-macrophage colony stimulating factor (GMCSF) and GMCSF receptor, and macrophage colony stimulating factor (MCSF) and MCSF receptor. Further exemplary linkers and / or chemistries that can be used to attach the two components include, but are not limited to, photocleavable biotin, a linker that provides a covalent bond between the solid support and the recognition element, avidin, Acrydite™ available from Integrated DNA Technologies, Inc., adenylation, azide (NHS ester), digoxigenin (NHS ester), cholesterol-TEG, I-Linker™ available from Integrated DNA Technologies, Inc., Uni-Link™ Amino available from Integrated DNA Technologies, Inc. Modifiers include 5'hexynyl, 5-octadiynyl dU, biotin dT, biotin-TEG, dual biotin, desthiobiotin-TEG, thiol modifier C3 SS, dithiol, and / or thiol modifier C6 SS.

[0068] Any suitable coupling chemistry can be used to couple the molecular recognition element and / or the nucleic acid sequence to the solid support. In some embodiments, the linker, the molecular recognition element and / or the nucleic acid sequence includes a labile chemical bond that can be used to separate the molecular recognition element or a portion thereof from the solid support and / or to separate the nucleic acid sequence or a portion thereof from the solid support. The labile chemical bond can be cleaved using a variety of techniques, including, but not limited to, the use of heat, light, and / or chemicals to cleave the labile chemical bond. For example, thermal energy can be applied to break or cleave the labile chemical bond, or light can be applied to break the labile chemical bond (e.g., a photocleavable bond). Thermal heating can be achieved by conductive contact with a hot surface, Joule heating of a solution, resistive heating of a substrate adjacent to the solid support, exposure to electromagnetic radiation and / or electromagnetic induction. In some embodiments, the labile chemical bond can be cleaved using ionizing radiation, chemicals, enzymes, electrochemical processes, changes in pH, and / or other suitable cleavage procedures. For example, the change in pH can be caused by an electrochemical reaction occurring on the surface of the region containing the solid support, or the change in conditions can be caused directly or indirectly by the activation of an initiator reagent, which itself can be activated by a physical change, such as heat, light, an electrochemical process, a change in pH, etc. In some embodiments, cleavage of the labile chemical bond can be performed before and / or during the PCR assay and / or reaction. In some embodiments, once the labile chemical bond is cleaved, the nucleic acid sequence is separated from the solid support so that PCR can occur and / or be initiated. In some embodiments, the PCR reaction does not occur until the nucleic acid sequence (e.g., the forward primer and / or the reverse primer) is released from the solid support to which it is attached. In some embodiments, the PCR reaction can occur while the nucleic acid sequence is attached to the solid support. In some embodiments, the components (e.g., the molecular recognition element and / or the nucleic acid sequence) can be attached to the solid support using the compounds, compositions, and / or methods described in U.S. Pat. No. 9,617,589, the entire contents of which are incorporated herein by reference.

[0069] In some embodiments, the molecular recognition element (e.g., an antibody) and / or the nucleic acid sequence (e.g., a primer or oligonucleotide) are attached to the solid support via a photocleavable or thermolabile bond. For example, in some embodiments, photocleavable biotin is used to attach the molecular recognition element and / or the nucleic acid sequence to the solid support. In some embodiments, the molecular recognition element and / or the nucleic acid sequence are attached to the solid support using one or more attachment chemistries, such as, but not limited to, those that utilize molecules with affinity for antibodies, such as Protein A, Protein G, Protein A / G, Protein L, etc.

[0070] In some embodiments, the nucleic acid sequence (e.g., the forward primer) can be covalently attached to the solid support, especially when hydrolysis probes or TaqMan probes are used for the detection chemistry, since the dye from the hydrolysis probe can still be detected even though the dsDNA amplicon is attached to the bead.

[0071] In some embodiments, the solid support comprises multiple linkers and / or is coated with one or more linkers. For example, the solid support may comprise streptavidin and one or more biotinylated molecular recognition elements bound and / or coated on at least a portion of the surface of the solid support, and / or one or more biotinylated nucleic acid sequences may be bound to the solid support via binding between streptavidin and biotin. In some embodiments, the solid support (e.g., beads) comprises about 3.5 amol of streptavidin per solid support, and each streptavidin binds about 1, 2, 3, or 4 biotinylated molecules. In some embodiments, the ratio of molecules of molecular recognition elements to molecules of nucleic acid sequences present on the solid support is about 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, or 1:4 (molecular recognition elements: nucleic acid sequences).

[0072] A solid support can include molecular recognition elements and / or nucleic acid sequences (i.e., one or more molecular recognition elements and / or one or more nucleic acid sequences) in an amount from about 1 molecule, 10 molecules, 50 molecules, 100 molecules, 200 molecules, 300 molecules, 400 molecules, 500 molecules, or 600 molecules to about 1000 molecules, 5000 molecules, 10000 molecules, 50000 molecules, 100000 molecules, 250000 molecules, 500000 molecules, 1000000 molecules, 10000000 molecules, 25000000 molecules, 50000000 molecules, 60000000 molecules, or more. In some embodiments, the solid support comprises a molecular recognition element and / or a nucleic acid sequence in an amount of about 10 ymol, 30 ymol, 50 ymol, 75 ymol, or 100 ymol to about 1 fmol, 2 fmol, 3 fmol, 4 fmol, 5 fmol, 6 fmol, 7 fmol, 8 fmol, 9 fmol, 10 fmol, 11 fmol, 12 fmol, 13 fmol, 14 fmol, or 15 fmol. In some embodiments, the solid support comprises a molecular recognition element and / or a nucleic acid sequence in an amount of about 0.1 zmol, 0.3 zmol, 0.5 zmol, 0.75 zmol, 1 zmol, 2 zmol, 3 zmol, 4 zmol, or 5 zmol to about 1 amol, 5 amol, 10 amol, 15 amol, 20 amol, 25 amol, 30 amol, 35 amol, or 40 amol. In some embodiments, the solid support comprises and / or has associated therewith molecular recognition elements in an amount of about 1 amol, in an amount ranging from about 1 zmol to about 100 amol, or in an amount ranging from about 100 ymol to about 10 fmol. In some embodiments, the solid support comprises and / or has associated therewith nucleic acid sequences (e.g., primers and / or single-stranded oligonucleotides) in an amount of about 3 amol, in an amount ranging from about 0.3 zmol to about 30 amol, or in an amount ranging from about 30 ymol to about 3 fmol.

[0073] The solid support may provide assay and / or reaction wells in which the molecular recognition elements are present in an amount ranging from about 1 nM to about 1 mM and / or the nucleic acid sequences are present in an amount ranging from about 300 pM or 1 nM or 3 nM to about 300 μM or 3 mM. In some embodiments, the solid support may provide assay and / or reaction wells in which the nucleic acid sequences (e.g., primers) are present in an amount ranging from about 1 μM to about 100 nM. The concentration of the molecular recognition elements and / or nucleic acid sequences may be the amount present on the solid support and / or the amount released from the solid support. In some embodiments, the concentration of the molecular recognition elements and / or nucleic acid sequences provided by the solid support in the assay and / or reaction wells may be sufficient to perform or carry out an amplification process (e.g., PCR) and / or to detect a target.

[0074] In some embodiments, the nucleic acid sequence may be attached and / or linked to a solid support, optionally via a linker, and may have a length of from about 5, 10, 20, or 25 nucleotides to about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides. In some embodiments, the nucleic acid sequence comprises at least 5 nucleotides to about 500 nucleotides (e.g., 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 12 nucleotides, 15 nucleotides, 18 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, 175 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, or 500 nucleotides). In some embodiments, the nucleic acid sequence is an oligonucleotide. In some embodiments, the nucleic acid sequence is a primer (e.g., a forward primer or a reverse primer) that can be used, for example, as a primer in a PCR assay, as a probe in a hybridization assay, and / or in a microarray. Primers useful in the present invention include, but are not limited to, amplification and / or detection primers. In some embodiments, the nucleic acid sequence comprises about 15 nucleotides to about 50 nucleotides or about 20 nucleotides to about 25 nucleotides. In some embodiments, the solid support of the present invention comprises at least one forward primer and / or at least one reverse primer.In some embodiments, the solid support of the invention comprises a forward primer and does not comprise a reverse primer. In some embodiments, the solid support of the invention comprises a forward primer and a reverse primer.

[0075] In some embodiments, the primers can be absorbed onto a solid support (to which a molecular recognition element (e.g., a capture antibody) may also be attached) such that the primers are mostly inside the solid support (e.g., hydrogel beads) and are not exposed on the outside of the solid support. Exemplary hydrogel beads include, but are not limited to, agarose beads (e.g., sepharose beads).

[0076] The primers present on the solid support may be associated with chemical species that prevent hybridization, such as, for example, specific nucleic acid sequences (optionally with modified bases) that have a melting temperature higher than the temperature at which the detection reagents are incubated, but lower than the annealing temperature at which PCR is carried out. Alternatively or in addition, peptides, proteins, RNA sequences, polymers and / or other reagents can be used to reduce non-specific binding and / or undesired hybridization to the primers on the solid support.

[0077] In some embodiments, the solid support comprises a self-avoiding primer derived from one or more modified bases, such as, for example, self-avoiding primers available from Firebird and other sources. In some embodiments, the self-avoiding molecular recognition system (SAMRS) is a nucleic acid tag on the forward primer and the detection reagent, and the reverse primer is regular DNA.

[0078] The forward primer and / or reverse primer on the solid support of the present invention and / or the detection reagent of the present invention can be configured to bind to a specific component at the appropriate time. For example, in some embodiments, the primer (e.g., forward primer) is prevented from binding to the nucleic acid tag of the detection reagent until after the first PCR cycle because the NA tag is single-stranded and no primer binding site is present until after the first PCR cycle. In some embodiments, the forward primer and reverse primer can be designed such that they are not available for base pairing at lower temperatures (e.g., during incubation with the detection reagent), but are available at higher temperatures (e.g., after washing away excess detection reagent). In some embodiments, the forward primer and / or reverse primer on the solid support of the present invention can be double-stranded (e.g., via a duplex or hairpin structure), and upon the denaturation step of PCR, one or more primers become single-stranded. One or more primers can be designed such that the double-stranded structure (e.g., duplex or hairpin) is not highly formed at the PCR annealing temperature, since the structure may inhibit PCR. In some embodiments, the forward primer and / or reverse primer on the solid support of the invention may become available after enzymatic cleavage. For example, the primer may be a portion of a duplex, and the competitor strand (reverse complementary to the primer) contains a cleavable bond. If the competitor strand is broken down into smaller portions, the melting temperature may be significantly lowered, so that there is no competition during PCR. For the cleavable bond, the following non-exclusive examples may be used: photocleavable bonds, specially incorporated nucleotides that can be acted upon by endonucleases or other enzymes, or RNA that can be acted upon by RNAses. In some embodiments, cleavage provides a primer with an extendable 3' end.

[0079] In some embodiments, the detection reagents of the invention can be protected and / or designed to minimize hybridization to primers on the solid support, for example, using techniques similar to those discussed above (e.g., dsDNA duplexes, hairpin structures) such that the binding site for the reverse primer is already base-paired.

[0080] According to some embodiments, a plurality of solid supports is provided, including a first plurality of solid supports and a second plurality of solid supports. Each solid support in the first plurality of solid supports comprises a first coding agent (e.g., a dye), a first nucleic acid sequence, and a first molecular recognition element (e.g., an antibody) that provides a first coding signal, and each solid support in the second plurality of solid supports comprises a second coding agent (e.g., a dye), a second nucleic acid sequence, and a second molecular recognition element (e.g., an antibody) that provides a second coding signal. The solid supports in the first plurality of solid supports and the solid supports in the second plurality of solid supports are distinct and / or distinguished because the first coding signal is different from the second coding signal, the first nucleic acid sequence is different from the second nucleic acid sequence, and / or the first molecular recognition element is different from the second molecular recognition element.

[0081] According to some embodiments, a detection reagent is provided that includes a molecular recognition element (e.g., an antibody) and a nucleic acid sequence. The nucleic acid sequence of the detection reagent is also referred to herein as a "nucleic acid tag" or "tag." The detection reagent can bind to a first portion of the target, and the solid support of the present invention can bind to a second portion of the target. In this manner, the detection reagent has a corresponding solid support by pairing with the solid support through the target. The nucleic acid tag of the detection reagent of the present invention has a portion configured to participate in a nucleic acid amplification process with a portion of the nucleic acid sequence attached (e.g., bound) to the corresponding solid support of the detection reagent. In some embodiments, the detection reagent of the present invention is a detection antibody that includes a nucleic acid tag.

[0082] The nucleic acid tag of the detection reagent can comprise from about 20, 30, 40, 50, or 60 nucleotides to about 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, or 300 nucleotides. In some embodiments, the nucleic acid tag comprises a hairpin loop.

[0083] In some embodiments, at least a portion of the nucleic acid tag is substantially identical to at least a portion of the nucleic acid sequence of the corresponding solid support of the detection reagent. In some embodiments, at least about 5 or more (e.g., 6, 7, 8, 9, 10, 15, 20 or more) consecutive nucleotides in the nucleic acid tag are substantially identical to at least about 5 or more (e.g., 6, 7, 8, 9, 10, 15, 20 or more) consecutive nucleotides in the nucleic acid sequence of the corresponding solid support of the detection reagent. In some embodiments, the sequence in the 5' region of the nucleic acid tag is substantially identical to the sequence in the nucleic acid molecule of the solid support. In some embodiments, the sequence in the 5' region of the nucleic acid tag is substantially identical to the nucleic acid sequence of the corresponding solid support of the detection reagent, and / or the sequence in the 3' region of the nucleic acid tag is complementary to a reverse primer that may be optionally present in the PCR master mix. In some embodiments, two or more detection reagents corresponding to a set of two or more different solid supports comprise a substantially identical sequence in the 3' region of the nucleic acid tag, which sequence may be complementary to the reverse primer.

[0084] Referring now to FIG. 6, an exemplary complex according to an embodiment of the present invention is shown, which includes a coded solid support, a target, and a detection reagent. The coded solid support is a coded bead functionalized with both a forward primer and an antibody (e.g., a capture antibody) that binds to the target. The detection reagent shown in FIG. 6 is an antibody that comprises a nucleic acid sequence, which forms a complex of the present invention by binding to the target. Thus, the captured target is labeled with a detection antibody that comprises a unique nucleic acid sequence, and at least a portion of the nucleic acid sequence (i.e., the nucleic acid tag) is amplified by the forward primer of the corresponding coded bead, optionally once the forward primer is released from the bead and the reverse primer is provided in a PCR master mix. The PCR product can then be detected by a fluorescent signal (e.g., from a dye that intercalates into dsDNA).

[0085] The detection reagents corresponding to the solid supports in one or more solid support sets each contain the same nucleic acid tag. The nucleic acid tag is different for the detection reagents corresponding to different solid supports. Referring again to FIG. 6, the nucleic acid tag of the detection reagent contains a sequence in the 5' region that is configured to participate in the nucleic acid amplification process with at least a part of the forward primer. Furthermore, as shown in FIG. 7, the nucleic acid tag contains a sequence in the 3' region that is complementary to the reverse primer, and this nucleic acid sequence is also present (and therefore common) in the different detection reagents corresponding to the different solid supports. Thus, the nucleic acid tag can be amplified by two primers, namely a unique forward primer and a common reverse primer. The unique forward primer can be substantially identical to the sequence in the 5' region of the nucleic acid tag. The reverse primer can be complementary to the sequence in the 3' region of all the nucleic acid tags and can be added to the PCR master mix.

[0086] In some embodiments, the nucleic acid sequence of the solid support and / or the nucleic acid tag of the detection reagent comprises single-stranded DNA. In some embodiments, the nucleic acid sequence of the solid support and / or the nucleic acid tag of the detection reagent comprises double-stranded DNA. In some embodiments, the nucleic acid sequence of the solid support and / or the nucleic acid tag of the detection reagent comprises RNA or hybridized DNA-RNA. In some embodiments, the nucleic acid sequence of the solid support and / or the nucleic acid tag of the detection reagent comprises one or more chemically modified bases, such as one or more chemically modified bases not normally found in nature. The one or more chemically modified bases may improve the specificity of the PCR reaction and / or reduce non-specific binding between nucleic acid sequences (e.g., oligonucleotides).

[0087] The nucleic acid tag can be attached or bound to the molecular recognition element in any manner and / or to any portion of the molecular recognition element. In some embodiments, the nucleic acid tag is covalently or non-covalently attached to the molecular recognition element. In some embodiments, the nucleic acid tag is attached to the molecular recognition element using a linker. The nucleic acid tag can be attached to one or more amino acids of the molecular recognition element (e.g., an antibody). In some embodiments, the molecular recognition element is an antibody or a fragment thereof, and the nucleic acid tag can be attached (e.g., covalently attached) to the fragment crystallizable (Fc) region of the antibody or a fragment thereof.

[0088] In some embodiments, the nucleic acid tag can be a chemically synthesized single-stranded DNA (ssDNA). In some embodiments, a double-stranded DNA (dsDNA) tag attached to a molecular recognition element provides a detection reagent that can result in reduced non-specific binding. In some embodiments, hybridization of dsDNA to a primer bound to a solid support is reduced by designing the complementary strand (e.g., to the primer or nucleic acid tag) to ensure that it remains annealed during the assay, optionally until its desired release. As will be appreciated by those skilled in the art, DNA strands can undergo melting and annealing (fraying) at a rate determined by the sequence, temperature, and chemical environment. This fraying of the ends can increase non-specific binding or undesired hybridization to other nucleic acid molecules (e.g., primers or probes bound to a solid support, etc.). To prevent hybridization of the complementary strand of the dsDNA tag to a forward primer bound to a solid support, the complementary strand can be shorter, i.e., lacking a sequence complementary to the forward primer. In this case the tag may be partially dsDNA and partially ssDNA.

[0089] In some embodiments, the nucleic acid tag can be a hairpin tag sequence and / or a sequence with a GC clamp. The hairpin sequence and / or the sequence with a GC clamp can reduce fraying of ends and / or undesired hybridization during incubation with detection reagents. In some embodiments, the nucleic acid tag can be an RNA sequence and reverse transcriptase can be used. The RNA sequence can include an RNA hairpin with an internal structure that can prevent hybridization during sample capture.

[0090] In some embodiments, the nucleic acid tag may be encapsulated by a protein, membrane, micelle, peptide, bacteriophage, etc. such that it cannot hybridize to a nucleic acid sequence (e.g., a primer) bound to a solid support until desired (e.g., after being sealed into a reaction well).

[0091] In some embodiments, the nucleic acid tag comprises a restriction enzyme site. The nucleic acid tag can be designed such that an enzyme can cleave the hairpin or GC clamp, which can be used to release the nucleic acid sequence for efficient PCR analysis. In some embodiments, the nucleic acid tag comprises a photocleavable bond or a chemically cleavable disulfide linker, which can be used to release all or a portion of the nucleic acid tag from the detection reagent.

[0092] Nucleic acid tags of the invention need not contain unique and common regions, for example as shown in Figure 7. In some embodiments, the nucleic acid tags of the detection reagents contain sequences that are wholly or partially unique, and a mixture of forward or reverse primers can be added to a master mix of such sequences.

[0093] In some embodiments, the nucleic acid tag is a conserved nucleic acid sequence in the nucleic acid tags of the different detection reagents, and includes at least one common region that is large enough so that part or all of the sequence can be detected by amplification with one or more primer pairs. The conserved nucleic acid sequence can be in the 5' and / or 3' regions located outside the unique region. The conserved region can be present in all of the nucleic acid tags for all of the two or more different detection reagents. Thus, the nucleic acid tags for the different detection reagents can be designed such that they can be amplified by a unique primer pair and by a universal primer pair that is common to the nucleic acid tags of all the detection reagents. This embodiment can be useful for troubleshooting during assay development, as non-specific binding and / or cross-reactivity can be easily visualized. In some embodiments, the nucleic acid tag of the present invention includes at least two nucleic acid sequences, one amplified by a unique reverse primer and one amplified by a universal reverse primer.

[0094] The nucleic acid tag can be attached to the molecular recognition element using any method known to one of skill in the art to provide a detection reagent, hi some embodiments, the nucleic acid tag is attached to the molecular recognition element using conventional chemical methods, click chemistry, genetic engineering, and / or molecular engineering methods.

[0095] In some embodiments, the molecular recognition elements of the detection reagents of the invention can be functionalized to nanoparticles functionalized with nucleic acid tags and, optionally, primers and / or probes for amplifying nucleic acid sequences bound to a solid support, however, in some embodiments, multiplexing primer sets with variable concentrations is avoided to reduce assay variability.

[0096] In some embodiments, where the detection reagent includes a molecular recognition element that comprises a nucleic acid, the nucleic acid tag may be provided by all or part of the molecular recognition element, i.e., the molecular recognition element itself may function as the nucleic acid tag.

[0097] According to some embodiments, a plurality of detection reagents is provided, the plurality of detection reagents including a first plurality of detection reagents, each detection reagent including a first molecular recognition element (e.g., an antibody) and a first nucleic acid tag, and a second plurality of detection reagents, each detection reagent including a second molecular recognition element (e.g., an antibody) and a second nucleic acid tag, the first nucleic acid tag and the second nucleic acid tag each including a first portion and a second portion, and the nucleic acid sequence of the first portion for the first nucleic acid tag is different from the nucleic acid sequence of the first portion for the second nucleic acid tag. In some embodiments, the nucleic acid sequences of the second portion for the first nucleic acid tag and the second nucleic acid tag are the same or substantially identical. In some embodiments, the first portion of the first nucleic acid tag and the second nucleic acid tag are in the 5' region of the respective tag, and the second portion is in the 3' region of the respective tag. A first nucleic acid tag can be attached (e.g., linked, bound, and / or fused) to a first molecular recognition element, and a second nucleic acid tag can be attached (e.g., linked, bound, and / or fused) to a second molecular recognition element.

[0098] According to some embodiments, compositions, kits, devices, and / or systems are provided that include the solid supports described herein and / or the detection reagents described herein. The solid supports and the detection reagents may form a complex when a target corresponding to the solid supports and the detection reagents is present. The compositions, kits, devices, and / or systems may include a plurality of solid supports, optionally including two or more different coded solid supports, and a plurality of detection reagents, optionally including two or more different detection reagents.

[0099] In some embodiments, bead sets can be prepared by encoding each set with a unique dye or a unique combination of fluorescent dyes, as shown by beads "A," "B," and "C" in FIG. 7. For example, bead set "A" is stained with a red fluorescent dye, while bead set "B" is stained with a blue fluorescent dye. In some embodiments, all sets are functionalized with streptavidin before each set is incubated with a different biotinylated capture antibody for a specific target molecule. Finally, each set is incubated with a unique biotinylated primer (such as a forward primer). In some embodiments, detection reagents are prepared by covalently attaching a unique nucleic acid tag to the detection antibody for each target. In some embodiments, the tag is a 60 nucleotide (nt) single-stranded DNA (ssDNA) with a unique sequence at the 5' end or region and a common sequence at the 3' end or region that is conserved among all tags. Each tag is amplified by two primers: a forward primer (different for each tag) and a common reverse primer. In some embodiments, the forward primer may contain a sequence that is identical or substantially identical to the sequence of the 5' region or 5' end of the tag attached to the detection antibody. Alternatively, the 3' end of the tag can be attached to the detection antibody. In some embodiments, a reverse primer that is complementary to the conserved 3' end or region of all tags can be added to the PCR master mix. In this way, bead set A has both capture antibody A and forward primer A that can amplify only nucleic acid tag A linked to detection antibody A. Bead set B has both capture antibody B and forward primer B that can amplify only nucleic acid tag B linked to detection antibody B. The primer added to the master mix functions as a reverse primer for both nucleic acid tag A and nucleic acid tag B, since the 3' end or region of the tag contains a conserved sequence.

[0100] In some embodiments, the solid support of the present invention does not contain a complementary sequence that can hybridize to the nucleic acid tag. Such hybridization can result in a significant pull-down of the detection reagent from the incubation mixture in the absence of target, resulting in a very high background signal. For this reason, in some embodiments, only the forward primer is attached to the solid support, since it is identical or substantially identical (but not complementary) in sequence to the 5' end or region of the tag. A reverse primer complementary to the tag can be added to the PCR master mix, so that PCR can be performed.

[0101] In some embodiments, the assay is performed in the presence of a mixture of solid supports for different targets, and the solid supports are incubated with the sample. The solid supports can capture the targets, and the sample matrix can be washed away. The captured targets can then be labeled to form complexes by incubating the solid supports with a mixture of detection antibody reagents corresponding to the solid supports. The complexes can be washed and / or loaded into an array (e.g., a microwell array sized such that most reaction wells only accommodate a single bead). After addition of a PCR master mix containing a common reverse primer complementary to the 3' end or region of all tags, each reaction can be optionally fluidically isolated, for example by sealing with immiscible oil. During the denaturation step of PCR, the forward primer attached to the solid support can be released into the isolated reaction wells, so that each well now contains a complete PCR primer set. For example, if detection antibody conjugate A is present, wells containing bead type A will be PCR positive, and if detection antibody conjugate B is present, wells containing bead type B will be PCR positive. However, reactions containing bead type A and non-specifically bound detection antibody conjugate B will not give a positive PCR result because the primers carried by bead type A cannot amplify the nucleic acid tag on detection antibody conjugate B (Figure 8). Thus, iPCR assays optimized as singleplex reactions require little reoptimization upon multiplexing, since the total detection antibody concentration that can be amplified and / or detected by each bead set does not change when additional label is added to the assay.

[0102] In some embodiments, the methods of the invention capture and / or result in the binding of about 0 to about 10 million detection reagents as complexes with their corresponding solid supports. For example, the methods of the invention capture and / or result in the binding of about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, The binding of 1600, 2000, 2200, 2500, 2750, 3000, 3500, 4000, 4500 or 5000 to about 10000, 25000, 50000, 75000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 5000000 or 10000000 detection reagents may be captured and / or may result.

[0103] In some embodiments, the compositions, kits, devices, and / or systems of the present invention further comprise an amplification agent. Exemplary amplification agents include, but are not limited to, reverse primers, ssDNA splints, and ligases. In some embodiments, the amplification agent has a nucleic acid sequence that is complementary to at least a portion of the nucleic acid sequence of the detection reagent and solid support, and optionally, a portion of the nucleic acid sequence is within the 3' region of the nucleic acid tag. The amplification agent can be separate from the solid support and / or the detection reagent. In some embodiments, the amplification agent is attached to the solid support, and optionally, the amplification agent is biotinylated.

[0104] The solid support of the invention and its corresponding detection reagent may be stored separately in the compositions, kits, devices, and / or systems of the invention. In some embodiments, the detection reagent and the amplification agent of the invention are stored separately in the compositions, kits, devices, and / or systems of the invention. In some embodiments, the solid support of the invention and the amplification agent are stored separately when the amplification agent is present and not attached to the solid support.

[0105] According to some embodiments, a method for detecting a target in a sample is provided, which may include combining a solid support of the invention with a sample containing the target to form a first composition comprising the solid support bound to the target.

[0106] As used herein, "combining" and "combine" refer to bringing two or more components into sufficient proximity and / or contact to bind and / or react, optionally under suitable conditions. The detection reagent of the present invention can be combined with a solid support bound to a target to form a complex including the solid support, the target, and the detection reagent, where the detection reagent includes a molecular recognition element that binds to the target and a nucleic acid tag. At least a portion of the nucleic acid tag and at least a portion of the nucleic acid sequence bound to the solid support are configured to participate in a nucleic acid amplification process. At least a portion of the nucleic acid tag is amplified, thereby allowing detection of the target in the sample. Any suitable amplification method can be used and / or performed, including but not limited to PCR and / or rolling circle amplification (RCA).

[0107] In some embodiments, the method may include one or more washing steps, optionally using an aqueous solution. One or more washing steps may be performed to wash the solid support bound to the target and / or to wash the complex. The one or more washing steps may remove unreacted and / or unbound components and / or components non-specifically bound to the complex.

[0108] In some embodiments, an amplification agent is used in the methods of the invention. The methods may include combining and / or reacting an amplification agent with a complex comprising a solid support, a target, and a detection reagent. If the amplification agent is attached to the solid support, the amplification agent may be released from the solid support prior to, during, and / or after the step of combining the amplification agent with the complex. In some embodiments, the amplification agent remains on the solid support. In some embodiments, the amplification agent is not attached to the solid support and is added to a composition comprising the complex.

[0109] In some embodiments, the amplification agent is a nucleic acid sequence (e.g., a primer) that can be optionally released from the solid support prior to the amplification step. In some embodiments, at least a portion of the nucleic acid sequence bound to and / or released from the solid support is amplified. In some embodiments, the amplification agent comprises a ssDNA splint and a ligase that react under suitable conditions to ligate two oligonucleotides together. In some embodiments, the solid support of the invention comprises an oligonucleotide (e.g., a ssDNA oligo), the detection reagent of the invention comprises an oligonucleotide (e.g., a ssDNA oligo), and in the presence of the ssDNA splint and the ligase, under suitable conditions (e.g., when the oligos are in close proximity to each other, e.g., when each binds to the same target), the two oligonucleotides can be ligated and / or extended, and then at least a portion of the oligonucleotides can be amplified. In some embodiments, the solid supports of the invention comprise oligonucleotides (e.g., ssDNA oligos) and the detection reagents of the invention comprise oligonucleotides (e.g., oligos having 3' overhangs that can hybridize to the ssDNA oligos of the solid support), and when in close proximity to each other, e.g., when each is bound to the same target, the two oligonucleotides can hybridize and be extended, after which at least a portion of the oligonucleotide can be amplified.

[0110] In some embodiments, a detection reagent of the invention is present in a composition that includes a corresponding solid support of the invention when the target and solid support are combined, hi some embodiments, a detection reagent of the invention, its corresponding solid support of the invention, and the target are combined simultaneously.

[0111] The method of the invention may include amplifying one or more of the same and / or different nucleic acid sequences. In some embodiments, the method of the invention includes polymerase chain reaction (PCR). In some embodiments, the method of the invention includes amplifying at least a portion of the nucleic acid tag of the detection reagent. In some embodiments, amplifying at least a portion of the nucleic acid tag of the detection reagent is performed using PCR. In some embodiments, amplifying at least a portion of the nucleic acid tag of the detection reagent does not involve PCR, but another nucleic acid amplification technique is used, such as, but not limited to, loop-mediated isothermal amplification (LAMP), rolling circle amplification, strand displacement amplification, transcription-mediated amplification and / or nucleic acid circuitry, where the enzymatically degraded tag can initiate a chain reaction driven by hybridization.

[0112] In some embodiments, the methods of the invention are performed using Proximity Ligation Assay (PLA). In some embodiments, the methods of the invention are performed using Binding Induced DNA Amplification (BINDA), Proximity Extension Assay (PEA) and / or Triple PLA. In some embodiments, the methods of the invention are multiplexed assays (e.g., immunopolymerase chain reaction (iPCR) assays) performed in a microfluidic device comprising a plurality of wells, the method further comprising isolating the solid support from one or more additional solid supports in at least one well of the plurality of wells.

[0113] The microfluidic device may or may not include a rigid or solid microwell array. In some embodiments, the solid support of the invention may be sequestered in droplets of aqueous PCR master mix in immiscible oil (e.g., droplets used in emulsion PCR, BioRad Digital Droplet PCR System, RainDance PCR System, or technology known as BEaMing). In some embodiments, the forward primer is attached (e.g., tethered) to the solid support of the invention (e.g., beads) via a covalent linkage or multiple biotin groups, so that the signal can be read by a flow cytometer after breaking of the emulsion. Certain isothermal amplification techniques can be used in emulsion / droplet format or in bulk solution where the amplicons remain attached to the solid support. In some embodiments, substrates, devices, and / or methods of the invention can include, but are not limited to, those described (e.g., substrates, devices, and / or methods) in U.S. Patent Application Publication No. 2015 / 0211048, International Application No. PCT / US2016 / 042913, International Application No. PCT / US2016 / 043463, and International Application No. PCT / US2016 / 055470, the entire contents of each of which are incorporated herein by reference. In some embodiments, methods of the invention include methods of delivering reagents and / or targets to reaction wells using beads (e.g., superparamagnetic beads), such as those described in U.S. Patent Application Publication No. 2015 / 0211048 and International Application No. PCT / US2016 / 042913, the entire contents of each of which are incorporated herein by reference.

[0114] In some embodiments, the compounds, compositions, kits, systems, devices, and / or methods of the invention may be used to perform any suitable reaction, such as, for example, assays involving biomolecules such as PCR reactions, spatial array-based assays, such as reconfigurable multi-element diagnostic (ReMeDx) assays, singleplex reactions on compact arrays (SiRCA), assays involving aqueous reaction compositions, assays performed on plastic substrates, and / or assays described in WO 2013 / 176767, the contents of which are incorporated herein by reference.

[0115] In some embodiments, the method of the invention includes preparing a solid support of the invention. The solid support of the invention can be formed by contacting a solid support (optionally including one or more linkers) with a molecular recognition element and a nucleic acid sequence at the same time and / or at different times. In some embodiments, the molecular recognition element and the nucleic acid sequence can be added in multiple increments to help provide a plurality of beads having substantially the same number (i.e., within ±20%) of molecular recognition elements and nucleic acid sequences in each bead in the plurality of beads. In some embodiments, the method of preparing a solid support of the invention includes contacting the solid support with one or more fillers, such as, but not limited to, biotin, biotinylated polymers, biotinylated nucleic acid polymers, biotinylated carbohydrates, biotinylated quantum dots or other nanoparticles, biotinylated peptides, and / or biotinylated proteins. The fillers can help control the amount of molecular recognition elements and / or nucleic acid sequences added to the solid support and / or can help reduce non-specific binding during the assay.

[0116] In some embodiments, undesired hybridization of detection reagents to nucleic acid sequences (e.g., primers) attached to solid supports can be reduced by using toehold displacement primers, hairpin primers, partially digestible hairpin primers, and / or blocking NA sequences. Primers can be tethered to the solid support at the 5' and / or 3' ends or both the 5' and 3' ends. In some embodiments, double-stranded primers can be used if appropriate conditions are provided.

[0117] In some embodiments, incubation and / or amplification conditions can be controlled in the methods of the invention such that hybridization of the detection reagent to the nucleic acid sequence attached to the solid support is not favored and PCR amplification can still occur during the detection stage of the method. For example, low melting temperature primers can be used under incubation conditions that support melting conditions (e.g., low salt or addition of betaine) such that the primers do not hybridize to the detection reagent. However, conditions that support a higher melting temperature can be used during PCR (e.g., higher salt or removal of betaine) so that the primers can efficiently amplify the nucleic acid sequence of the detection reagent.

[0118] In the methods of the invention, the target can be detected using any method known to one of skill in the art, such as, for example, visual observation and / or chemical or biochemical assays. In some embodiments, the methods of the invention include detecting the target by detecting an assay signal. For example, the assay signal can be a fluorescent signal, such as from a dye that intercalates into dsDNA and / or from hydrolysis of a probe that includes a quencher and a dye.

[0119] In some embodiments, fluorescence is used for detection. In some embodiments, the methods of the invention include and / or provide a fluorescent readout. In some embodiments, the methods of the invention include and / or provide an electronic readout by hybridization of the amplicons to a solid-state sensor, sequencing of the amplicons by methods such as pyrosequencing, and / or formation of a precipitate such as that seen during LAMP amplification.

[0120] The methods of the invention can improve the specificity of an assay and / or reduce the background signal for an assay. In some embodiments, the methods of the invention reduce the observed cross-reactivity between a solid support and a non-target molecule (e.g., incorrect antigen). As used herein, "observed cross-reactivity" refers to a detected signal associated with an interaction between a solid support and a non-target molecule (e.g., incorrect antigen). In some embodiments, the detected signal can be observed visually. In some embodiments, the methods of the invention reduce the observed non-specific binding of a solid support, such as, for example, a fluorescent signal observed in a false positive as shown in FIG. 5. In some embodiments, the methods of the invention improve the signal-to-noise ratio for an assay, improve the sensitivity for an assay, and / or reduce the observed cross-reactivity for an assay. In some embodiments, the compounds, compositions, devices, kits, systems, and / or methods of the invention improve a multiplexed immuno-PCR (iPCR) assay.

[0121] In some embodiments, cross-reactivity and / or background signal observed for targets can be reduced by using different nucleic acid sequences for each target and / or by performing multiplexed probe-based qPCR where each probe is labeled with a spectrally distinct fluorescent dye, in which case the increase in fluorescence observed from probe hydrolysis can be referenced to the coding of the bead to determine if the correct complex has been formed.

[0122] In some embodiments, the sequences of the NA tags used in the detection reagents are carefully selected so that the amplicons resulting from correct PCR amplification have distinct, optionally different, melting temperatures, allowing these sequences to be distinguished from non-specific amplification products and, optionally, from each other. The use of amplicons with different melting temperatures can be used as a means of coding to distinguish one bead population from another and / or as a means of supporting a coding signal. The methods of the present invention can reduce the background signal for an assay by about 0.5, 1, 1.5, 2, 2.5, or 3 orders of magnitude compared to a control method (e.g., iPCR using a known solid support and detection reagent).

[0123] The compounds, compositions, kits, devices, systems and / or methods of the invention can be used in multiplexed immunoassay panels. Some embodiments of the invention may be useful in qualitative and / or quantitative medical and / or veterinary diagnostic testing, forensic testing, biohazard identification, environmental monitoring, research and development, and / or the like. In some embodiments, the ability to multiplex molecular recognition element (e.g., antibody) pairs that would otherwise exhibit high levels of cross-reactivity may enable new diagnostic tests not currently realized.

[0124] The invention will now be described in more detail in the following non-limiting examples. EXAMPLES

[0125] Example 1 Single-molecule bead-based immunoassays were developed using solid supports and detection reagents as described herein (referred to as "Protein SiRCA technology"). These assays were compared to similar iPCR assays to show how Protein SiRCA technology can reduce background from non-specific binding and antibody cross-reactivity.

[0126] First, the iPCR assay reagents were prepared. Bead sets for the cytokines IL-6, IL-7, IL-8, IL-10, and TNF-α were prepared by functionalizing 3 μm ProMag COOH-modified superparamagnetic beads with the encoding dye and capture antibody. Detection antibody conjugates were generated by attaching the same 60 nucleotide ssDNA tag to the relevant detection antibody (Figure 3) using the Protein-Oligo Conjugation Kit from TriLink Biotechnologies. iPCR assays were performed as shown in Figure 4. Singleplex assays showed excellent sensitivity (Figure 9A). 5plex assays showed excellent sensitivity for most bead sets, with measurements performed near the limit of detection (LOD), typically around 1 pg / mL or less (Figure 9B). However, when multiplexed, the background signal increased significantly for all bead sets, especially for IL-8 (Figures 9B and 9C).

[0127] Protein Sirca beads were prepared by incubating streptavidin-coated Dynabeads with biotinylated capture antibodies (Figure 10). Antibodies were biotinylated in-house using the Chromalink Biotin Antibody Labeling Kit from TriLink Biotechnologies. The beads were then coded by incubating the antibody-labeled beads with a biotinylated forward primer covalently labeled with a fluorescent dye (Figure 10). Three sets of beads were prepared for the cytokines IL-7, IL-8, and IL-10. Plots of the fluorescence intensity at the two coding wavelengths for a representative bead from each set are shown in Figure 11. The coding clusters for the three iPCR beads with covalently attached dyes are also shown in Figure 11. The tight clustering of each bead set allowed precise identification of each bead type in multiplexed experiments.

[0128] The percent positives of Protein SiRCA beads were compared to conventional iPCR beads in a singleplex assay. iPCR beads differ from Protein SiRCA beads in that they are not functionalized with streptavidin or coated with primers that are covalently linked to an encoding dye and capture antibody. iPCR and Protein SiRCA beads for each target were incubated with either serum (blank) or 90 fM of the target cytokine in serum (positive control). Since the goal of this initial study was to measure the sensitivity or activity of the capture antibodies on the different assay beads, each bead set was incubated with the appropriate antibody conjugate developed for iPCR (i.e., all detection conjugates had the same NA tag). Both primers were added to the PCR master mix. Figure 12 shows the signals obtained for serum blank and each cytokine at 90 fM. In general, Protein SiRCA beads behaved very similarly to the iPCR beads in terms of signal to background, with Protein SiRCA beads showing slightly higher signal and slightly higher background for these assays.

[0129] Next, detection antibody conjugates were prepared for Protein SiRCA. In iPCR, the same NA tag is used for each antibody conjugate. In Protein SiRCA, a different NA tag is used for each conjugate, as shown in Figure 7. The activity of iPCR and Protein SiRCA conjugates was compared using iPCR beads lacking the forward primer in singleplex assays. To do so, primers for each tag were added to the master mix of each singleplex assay. Comparison of the activity of detection antibody conjugates in otherwise identical experiments showed that all Protein SiRCA conjugates were less sensitive than the iPCR conjugates (Figure 13). Characterization of the conjugates revealed that a significant proportion of the antibodies lacked the NA tag, since all Protein SiRCA antibodies were underlabeled. These tag-less antibodies were able to bind the captured target molecules but did not produce a detectable (convertible) PCR signal. The difference in labeling efficiency between the iPCR conjugate and the Protein Sirca conjugate may be due to batch variations in the purchased antibody stock or degradation of reagents in the conjugation kit (Protein-Oligo Conjugation Kit, TriLink Biotechnologies) since the iPCR conjugate and the Protein Sirca conjugate were prepared at different times. The differences observed between the conjugates may be due to variability in the preparation of the antibody conjugates and are not an inherent property of the Protein Sirca conjugate.

[0130] To account for differences in bead type, coding scheme, antibody attachment to beads, primer delivery, antibody conjugate labeling efficiency, and PCR amplification efficiency of different NA tag sequences, baseline singleplex assays were performed for iPCR and Protein SiRCA (Figure 14). The background signals for IL-7 and IL-8 in singleplex assays are slightly higher for Protein SiRCA, while the IL-10 Protein SiRCA has a lower background signal.

[0131] Next, a 3-plex iPCR assay containing IL-7, IL-8, and IL-10 iPCR beads was performed using the three iPCR antibody conjugates. Beads for all three cytokines were mixed together and then incubated with buffer (blank) or 90 fM of each cytokine. A single set of forward and reverse primers for the common iPCR NA tag was provided in the PCR master mix. As expected from previous studies, IL-8 showed very high background signal. Such a high percentage of positive wells indicates that there is likely some non-specifically bound detection antibody complex per bead. When 90 fM of either IL-7 or IL-10 was spiked into the sample buffer, a significant increase in assay signal was observed for the appropriate bead set. However, the addition of 90 fM IL-8 to the sample buffer did not result in a significant increase in signal above background for the IL-8 beads, indicating that this concentration is below the detection limit in the 3-plex iPCR assay (Figure 15). A comparison chart of the four different assay conditions (blank and 90 fM of each cytokine) for each bead type is shown in Figure 15e. The high signal from the IL-8 beads may be due to non-specific binding of other detection antibody conjugates.

[0132] The same 3-plex assay was repeated using Protein SiRCA beads and detection antibody conjugates for IL-7, IL-8 and IL-10. Beads for all three cytokines were mixed together and then incubated with buffer (blank) or 90 fM of each cytokine (Figure 16). Since the unique forward primer for each conjugate tag was delivered to the well by the bead itself, only the common reverse primer was provided in the PCR master mix. A comparison chart showing the signal from each bead set for the four experiments (blank and 90 fM of each cytokine) is shown in Figure 16. A discernible increase in signal above background is observed for all beads when the respective analyte is present.

[0133] A direct comparison of iPCR and Protein SiRCA assays is shown in Figure 17. In the singleplex assay data, IL-7 shows a lower signal to background in Protein SiRCA compared to iPCR, which may be due to issues with the Protein SiRCA antibody conjugate. IL-10 shows a significant improvement in signal to background in Protein SiRCA compared to iPCR. However, the most significant improvement is seen with the IL-8 beads. The background signal of the Protein SiRCA IL-8 beads is more than an order of magnitude lower than that seen with iPCR, allowing enough signal to background to easily distinguish the difference between 90 fM IL-8 and blank.

[0134] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The present invention is defined by the appended claims, including equivalents thereof. The entire contents of all publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference for their teachings relevant to the sentence and / or paragraph in which the reference is set forth.

Claims

1. (i) Below: Coding agents (e.g., dyes), a first nucleic acid sequence (e.g., an oligonucleotide and / or a primer), and A first molecular recognition element (e.g., an antibody) a solid support (e.g., a bead) comprising (ii) a detection reagent comprising a second molecular recognition element (e.g., an antibody) and a second nucleic acid sequence; and A kit comprising: A kit, wherein at least a portion of the first nucleic acid sequence and at least a portion of the second nucleic acid sequence are configured to participate in a nucleic acid amplification process.

2. 2. The kit of claim 1, wherein the solid support further comprises a linker (e.g., one member of a specific binding pair), the linker being on a surface of the solid support, and optionally the solid support being coated and / or functionalized with a plurality of linkers.

3. The kit of claim 1 or 2, wherein the first molecular recognition element and / or the first nucleic acid sequence is attached (e.g., covalently and / or non-covalently) to the linker (e.g., one member of a specific binding pair).

4. 4. The kit of claim 1, wherein the linker is streptavidin and the first molecular recognition element is a biotinylated molecular recognition element, and the first molecular recognition element is attached to the solid support via binding of streptavidin and biotin.

5. 5. The kit of claim 1, wherein the linker is streptavidin, the first nucleic acid sequence is biotinylated, and the first nucleic acid sequence is attached to the solid support via binding of streptavidin and biotin.

6. The kit of any one of claims 1 to 5, wherein the first nucleic acid sequence comprises from about 10, 20 or 25 nucleotides to about 30, 40, 50 or 60 nucleotides.

7. 7. The kit of any one of claims 1 to 6, wherein the second nucleic acid sequence comprises from about 20, 30, 40, 50 or 60 nucleotides to about 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280 or 300 nucleotides.

8. 8. The kit of claim 1, wherein the second nucleic acid sequence comprises a sequence that is substantially identical (e.g., about 5 or more contiguous nucleotides) to a sequence in the first nucleic acid sequence, and optionally, the sequence that is substantially identical to a sequence in the first nucleic acid sequence is present in the 5' region of the second nucleic acid sequence.

9. The kit of any one of claims 1 to 8, wherein the first nucleic acid sequence and / or the second nucleic acid sequence comprises single-stranded DNA.

10. The kit of any one of claims 1 to 9, wherein the first nucleic acid sequence and / or the second nucleic acid sequence comprises double-stranded DNA.

11. The kit according to any one of claims 1 to 10, wherein the first nucleic acid sequence and / or the second nucleic acid sequence is RNA.

12. 12. The kit of any one of claims 1 to 11, wherein the first nucleic acid sequence and / or the second nucleic acid sequence comprise one or more chemically modified bases (e.g. one or more chemically modified bases not normally found in nature), optionally wherein the chemically modified bases improve specificity of a PCR reaction and / or reduce non-specific binding between nucleic acid sequences (e.g. oligos).

13. The kit of any one of claims 1 to 12, wherein the second nucleic acid sequence is covalently attached to the second molecular recognition element.

14. 14. The kit of any one of claims 1 to 13, wherein the second molecular recognition element is an antibody and the second nucleic acid sequence is attached to one or more amino acids of the antibody, and optionally, the second nucleic acid sequence is covalently attached to a fragment crystallizable (Fc) region of the antibody.

15. The kit of any one of claims 1 to 14, further comprising amplification agents, such as a reverse primer, a ssDNA splint and a ligase.

16. 16. The kit of any one of claims 1 to 15, wherein the amplification agent has a nucleic acid sequence that is complementary to a portion of the second nucleic acid sequence, optionally wherein the portion of the second nucleic acid sequence is in the 3' region of the second nucleic acid sequence.

17. The kit of any one of claims 1 to 16, wherein the amplification agent is attached to the solid support, and optionally the amplification agent is biotinylated.

18. The kit according to any one of claims 1 to 17, wherein the solid support and the detection reagent are stored separately, and / or the detection reagent and, if present, the amplification agent are stored separately.

19. The kit of any one of claims 1 to 18, wherein the solid support and the amplification agent are stored separately when the amplification agent is present and not attached to the solid support.

20. Coding agents (e.g., dyes), Nucleic acid sequences, and Molecular recognition elements (e.g., antibodies) A solid support (e.g., a bead), comprising:

21. 21. The solid support of claim 20, further comprising a linker (e.g., one member of a binding pair), said linker being on a surface of said solid support, and optionally said solid support being coated and / or functionalized with a plurality of linkers.

22. 22. The solid support of claim 20 or 21, wherein the molecular recognition element and / or the nucleic acid sequence is attached to the linker.

23. 23. The solid support of any one of claims 20 to 22, wherein the linker is streptavidin and the molecular recognition element is a biotinylated molecular recognition element, and the molecular recognition element is attached to the solid support via binding of streptavidin and biotin.

24. 24. The solid support of any one of claims 20 to 23, wherein the linker is streptavidin and the nucleic acid sequence is biotinylated and attached to the solid support via binding of streptavidin and biotin.

25. The solid support of any one of claims 20 to 24, wherein the nucleic acid sequence comprises from about 10, 20 or 25 nucleotides to about 30, 40, 50 or 60 nucleotides.

26. a first coding agent (e.g., a dye) that provides a first coding signal; a first nucleic acid sequence, and A first molecular recognition element (e.g., an antibody) a first plurality of solid supports, each solid support comprising: a second coding agent (e.g., a dye) that provides a second coding signal; a second nucleic acid sequence, and A second molecular recognition element (e.g., an antibody) a second plurality of solid supports, each solid support comprising A plurality of solid supports comprising: A plurality of solid supports, wherein the first coded signal is different from the second coded signal, the first nucleic acid sequence is different from the second nucleic acid sequence, and the first molecular recognition element is different from the second molecular recognition element.

27. a first molecular recognition element (e.g., an antibody); and First Nucleic Acid Sequence a first plurality of detection reagents, each detection reagent comprising: a second molecular recognition element (e.g., an antibody); and Second Nucleic Acid Sequence a second plurality of detection reagents, each detection reagent comprising: A plurality of detection reagents comprising: the first nucleic acid sequence and the second nucleic acid sequence each include a first portion and a second portion; a nucleic acid sequence of the first portion for the first nucleic acid sequence is different from a nucleic acid sequence of the first portion for the second nucleic acid sequence, and a nucleic acid sequence of the second portion for the first nucleic acid sequence and the second nucleic acid sequence are the same.

28. 28. The plurality of detection reagents of claim 27, wherein the first portion for the first nucleic acid sequence and the second nucleic acid sequence is in the 5' region of each sequence and the second portion is in the 3' region of each sequence.

29. 1. A method for detecting a target in a sample, comprising: combining a solid support of the kit according to any one of claims 1 to 19, a solid support according to any one of claims 20 to 25, or a plurality of solid supports according to claim 26 with a sample comprising a target to form a first composition comprising the solid support bound to the target; Optionally, washing the target-bound solid support (e.g., with an aqueous solution); combining a detection reagent with the solid support having the bound target to form a complex comprising the solid support, the target, and the detection reagent, wherein the detection reagent comprises a second molecular recognition element and a second nucleic acid sequence, wherein at least a portion of the first nucleic acid sequence and at least a portion of the second nucleic acid sequence are configured to participate in a nucleic acid amplification process; Optionally, washing the complex (e.g., with an aqueous solution); Optionally, combining the complex with amplification agents (e.g., a reverse primer, a ssDNA splint, and a ligase, etc.); Optionally, releasing the first nucleic acid sequence from the solid support; and detecting a target in said sample by amplifying at least a section of said second nucleic acid sequence; A method comprising:

30. 30. The method of claim 29, further comprising combining the amplification agent with the complex, wherein combining the amplification agent with the complex comprises releasing the amplification agent from the solid support.

31. 31. The method of claim 29 or 30, wherein the detection reagent is present in the first composition.

32. The method of any one of claims 29 to 31, wherein the amplification agent is present in the first composition, and optionally, the amplification agent (e.g., a reverse primer) is attached to the solid support.

33. The method according to any one of claims 29 to 32, wherein the amplifying step is carried out using the polymerase chain reaction.

34. The method of any one of claims 29 to 33, which is carried out using a proximity ligation assay or a proximity extension assay.

35. 35. The method of any one of claims 29 to 34, further comprising detecting the target by detecting an assay signal (e.g. a fluorescent signal, such as a fluorescent signal from a dye that intercalates into dsDNA or from hydrolysis of a probe comprising a quencher and a dye).

36. 36. The method of any one of claims 29 to 35, wherein the multiplexed assay (e.g. an immunopolymerase chain reaction (iPCR) assay) is performed in a microfluidic device comprising a plurality of wells, and further comprising isolating the solid support from one or more further solid supports in at least one well of the plurality of wells.

37. 1. A method for improving specificity and / or reducing background signal for an assay, comprising: Providing a kit solid support according to any one of claims 1 to 19, a solid support according to any one of claims 20 to 25, or a plurality of solid supports according to claim 26; performing an assay in the presence of said solid support or said plurality of solid supports thereby improving specificity and / or reducing background signal associated with said assay; A method comprising:

38. 38. The method of claim 37, wherein the assay is a multiplexed immunoPCR (iPCR) assay.

39. 39. The method of any one of claims 29 to 38, which reduces observed cross-reactivity between said solid support or a solid support in said plurality of solid supports and non-target molecules (e.g. wrong antigens).

40. 40. The method of any one of claims 29 to 39, wherein the method reduces observed non-specific binding of said solid support or a solid support in said plurality of solid supports.

Images