Compounds, compositions and methods for improved assays

Abstract

The present invention relates to compounds, compositions and methods for improved assays. Compounds, compositions, kits, systems, devices, and methods are provided for improving assays, such as multiplex PCR assays (e.g., multiplex immune PCR assays). In accordance with some embodiments of the invention, solid supports (e.g., beads) may be provided. The solid support may comprise an encoding agent (e.g., a dye), a nucleic acid sequence (e.g., an oligonucleotide and / or primer), and a molecular recognition element (e.g., an antibody). In accordance with some embodiments of the invention, detection reagents may be provided. The detection reagent may include a molecular recognition element (e.g., an antibody) and a nucleic acid tag. In some embodiments, at least a portion of the nucleic acid sequence of the solid support and at least a portion of the nucleic acid tag of the detection reagent are configured to participate in a nucleic acid amplification process. The solid support and the detection reagent can be bound to the same target, thereby forming a reagent pair.

Description

[0001] This application is a divisional application of an invention patent application with an application date of September 24, 2019, application number 201980036497.3, and invention name “Compounds, compositions and methods for improving determinations”. Statement of Federal Support This invention was made with Government support under Grant No. HR0011-12-2-0001 awarded by the United States Department of Defense (DARPA). Technical Field The present invention relates to compounds, compositions and methods for improving assays such as multiplex immuno-PCR assays. Background Art Immunoassays of target molecules (such as proteins) generally rely on paired molecular recognition elements (such as antibodies or aptamers) to first capture and then label the target molecules so that they can be quantified. Generally, the capture antibody is bound to a solid support, such as the wall of a microwell plate or the surface of a microbead. The sample is incubated with the antibody-coated surface to capture the target molecule. After the washing step of removing the sample matrix, the captured target molecule is labeled with a detection recognition element (such as an antibody or aptamer), which can be directly conjugated to a reporter molecule (label) or a linking agent (such as biotin), which allows subsequent coupling to the label. Alternatively, the detection recognition element can be detected with a second antibody, which is detected by a label. The label can include nanoparticles, dyes, enzymes or nucleic acids (NA) that can be detected by direct observation or by chemical or biochemical assays.

[0004] In applications where high sensitivity is not required, the label can be a gold nanoparticle (popular in lateral flow assays) or a directly detectable fluorescent dye. Luminex Corp. has commercialized a popular immunoassay using direct labeling. In this assay, a coded microbead set is used to perform a multiple immunoassay. Each bead set is encoded by dyeing the beads with a unique dye combination. Each bead set is then functionalized with different affinity reagents (e.g., antibodies, antigens, etc.). The beads can be mixed together to form a multiple bead library. During the assay, the beads are incubated with the sample to capture the target molecule if the target is present. The beads are washed and then incubated with a detection antibody, which is labeled with a reporter dye or biotinylated so that it can be subsequently labeled with a reporter dye conjugated with streptavidin. Flow cytometry or fluorescence microscopy is used to determine both the combination of the coding dye (to identify the bead type) and the assay signal (i.e., the signal from the reporter dye) of each bead. The concentration of the target molecule in the sample is determined by comparing the average measured assay signal of each bead type with the standard curve. In this method, the captured target molecules are labeled with approximately 1-5 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 increase assay sensitivity. ELISA uses an affinity reagent pair to capture a target molecule and labels the target molecule with an enzyme (such as horseradish peroxidase, alkaline phosphatase, glucuronidase, or β-galactosidase). The enzyme is then used to convert the substrate into a detectable product in a concentration-dependent manner, allowing the concentration of the target molecule to be determined ( Figure 1 ). This method is generally more sensitive than direct labeling because 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 microplate, and an analog signal is measured using a reader (such as a fluorescent plate reader). Multiplexing can be done spatially, where the sample is split between different wells of a microplate containing different capture antibodies and to which different detection antibodies are added. Single-pot multiplexing is also possible, but a different enzyme and substrate pair is required for each target, which adds additional complexity that is generally not practical. Alternatively, the capture antibody can be attached to microbeads or magnetic microbeads for easier handling or improved workflow ( Figure 1 ).

[0007] In addition to ELISA, immuno-PCR (iPCR) can also be used to quantify proteins and other molecules. This method uses affinity reagents in a similar manner to ELISA, but unlike ELISA, the detection reagent (e.g., antibody) is not labeled with an enzyme but with a nucleic acid (NA) tag. The NA tag can be covalently linked to the antibody (forming a conjugate) or linked by an interaction such as a biotin-streptavidin bond. The tag is usually amplified by quantitative PCR (qPCR) to determine the concentration of the target molecule. Similar to ELISA, iPCR can be performed in the form of an analog signal using a microplate coated with a capture antibody. The immune complex can remain intact, or it can be separated to release the NA tag before performing PCR. Digital PCR can be used to detect the released tags in the form of droplets or microwell arrays, thereby obtaining a higher degree of precision quantification. Multiplexing can be achieved by shunting samples between single reactions, or by using multiple unique tag sequences with different color probe sets in a single reaction.

[0008] Similar to ELISA, iPCR can also be performed using capture antibodies bound to beads ( Figure 2 ). Different sets of encoded beads are functionalized with separate capture antibodies so that the identity of the capture antibody bound to each bead can be decoded from an optical signal (e.g., fluorescence from multiple dyes at different concentrations attached to or embedded in the beads). Different sets of beads 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 sample to capture target molecules, washed to remove sample matrix, and then labeled with detection antibodies ( Figure 4 ). If the detection antibody is covalently linked to a nucleic acid tag (i.e., a detection antibody-NA conjugate or "detection conjugate"), the immune complex is intact. However, if the detection antibody is biotinylated, the beads are incubated successively with streptavidin and the biotinylated NA tag. Quantitative PCR is used to determine the number of NA tags and thus the concentration of the initial sample. Multiplex assays are more complex than singleplex assays because different capture and detection antibody pairs are required for each target molecule.

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

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

[0012] In all of the 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 assay will have poor sensitivity because the captured target molecules will not be effectively labeled. Increasing the detection antibody concentration improves sensitivity because more captured target molecules are labeled, but this may also increase the background signal of the assay. The increase in background signal is typically caused by nonspecific binding (NSB) of the detection reagent to the microwell or microbead surface, interaction with the capture antibody, or interaction with the bead-encoded dye in bead-based immunoassays. Unfortunately, it is not possible to distinguish between antibodies derived from properly formed immune complexes ( Figure 5 , the positive signal of complex A) is related to nonspecific interactions (such as capture antibody and detection antibody ( Figure 5 , complex B), capture antibody and NA tag ( Figure 5 , complex C) or NA tag and bead surface ( Figure 5 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 can occur between the detection antibody and other proteins in the sample matrix that may have specifically or non-specifically bound to the beads or capture antibody.

[0013] The above-mentioned NSB interactions become more severe when single-plex assays are combined into multiplex panels (which usually require re-optimization of detection antibody concentration and other assay parameters). The main reason for the increase of NSB in multiplex assays is that as more target molecules are added to the assay, the total detection antibody concentration also increases, which generally increases the signal observed due to NSB. In addition, cross-reactivity between antibodies and off-target molecules may become a greater problem in multiplex assay formats. There are many forms of cross-reactions that can affect the assay signal. For example, an antigen can bind to the wrong (off-target) capture antibody and then be detected by its own detection antibody, which will result in a false-positive signal. In a similar example, an antigen can bind to the wrong (off-target) capture antibody but cannot be detected by the detection antibody. In this case, the wrongly captured antigen can block (i.e., competitively inhibit) the binding of the true target and subsequent detection, resulting in a false-negative signal.

[0014] Although the signal due to cross-reactivity and NSB generation can sometimes be reduced for lower sensitivity, direct labeling assays or batch analog assays, this is a much more significant problem for single molecule assays, as even a single NSB event per bead can be detrimental to the performance of the digital assay. High-sensitivity multiplexed assays are generally limited to about 5-10 different targets, as cross-reactivity between NSBs and detection chemistries significantly reduces the signal-to-noise ratio and increases the limit of detection.

[0015] In addition to the problems highlighted above, multiplex PCR often suffers from off-sequence amplification and competition between primer and probe sets.To achieve highly sensitive iPCR assays at higher multiplexing levels, an alternative approach is needed. Summary of the invention Aspects of the invention relate to compounds, compositions, kits, systems, devices and methods for improving assays such as multiplex PCR assays (eg, multiplex immuno-PCR assays).

[0017] One aspect of the present invention relates to a kit comprising: (i) a solid support (e.g., beads), wherein the solid support comprises: an encoding agent (e.g., a dye); a first nucleic acid sequence (e.g., an oligonucleotide and / or a 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 are configured to 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: an encoding agent (eg, a dye); a nucleic acid sequence; and a molecular recognition element (eg, an antibody).

[0019] Another aspect of the invention relates to a plurality of solid supports comprising: (i) a first plurality of solid supports, each of the first plurality of solid supports comprising: a first coding agent (e.g., a dye) providing a first coding signal; a first nucleic acid sequence; and a first molecular recognition element (e.g., an antibody); and (ii) a second plurality of solid supports, each of the second plurality of solid supports comprising: a second coding agent (e.g., a dye) providing a second coding signal; a second nucleic acid sequence; and a second molecular recognition element (e.g., an antibody), 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 present invention relates to a plurality of detection reagents, comprising: (i) a first plurality of detection reagents, and each of the first plurality of detection reagents comprises a first molecular recognition element (e.g., an antibody) and a first nucleic acid sequence; and (ii) a second plurality of detection reagents, and each of the second plurality of detection reagents comprises a second molecular recognition element (e.g., an antibody) and a second nucleic acid sequence; wherein the first and second nucleic acid sequences each comprise a first portion and a second portion; and wherein the nucleic acid sequence for the first portion of the first nucleic acid sequence is different from the nucleic acid sequence for the first portion of the second nucleic acid sequence. In some embodiments, the nucleic acid sequences for the second portions of the first and second nucleic acid sequences are identical.

[0021] Another aspect of the present invention relates to a method for detecting a target in a sample, the method comprising: combining a solid support as described herein and a sample comprising a target to form a first composition comprising a target-bound solid support; optionally washing the target-bound solid support (e.g., with an aqueous solution); combining a detection reagent and the target-bound solid support to form a complex comprising a solid support, a 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 the complex (e.g., with an aqueous solution); optionally combining an amplification agent (e.g., a reverse primer, a ssDNA splint, and a ligase, etc.) and the complex; optionally releasing the nucleic acid sequence from the solid support; and amplifying at least a portion of the second nucleic acid sequence, thereby detecting the target in the sample.

[0022] Another aspect of the present invention relates to a method for improving the specificity of an assay and / or reducing the background signal of an assay, the method 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 the solid support or the plurality of solid supports, thereby improving the specificity of the assay and / or reducing the background signal of the assay.

[0023] It is noted that aspects of the invention described with respect to one embodiment may be incorporated into different embodiments, although not specifically described with respect thereto. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination. The applicant reserves the right to change any initially filed claims and / or submit any new claims accordingly, including the right to amend any initially filed claims to be subordinate to and / or incorporate any features of any other one or more claims, although protection was not initially claimed in this manner. These and other objects and / or aspects of the invention are explained in detail in the specification set forth below. By reading the accompanying drawings and the subsequent detailed description of the preferred embodiments, those of ordinary skill in the art will be aware of other features, advantages and details of the invention, and this description is only an illustration of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Schematic diagram of the complex formed by an example affinity reagent pair for ELISA.

[0025] Figure 2 Schematic diagram of the complex formed by an example affinity reagent pair for iPCR.

[0026] Figure 3 Schematic diagram of 3 different encoded bead sets functionalized with separate capture antibodies and each bead set corresponds to a different detection antibody.

[0027] Figure 4 Schematic diagram of example method steps in a bead-based iPCR assay.

[0028] Figure 5 Schematic representation of interactions that can provide true positives or false positives in an immunoassay.

[0029] Figure 6 Schematic diagram of example composites formed according to some embodiments of the present invention.

[0030] Figure 7 is a schematic diagram of bead sets prepared by encoding each set with a unique dye or combination of dyes, and according to some embodiments of the present invention, the corresponding detection reagents for each set include different nucleic acid tags.

[0031] Figure 8Schematic diagrams of example complexes that are correctly formed according to some embodiments of the present invention (i.e., the capture antibody on the bead binds to the appropriate target, and the corresponding detection reagent also binds to the target) (which results in a fluorescent signal) and incorrectly formed complexes (i.e., the capture antibody on the bead binds to a detection reagent that is not the corresponding detection reagent of the bead) (which does not produce a fluorescent signal).

[0032] Fig.9A is a graph showing the percentage of positive beads presented for different targets in singleplex assays.

[0033] Fig. 9B Figure 2 is a graph showing the percentage of positive beads presented for different targets in a multiplex assay.

[0034] Fig. 9C To show the Fig.9A and 9B Figures refer to graphs of background signals for singleplex and multiplex assays.

[0035] Fig.10 is a schematic diagram of a method for preparing a solid support according to some embodiments of the present invention.

[0036] Fig.11 Graphs showing the fluorescence intensity of the encoding dye bound to representative beads from different bead sets.

[0037] Fig.12 A graph is provided comparing the percentage of positive beads for conventional iPCR beads to the percentage of positive beads prepared according to some embodiments of the present invention. iPCR dAb conjugates were used for all assays and 1 set of primers was included in the PCR master mix.

[0038] Fig.13 A graph is provided comparing the activity of conventional iPCR detection reagent conjugates to the activity of detection reagents prepared according to some embodiments of the present invention. iPCR beads were used for all assays and 1 set of primers was included in the PCR master mix.

[0039] Fig.14 A graph is provided 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.

[0040] Fig.15 Graphs showing the signals obtained for each bead type in a triplex iPCR assay and graphs comparing different assay conditions are provided.

[0041] Fig.16Graphs showing the signals obtained for each bead type in a triplicate assay according to some embodiments of the invention and graphs comparing different assay conditions are provided.

[0042] Fig.17 supply Fig.15 Comparison chart of triplex iPCR assays and Fig.16 A comparative chart of triplex assays of some embodiments of the present invention. DETAILED DESCRIPTION The present invention is now described more fully below with reference to the accompanying drawings in which embodiments of the invention are shown. However, the present invention can be implemented 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.

[0044] The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The singular forms "a", "an" and "the" as used in the description of the present invention and the appended claims are also intended to include plural forms, unless the context clearly indicates otherwise.

[0045] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meanings as those of ordinary skill in the art to which the present invention belongs. It will be further understood that terms such as those defined in common dictionaries should be interpreted as having the same meaning as that in the context of the present application and the related art, and unless clearly defined as such herein, should not be interpreted in an idealized or overly formal sense. The terms used herein in the description of the present invention are only for the purpose of describing a specific embodiment, and are not intended to limit the present invention. All disclosures, patent applications, patents and other references mentioned herein are combined in their entirety by reference. In the event of a conflict of terms, this specification shall prevail.

[0046] Furthermore, "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as no combination when interpreted in the alternative ("or").

[0047] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. In addition, the present invention also contemplates that any feature or combination of features set forth herein can be excluded or omitted in some embodiments of the present invention. For example, if the specification indicates that a composite comprises components A, B, and C, it is specifically intended that any one or combination of A, B, or C can be omitted and abandoned.

[0048] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be interpreted to cover the recited materials or steps "and those which do not materially affect the basic and novel characteristics of the claimed invention." See In reHerz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in original); see also MPEP §2111.03. Therefore, as used herein, the term "consisting essentially of" should not be interpreted as equivalent to "comprising."

[0049] It should also be understood that the terms "example," "exemplary," and grammatical variations thereof as used herein are intended to refer to non-limiting examples and / or variant embodiments discussed herein, and are not intended to imply that one or more embodiments discussed herein are preferred over one or more other embodiments.

[0050] As used herein, the term "about" when referring to a measurable value such as an amount or concentration, is meant to include variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value as well as the specified value. For example, "about X" (where X is a measurable value) is meant to include X and variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. The ranges of measurable values ​​provided herein may include any other ranges and / or individual values ​​therein.

[0051] As used herein, the terms "increase," "increase," "enhance," "increase," "enhance," "enhance" and the like mean an increase in the specified parameter by at least about 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.

[0052] As used herein, the terms "reduce," "decrease," "lower," "minimize," "inhibit," and the like refer to a decrease in a specified parameter by at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100%.

[0053] It will be understood that when an element is referred to as being "on," "engaged" to, "connected" to, "coupled" to, "contacting" or the like, it may be directly on, engaged to, connected to, coupled to, or contacting another element, or there may be intervening elements. In contrast, when an element is referred to as being, for example, "directly on," "directly engaged" to, "directly connected" to, "directly coupled" to, or "directly contacting" another element, there may be no intervening elements. Those skilled in the art will also appreciate that reference to a structure or feature being disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature.

[0054] For ease of description, spatial relative terms such as "below", "below", "below", "above", "above", etc. may be used herein to describe the relationship between an element or feature and another element or feature as shown in the figure. It will be understood that, in addition to the positioning depicted in the accompanying drawings, spatial relative terms are also intended to cover different positioning of the device in use or operation. For example, if the device in the accompanying drawings is inverted, the elements described as "below" or "below" other elements or features will be positioned as "above" other elements or features. Therefore, the exemplary term "below" can cover both "above" and "below" positioning. The device can be positioned in other ways (rotated 90 degrees or other positioning), and the spatial relative descriptors used herein are interpreted accordingly. Similarly, unless otherwise specifically indicated, the terms "upward", "downward", "vertical", "horizontal", etc. are used herein only for the purpose of explanation.

[0055] It will be understood that although the terms "first", "second", etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Therefore, the "first" element discussed below may also be referred to as the "second" element without departing from the teachings of the present invention. Unless otherwise specifically indicated, the order of operations (or steps) is not limited to the order presented in the claims or drawings.

[0056] 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. The term also encompasses RNA / DNA hybrids. In some embodiments, the RNA and / or DNA may contain chemically modified bases, such as those not typically found in nature, and the chemically modified bases may increase the specificity of an assay (e.g., a PCR reaction) and / or reduce nonspecific binding between other RNAs and / or DNAs. When dsRNA is produced synthetically, less common bases (such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc.) may be used for antisense, dsRNA and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are effective antisense inhibitors of gene expression. Other modifications may also be made, such as modifications to the 2'-hydroxyl group in the phosphodiester backbone or the ribose group of the RNA.

[0057] The terms "nucleotide sequence" and "nucleic acid sequence" used herein are used interchangeably, and refer to a heteropolymer of nucleotides or a sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule, and include 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 can 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. The nucleic acid molecules and / or nucleotide sequences provided herein are presented in a 5' to 3' direction from left to right, and are represented using a standard encoding representation, which represents the nucleotide characters set forth in the U.S. Sequence Rules 37CFR§§1.821-1.825 and the World Intellectual Property Organization (WIPO) standard ST.25. "5' region" used herein refers to the region closest to the 5' end in a polynucleotide. Thus, for example, an element in a 5' region of a polynucleotide may be located anywhere from the first nucleotide at the 5' end of the polynucleotide to a nucleotide midway in the polynucleotide. "3' region" as used herein refers to the region closest to the 3' end of a polynucleotide. Thus, for example, an element in a 3' region of a polynucleotide may be located anywhere from the first nucleotide at the 3' end of the polynucleotide to a nucleotide midway in the polynucleotide.

[0058] As used herein, the terms "complementary" or "complementarity" refer 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", in which only certain nucleotides bind, or it can be complete when there is complete complementarity between single-stranded molecules. The degree of complementarity between nucleic acid chains has a significant effect on the efficiency and strength of hybridization between nucleic acid chains.

[0059] As used herein, "complementary" may mean 100% complementarity to 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. complementary).

[0060] A "part" or "segment" of a nucleic acid sequence of the present invention is understood to mean a portion or "segment" of a nucleic acid sequence of the present invention that is reduced in length (e.g., reduced by 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 the reference nucleic acid sequence. The invention relates to a nucleic acid sequence 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 nucleic acid sequence. Such nucleic acid portions of the invention may, where appropriate, be included in any portion of a larger polynucleotide of which it is a component.

[0061] A "fragment" of an amino acid sequence of the invention is understood to mean a fragment that is reduced in length (e.g., reduced by 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 a reference amino acid sequence. The amino acid sequence of the present invention may be a fragment of a peptide or a polypeptide of the present invention and may be a fragment of a polypeptide of the present invention ...

[0062] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or peptide sequences are invariant over the entire comparison window of components, such as nucleotides or amino acids. "Identity" can be readily calculated by known methods, including, but not limited to, those described in 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, ed.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., ed.) Stockton Press, New York (1991).

[0063] 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.

[0064] As used herein, the phrases "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 having 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 present invention, basic identity exists in the region of continuous nucleotides of nucleotide sequence of the present invention, and this region length is about 4 nucleotides-about 30 nucleotides, about 5 nucleotides-about 20 nucleotides, about 16 nucleotides-about 30 nucleotides, about 18 nucleotides-about 25 nucleotides, about 30 nucleotides-about 40 nucleotides, about 50 nucleotides-about 60 nucleotides, about 70 nucleotides-about 80 nucleotides, about 90 nucleotides-about 100 nucleotides or more nucleotides and any range therein, until the total length of sequence. In some embodiments, nucleotide sequence can be substantially the same on at least about 5,10,15,20 or 25 continuous nucleotides. In some embodiments, nucleotide sequence can be substantially the same on at least about 5 continuous nucleotides. In some embodiments, substantially identical nucleotide or protein sequence performs substantially the same function with its substantially identical nucleotide or protein sequence.

[0065] In order to perform sequence comparison, a sequence is generally used as a reference sequence for comparison of a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input to a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters.

[0066] Optimal alignment of sequences for aligning a comparison window is well known to those skilled in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA, available as Wisconsin (Accelrys Inc., San Diego, CA)). The "identity score" of the aligned segments of the test sequence and the 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 segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The percent sequence identity is expressed as the identity score multiplied by 100. The comparison of one or more polynucleotide sequences can be to a full-length polynucleotide sequence or a portion thereof or to a longer polynucleotide sequence. For the 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.

[0067] When two nucleotide sequences hybridize to each other under stringent conditions, the two sequences may also be considered to be substantially complementary. In some representative embodiments, two nucleotide sequences that are considered to be substantially complementary hybridize to each other under highly stringent conditions.

[0068] In the context of nucleic acid hybridization experiments such as Southern and Northern hybridization, "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. For an extensive guide to nucleic acid hybridization, see 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 assay", Elsevier, New York (1993). Typically, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (Tm) for a specific sequence at a specified ionic strength and pH.

[0069] The temperature (under specified 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 of a particular probe. In Southern or Northern blotting, an example of stringent hybridization conditions for hybridization of complementary nucleotide sequences with more than 100 complementary residues on a filter is 50% formamide and 1 mg heparin at 42°C for overnight hybridization. An example of highly stringent washing conditions is 0.15M NaCl at 72°C for about 0.15 minutes. An example of stringent washing conditions is 0.2x saline-sodium citrate (SSC) washing at 65°C for 15 minutes (for a description of SSC buffer, see Sambrook below). Typically, a low stringency wash is performed before a highly stringent wash to remove background probe signals. For example, for a duplex of more than 100 nucleotides, an example of a moderately stringent wash is 1x SSC at 45°C for 15 minutes. For duplexes of, for example, more than 100 nucleotides, an example of a low stringency wash is 4-6x SSC at 40°C for 15 minutes. For short probes (e.g., about 10-50 nucleotides), stringent conditions generally involve a salt concentration of less than about 1.0 M Na ion, generally about 0.01-1.0 M Na ion concentration (or other salts) at pH 7.0-8.3, and a temperature of generally at least about 30°C. Stringent conditions may also be obtained by adding destabilizing agents such as formamide. Typically, a signal-to-noise ratio of 2x (or higher) of the signal-to-noise ratio observed for an unrelated probe in a particular hybridization assay indicates that a specific hybridization is detected.

[0070] The following are examples of hybridization / washing condition sets that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present invention. In one embodiment, the reference nucleotide sequence is hybridized to the "test" nucleotide sequence at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and washed with 2XSSC, 0.1% SDS at 50°C. In another embodiment, the reference nucleotide sequence is hybridized to the "test" nucleotide sequence at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and washed with 1XSSC, 0.1% SDS at 50°C, or hybridized to the "test" nucleotide sequence at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and washed with 0.5X SSC, 0.1% SDS at 50°C. In still other embodiments, the reference nucleotide sequence is hybridized to the "test" nucleotide sequence at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA and washed with 0.1X SSC, 0.1% SDS at 50°C, or hybridized to the "test" nucleotide sequence at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA and washed with 0.1X SSC, 0.1% SDS at 65°C.

[0071] The term "target" as used herein refers to any target analyte that may be present in a sample. Example 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 analogs. The target may be one or more analyte molecules. In some embodiments, the target may be present in a sample, optionally wherein the sample is obtained from a source (e.g., a subject, a water supply, a food supply, etc.). The target and / or sample may include one or more polar metabolites, such as amino acids and / or charged molecules, peptides, and / or proteins. The target and / or sample may include one or more chemical and / or biological compounds extracted from biological fluids, tissues, blood, serum, urine, cell growth medium, lysed cells, beverages, and / or food, etc. In some embodiments, the target may come from and / or be present in environmental samples such as water, air, and / or soil samples. In some embodiments, the target may come from and / or be present in a forensic sample. In some embodiments, the target and / or sample may include a merged sample comprising a mixture of two or more samples.

[0072] As used herein, "contact," "contacting," "contacted," and grammatical variations thereof, refer to bringing two or more components together or into close proximity such that a desired reaction (e.g., binding, transcriptional control, genome editing, nicking, cleavage, and / or amplification of nucleic acids) can occur under appropriate conditions.

[0073] Embodiments of the present invention can solve and / or reduce one or more problems in assays (e.g., multiplex iPCR assays), such as background signal problems, such as those caused by non-specific binding (NSB) and / or antibody cross-reactivity problems. Embodiments of the present invention can include and / or utilize a combination of unique nucleic acid tags and provide solid support (e.g., bead-based delivery to form spatial multiplex PCR reactions, which can avoid the common complications associated with single reaction, multiplex PCR.

[0074] According to some embodiments of the present invention, solid supports are provided.Terms used herein " solid supports " include one or more objects, such as microbeads, chips, plates, slides, pins, plate holes, beads, microspheres, nanoparticles, micropores or can be used to provide nucleic acid sequences and molecular recognition elements and / or can be coupled to coding agents (such as fluorescent compounds, chemiluminescent compounds, radioactive elements and / or enzymes) and / or dynamic elements or other components marked with coding agents and / or dynamic elements.Nucleic acid sequences, molecular recognition elements, coding agents and / or dynamic elements can be directly or indirectly connected to or embedded (partially or completely) in solid supports, such as by covalent attachment, non-covalent attachment and / or physical merging.In some embodiments, solid supports can be printed arrays on solid surfaces.In some embodiments, solid supports can be microspheres.In some embodiments, solid supports can be solid surfaces, contact samples such as chemical and / or biological components (such as tissue samples) and and molecular recognition elements on solid supports (optionally including one or more coding agents) can be connected to a part of sample and / or target therein. The molecular recognition element may comprise 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 comprise unstable reagents and / or support bonds, each of which may allow a component (e.g., a nucleic acid sequence) to be cleaved from the solid support.

[0075] In some embodiments, one or more coding agents may be connected to a solid support (e.g., beads or microspheres) to provide a coded solid support (e.g., coded beads or coded microspheres). In some embodiments, one or more coding agents may be connected to a molecular recognition element (e.g., an antibody) to provide a coded molecular recognition element (e.g., an coded antibody), and the recognition element may be connected to a solid support, thereby providing a coded solid support. In some embodiments, the coded molecular recognition element may be connected to a particle, such as a nanoparticle. In some embodiments, the method of the present invention may include providing or binding one or more coding agents and / or coded molecular recognition elements to a solid support. In some embodiments, the solid support and / or the coded solid support is as described in International Publication No. WO 2017 / 112025, the contents of which are incorporated herein by reference in their entirety.

[0076] The term "bead" or "beads" as used herein refers to a solid phase member, such as a particle, microparticle or microsphere, generally a magnetic microsphere, which may be a porous, superficially porous or non-porous material, such as a polymer, plastic, glass, silica, metal or semi-metal oxide (including but not limited to alumina, titanium oxide, zirconium oxide or other oxides), quantum dots, metal particles and / or the like suitable for use in a reaction well. In some embodiments, a multiplex reusable bead set comprising a plurality of beads (e.g., microspheres) may be provided, the plurality of beads being uniquely encoded to distinguish one bead population from another bead population.

[0077] In some embodiments, the solid support is a bead, such as a magnetic or superparamagnetic bead. In some embodiments, the solid support is not a magnetic or superparamagnetic bead. In some embodiments, the solid support is porous and / or has a bead of a coating that increases its surface area. In some embodiments, the solid support is a bead made by producing an aggregate of micron particles and / or nanoparticles, and optionally some or all of the micron particles and / or nanoparticles are functionalized so that it can be used to carry reagents such as capture affinity reagents, primers, probes, blockers, polymerases, reverse transcriptases and / or other enzymes.

[0078] In some embodiments, the solid support (e.g., beads) 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 connected to or at least partially embedded in the solid support. In some embodiments, the nucleic acid sequence and / or molecular recognition element of the solid support comprises a coding agent. The molecular recognition element may comprise or may 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.

[0079] As used herein, the term "coding agent" refers to one or more reagents (e.g., chemicals, proteins, etc.) associated with (e.g., applied to, connected to, bound to, compounded, used to manufacture or produce, etc.) a solid support and / or a material of a solid support and / or a material in contact with a solid support, which provides and / or generates a coding signal for the corresponding solid support (which may be all or a portion thereof). In some embodiments, one or more coding agents provide and / or generate a detectable coding signal that allows a population or subpopulation of solid supports (e.g., beads) to be distinguished. For example, in some embodiments, one or more coding agents may provide and / or generate a detectable coding signal for a single solid support (e.g., bead) to which one or more coding agents are associated (e.g., connected) and / or one or more coding agents may provide and / or generate a detectable coding signal for a specific portion of a solid support to which one or more coding agents are attached (e.g., a portion of a solid support to which a molecular recognition element comprising one or more coding agents is bound).

[0080] The coding 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 by a material (e.g., compound) associated with the solid support. In some embodiments, the coding signal may be the size and / or shape of the solid support. In some embodiments, the coding 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, connected to, bound to, compounded, used to manufacture or generate, etc.) a solid support and / or generated by a solid support. The detectable coding signal may be optically and / or electronically detectable, which can be visually perceived by the human eye and / or read, detected, and / or obtained electronically. The detectable coding signal may include intensity, color (e.g., hue, color intensity, and / or color value), color and intensity, and / or size, shape changes, and / or radioactivity generally at or above a defined threshold. In some embodiments, the detectable coding signal includes luminescence intensity, which may include fluorescence, phosphorescence, and / or chemiluminescence intensity.

[0081] The coded signal of the corresponding solid support (which may be all or part thereof) may be detectable (e.g., optical, electronic, electrochemical, electrostatic, magnetic, etc.) or may be undetectable. In some embodiments, the coded signal of the corresponding solid support (which may be all or part thereof) may vary. For example, the coded signal of the corresponding solid support may vary from detectable to undetectable, from undetectable to detectable, from a larger value to a lower value (e.g., from a larger signal amplitude, fluorescence intensity value, diameter, etc.) and / or from a lower value to a larger value. The change of the coded signal of the corresponding 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 coded signal of the solid substrate may vary 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 is in contact). For example, the coded signal of the solid support can vary and / or can 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 caused by isotopic decay, and combinations of the above.

[0082] The term "decoding" refers to the identification of different groups of corresponding samples (comprising, for example, samples of solid supports and / or samples comprising a variety of coded molecular recognition elements) based at least in part on (generally, electronics / programs) on coded signals from coded solid supports and / or coded molecular recognition elements. Detection and / or identification of one or more coded signals may allow samples to be decoded to determine the identity and / or detection target of a specific solid support population.

[0083] 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 may modify the coded signal of the corresponding solid support (which may be all or part thereof) at one time point compared to the coded signal at a different (e.g., earlier) time point, and may be detectable (e.g., optically and / or electrically detectable). A "dynamic element" may 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 may be a physical and / or chemical change that affects a solid support (e.g., a change may be a change in the charge and / or color of the solid support and / or a change in the size and / or shape of the solid support). Therefore, the dynamic element may be a solid support and / or a compound connected and / or associated with a solid support. In some embodiments, the physical and / or chemical change is a physical and / or chemical change that affects the encoding agent associated with the solid support (e.g., the change may be a change in the stability of the linker that binds the encoding agent to the solid support, the solvent accessibility of the encoding agent, and / or the presence and / or absence of a quencher). Thus, the dynamic element may be an encoding agent and / or a compound (e.g., a quencher) connected to and / or associated with an encoding agent. The physical and / or chemical change may be reversible or irreversible. In some embodiments, the dynamic element may be an encoding agent. In some embodiments, the dynamic element and / or encoding agent is as described in International Publication No. WO 2017 / 112025, the contents of which are incorporated herein by reference in their entirety.

[0084] The encoding of the solid support can be achieved by any method known to those skilled in the art (e.g., using color, fluorescent color, size, shape, changing properties (such as thermally unstable or photolabile dyes), and / or any combination thereof). In some embodiments, the solid support can include an encoding agent covalently or non-covalently attached to the solid support. In some embodiments, the dye is embedded in the solid support (e.g., beads), such as via solvent swelling and entrapment.

[0085] In some embodiments, the solid support comprises one or more joints that may be the same or different. One or more joints may connect and / or bind (e.g., covalently or non-covalently) a molecular recognition element and / or a nucleic acid sequence to a solid support. For example, a joint may be used to connect or bind one or more molecular recognition elements to a solid support and / or the same or different joints may be used to connect or bind one or more nucleic acid sequences to a solid support. "Joints" as used herein refer to any molecule, part and / or functional group that may be used to connect or bind a component (e.g., a solid support) to another component (e.g., a molecular recognition element). Example joints include, but are not limited to, chemical moieties and / or compounds, such as amines (e.g., amine derivatives, isocyanates, isothiocyanates, iodoacetamide, azides, etc.), acids (e.g., acid derivatives, N-hydroxysuccinimide esters, hydrazides, etc.), aldehydes, sulfonyl chlorides, sulfonyl hydrazides, epoxides, hydroxyls, thiols and / or maleimides and / or biomolecules (e.g., proteins, peptides, DNA and / or RNA). In some embodiments, joints are biomolecules that are components of specific binding pairs. "Specific binding pair" and "ligand-receptor binding pair" are used interchangeably herein and refer to two different molecules, one of which has a specific spatial or polar organization on the surface or within a cavity of the molecule that specifically attracts or binds to the other molecule, resulting in an area where the two molecules have affinity for each other. The components of the specific binding pair may be referred to as a ligand and a receptor (anti-ligand). The terms ligand and receptor are intended to encompass the entire ligand or receptor or a portion thereof sufficient 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, such as epidermal growth factor and epidermal growth factor receptor, tumor necrosis factor-α and tumor necrosis factor receptor, and interferons and interferon receptors; 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. Other exemplary linkers and / or chemicals that can be used to connect the two components include, but are not limited to, photocleavable biotin, linkers that provide a covalent bond between the solid support and the recognition element, avidin, Acrydite commercially available from Integrated DNA Technologies, Inc. TM , adenylation, azide (NHS ester), digoxigenin (NHS ester), cholesterol-TEG, I-Linker commercially available from Integrated DNA Technologies Inc. TM, Uni-Link commercially available from Integrated DNA Technologies, Inc. TM AminoModifier, 5' Hexynyl, 5-Octadiynyl dU, Biotin dT, Biotin-TEG, Dual Biotin, Desthiobiotin-TEG, Sulfhydryl Modifier C3 SS, Dithiol and / or Sulfhydryl Modifier C6 SS.

[0086] Any suitable coupling chemistry can be used to bind molecular recognition elements and / or nucleic acid sequences to solid supports. In some embodiments, joints, molecular recognition elements and / or nucleic acid sequences include unstable chemical bonds, which can be used to separate molecular recognition elements or a part thereof from solid supports and / or to separate nucleic acid sequences or a part thereof from solid supports. Unstable chemical bonds can be cracked using a variety of techniques, including, but not limited to, using heat, light and / or chemicals to crack unstable chemical bonds. For example, thermal energy can be applied to destroy or crack unstable chemical bonds, or light can be applied to destroy unstable chemical bonds (such as photo-cleavable bonds). Thermal heating can be achieved by conducting contact with a hot surface, Joule heating of a solution, resistive heating of a substrate adjacent to a solid support, exposure to electromagnetic radiation and / or electromagnetic induction. In some embodiments, unstable chemical bonds can be cracked using changes in ionizing radiation, chemicals, enzymes, electrochemical processes, pH and / or other suitable cracking operations. For example, pH changes can be caused by electrochemical reactions occurring on the surface of the region comprising the solid support, or changes in conditions can be caused directly or indirectly by the activation of initiator reagents that can be activated by physical changes such as heat, light, electrochemical processes, pH changes, etc. In some embodiments, the cracking of unstable chemical bonds can be performed before and / or during PCR determination and / or reaction. In some embodiments, the unstable chemical bonds are cracked to separate the nucleic acid sequence from the solid support so that PCR can occur and / or initiate. In some embodiments, PCR reaction does not occur until the nucleic acid sequence (such as forward primer and / or reverse primer) is released from the solid support to which it is connected. In some embodiments, PCR reaction can occur when the nucleic acid sequence is connected to the solid support. In some embodiments, the components (such as molecular recognition elements and / or nucleic acid sequences) can be connected to the solid support using compounds, compositions and / or methods as described in U.S. Patent No. 9617589, the contents of which are incorporated herein by reference in their entirety.

[0087] In some embodiments, molecular recognition elements (e.g., antibodies) and / or nucleic acid sequences (e.g., primers or oligonucleotides) are connected to a solid support via a photocleavable or thermally unstable bond. For example, in some embodiments, photocleavable biotin is used to connect molecular recognition elements and / or nucleic acid sequences to a solid support. In some embodiments, one or more connection chemicals are used to connect molecular recognition elements and / or nucleic acid sequences to a solid support, such as, but not limited to, molecules with affinity for antibodies such as protein A, protein G, protein A / G, protein L, etc.

[0088] In some embodiments, the nucleic acid sequence (eg, forward primer) can be attached to the solid support by a covalent bond, especially if a hydrolysis probe or TaqMan probe is used for the detection chemistry, since the dye from the hydrolysis probe can still be detected even if the dsDNA amplicon is attached to the beads.

[0089] In some embodiments, the solid support comprises a variety of joints and / or is coated with one or more joints. For example, the solid support may include streptavidin that is bound to and / or coated on at least a portion of the surface of the solid support, and one or more biotinylated molecular recognition elements and / or one or more biotinylated nucleic acid sequences can be bound to the solid support through the combination of 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 is bound to about 1, 2, 3 or 4 biotinylated molecules. In some embodiments, the ratio of the molecular recognition element molecules to the nucleic acid sequence molecules present on the solid support is about 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3 or 1: 4 (molecular recognition element: nucleic acid sequence).

[0090] Solid support can comprise amount and be about 1,10,50,100,200,300,400,500 or 600 molecules (i.e. molecular recognition elements and / or nucleic acid sequences)-about 1000,5000,10000,50000,100000,250000,500000,1000000,1000000,25000000,50000000,60000000 or more molecular recognition elements and / or nucleic acid sequences.In some embodiments, solid support comprises amount and be about 10,30,50,75 or 100 μ mol-about 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15 fmol molecular recognition elements and / or nucleic acid sequences. In some embodiments, solid support comprises and / or the molecular recognition element and / or the nucleic acid sequence of about 0.1,0.3,0.5,0.75,1,2,3,4 or 5zmol-about 1,5,10,15,20,25,30,35 or 40amol.In some embodiments, solid support comprises and / or the molecular recognition element and / or the nucleic acid sequence of about 1amol, about 1zmol-about 100amol, or about 100ymol-about 10fmol.In some embodiments, solid support comprises and / or the molecular recognition element of about 3amol, about 0.3zmol-about 30amol or about 30ymol-about 3fmol.

[0091] Solid support can provide to measure and / or reaction well with the molecular recognition element existing in the amount in the range of about 1nM-about 1mM and / or the nucleic acid sequence existing in the amount in the range of about 300pM or 1 or 3nM-about 300μM or 3mM. In some embodiments, solid support can provide to measure and / or reaction well with the nucleic acid sequence (such as primer) existing in the amount in the range of about 1μM-about 100nM. The concentration of molecular recognition element and / or nucleic acid sequence can be the amount existing on solid support and / or released from solid support. In some embodiments, the concentration of molecular recognition element and / or nucleic acid sequence provided by solid support in measure and / or reaction well can be enough to perform or carry out amplification process (such as PCR) and / or detect target.

[0092] In some embodiments, the nucleic acid sequence can be optionally linked and / or bound to a solid support via a linker and can 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, 6, 7, 8, 9, 10, 12, 15, 18, 20, 21, 22, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 nucleotides). In some embodiments, the nucleic acid sequence is an oligonucleotide. In some embodiments, the nucleic acid sequence is a primer (such as a forward primer or a reverse primer), which can be used, for example, as a primer in a PCR assay, as a probe in a hybrid assay and / or a microarray. Primers that can be used for the present invention include, but are not limited to, those primers for amplification and / or detection. In some embodiments, the nucleic acid sequence comprises about 15 nucleotides-about 50 nucleotides or about 20 nucleotides-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 present invention comprises a forward primer and does not comprise a reverse primer. In some embodiments, the solid support of the present invention comprises a forward primer and a reverse primer.

[0093] In some embodiments, the primer can be absorbed into a solid support (e.g., a hydrogel bead) such that the primer is primarily within the solid support and is not exposed to the exterior of the solid support (where a molecular recognition element (e.g., a capture antibody) can be bound). Exemplary hydrogel beads include, but are not limited to, agarose beads (e.g., Sepharose beads).

[0094] Primers present on a solid support may be associated with chemical species that prevent hybridization, such as certain nucleic acid sequences (optionally with modified bases) whose melting temperatures are above the temperature at which the detection reagents are incubated but below the annealing temperature at which PCR is performed. Alternatively or additionally, peptides, proteins, RNA sequences, polymers and / or other agents may be used to reduce nonspecific binding and / or unwanted hybridization to primers on a solid support.

[0095] In some embodiments, the solid support comprises self-avoiding primers derived from one or more modified bases such as those available from Firebird and other sources. In some embodiments, the self-avoiding molecular recognition system (SAMRS) is the nucleic acid tag for the forward primer and the detection reagent, while the reverse primer is normal DNA.

[0096] 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 combine certain components at the appropriate time. For example, in some embodiments, since the NA tag is single-stranded and the primer binding site does not exist until after the first PCR cycle, the primer (such as forward primer) is prevented from combining with the nucleic acid tag of the detection reagent until after the first PCR cycle. In some embodiments, the forward primer and reverse primer can be designed so that it is not available for base pairs at a lower temperature (such as during incubation with the detection reagent), but becomes available at a higher temperature (such as the time after the excessive detection reagent is washed away). In some embodiments, the forward primer and / or reverse primer on the solid support of the present invention can be double-stranded (such as through a duplex or hairpin structure), and the primer becomes single-stranded after the denaturation step of PCR. Primers can be designed so that double-stranded structures (such as duplexes or hairpins) will not be significantly formed at the PCR annealing temperature because they can inhibit PCR. In some embodiments, the forward primer and / or reverse primer on the solid support of the present invention can become available after enzymatic cleavage. For example, the primer can be part of a duplex, and the competing strand (the reverse complement of the primer) contains a cleavable bond. Degradation of the competing strand into smaller parts can significantly reduce the melting temperature so that it will not compete during PCR. For cleavable bonds, the following non-exclusive examples can 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 RNA enzymes. In some embodiments, cleavage provides a primer with an extendable 3' end.

[0097] In some embodiments, detection reagents of the present invention can be protected and / or designed to minimize hybridization with primers on a solid support. For example, techniques similar to those discussed above (e.g., dsDNA duplexes, hairpin structures) can be used so that the reverse primer binding site has base pairing.

[0098] According to some embodiments, a variety of solid supports are provided, which include a first plurality of solid supports and a second plurality of solid supports. Each solid support in the first plurality of solid supports includes: a first coding agent (e.g., a dye) providing a first coding signal; a first nucleic acid sequence; and a first molecular recognition element (e.g., an antibody), and each solid support in the second plurality of solid supports includes: a second coding agent (e.g., a dye) providing a second coding signal; a second nucleic acid sequence; and a second molecular recognition element (e.g., an antibody). Since 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, the solid support in the first plurality of solid supports is distinguished and / or distinguished from the solid support in the second plurality of solid supports.

[0099] According to some embodiments, a detection reagent is provided, which comprises 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 as a "nucleic acid tag" or "tag" in this article. The detection reagent can be bound to the first part of the target, and the solid support of the present invention can be bound to the second part of the target. In this way, the detection reagent has a corresponding solid support because it is paired with the solid support through the target. The nucleic acid tag used for the detection reagent of the present invention has a portion configured to participate in the nucleic acid amplification process, and a portion of the nucleic acid sequence is connected (e.g., bound) to the corresponding solid support for the detection reagent. In some embodiments, the detection reagent of the present invention is a detection antibody comprising a nucleic acid tag.

[0100] The nucleic acid tag of the detection reagent can comprise 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.

[0101] 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 for 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 for 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 for the detection reagent and / or the sequence in the 3' region of the nucleic acid tag is complementary to the reverse primer optionally present in the PCR master mix. In some embodiments, two or more detection reagents corresponding to two or more different solid support groups include substantially identical sequences in the 3' region of the nucleic acid tag, and the sequence can be complementary to the reverse primer.

[0102] Reference Figure 6 , shows an exemplary complex of an embodiment of the present invention, the complex comprising an encoded solid support, a target and a detection reagent. The encoded solid support is an encoded bead functionalized with a forward primer and an antibody (eg, a capture antibody) that binds to the target. Figure 6The detection reagent shown is an antibody containing a nucleic acid sequence, and the antibody binds to the target to form a complex of the present invention. Thus, the captured target is labeled with a detection antibody containing a unique nucleic acid sequence, and at least a portion of the nucleic acid sequence (i.e., the nucleic acid tag) is amplified by a forward primer of the correspondingly encoded bead (optionally when the forward primer is released from the bead) and a reverse primer provided in the PCR master mix. The PCR product can then be detected by a fluorescent signal (e.g., from a dsDNA intercalating dye).

[0103] The detection reagents corresponding to the solid supports in one or more solid support groups each contain the same nucleic acid tag. For detection reagents corresponding to different solid supports, the nucleic acid tags are different. Figure 6 , the nucleic acid tag of the detection reagent comprises in the 5' region a sequence configured to participate in the nucleic acid amplification process performed with at least a portion of the forward primer. In addition, the nucleic acid tag comprises in the 3' region a sequence complementary to the reverse primer, and the nucleic acid sequence is also present in (and therefore shared by) different detection reagents corresponding to different solid supports, such as Figure 7 As shown. Thus, the nucleic acid tag can be amplified by two primers: a unique forward primer and a shared 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 nucleic acid tags and can be added to the PCR master mix.

[0104] 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 usually found in nature. One or more chemically modified bases can improve the specificity of the PCR reaction and / or reduce the non-specific binding between nucleic acid sequences (e.g., oligonucleotides).

[0105] The nucleic acid tag can be connected or bound to the molecular recognition element and / or connected or bound to any part of the molecular recognition element in any way. In some embodiments, the nucleic acid tag is covalently or non-covalently connected to the molecular recognition element. In some embodiments, a joint is used to connect the nucleic acid tag to the molecular recognition element. The nucleic acid tag can be connected to one or more amino acids of a 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 connected (e.g., covalently connected) to the crystallizable fragment (Fc) region of the antibody or its fragment.

[0106] In some embodiments, the nucleic acid tag may be a chemically synthesized single-stranded DNA (ssDNA). In some embodiments, a double-stranded DNA (dsDNA) tag connected to a molecular recognition element provides a detection reagent that can provide reduced non-specific binding. In some embodiments, by designing (e.g., with a primer or with a nucleic acid tag) complementary chain to ensure that it remains annealed during the assay, optionally until its desired release, to mitigate the hybridization of dsDNA with a primer bound to a solid support. As will be appreciated by those skilled in the art, the DNA chain can undergo melting and annealing (fraying) at a rate determined by sequence, temperature, and chemical environment. This fraying of the end can increase non-specific binding or undesirable hybridization with other nucleic acid molecules (such as primers or probes bound to a solid support). In order to prevent the complementary chain of the dsDNA tag from hybridizing with the forward primer bound to a solid support, the complementary chain can be shorter, i.e., lacking a sequence complementary to the forward primer. In this case, the tag may be part of the dsDNA and part of the ssDNA.

[0107] In some embodiments, the nucleic acid tag may be a hairpin tag sequence and / or a sequence with a GC clip. The hairpin sequence and / or the sequence with a GC clip can reduce the flaring and / or undesirable hybridization of the end during incubation with the detection reagent. In some embodiments, the nucleic acid tag may be an RNA sequence, and a reverse transcriptase may be used. The RNA sequence may include an RNA hairpin with an internal structure that prevents hybridization during sample capture.

[0108] In some embodiments, the nucleic acid tag may be encapsulated by a protein, membrane, micelle, peptide, phage, etc., so that it cannot hybridize to a nucleic acid sequence (e.g., a primer) bound to a solid support until desired (e.g., after sealing in a reaction well).

[0109] In some embodiments, the nucleic acid tag comprises a restriction enzyme site. Nucleic acid tags comprising restriction enzyme sites can be designed so that the enzyme cleaves the hairpin or GC clamp, and this 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 part of the nucleic acid tag from the detection reagent.

[0110] The nucleic acid tags of the present invention do not need to contain unique regions and common regions, such as Figure 7 In some embodiments, the nucleic acid tag of the detection reagent comprises a completely or partially unique sequence, and a mixture of forward or reverse primers can be added to a master mix for such a sequence.

[0111] In some embodiments, the nucleic acid tag comprises at least one common region, which is a conserved nucleic acid sequence in the nucleic acid tags of different detection reagents, and it is large enough to detect some or all sequences by amplification with one or more primers. The conserved nucleic acid sequence may be present in the 5' region and / or in the 3' region outside the unique region. For all two or more different detection reagents, the conserved region may be present in all nucleic acid tags. Therefore, nucleic acid tags for different detection reagents can be designed so that they can be amplified by unique primer pairs and universal primer pairs shared by the nucleic acid tags of all detection reagents. This embodiment can be used for troubleshooting during assay development so that non-specific binding and / or cross-reactivity can be easily visualized. In some embodiments, the nucleic acid tag of the present invention comprises at least two nucleic acid sequences, one amplified by a unique reverse primer and the other amplified by a universal reverse primer.

[0112] Any method known to those skilled in the art can be used to connect nucleic acid tags to molecular recognition elements to provide detection reagents. In some embodiments, nucleic acid tags are connected to molecular recognition elements using traditional chemical methods, click chemistry, genetic engineering and / or molecular engineering methods.

[0113] In some embodiments, the molecular recognition element of the detection reagent of the present invention can be functionalized as a nanoparticle functionalized with a nucleic acid tag and optionally amplified primers and / or probes for the nucleic acid sequence bound to a solid support. However, in some embodiments, multiple primer sets with variable concentrations are avoided to reduce assay variations.

[0114] In some embodiments, when the detection reagent comprises a molecular recognition element comprising a nucleic acid, the nucleic acid tag may be provided by all or part of the molecular recognition element. That is, the molecular recognition element itself may serve as a nucleic acid tag.

[0115] According to some embodiments, a plurality of detection reagents are provided, which include a first plurality of detection reagents, wherein each detection reagent in the first plurality of detection reagents comprises: a first molecular recognition element (e.g., an antibody) and a first nucleic acid tag; and a second plurality of detection reagents, wherein each detection reagent in the second plurality of detection reagents comprises: a second molecular recognition element (e.g., an antibody) and a second nucleic acid tag; wherein the first and second nucleic acid tags each comprise a first portion and a second portion; and wherein the nucleic acid sequence of the first portion of the first nucleic acid tag is different from the nucleic acid sequence of the first portion of the second nucleic acid tag. In some embodiments, the nucleic acid sequences of the second portions for the first and second nucleic acid tags are the same or substantially the same. In some embodiments, the first portion for the first and second nucleic acid tags is in the 5' region of the corresponding tag and the second portion is in the 3' region of the corresponding tag. The first nucleic acid tag may be connected to (e.g., joined to, bound to, and / or fused to) the first molecular recognition element, and the second nucleic acid tag may be connected to (e.g., joined to, bound to, and / or fused to) the second molecular recognition element.

[0116] According to some embodiments, compositions, kits, devices and / or systems comprising solid supports as described herein and / or detection reagents as described herein are provided. When there is a target corresponding to the solid support and the detection reagent, the solid support and the detection reagent can form a complex. Compositions, kits, devices and / or systems can include multiple solid supports optionally including two or more differently coded solid supports and multiple detection reagents optionally including two or more different detection reagents.

[0117] In some embodiments, bead sets can be prepared by encoding each set with a unique dye or unique combination of fluorescent dyes, such as Figure 7, "A", "B" and "C" in the beads. For example, bead group "A" can be stained with a red fluorescent dye, while bead group "B" is stained with a blue fluorescent dye. In some embodiments, all groups are functionalized with streptavidin before each group is incubated with a different biotinylated capture antibody for a specific target molecule. Finally, each group is incubated with a unique biotinylated primer (e.g., a forward primer). In some embodiments, the detection reagent is prepared by covalently linking a unique nucleic acid tag to a detection antibody for each target. In some embodiments, the tag is a 60 nucleotide (nt) single-stranded DNA (ssDNA) having a unique sequence at the 5' end or region, and a common sequence conserved among all tags at the 3' end or region. 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 a sequence in the 5' region or 5' end of the tag attached to the detection antibody. Alternatively, the 3' end of the tag may be connected to the detection antibody. In some embodiments, a reverse primer complementary to the conserved 3' end or region of all tags may be added to the PCR master mix. In this way, bead group A will carry both capture antibody A and forward primer A that can only amplify nucleic acid tag A connected to detection antibody A. Bead group B will carry both capture antibody B and forward primer B that can only amplify nucleic acid tag B connected to detection antibody B. Since the 3' end or region of the tag contains a conserved sequence, the primer added to the master mix is ​​used as a reverse primer for both nucleic acid tag A and nucleic acid tag B.

[0118] In some embodiments, the solid support of the present invention does not include a complementary sequence that can hybridize with a nucleic acid tag. In the absence of a target, this hybridization can cause a detection reagent to be significantly pulled down from the incubation mixture, thereby producing a very high background signal. For this reason, in some embodiments, since the forward primer is identical or substantially identical (not complementary) to the 5' end or region of the tag in sequence, only the forward primer is connected to the solid support. A reverse primer complementary to the tag can be added to the PCR master mix so that PCR can be performed.

[0119] In some embodiments, the assay is performed in the presence of a mixture of solid supports for different targets, and the solid support is incubated with the sample. The solid support can capture the target, and the sample matrix can be washed away. The solid support can then be incubated with a mixture of a plurality of detection antibody reagents corresponding to the solid support to mark the captured target to form a complex. The complex can be washed and / or loaded into an array (e.g., sized so that most reaction wells contain only a single bead microwell array). Optionally, before each reaction is separated by fluid, for example, by sealing with immiscible oil, a PCR master mixture containing a common reverse primer complementary to the 3' end or region of all tags is added. During the denaturation step of PCR, the forward primer connected to the solid support can be released into the separated reaction wells so that each well now contains a complete set of PCR primers. For example, if there is a detection antibody conjugate A, the well containing the A-type beads will be PCR positive; and if there is a detection antibody conjugate B, the well containing the B-type beads will be PCR positive. However, reactions containing Type A beads and non-specifically bound Detection Antibody Conjugate B will not be PCR positive because the primers carried by Type A beads cannot amplify the nucleic acid tag on Detection Antibody Conjugate B ( Figure 8 ). In this way, an iPCR assay optimized as a singleplex reaction may require less re-optimization when multiplexed, since the total detection antibody concentration that can be amplified and / or detected by each bead set does not change as additional targets are added to the assay.

[0120] In some embodiments, the methods of the invention capture and / or cause about 0 to about 10,000,000 detection agents to bind as complexes to their respective solid supports. For example, the methods of the invention can capture and / or cause 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, 1600, 2000, 2200 or about 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 1,000,000, 5,000,000, or 10,000,000 detection agents as complexes bound to their corresponding solid supports.

[0121] In some embodiments, the compositions, kits, devices and / or systems of the present invention further comprise an amplifier. Exemplary amplifiers include, but are not limited to, reverse primers, ssDNA splints, and ligases, etc. In some embodiments, the amplifier has a nucleic acid sequence complementary to at least a portion of the nucleic acid sequence of the detection reagent and the solid support, optionally wherein the portion of the nucleic acid sequence is in the 3' region of the nucleic acid tag. The amplifier can be separated from the solid support and / or the detection reagent. In some embodiments, the amplifier is connected to the solid support, optionally wherein the amplifier is biotinylated.

[0122] The solid supports of the present invention and their corresponding detection reagents can be stored separately in the compositions, kits, devices and / or systems of the present invention. In some embodiments, the detection reagents of the present invention and the amplification agents are stored separately in the compositions, kits, devices and / or systems of the present invention. In some embodiments, the solid supports of the present invention and the amplification agents are stored separately when the amplification agents are present and not connected to the solid supports.

[0123] According to some embodiments, a method of detecting a target in a sample is provided. The method may include combining a solid support of the present invention and a sample comprising the target to form a first composition comprising a target-bound solid support.

[0124] "Combining" and "combine" as used herein refer to bringing two or more components close enough and / or in contact to optionally bind and / or react when 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 comprising a solid support, a target and a detection reagent, wherein the detection reagent comprises a molecular recognition element bound 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, such as (but not limited to) PCR and / or rolling circle amplification (RCA).

[0125] 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. One or more washing steps may remove unreacted and / or non-bound components and / or those that are non-specifically bound to the complex.

[0126] In some embodiments, an amplifier is used in the method of the present invention. The method may include merging and / or reacting an amplifier and a complex comprising a solid support, a target, and a detection agent. Before, during, and / or after the step of merging the amplifier and the complex, when the amplifier is connected to the solid support, the amplifier can be released from the solid support. In some embodiments, the amplifier is maintained on the solid support. In some embodiments, the amplifier is not connected to the solid support, but is added to the composition comprising the complex.

[0127] In some embodiments, the amplification agent is a nucleic acid sequence (e.g., a primer) that is optionally released from a 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 a solid support is amplified. In some embodiments, the amplification agent comprises a ssDNA splint and a ligase that reacts under suitable conditions to connect two oligonucleotides together. In some embodiments, the solid support of the present invention comprises an oligonucleotide (e.g., an ssDNA oligonucleotide), and the detection reagent of the present invention comprises an oligonucleotide (e.g., an ssDNA oligonucleotide), and when in the presence of a ssDNA splint and a ligase and suitable conditions (e.g., when the oligonucleotides are adjacent to each other, such as when each oligonucleotide is bound to the same target), the two oligonucleotides are connected and / or extended, and then at least a portion of the oligonucleotide can be amplified. In some embodiments, a solid support of the invention comprises an oligonucleotide (e.g., a ssDNA oligonucleotide), and a detection reagent of the invention comprises an oligonucleotide (e.g., an oligonucleotide having a 3' overhang that can hybridize to the ssDNA oligonucleotide of the solid support), and when in close proximity to each other, such as when each oligonucleotide binds to the same target, the two oligonucleotides hybridize, extend, and then at least a portion of the oligonucleotides can be amplified.

[0128] In some embodiments, when the target and the solid support are combined, the detection reagent of the invention is present in a composition comprising the corresponding solid support of the invention. In some embodiments, the detection reagent of the invention, its corresponding solid support of the invention and the target are combined simultaneously.

[0129] The method of the present invention may include amplifying one or more nucleic acid sequences that are identical and / or different. In some embodiments, the method of the present invention includes polymerase chain reaction (PCR). In some embodiments, the method of the present invention includes amplifying at least a portion of the nucleic acid tag of the detection reagent. In some embodiments, at least a portion of the nucleic acid tag of the amplification detection reagent is performed using PCR. In some embodiments, at least a portion of the nucleic acid tag of the amplification detection reagent does not involve PCR, and 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 loops, wherein the enzymatically degraded tag can initiate a chain reaction driven by hybridization.

[0130] In some embodiments, the method of the present invention is carried out using proximity ligation assay (PLA). In some embodiments, the method of the present invention is carried out using binding induced DNA amplification (BINDA), proximity extension assay (PEA) and / or triple PLA. In some embodiments, the method of the present invention is a multiple determination (e.g., immunopolymerase chain reaction (iPCR) assay) carried out in a microfluidic device comprising a plurality of holes, and the method further includes separating a solid support from one or more other solid supports and at least one hole of the plurality of holes.

[0131] Microfluidic device may or may not include rigidity or solid microwell array.In some embodiments, solid support of the present invention can be separated in the droplet of aqueous PCR master mixture in immiscible oil (such as those used in emulsion PCR, BioRad Digital Droplet PCR system, RainDance PCR system or the technology called BEaMing).In some embodiments, forward primer is combined (such as tethered) to solid support of the present invention (such as bead) through covalent bond or multiple biotin groups, makes it possible to read signal by flow cytometer after demulsification.Some isothermal amplification techniques can be used in emulsion / droplet form, or if amplicon remains connected to solid support, then the form of bulk solution can be used. In some embodiments, the substrates, devices and / or methods of the present invention may include, but are not limited to, those described in U.S. Application Publication No. 2015 / 0211048, International Application No. PCT / US2016 / 042913, International Application No. PCT / US2016 / 043463, and International Application No. PCT / US2016 / 055407 (e.g., substrates, devices, and / or methods), the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the methods of the present invention include methods of delivering reagents and / or targets to reaction wells using beads (e.g., superparamagnetic beads), such as, for example, those described in U.S. Application Publication No. 2015 / 0211048 and International Application No. PCT / US2016 / 042913, the contents of each of which are incorporated herein by reference in their entirety.

[0132] In some embodiments, the compounds, compositions, kits, systems, devices and / or methods of the invention can be used to perform any suitable reaction, such as (but not limited to) assays involving biomolecules, such as PCR reactions; assays based on spatial arrays, such as reconfigurable multi-element diagnostic (ReMeDx) assays, singleplex reactions in a compact array (SiRCA); assays comprising aqueous reaction compositions; assays performed on plastic substrates and / or assays described in International Publication No. WO 2013 / 176767, the contents of which are incorporated herein by reference.

[0133] In some embodiments, the method of the present invention includes preparing the solid support of the present invention. The solid support of the present invention can be formed by contacting the solid support (optionally comprising one or more joints) with the molecular recognition element and the nucleic acid sequence simultaneously and / or at different times. In some embodiments, the molecular recognition element and the nucleic acid sequence can be added at separate times to help provide a plurality of beads with substantially the same number (i.e., within ± 20%) of molecular recognition elements and nucleic acid sequences on each respective bead in the plurality of beads. In some embodiments, the method for preparing the solid support of the present 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. 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.

[0134] In some embodiments, undesired hybridization of detection reagents with nucleic acid sequences (e.g., primers) attached to a solid support can be reduced by using toe-hold displacement primers, hairpin primers, partially digestible hairpin primers, and / or blocking NA sequences. Primers can be tethered to a solid support at the 5' and / or 3' ends or at both 5' and 3' ends. In some embodiments, double-stranded primers can be used if appropriate conditions are provided.

[0135] In some embodiments, the incubation conditions and / or amplification conditions can be controlled in the methods of the present invention so as to be unfavorable for hybridization of the detection reagent with the nucleic acid sequence attached to the solid support, but PCR amplification can still occur subsequently during the detection phase of the method. For example, low melting temperature primers can be used under incubation conditions that are favorable for the melt state (e.g., low salt or addition of betaine) so that the primers do not hybridize with the detection reagent. However, conditions that are favorable for higher melting temperatures (e.g., higher salt or removal of betaine) can be used during PCR so that the primers can effectively amplify the nucleic acid sequence of the detection reagent.

[0136] Any method known to those skilled in the art can be used to detect the target in the methods of the present invention, such as by visual observation and / or chemical or biochemical assays. In some embodiments, the methods of the present invention include detecting the target by detecting an assay signal. The assay signal can be, for example, a fluorescent signal, such as a fluorescent signal from a dsDNA intercalating dye and / or from the hydrolysis of a probe containing a quencher and a dye.

[0137] In some embodiments, fluorescence is used for detection. In some embodiments, the methods of the present invention include and / or provide for fluorescence readout. In some embodiments, the methods of the present invention include and / or provide for electronic readout by hybridizing the amplicon to a solid-state sensor, sequencing the amplicon by methods such as pyrophosphate sequencing and / or forming a precipitate (such as that found during LAMP amplification).

[0138] The methods of the present invention can improve the specificity of the assay and / or can reduce the background signal of the assay. In some embodiments, the methods of the present invention reduce the cross-reactivity observed between the solid support and the non-target molecule (e.g., false antigen). As used herein, "observed cross-reactivity" refers to the detected signal associated with the interaction between the solid support and the non-target molecule (e.g., false antigen). In some embodiments, the detected signal can be observed visually. In some embodiments, the methods of the present invention reduce the observed non-specific binding of the solid support, such as Figure 5 As shown, the fluorescent signal observed with a false positive. In some embodiments, the methods of the present invention improve the signal-to-noise ratio of the assay, improve the sensitivity of the assay and / or reduce the cross-reactivity observed in the assay. In some embodiments, the compounds, compositions, devices, kits, systems and / or methods of the present invention improve multiplex immuno-PCR (iPCR) assays.

[0139] 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 reduced cross-reactivity and / or background signal observed for multiplexed probe-based qPCR, where each probe is labeled with a spectrally distinct fluorescent dye. In this case, the increase in fluorescence observed from probe hydrolysis can be referenced to the bead's code to determine whether the correct complex has been formed.

[0140] In some embodiments, the sequence of the NA tag used for the detection reagent is carefully selected so that the amplicons produced by the correct PCR amplification will have discrete melting temperatures, optionally with different melting temperatures, so that they can be distinguished from non-specific amplification products and optionally from each other. The use of different melting temperature amplicons can be used as a means of encoding to distinguish one bead population from another and / or as a means of confirming the encoded signal. Compared with control methods (e.g., iPCR using known solid supports and detection reagents), the methods of the present invention can reduce the background signal of the assay by about 0.5, 1, 1.5, 2, 2.5 or 3 orders of magnitude.

[0141] The compounds, compositions, kits, devices, systems and / or methods of the present invention can be used for multiple immunoassay panels. Some embodiments of the present invention can be used for qualitative and / or quantitative medical and / or veterinary diagnostic tests, forensic testing, identification of biohazard materials, environmental monitoring, research and development and / or similar uses. In some embodiments, the ability to multiplex molecular recognition elements (e.g., antibodies) that would otherwise exhibit high levels of cross-reactivity can enable new diagnostic tests that are not currently available.

[0142] The invention is explained in more detail in the following non-limiting examples. Example

[0143] Example 1 Single-molecule bead-based immunoassays (referred to as "Protein SiRCA technology") were developed using the solid supports and detection reagents described herein. These assays were compared to similar iPCR assays to show how Protein SiRCA technology can reduce background from nonspecific binding and antibody cross-reactivity.

[0144] First, iPCR assay reagents were prepared. Bead sets for cytokines IL-6, IL-7, IL-8, IL-10, and TNF-α were prepared by functionalizing 3 μm ProMag COOH-modified superparamagnetic beads with encoding dyes and capture antibodies. Detection antibody conjugates were prepared by attaching the same 60nt ssDNA tag to the relevant detection antibody using the TriLink Biotechnologies Protein-Oligo Conjugation Kit ( Figure 3 ). iPCR assay as Figure 4 The single-plex assay showed excellent sensitivity ( Fig.9A In the 5-plex assay, measurements close to the limit of detection (LOD) (generally at or below about 1 pg / mL) showed excellent sensitivity for most bead sets ( Fig. 9B However, upon multiplexing, background signal increased significantly for all bead groups, especially for IL-8 ( Fig. 9B and Fig. 9C ).

[0145] Protein SiRCA beads were prepared by incubating streptavidin-coated Dynabeads with biotinylated capture antibodies ( Fig.10The antibodies were biotinylated in-house using the TriLink Biotechnologies Chromalink BiotinAntibody Labeling Kit. The antibody-labeled beads were then incubated with a biotinylated forward primer covalently labeled with a fluorescent dye ( Fig.10 ) to encode beads. Three sets of beads were prepared for the cytokines IL-7, IL-8 and IL-10. Fig.11 Fluorescence intensity plots at two encoding wavelengths are shown for representative beads from each group. Fig.11 Also shown are coded clusters of the three iPCR beads with covalently attached dyes. The tight clustering of each bead set enables accurate identification of each bead type in multiplex experiments.

[0146] In the monoplex assay, the positive percentage of Protein SiRCA beads was compared with that of conventional iPCR beads. The difference between iPCR beads and Protein SiRCA beads is that iPCR beads are not functionalized with streptavidin or coated with primers; they are covalently linked to coding dyes and capture antibodies. iPCR and Protein SiRCA beads of each target were incubated with serum (blank) or 90fM target cytokines in serum (positive control). Since the purpose of this initial test is to determine the sensitivity or activity of the capture antibody to different assay beads, each bead group was incubated with the appropriate antibody conjugate developed for iPCR (i.e., all detection conjugates have the same NA tag). Both primers were added to the PCR master mix. Fig.12 The resulting signals are shown for serum blank and 90 fM of each cytokine. In general, the Protein SiRCA beads performed very similarly to the iPCR beads in terms of signal to background ratio, with the Protein SiRCA beads showing slightly higher signal and slightly higher background for these assays.

[0147] Next, prepare the detection antibody conjugates for Protein SiRCA. In iPCR, use the same NA tag for each antibody conjugate. Figure 7 As shown, in Protein SiRCA, different NA tags were used for each conjugate. iPCR beads lacking a forward primer were used in the single-plex assay to compare the activity of the iPCR and Protein SiRCA conjugates. Therefore, primers for each tag were added to the master mix of each single-plex assay. In otherwise identical experiments, comparison of the activity of the detection antibody conjugates showed that all Protein SiRCA conjugates were less sensitive than the iPCR conjugates ( Fig.13). Characterization of the conjugates showed that the Protein SiRCA antibodies were all under-labeled, such that a large fraction of the antibodies lacked the NA tag. These tag-free antibodies can bind to the captured target molecules, but will not produce a detectable (convertible) PCR signal. The difference in labeling efficiency between the iPCR and Protein SiRCA conjugates is likely due to batch variability of the purchased antibody stock solution or aging reagents in the conjugation kit (TriLink Biotechnologies Protein-Oligo Conjugation Kit), as the iPCR and Protein SiRCA conjugates were prepared at different times. The differences observed between the conjugates are likely due to variability in the preparation of the antibody conjugates rather than inherent characteristics of the Protein SiRCA conjugates.

[0148] To account for differences in bead type, encoding scheme, antibody-to-bead attachment, 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 ( Fig.14 ). In the singleplex assay, the background signal of IL-7 and IL-8 was slightly higher in Protein SiRCA, but the background signal of IL-10 Protein SiRCA was lower.

[0149] Next, IL-7, IL-8 and IL-10 were measured with triple iPCR containing iPCR beads using 3 kinds of iPCR antibody conjugates. The beads for all 3 kinds of cytokines were mixed together and then incubated with buffer (blank) or 90fM of each cytokine. A single set of forward and reverse primers for common iPCR NA tags was provided in the PCR master mix. As expected from early work, IL-8 showed a very high background signal; This high percentage of positive wells showed that there were probably several non-specifically bound detection antibody complexes per bead. When 90fM IL-7 or IL-10 were incorporated into sample buffer, a significant increase in assay signal was observed in the appropriate bead group. However, adding 90fM IL-8 to the sample buffer did not result in a significant increase in the signal in the IL-8 beads exceeding the background, indicating that the concentration was below the detection limit ( Fig.15 ). Comparison of 4 different assay conditions (blank and 90 fM of each cytokine) for each bead type is shown in Figure 2. Fig.15 The high signal from the IL-8 beads is most likely due to nonspecific binding of other detection antibody conjugates.

[0150] The same triplicate assay was repeated using Protein SiRCA beads and detection antibody conjugates for IL-7, IL-8, and IL-10. Beads for all 3 cytokines were mixed together and then incubated with either buffer (blank) or 90 fM of each cytokine ( Fig.16 ). Only a common reverse primer was provided in the PCR master mix, as the unique forward primer for each conjugate tag was delivered to the wells by the beads themselves. A comparison of the signals from each bead set for 4 experiments (blank and 90 fM of each cytokine) is shown. Fig.16 A discernible increase in signal over background was seen for all beads when their corresponding analyte was present.

[0151] Direct comparison of iPCR and Protein SiRCA assays Fig.17 As shown. For the single-plex assay data, IL-7 showed a lower signal-to-background ratio in Protein SiRCA compared to iPCR, most likely due to issues with the Protein SiRCA antibody conjugate. IL-10 showed a significant improvement in signal-to-background ratio in Protein SiRCA compared to iPCR. However, the most significant improvement was seen in the IL-8 beads. However, the most significant improvement was seen in the IL-8 beads. The background signal for the Protein SiRCA IL-8 beads was more than an order of magnitude lower than that seen in iPCR, which allowed for sufficient signal-to-background ratio to easily discern the difference between 90fM IL-8 and the blank.

[0152] The foregoing is illustrative of the present invention and should not be construed as limiting the present invention. The present invention is defined by the following claims, including equivalents of the claims. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entirety for teachings relevant to the sentence and / or paragraph in which the reference is presented.

Claims

1. A kit comprising: (i) a solid support (e.g., a bead), wherein the solid support comprises: Coding agents (e.g. dyes); a first nucleic acid sequence (eg, an oligonucleotide and / or a primer); and a first molecular recognition element (eg, 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 are configured to participate in a nucleic acid amplification process.

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

3. The kit of any preceding claim, wherein the first molecular recognition element and / or the first nucleic acid sequence is linked (eg, covalently and / or non-covalently) to the linker (eg, one member of the specific binding pair).

4. The kit of any preceding claim, 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 the binding of streptavidin and biotin.

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

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

7. The kit of any preceding claim, 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. The kit of any preceding claim, wherein the second nucleic acid sequence comprises a sequence (e.g., about 5 or more consecutive nucleotides) substantially identical to a sequence in the first nucleic acid sequence, optionally wherein the sequence 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 preceding claim, wherein the first nucleic acid sequence and / or the second nucleic acid sequence comprises single-stranded DNA.

10. The kit of any preceding claim, wherein the first nucleic acid sequence and / or the second nucleic acid sequence comprises double-stranded DNA.

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