Kits for detecting one or more target nucleic acid analytes in a sample and methods for making and using same - Patents.com

Abstract

Oligonucleotides, methods, and kits for detecting, identifying, or quantifying one or more target analytes in a sample, as well as methods for immobilizing oligonucleotides on a support surface, are provided.

Description

[Technical Field]

[0001] SUMMARY The present disclosure relates to kits for detecting one or more target analytes in a sample, and methods for making and using the same.

[0002] Sequence Listing This specification contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy, created on April 17, 2020, is named 0076-0006WO1_SL.txt and is 431,364 bytes in size. [Background technology]

[0003] A single nucleotide polymorphism (SNP) refers to a single-base variation in an organism's genome, resulting in two or more distinct nucleotide residues (alleles), each occurring in a significant portion (>1%) of the population. SNPs are the most frequent form of sequence variation between individuals and are involved in the etiology of many genetic disorders. Wang et al. (1998), Large-Scale Identification, Mapping, and Genotyping of Single-Nucleotide Polymorphisms in the Human Genome, Science, 280:1077-1082. There are an estimated 10 million SNPs in the human genome, which can occur in coding and noncoding regions. Kruglyak et al. (2001), Variation is the Spice of Life, Nat. Genet., 27:234-236. While many SNPs do not affect cellular function, others are associated with inherited traits, genetic disorders, age-related diseases, and responses to drug and environmental factors.

[0004] Genotyping assays are genetic tests used to detect the presence of nucleotide sequences in a sample, and can be used to detect the presence of SNPs or other sequence variations, including, but not limited to, deletions, insertions, duplications, and translocations. High-density oligonucleotide arrays allow for the simultaneous interrogation of many nucleotide sequences using hundreds of thousands of probes arrayed on a chip.

[0005] Because large-scale analysis of nucleotide sequences in samples is necessary to correlate sequences with disease or susceptibility to disease, to link sequences to individual variability in drug response, or to conduct population studies, there remains a need for kits for identifying nucleotide sequences in samples. Summary of the Invention

[0006] The present invention relates to a kit for identifying, detecting, or quantifying one or more target analytes in a sample, as well as methods for making and using the same. In one embodiment, the target analyte comprises a nucleic acid sequence. In one embodiment, the target analyte comprises a polypeptide sequence. In one embodiment, the method or kit includes one or more capture molecules that are immobilized or can be immobilized to individual binding domains on a support surface. In one embodiment, a set of two or more non-cross-reactive capture oligonucleotides is provided.

[0007] In one embodiment, the set of capture oligonucleotides is a subset of the set of parent capture oligonucleotides, wherein one or more capture oligonucleotides in the set comprise a nucleotide sequence having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of one or more nucleotide sequences from the parent set, and the set of parent capture oligonucleotides is (a) a capture set 1 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 1-64; (b) a capture set 2 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 65-122; (c) a capture set 3 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 123-186; (d) a capture set 4 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 187-250; (e) capture set 5, comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 251-308; (f) capture set 6, comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 309-372; (g) capture set 7 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 373 to 436; (h) capture set 8 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 437 to 494; (i) a capture set 9 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 495 to 558; (j) a capture set 10 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 559 to 662; (k) a capture set 11 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 623 to 680; (l) Capture set 12, which includes a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 681 to 744.

[0008] In another aspect, a set of two or more non-cross-reactive capture oligonucleotides is provided, wherein the set of capture oligonucleotides is a subset of a set of parent capture oligonucleotides, wherein one or more capture oligonucleotides in the set comprise a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to one or more nucleotide sequences from the parent set, and wherein the set of parent capture oligonucleotides is (a) a capture set 1 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 1-64; (b) a capture set 2 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 65-122; (c) a capture set 3 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 123-186; (d) a capture set 4 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 187-250; (e) capture set 5, comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 251-308; (f) capture set 6, comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 309-372; (g) capture set 7 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 373 to 436; (h) capture set 8 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 437 to 494; (i) a capture set 9 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 495 to 558; (j) a capture set 10 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 559 to 662; (k) a capture set 11 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 623 to 680; (l) Capture set 12, which includes a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 681 to 744.

[0009] In another embodiment, a set of two or more non-cross-reactive capture oligonucleotides is provided, wherein the set of capture oligonucleotides is a subset of a set of parent capture oligonucleotides, the set of parent capture oligonucleotides comprising: (a) a capture set 1 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 1-64; (b) a capture set 2 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 65-122; (c) a capture set 3 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 123-186; (d) a capture set 4 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 187-250; (e) capture set 5, comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 251-308; (f) capture set 6, comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 309-372; (g) capture set 7 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 373 to 436; (h) capture set 8 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 437 to 494; (i) a capture set 9 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 495 to 558; (j) a capture set 10 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 559 to 662; (k) a capture set 11 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 623 to 680; (l) Capture set 12, which includes a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 681 to 744.

[0010] In one embodiment, the set of two or more non-cross-reactive capture oligonucleotides comprises: (a) a capture oligonucleotide having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-64; (b) a capture oligonucleotide comprising a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 1-64; and (c) a capture oligonucleotide having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 1-64; and (d) a capture oligonucleotide comprising a sequence selected from SEQ ID NOs: 1 to 64; and (e) a capture oligonucleotide selected from any of (a) to (d), and one or more capture oligonucleotides selected from:

[0011] In one embodiment, the set of two or more non-cross-reactive capture oligonucleotides comprises: (a) a capture oligonucleotide comprising a sequence having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-10; (b) a capture oligonucleotide comprising a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 1-10; and (c) a capture oligonucleotide having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 1-10; and (d) a capture oligonucleotide comprising a sequence selected from SEQ ID NOs: 1 to 10; and (e) a capture oligonucleotide selected from any of (a) to (d), and one or more capture oligonucleotides selected from:

[0012] In one aspect, a set of non-cross-reactive capture oligonucleotides immobilized on an array is provided, the set of non-cross-reactive oligonucleotides satisfying the following requirements: (a) a GC content of about 40% to about 50%; (b) an AG content of about 30% to about 70%; (c) a CT content of about 30% to about 70%; (d) the maximum string of nucleotide repeats in the sequence of three or fewer; (e) the absence of undesired oligonucleotide-oligonucleotide interactions with strings of more than seven consecutive complementary base pair matches; and (f) the absence of undesired oligonucleotide-oligonucleotide interactions with stretches of 18 or fewer consecutive bases; (i) The terminal bases at both ends are complementary to each other, (ii) no interactions where the sum of complementary base pair matches minus the sum of mismatches is greater than 7; and (g) There is no sequence of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs or more that matches a sequence or the complement of a sequence in a genome or in nature, or both; (h) the difference in hybridization free energy of the sequences with their complements is less than about 1 kCal / mol, about 2 kCal / mol, about 3 kCal / mol, or about 4 kCal / mol; (i) there are no predicted hairpin loops with four or more consecutive matches within the stem; and (j) There are four or more consecutive matches within the stem, and there are no predicted hairpin loops with a loop size of more than six bases.

[0013] In one embodiment, the set of two or more non-cross-reactive capture oligonucleotides comprises one or more capture oligonucleotides having a reactive functional group. In one embodiment, the reactive functional group is attached to the capture oligonucleotide via a linker. In one embodiment, the reactive functional group comprises a thiol group.

[0014] In one embodiment, one or more of the oligonucleotides are immobilized to a surface via a reactive functional group. In one embodiment, the surface comprises an electrode surface. In one embodiment, the electrode comprises a carbon-based electrode. In one embodiment, the electrode comprises a carbon ink electrode.

[0015] In one embodiment, one or more non-reactive capture oligonucleotides are at least 20 nucleotides in length. In one embodiment, one or more non-reactive capture oligonucleotides are at least 24 nucleotides in length. In one embodiment, one or more non-reactive capture oligonucleotides are at least 36 nucleotides in length.

[0016] In one embodiment, a kit is provided that includes two or more sets of non-cross-reactive capture oligonucleotides. In one embodiment, the kit includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 sets of non-cross-reactive capture oligonucleotides, and up to 64 sets of non-cross-reactive capture oligonucleotides. In one embodiment, the kit includes at least 10 non-cross-reactive capture oligonucleotides.

[0017] In one aspect, a set of two or more non-cross-reactive oligonucleotide tags is provided, wherein the set of oligonucleotide tags is a subset of a set of parent oligonucleotide tags, wherein one or more oligonucleotide tags in the set comprise a nucleotide sequence having at least 20, 21, 22, 23 or 24 contiguous nucleotides of one or more nucleotide sequences from the parent set, wherein the parent set is (a) tag set 1 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 745 to 808; (b) tag set 2 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 809 to 866; (c) tag set 3 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 867 to 930; (d) tag set 4 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 931 to 994; (e) tag set 5 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 995 to 1052; (f) tag set 6 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1053 to 1116; (g) tag set 7 including an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1117 to 1180; (h) tag set 8 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1181 to 1238; (i) tag set 9 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1239 to 1302; (j) a tag set 10 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1303 to 1366; (k) tag set 11 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1367 to 1424; (l) tag set 12 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1425 to 1488.

[0018] In one aspect, a set of two or more non-cross-reactive oligonucleotide tags is provided, wherein the set of oligonucleotide tags is a subset of a set of parent oligonucleotide tags, wherein one or more oligonucleotide tags in the set comprise a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to one or more nucleotide sequences from the parent set, wherein the parent set is (a) tag set 1 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 745 to 808; (b) tag set 2 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 809 to 866; (c) tag set 3 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 867 to 930; (d) tag set 4 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 931 to 994; (e) tag set 5 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 995 to 1052; (f) tag set 6 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1053 to 1116; (g) tag set 7 including an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1117 to 1180; (h) tag set 8 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1181 to 1238; (i) tag set 9 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1239 to 1302; (j) a tag set 10 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1303 to 1366; (k) tag set 11 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1367 to 1424; (l) tag set 12 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1425 to 1488.

[0019] In one aspect, a set of two or more non-cross-reactive oligonucleotide tags is provided, wherein the set of oligonucleotide tags is a subset of a set of parent oligonucleotide tags, the parent set comprising: (a) tag set 1 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 745 to 808; (b) tag set 2 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 809 to 866; (c) tag set 3 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 867 to 930; (d) tag set 4 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 931 to 994; (e) tag set 5 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 995 to 1052; (f) tag set 6 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1053 to 1116; (g) tag set 7 including an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1117 to 1180; (h) tag set 8 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1181 to 1238; (i) tag set 9 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1239 to 1302; (j) a tag set 10 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1303 to 1366; (k) tag set 11 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1367 to 1424; (l) tag set 12 comprising an oligonucleotide tag having a nucleotide sequence selected from SEQ ID NOs: 1425 to 1488.

[0020] In one embodiment, the set of two or more non-cross-reactive oligonucleotide tags comprises: (a) an oligonucleotide tag comprising a sequence having at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 745 to 808; (b) an oligonucleotide tag comprising a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 745 to 808; (c) an oligonucleotide tag having at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 745-808; (d) an oligonucleotide tag comprising a sequence selected from SEQ ID NOs: 745 to 808; (e) An oligonucleotide tag selected from any of (a) to (d), and one or more oligonucleotide tags selected from:

[0021] In one embodiment, the set of two or more non-cross-reactive oligonucleotide tags comprises: (a) an oligonucleotide tag comprising a sequence having at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence selected from SEQ ID NOs: 745 to 754; (b) an oligonucleotide tag comprising a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 745 to 754; (c) an oligonucleotide tag having at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 745-754; (d) an oligonucleotide tag comprising a sequence selected from SEQ ID NOs: 745 to 754; and (e) An oligonucleotide tag selected from any of (a) to (d), and one or more oligonucleotide tags selected from:

[0022] In one embodiment, the set comprises two or more non-cross-reactive oligonucleotide tags, wherein one or more oligonucleotide tags in the set hybridize to less than 0.05% of the non-complementary capture oligonucleotides compared to complementary capture oligonucleotides.

[0023] In one embodiment, one or more oligonucleotide tags in the set comprise a label. In one embodiment, the label is attached to the oligonucleotide tag via a linker. In one embodiment, the label is selected from radioactive, fluorescent, chemiluminescent, electrochemiluminescent, light absorption, light scattering, electrochemical, magnetic, and enzymatic labels. In one embodiment, the label comprises an electrochemiluminescent label. In one embodiment, the label is selected from radioactive, fluorescent, colorimetric, antigenic, and enzymatic labels. In one embodiment, the label comprises biotin or a hapten. In one embodiment, the label comprises biotin, fluorescein, or digoxigenin. In one embodiment, the label comprises an organometallic complex comprising a transition metal. In one embodiment, the transition metal comprises ruthenium. In one embodiment, the label comprises an MSD SULFO-TAG™ label. In one embodiment, the label comprises a primary binding reagent that is a binding partner of a secondary binding reagent. In one embodiment, the secondary binding reagent comprises biotin, streptavidin, avidin, or an antibody. In one embodiment, the primary binding reagent comprises an oligonucleotide and the secondary binding reagent comprises an oligonucleotide that is complementary to the primary binding reagent.

[0024] In one embodiment, a kit is provided that comprises two or more sets of non-cross-reactive oligonucleotide tags.In one embodiment, the kit comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 sets of non-cross-reactive oligonucleotide tags, and up to 64 sets of non-cross-reactive oligonucleotide tags.In one embodiment, the kit comprises at least 10 sets of non-cross-reactive oligonucleotide tags.

[0025] In one aspect, a method for immobilizing an oligonucleotide on a carbon-based support surface is provided. In one aspect, the oligonucleotide comprises a thiol group. In one aspect, the method comprises: (a) printing one or more droplets containing oligonucleotides onto a carbon-based support surface; (b) allowing the droplet to spread on a surface; (c) drying the droplets to form dried droplets; (d) immobilizing the oligonucleotides on a carbon-based support surface via thiol groups; (e) washing the dried droplets with a washing solution containing a thiol-containing compound to remove any unimmobilized oligonucleotides.

[0026] In one aspect, a method for producing a carbon-based support surface having one or more immobilized oligonucleotides is provided. In one aspect, the method comprises: (a) printing one or more droplets containing thiol-containing oligonucleotides onto a carbon-based support surface; (b) allowing the droplet to spread on a surface; (c) drying the droplets to form dried droplets; (d) immobilizing the oligonucleotides on a carbon-based support surface via thiol groups; (e) washing the dried droplets with a washing solution containing a thiol-containing compound to remove any unimmobilized oligonucleotides.

[0027] In one aspect, the carbon-based support surface comprises a carbon-based electrode. In one aspect, the oligonucleotides are covalently attached to the carbon-based support surface via thiol groups. In one aspect, the method comprises printing an array of droplets comprising capture oligonucleotides onto a plurality of binding domains on the carbon-based support surface.

[0028] In one aspect, a multi-well plate is provided, (a) one or more wells with one or more carbon-based electrodes; (b) a set of one or more non-cross-reactive capture oligonucleotides selected from the set of parent non-cross-reactive capture oligonucleotides described herein, wherein the one or more non-cross-reactive capture oligonucleotides are immobilized on one or more carbon-based electrodes.

[0029] In one aspect, a method of manufacturing a multi-well plate is provided, the method comprising: (a) printing one or more droplets containing thiol-containing oligonucleotides onto a carbon-based electrode; (b) allowing the droplet to spread on a surface; (c) drying the droplets to form dried droplets; (d) immobilizing oligonucleotides to a carbon-based electrode via thiol groups; (e) washing the dried droplets with a washing solution containing a thiol-containing compound to remove any unimmobilized oligonucleotides.

[0030] In one aspect, the carbon-based support surface comprises a carbon-based electrode. In one aspect, the oligonucleotides are covalently attached to the carbon-based support surface via thiol groups. In one aspect, the method comprises printing an array of droplets comprising capture oligonucleotides onto a plurality of binding domains on the support surface.

[0031] In one aspect, a method is provided for immobilizing an array of capture oligonucleotides on the surface of one or more carbon-based electrodes. In one aspect, the method comprises: (a) printing an array of droplets comprising one or more capture oligonucleotides onto a plurality of binding domains on the surface of one or more carbon-based electrodes, wherein one or more capture oligonucleotides comprise a thiol group; (b) allowing the droplet to spread on a surface; (c) drying the droplets to form dried droplets; (d) incubating the dried droplets for a time sufficient to immobilize one or more oligonucleotides on the surface of the carbon-based electrode via the thiol groups; (e) washing the dried droplets with a washing solution containing a thiol-containing compound to remove excess non-immobilized oligonucleotides.

[0032] In one embodiment, the capture oligonucleotides printed in one binding domain of the array have a different sequence than the capture oligonucleotides printed in other binding domains within the array. In one embodiment, each binding domain contains less than about 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% contaminating capture oligonucleotides. In one embodiment, each binding domain contains less than about 0.05% contaminating capture oligonucleotides.

[0033] In one embodiment, the method includes printing an array onto a plurality of carbon-based electrodes. In one embodiment, one or more carbon-based electrodes comprise a plurality of binding domains. In one embodiment, the carbon-based electrodes comprise carbon ink electrodes. In one embodiment, the one or more carbon-based electrodes are in a multi-well plate.

[0034] In one embodiment, the droplets contain a surfactant. In one embodiment, the thiol-containing compound is water-soluble and has a molecular weight of less than about 200 g / mol, 175 g / mol, 150 g / mol, or 125 g / mol. In one embodiment, the thiol-containing compound is selected from cysteine, cysteamine, dithiothreitol, 3-mercaptoproprionate, 3-mercapto-1-propanesulfonic acid, and combinations thereof. In one embodiment, the thiol-containing compound contains cysteine. In one embodiment, the wash solution contains about 5 mM to about 750 mM cysteine, about 10 mM to about 500 mM cysteine, or about 25 mM to about 75 mM cysteine. In one embodiment, the wash solution contains a pH buffering component, a surfactant, or a combination thereof, and has a pH of about 7 to 9. In one embodiment, the pH buffering component contains Tris, the surfactant contains Triton X-100, and the pH is about 8.0.

[0035] In one aspect, the method includes packaging the carbon-based electrode in a dry package. In one aspect, the carbon-based electrode is packaged in a dry package before the washing step. In one aspect, the carbon-based electrode is packaged in a dry package after the washing step.

[0036] In one aspect, a kit is provided, comprising: (a) one or more support surfaces; (b) a set of one or more non-cross-reactive capture oligonucleotides selected from the set of parent non-cross-reactive capture oligonucleotides, wherein the one or more non-cross-reactive capture oligonucleotides are immobilized on one or more support surfaces.

[0037] In one embodiment, the one or more support surfaces provided in the kit comprise a carbon-based support surface, hi one embodiment, the one or more support surfaces comprise a carbon-based electrode.

[0038] In one aspect, a kit is provided, comprising: (a) one or more carbon-based electrodes having one or more surfaces; (b) a set of one or more non-cross-reactive capture oligonucleotides selected from the set of parent non-cross-reactive capture oligonucleotides, wherein the one or more non-cross-reactive capture oligonucleotides are immobilized on the surface of one or more carbon-based electrodes.

[0039] In one embodiment, the kit includes one or more carbon ink electrodes.

[0040] In one embodiment, the kit comprises one or more non-cross-reactive capture oligonucleotides immobilized on one or more support surfaces in an array. In one embodiment, the one or more non-cross-reactive capture oligonucleotides are immobilized on one or more binding domains. In one embodiment, two or more non-cross-reactive capture oligonucleotides are immobilized on two or more unique binding domains, and the sequences of the capture oligonucleotides immobilized on each unique binding domain are the same.

[0041] In one aspect, the kit comprises the following components: (a) a labeled oligonucleotide probe comprising a sequence complementary to a target sequence in a nucleic acid of interest; (b) one or more blocking probes; (c) one or more nucleoside triphosphates; (d) one or more labeled nucleoside triphosphates; (e) one or more labeled dideoxynucleoside triphosphates; and (f) one or more ligases; (g) one or more polymerases; and

[0042] In one embodiment, the kit includes a plurality of labeled oligonucleotide probes comprising a first sequence complementary to a target sequence in a nucleic acid of interest and an oligonucleotide tag having a sequence complementary to the sequence of a capture oligonucleotide.

[0043] In one aspect, a kit is provided for detecting, identifying, or quantifying one or more target nucleotide sequences in a sample, wherein the one or more target nucleotide sequences comprise a polymorphic nucleotide, and the kit comprises: (i) a targeting probe comprising a single-stranded oligonucleotide tag that is complementary to at least a portion of a capture oligonucleotide immobilized on a support surface and a first nucleic acid sequence that is complementary to a first region of a target nucleotide sequence in a sample; (ii) at least one pair of oligonucleotide probes comprising a label and a detection probe comprising a second nucleic acid sequence that is complementary to a second region of the target nucleotide sequence adjacent to a first region to which the first nucleic acid sequence of the targeting probe sequence is complementary, wherein the targeting or detection probe comprises a terminal 3' or 5' nucleotide located above the polymorphic nucleotide of the target nucleotide sequence.

[0044] In one embodiment, the targeting probe has a terminal 3' nucleotide that is complementary to a region of the target nucleotide sequence adjacent to the region to which the 5' terminal nucleotide of the detection probe is complementary. In one embodiment, the terminal 3' nucleotide of the targeting probe is complementary to a polymorphic nucleotide of the target nucleotide sequence.

[0045] In one embodiment, the kit comprises a first and a second targeting probe that bind to a target nucleotide sequence, the first and second targeting probes differing only in the terminal 3' nucleotide. In one embodiment, the first targeting probe is complementary to the wild-type sequence and the second targeting probe is complementary to the mutant sequence.

[0046] In one embodiment, the kit comprises a plurality of pairs of oligonucleotide probes for a plurality of target nucleotide sequences.

[0047] In one embodiment, the kit includes a detection probe having a label attached to the 3' end.

[0048] In one aspect, a kit is provided for detecting, identifying, or quantifying one or more target nucleotide sequences in a sample, wherein the one or more target nucleotide sequences comprise a polymorphic nucleotide, and the kit comprises: (a) a single-stranded oligonucleotide tag that is complementary to at least a portion of a capture oligonucleotide immobilized on a support surface; (b) a targeting nucleic acid sequence that is complementary to a target nucleotide sequence in the sample; and (c) a label; and one or more targeting probes comprising:

[0049] In one aspect, the kit comprises: (a) a polymerase; (b) one or more dideoxynucleotide triphosphates (ddNTPs); and

[0050] In one embodiment, the kit comprises an oligonucleotide tag attached to the 5' end of a targeting probe, wherein the targeting nucleic acid sequence comprises a 3' end that is complementary to a nucleotide adjacent to a polymorphic nucleotide of one or more target nucleotide sequences in a sample. In one embodiment, the oligonucleotide tag is attached to the 5' end of the targeting probe, wherein the targeting nucleic acid sequence comprises a terminal 3' nucleotide that is complementary to a polymorphic nucleotide of one or more target nucleotide sequences in a sample.

[0051] In one aspect, the kit comprises a plurality of probes comprising targeting nucleic acid sequences complementary to a plurality of target nucleotide sequences in the sample.

[0052] In one embodiment, the kit comprises a labeled nucleoside triphosphate. In one embodiment, the kit comprises a labeled nucleoside triphosphate and a secondary binding reagent. In one embodiment, the kit comprises a labeled nucleoside triphosphate comprising a binding partner of the secondary binding reagent. In one embodiment, the kit comprises a secondary binding reagent comprising avidin, streptavidin, or an antibody, and the labeled nucleoside triphosphate comprises a biotin or hapten label.

[0053] In one aspect, the kit comprises a labeled nucleoside triphosphate having a radioactive, fluorescent, chemiluminescent, electrochemiluminescent, light absorption, light scattering, electrochemical, magnetic, or enzymatic label. In one aspect, the nucleoside triphosphate is labeled with an electrochemiluminescent label.

[0054] In one embodiment, the kit comprises a labeled dideoxynucleotide triphosphate complementary to a polymorphic nucleotide of a target nucleotide sequence.

[0055] In one aspect, the kit comprises the following components: (a) a hybridization buffer; (b) a binding buffer; (c) Signs; (d) a secondary binding reagent; (d) a read buffer; (e) a unique kit identifier.

[0056] In one embodiment, the hybridization buffer comprises a nucleic acid denaturant. In one embodiment, the nucleic acid denaturant comprises formamide. In one embodiment, the hybridization buffer is provided as two separate components that can be combined to form the hybridization buffer. In one embodiment, the binding buffer comprises a surfactant. In one embodiment, the read buffer comprises an electrochemiluminescence read buffer. In one embodiment, the electrochemiluminescence read buffer comprises one or more electrochemiluminescence coreactants selected from a tertiary amine, tripropylamine, and N-butyldiethanolamine.

[0057] In one embodiment, the kit comprises one or more non-cross-reactive capture oligonucleotides immobilized to one or more binding domains on the surface of one or more carbon-based electrodes. In one embodiment, one or more capture oligonucleotides comprise a thiol group and are covalently bound to the carbon-based surface via the thiol group. In one embodiment, the capture oligonucleotides are attached to the thiol group via a linker. In one embodiment, the capture oligonucleotides are covalently attached to the support surface. In one embodiment, the non-cross-reactive capture oligonucleotides are immobilized on an array. In one embodiment, the non-cross-reactive capture oligonucleotides are immobilized on a bead array.

[0058] In one aspect, the kit comprises: (a) a capture oligonucleotide having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-64; (b) a capture oligonucleotide comprising a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 1-64; and (c) a capture oligonucleotide comprising a sequence selected from SEQ ID NOs: 1 to 64; and (d) a capture oligonucleotide selected from any of the foregoing sets; and a set of two or more non-cross-reactive capture oligonucleotides selected from.

[0059] In one aspect, the kit comprises: (a) a capture oligonucleotide comprising a sequence having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-10; (b) a capture oligonucleotide comprising a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 1-10; and (c) a capture oligonucleotide comprising a sequence selected from SEQ ID NOs: 1 to 10; and (d) a capture oligonucleotide selected from any of the foregoing sets; and a set of two or more non-cross-reactive capture oligonucleotides selected from.

[0060] In one embodiment, the oligonucleotide tags in the kit comprise 24 or more nucleotides. In one embodiment, the oligonucleotide tags comprise 36 or more nucleotides.

[0061] In one embodiment, the oligonucleotide tag binds to less than 0.05% of the non-complementary capture oligonucleotides compared to the complementary capture oligonucleotides.

[0062] In one embodiment, the kit comprises two or more electrodes having one or more binding domains.

[0063] In one embodiment, the kit includes two or more capture oligonucleotides immobilized to two or more unique binding domains, and the sequences of the capture oligonucleotides immobilized to each unique binding domain are the same. In one embodiment, the support surface includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 different capture oligonucleotides immobilized to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 unique binding domains. In one embodiment, one or more binding domains include at least some capture oligonucleotides that are not covalently bound to the electrode surface via thiol groups. In one embodiment, one or more binding domains include more than 10%, 15%, 20%, 25%, 50%, or 75% of the capture oligonucleotides that are not covalently bound to the support surface via thiol groups. In one embodiment, the binding domain comprises less than about 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of contaminating capture oligonucleotides.

[0064] In one embodiment, the kit includes a support surface comprising a multiwell plate. In one embodiment, one or more wells of the multiwell plate include one or more electrodes. In one embodiment, one or more electrodes include carbon-based electrodes. In one embodiment, one or more electrodes include carbon ink. In one embodiment, one or more electrodes in one or more wells of the multiwell plate include one or more binding domains. In one embodiment, one or more capture oligonucleotides are immobilized to one or more binding domains on one or more electrodes.

[0065] In one embodiment, the kit includes a cleaning solution containing a thiol-containing compound. In one embodiment, the thiol-containing compound is water-soluble and has a molecular weight of less than about 200 g / mol, 175 g / mol, 150 g / mol, or 125 g / mol. In one embodiment, the thiol-containing compound is selected from cysteine, cysteamine, dithiothreitol, 3-mercaptoproprionate, 3-mercapto-1-propanesulfonic acid, and combinations thereof. In one embodiment, the thiol-containing compound includes cysteine. In one embodiment, the thiol-containing compound includes a zwitterion. In one embodiment, the cleaning solution includes an aqueous solution. In one embodiment, the aqueous cleaning solution includes about 5 mM to 750 mM cysteine, about 10 mM to about 500 mM cysteine, or about 25 mM to about 75 mM cysteine. In one embodiment, the cleaning solution includes a pH buffering component, a surfactant, or a combination thereof. In one embodiment, the pH buffer contains Tris, and the surfactant contains Triton X-100. In one embodiment, the washing solution has a pH of 7 to 9. In one embodiment, the washing solution has a pH of about 8. In one embodiment, the washing solution contains about 15 mM to about 25 mM Tris, about 0.05% to about 0.15% Triton X-100, and about 5 mM to 750 mM cysteine, and has a pH of about 8.0. In one embodiment, the washing solution contains about 15 mM to about 25 mM Tris, about 0.05% to about 0.15% Triton X-100, and about 25 mM to about 75 mM cysteine, and has a pH of about 8.0. In one embodiment, the washing solution contains about 20 mM Tris, about 0.1% Triton X-100, and about 50 mM cysteine, and has a pH of about 8.0. In one embodiment, one or more components of the wash solution are provided in dry form. In one embodiment, the kit includes a liquid diluent for reconstituting the components of the wash solution.

[0066] In one embodiment, the kit comprises one or more blocking probes. In one embodiment, the kit comprises one or a pair of blocking probes, each pair of blocking probes comprising: (a) a first blocking probe that comprises a sequence identical to that of the targeting probe but does not comprise a single-stranded oligonucleotide tag; (b) a second blocking probe having a sequence identical to that of the detection probe but containing no label.

[0067] In one embodiment, the kit includes at least one pair of blocking probes for each pair of oligonucleotide probes.

[0068] In one embodiment, the kit includes a label selected from radioactive, fluorescent, chemiluminescent, electrochemiluminescent, light absorption, light scattering, electrochemical, magnetic, and enzymatic labels. In one embodiment, the label includes an electrochemiluminescent label. In one embodiment, the label includes an organometallic complex comprising a transition metal. In one embodiment, the transition metal includes ruthenium. In one embodiment, the label includes an MSD SULFO-TAG™ label. In one embodiment, the label includes a binding partner of a secondary binding reagent. In one embodiment, the secondary binding reagent includes biotin, streptavidin, avidin, or an antibody. In one embodiment, the label includes a hapten selected from biotin, fluorescein, and digoxigenin. In one embodiment, the label includes a primary binding agent including a first oligonucleotide sequence, and the secondary binding reagent includes a second oligonucleotide sequence that is complementary to the first oligonucleotide sequence of the primary binding agent.

[0069] In one embodiment, a method for identifying, detecting, or quantifying a target analyte is provided. In one embodiment, the method includes: providing an array having one or more carbon-based electrodes with one or more surfaces and one or more non-cross-reactive capture oligonucleotides immobilized to one or more binding domains on one or more surfaces of the one or more carbon-based electrodes; contacting the array with a composition containing one or more target analytes, wherein the one or more target analytes are linked to oligonucleotide tags that are complementary to at least a portion of the capture oligonucleotides immobilized on the support surface and a label; incubating the composition of one or more target analytes under conditions in which the oligonucleotide tags hybridize to their complementary capture oligonucleotides to form hybridization complexes; and identifying, detecting, or quantifying the target analytes based on the presence or absence of the label at the array location.

[0070] In one embodiment, the method includes contacting an array with a composition comprising a plurality of target analytes, each target analyte bound to an oligonucleotide tag complementary to a different capture oligonucleotide, and the target analytes can be identified, detected, or quantified based on the binding of the oligonucleotide tag at the array location. In one embodiment, less than about 0.05% of the oligonucleotide tags bind to non-complementary capture oligonucleotides on the array compared to their corresponding complementary capture oligonucleotides. In one embodiment, the oligonucleotide tags comprise at least 12, 24, or 36 nucleotides. In one embodiment, the one or more electrodes comprise carbon ink electrodes. In one embodiment, the one or more electrodes are comprised in a multiwell plate. In one embodiment, each well of the multiwell plate comprises an electrode. In one embodiment, the electrode of each well of the multiwell plate comprises an array of capture oligonucleotides immobilized to a plurality of binding domains. In one embodiment, the one or more target analytes are linked to the oligonucleotide tags via a binding partner. In one embodiment, the binding partner comprises an antibody that specifically binds to the target analyte. In one embodiment, the binding partner comprises an oligonucleotide sequence complementary to the oligonucleotide sequence of the target analyte. In one embodiment, the oligonucleotide tag and the binding partner are different regions of a single oligonucleotide strand. In one embodiment, one or more target analytes comprise a nucleic acid sequence and the binding partner comprises an oligonucleotide sequence that is complementary to a target sequence in the nucleic acid sequence of interest.

[0071] In one embodiment, the method comprises incubating a composition of one or more target analytes at a temperature of about 27° C. to about 47° C., a formamide concentration of about 21% to about 41%, a salt concentration of about 300 mM to about 500 mM, and a pH of about 7.5 to about 8.5, so that the oligonucleotide tags hybridize to their complementary capture oligonucleotides to form hybridization complexes. In one embodiment, the incubation comprises a temperature of about 37° C., a formamide concentration of about 31%, a salt concentration of about 400 mM, and a pH of 8.0.

[0072] In one embodiment, one or more target analytes are labeled with a primary binding reagent. (a) a targeting probe comprising a single-stranded oligonucleotide tag that is complementary to at least a portion of a capture oligonucleotide immobilized on a support surface and a first nucleic acid sequence that is complementary to a first region of a target nucleotide sequence in a sample; (b) incubated with one or more paired oligonucleotide probes comprising a label and a detection probe comprising a second nucleic acid sequence that is complementary to a second region of the target nucleotide sequence; Incubation includes incubation under conditions under which the detection probes bind to their corresponding target analytes.

[0073] In one embodiment, the targeting probe and the detection probe can simultaneously bind to the target analyte to form a sandwich complex.In one embodiment, the targeting probe and the detection probe have nucleotide sequences that are complementary to adjacent sequences of the target nucleotide sequence.In one embodiment, the targeting probe has a nucleic acid sequence with a 3' end that hybridizes to the target nucleotide sequence adjacent to the 5' end of the detection probe.In one embodiment, the 3' nucleotide of the targeting probe is complementary to the polymorphic nucleotide in the target nucleotide sequence.

[0074] In one embodiment, the method comprises incubating a hybridization complex in the presence of a nucleic acid ligase under conditions in which the nucleic acid ligase ligates the targeting and detection probes to form a reaction product. In one embodiment, the method comprises exposing the reaction product to denaturing conditions that dissociate the reaction product from the target nucleotide sequence. In one embodiment, the method comprises adding one or more blocking probes to the reaction product. In one embodiment, the method comprises ligating one or more target analytes to a (a) a single-stranded oligonucleotide tag that is complementary to at least a portion of a capture oligonucleotide immobilized on a support surface; (b) a targeting nucleic acid sequence that is complementary to a target nucleotide sequence in the sample; and (c) incubating with a targeting probe comprising a label;

[0075] In one embodiment, the method comprises incubating one or more target analytes with a targeting probe under conditions in which the targeting nucleic acid sequence of the targeting probe hybridizes to its complementary sequence on the target nucleotide sequence to form a hybridization complex. In one embodiment, the method comprises incubating the hybridization complex in the presence of a nucleic acid polymerase under conditions in which the polymerase extends the first probe sequence to form an extended sequence. In one embodiment, the method comprises incubating the polymerase with one or more labeled nucleoside triphosphates, wherein the extended sequence comprises the labeled nucleoside triphosphates.

[0076] In one embodiment, the method comprises incubating a polymerase with one or more unlabeled nucleoside triphosphates, wherein the extension sequence is longer than one nucleotide and comprises an unlabeled nucleoside triphosphate. In one embodiment, the labeled nucleoside triphosphate comprises a chain-terminating nucleoside triphosphate, and the extension sequence is one nucleotide in length. In one embodiment, the labeled nucleoside triphosphate comprises a labeled dideoxynucleoside triphosphate. In one embodiment, the first nucleic acid sequence of the targeting probe has a 3' end that is complementary to the nucleotide adjacent to the polymorphic nucleotide in the nucleic acid of interest, and the polymerase adds a nucleotide to the 3' end of the targeting nucleic acid sequence.

[0077] In one embodiment, the method includes washing the array with a wash buffer after the incubation step. In one embodiment, washing includes immersing the array in the wash buffer under high stringency conditions. In one embodiment, the high stringency conditions include a temperature of about 27°C to about 47°C, a formamide concentration of about 21% to about 41%, a salt concentration of about 300 mM to about 500 mM, and a pH of 7.5 to 8.5. In one embodiment, the high stringency conditions include a temperature of about 37°C, a formamide concentration of about 31%, a salt concentration of about 400 mM, and a pH of 8.0. In one embodiment, the method includes immersing the array under high stringency conditions for at least 5, 10, 30, or 60 minutes. In another embodiment, the high stringency conditions include low salt conditions, e.g., a buffer having a salt concentration of less than about 40 mM, 20 mM, 15 mM, or 10 mM. In one embodiment, high stringency conditions include low salt conditions such as 0.1x PBS at 37°C.

[0078] In one aspect, the label comprises an electrochemiluminescent label, and the method comprises contacting an electrode with an electrochemiluminescent read buffer comprising an electrochemiluminescent coreactant and generating an assay signal by applying a potential to the electrode. In one aspect, the coreactant is selected from a tertiary amine, tripropylamine, N-butyldiethanolamine, and combinations thereof. In one aspect, the method comprises imaging the assay signal to determine the assay signal associated with each capture oligonucleotide. [Brief explanation of the drawings]

[0079] [Figure 1A] FIG. 1 is a schematic diagram of the oligonucleotide ligation assay (OLA) hybridization step. [Figure 1B] FIG. 1 is a schematic diagram of the OLA ligation steps. [Figure 1C] FIG. 1 is a schematic diagram of the OLA detection steps. [Figure 1D] FIG. 1 is a schematic diagram of an OLA probe mismatch where no hybridization occurs. [Figure 2A]FIG. 1 is a schematic diagram of a primer extension assay (PEA) in which a labeled ddNTP is attached to the 3′ end of the probe. [Figure 2B] FIG. 1 is a schematic diagram of a PEA in which an unlabeled ddNTP is added to the 3′ end of the probe. [Figure 3] 1 is a graph showing the effect of varying the length of the linker (or spacer) between the capture oligonucleotide and the electrode on hybridization of the probe to the capture oligonucleotide and detection using electrochemiluminescence. [Figure 4] 1 is a graph showing the effect of different capture oligonucleotide array washing conditions on the measured cross-reactivity of oligonucleotide probes specific for one element of the array. [Figure 5] 1 is a graph comparing the electrochemiluminescent OLA assay signal of BRAF mutations as a function of the concentration of nucleic acid template containing the target BRAF gene region, and comparing the signal generated with mutant and wild-type sequences. [Figure 6] 1 is a graph showing the assay signal generated by a panel of electrochemiluminescent OLAs as a function of the concentration of their specific target sequences. [Figure 7] 1 is a graph showing that the inclusion of a blocking oligonucleotide can reduce the bridging background signal of a panel of electrochemiluminescent OLAs. [Figure 8] 10 is a graph showing that the background increase in electrochemiluminescence OLA due to nonspecific binding of probes to capture oligonucleotides can be reduced by including a blocking oligonucleotide or adding a high stringency high temperature soak step. [Figure 9] The actual percentage of mutant sequences in electrochemiluminescence OLA obtained from PCR-amplified genomic DNA extracted from a mixture of mutant and wild-type cells is shown relative to the predicted percentage of mutant BRAF and NRAS sequences. [Figure 10]Electrochemiluminescent PEA assay signal for the BRAF 1799T>A mutation is shown as a function of the concentration of template nucleic acid representing the mutant and wild-type sequences, demonstrating that the assay is specific for the mutant sequence. [Figure 11] 1 is a graph showing that a panel of electrochemiluminescent PEAs of BRAF and NRAS SNPs showed a linear response to input DNA concentration. [Figure 12] Shown is the predicted percentage of mutant BRAF 1799T>A sequences versus the actual percentage of mutant sequences using the electrochemiluminescent BRAF 1799T>A OLA assay measuring PCR-amplified genomic DNA extracted from a mixture of mutant and wild-type cells. [Figure 13] The predicted percentage of mutant NRAS 181C>A sequences versus the actual percentage of mutant sequences using the electrochemiluminescent NRAS 181C>A OLA assay measuring PCR-amplified genomic DNA extracted from a mixture of mutant and wild-type cells is shown. [Figure 14] Shown is the predicted percentage of mutant 182A>T sequences versus the actual percentage of mutant sequences using an electrochemiluminescent 182A>T OLA assay measuring PCR-amplified genomic DNA extracted from a mixture of mutant and wild-type cells. [Figure 15] As described in embodiments herein, an oligonucleotide ligation amplification (OLA) assay is presented for the detection, identification, and / or quantification of target nucleotide sequences, e.g., therapeutic oligonucleotides, in samples that may contain oligonucleotide metabolites. [Figure 16] As described in embodiments herein, direct hybridization methods are presented for the detection, identification, and / or quantification of target nucleotide sequences, e.g., therapeutic oligonucleotides, in samples that may contain oligonucleotide metabolites. [Figure 17]As described in embodiments herein, a nuclease protection assay (NPA) with a direct surface coating for the detection, identification, and / or quantification of target nucleotide sequences, e.g., therapeutic oligonucleotides, in samples that may contain oligonucleotide metabolites is presented. [Figure 18] As described in embodiments herein, a hybridization / protection assay is provided for the detection, identification, and / or quantification of target nucleotide sequences, e.g., therapeutic oligonucleotides, in a sample that may contain oligonucleotide metabolites. [Figure 19] A sandwich assay for the detection, identification, and / or quantification of antibodies, eg, anti-drug antibodies (ADAs), in a sample is presented. [Figure 20] Shown is a schematic diagram of a targeting probe and a detection probe bridged by a positive control oligonucleotide containing a nucleotide sequence complementary to the ASO sequence. [Figure 21] 19 shows a modification of the sandwich assay shown in FIG. 19 for the detection, identification, and / or quantification of antibodies, eg, anti-drug antibodies (ADA), in a sample. DETAILED DESCRIPTION OF THE INVENTION

[0080] A.Definition Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those skilled in the art. Furthermore, unless the context requires otherwise, the singular includes the plural, the plural includes the singular, e.g., "a" or "an," the plural includes, e.g., "one or more" or "at least one," and the term "or" can mean "and / or" unless expressly indicated to refer only to alternatives or unless the alternatives are mutually exclusive. The terms "comprising," "including," and "including" are not limiting. Any type of range provided herein includes all values ​​within the specific range described, as well as the values ​​related to the endpoints of the specific range.

[0081] As used herein, the term "about" is used to modify, for example, the amount, concentration, volume, processing temperature, processing time, yield, flow rate, pressure, and ranges of components in a composition, and is used in describing the present invention. The term "about" refers to the variability of numerical values ​​that arise, for example, from typical measuring and processing procedures used to make a compound, composition, concentrate, or formulation, from accidental errors in these procedures, from differences in the manufacture, source, or purity of starting materials or components used to carry out the method, and other similar considerations. The term "about" also encompasses amounts that differ due to aging of a formulation having a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation having a particular initial concentration or mixture. When modified with the term "about," the claims appended hereto include such equivalents.

[0082] As used herein, ranges expressed using the word "between" include the endpoints of the range. Thus, for example, a range of 50°C to 70°C includes 50°C to 70°C, i.e., it includes the endpoints of 50°C and 70°C.

[0083] Generally, the nomenclature used in connection with cell and tissue culture, molecular biology, and the chemistry and hybridization of proteins and oligonucleotides or polynucleotides described herein, and the techniques thereof, are well known and commonly used in the art.Amino acids herein can be referred to by their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.Similarly, nucleotides can be referred to by their commonly accepted one-letter codes.

[0084] A "target analyte" may include any molecule of interest that can be detected and analyzed by the methods and kits described herein, including biological molecules such as nucleic acids, proteins, carbohydrates, sugars, and lipids. In one embodiment, the target analyte is a target nucleotide sequence. In another embodiment, the target analyte is a protein. In one embodiment, the target analyte is a DNA-binding protein. The term "target analyte" can refer to an entire molecule of interest, or a segment or portion of a molecule of interest. In one embodiment, the target analyte includes a modified molecule, e.g., a labeled, cleaved, or chemically or enzymatically treated version of a molecule of interest.

[0085] A "target nucleotide sequence" can include any nucleotide sequence of interest, including, but not limited to, sequences found in the DNA or RNA of prokaryotic or eukaryotic DNA organisms. These can include single- or double-stranded DNA, single- or double-stranded RNA, DNA / RNA hybrids, or DNA / RNA mosaics. Target nucleotide sequences can include miRNA, therapeutic RNA, mRNA, RNA viruses, or combinations thereof. In the case of double-stranded nucleotide sequences, the target nucleotide sequence can be identified on either strand. The target nucleotide sequence can require extraction, e.g., nuclear DNA or viral genomic DNA or RNA, or can be directly manipulated in a sample, e.g., cell-free fetal or cell-free tumor DNA in serum or plasma, or circulating therapeutic oligonucleotides. The target nucleotide sequence can be directly isolated from a biological sample or can include amplified sequences from a biological sample. Amplification methods are known and include but are not limited to polymerase chain reaction (PCR), whole genome amplification (WGA), reverse transcription followed by polymerase chain reaction (RT-PCR), strand displacement amplification (SDA) or rolling circle amplification (RCA).The polymerase suitable for the amplification method herein includes, for example, Taq, Phi, Bst and Vent-exo for DNA amplification, and for example, T7 RNA polymerase for RNA amplification.

[0086] The target nucleotide sequence can be an oligonucleotide, e.g., a therapeutic oligonucleotide. As used herein, "therapeutic oligonucleotide" refers to an oligonucleotide that can interact with a biomolecule to provide a therapeutic effect. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). ASOs can affect RNA processing and / or regulate protein expression. ASOs are single-stranded oligonucleotides that bind to single-stranded RNA and inactivate it. ASOs are typically single-stranded oligonucleotides that are about 5, 10, 15, 20, or 25 nucleotides to about 30, 35, 40, 45, or 50 nucleotides in length. In one embodiment, the ASO binds to the messenger RNA (mRNA) of a gene, thereby inactivating the gene. In one embodiment, the gene is a disease gene. Thus, the ASO can inactivate the mRNA of the disease gene to prevent or ameliorate the production of a protein that causes a particular disease. In one embodiment, the ASO comprises DNA, RNA, or a combination thereof. Therapeutic oligonucleotides and ASOs are further described in, for example, Goodchild, Methods Mol Biol 764:1-15 (2011), Smith et al., Ann Rev Pharmacol Toxicol 59:605-630 (2019), and Stein et al., Mol Ther 25(5):1069-1075 (2017).

[0087] In one embodiment, the target analyte is an anti-drug antibody (ADA). As used herein, "anti-drug antibody" or "ADA" refers to an antibody induced in vivo in an organism against a biopharmaceutical product. ADA can be induced against biopharmaceuticals such as therapeutic polypeptides, including but not limited to proteins and antibodies, as well as therapeutic oligonucleotides, including but not limited to antisense oligonucleotides (ASOs), short interfering RNAs, microRNAs, and synthetic guide strands of CRISPR / Cas. ADA can include any antibody isotype capable of binding to a biopharmaceutical product, referred to as binding antibodies, and can also include a subpopulation of binding antibodies capable of inhibiting the functional activity of a therapeutic product, referred to as neutralizing antibodies. Detection of ADA can be an important measure of immunogenicity and can affect both the safety and efficacy of a biopharmaceutical product.

[0088] A target nucleotide sequence, such as a therapeutic oligonucleotide, in a sample may degrade, i.e., shorten, over time due to various factors, such as the presence of nucleases, temperature, pH, and salt concentration. The degradation products of a target nucleotide sequence are also referred to as oligonucleotide metabolites. In one embodiment, an oligonucleotide metabolite is 1 or more nucleotides, 2 or more nucleotides, 3 or more nucleotides, 4 or more nucleotides, 5 or more nucleotides, 6 or more nucleotides, 7 or more nucleotides, 8 or more nucleotides, 9 or more nucleotides, 10 or more nucleotides, 15 or more nucleotides, or 20 or more nucleotides shorter than the target nucleotide sequence. In one embodiment, an oligonucleotide metabolite is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% shorter than the target nucleotide sequence. In one embodiment, a sample of the present disclosure includes a target nucleotide sequence, e.g., a therapeutic oligonucleotide, and one or more oligonucleotide metabolites, e.g., a therapeutic oligonucleotide metabolite.

[0089] In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In certain embodiments, degradation of the therapeutic oligonucleotide in a sample indicates a pharmacodynamic response to the therapeutic oligonucleotide. Degraded or truncated therapeutic oligonucleotides, also referred to herein as therapeutic oligonucleotide metabolites, may lose therapeutic efficacy. The methods of the present disclosure can be used to measure the amount of a target nucleotide sequence, e.g., a therapeutic oligonucleotide, compared to an oligonucleotide metabolite, e.g., a therapeutic oligonucleotide metabolite. In one embodiment, the methods of the present disclosure are used to determine the pharmacokinetic parameters of a target nucleotide sequence, e.g., a therapeutic oligonucleotide. In one embodiment, the pharmacokinetic parameters of a target nucleotide sequence, e.g., a therapeutic oligonucleotide, are determined by measuring the rate and / or amount of degradation of the target nucleotide sequence, e.g., a therapeutic oligonucleotide, in a biological environment, e.g., a patient. In one embodiment, the pharmacokinetic parameters measured are clearance, volume distribution, plasma concentration, half-life, peak time, peak concentration, availability, or a combination thereof. Further discussion of the measurement and interpretation of pharmacokinetic parameters can be found, for example, in Benet, Eur J Respir Dis Suppl 134:45-61 (1984) and Le et al., "Overview of Pharmacokinetics," Merck Manual Professional Version, revised May 2019. Oligonucleotide metabolites present in a sample may also interfere with the detection, identification, and / or quantification of target nucleotide sequences in the sample. Therefore, it may be desirable to remove oligonucleotide metabolites from a sample. Therefore, the method of the present disclosure can also be used to reduce and / or remove oligonucleotide metabolites from a sample, for example, to obtain a more accurate measurement of the amount of target nucleotide sequence.

[0090] "Polymerase chain reaction" or "PCR" refers to a technique used to amplify a target nucleotide sequence that involves repeated cycles of three steps: (1) denaturation, in which a double-stranded DNA template is heated to separate the strands; (2) annealing, in which primers bind to regions flanking the target DNA sequence; and (3) extension, in which a DNA polymerase extends the 3' end of each primer along the template strand. PCR can utilize a thermostable DNA polymerase, such as Taq polymerase.

[0091] "Nucleotide" refers to a monomeric unit containing a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. Nucleotides include ATP, UTP, CTG, and GTP, which are found in RNA; deoxyribonucleoside triphosphates, most commonly dATP, dCTP, dGTP, and dTTP, which are found in DNA; and ribonucleoside triphosphates, such as dideoxyribonucleoside triphosphates (ddNTPs), which lack the 3'-OH required for polymerase-mediated extension, e.g., ddATP, ddCTP, ddGTP, and ddTTP.

[0092] "Oligonucleotide" or "oligo" refers to a nucleic acid having a nucleotide sequence of about 5 to about 100, about 10 to about 50, or about 10 to about 25 nucleotides in length, or at least about 10, 15, 20, 25, 30, 35, 40, 45, or 50 up to about 50, 75, or 100 nucleotides in length. Oligonucleotides, including but not limited to probe, primer, tag, or capture oligonucleotides described herein, can be prepared using known methods, including, for example, the phosphoramidite method described by Beaucage and Carruthers (1981) Deoxynucleoside phosphoramidites—a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett., 22(2):1859-1862, or the triester method by Matteucci and Caruthers (1981) Synthesis of deoxynucleotides on a polymer support. J. Am. Chem. Soc., 103(11):3185-3191.

[0093] For example, the nucleotides and nucleic acids of the present disclosure, including those in the target sequences or oligonucleotide reagents of the present disclosure, may contain structural analogs, including non-natural chemical structures that can also participate in hybridization reactions. In one example, a nucleotide or nucleic acid may contain a chemical modification that links it to a label or provides a reactive functional group that can be linked to a label, for example, through the use of an amine- or thiol-modified nucleotide base, phosphate, or sugar. The term "reactive functional group" refers to an atom or related group of atoms that can undergo further chemical reaction, for example, to form a covalent bond with another functional group. Examples of reactive functional groups include, but are not limited to, amino, thiol, hydroxy, and carbonyl groups. In one embodiment, the reactive functional group includes a thiol group. Labels that can be linked to nucleotides or nucleic acids via these chemical modifications include, but are not limited to, detectable moieties such as biotin, haptens, fluorophores, and electrochemiluminescence (ECL) labels.

[0094] In another embodiment, nucleotides can be modified to prevent enzymatic or chemical extension of the nucleic acid chain in which they are incorporated, for example, by replacing the ribose or deoxyribose group with dideoxyribose. In another example, the backbone components linking the nucleotide bases (e.g., sugar or phosphate groups) together can be modified or replaced, for example, by using peptide nucleic acids (PNAs) or by incorporating ribose analogs such as those found in 2'-O-methyl-substituted RNA, locked nucleic acids, bridged nucleic acids, and morpholino nucleic acids. These "backbone" analogs can be present at one, some, or all of the backbone linkages of a nucleic acid or oligonucleotide and can provide certain advantages, such as hybridization products with improved binding stability or stability to nucleases. Another example of nucleotide and nucleic acid structural analogs can include unnatural nucleotide bases. Unnatural (also called "non-canonical" bases) can hybridize with natural (canonical) bases or with other unnatural bases.

[0095] "Isolated" refers to an oligonucleotide or nucleic acid sequence that is substantially or essentially free from a target analyte, e.g., a polypeptide or protein, or other sequences or components that normally accompany or interact with it in its naturally occurring environment. In one aspect, an isolated nucleotide sequence includes components or sequences that are not found in the nucleic acid sequence in its natural environment. The term "isolated" also includes non-natural or recombinantly produced oligonucleotide or protein sequences, since such non-natural or recombinantly produced sequences are not found in nature. In particular, non-natural or recombinantly produced oligonucleotides may have immediately adjacent sequences that are not found in nature.

[0096] As used herein, the term "variant" refers to a polypeptide or oligonucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference polypeptide or oligonucleotide sequence, or that contains at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive amino acids or nucleotides of the reference sequence.

[0097] The term "identical" means that two polynucleotide or two polypeptide sequences contain identical nucleic acid bases or identical amino acid residues, respectively, at the same positions over the comparison window. The term "percent sequence identity" can be determined by comparing two aligned sequences over the comparison window, determining the number of positions where identical nucleic acid bases or amino acid residues occur in both sequences, calculating the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to calculate the percentage of sequence identity. The comparison window may include a full-length sequence or a portion of a larger sequence. Various methods and algorithms are known for determining percent identity between two sequences, including, but not limited to, MEGALIGN (DNASTAR, Inc., Madison, Wis.), FASTA, BLAST, or ENTREZ.

[0098] "Capture oligonucleotide" refers to an oligonucleotide reagent that can be immobilized on a support surface and is designed to hybridize to (and thus capture on) a complementary oligonucleotide. In one embodiment, the capture oligonucleotide is a single-stranded sequence that can selectively hybridize to a single-stranded oligonucleotide tag present on a target reaction product, e.g., under stringent hybridization conditions. In one embodiment, the target reaction product is generated by oligonucleotide ligation using the target nucleotide sequence as a template. In another embodiment, the target reaction product is generated by primer extension using the target nucleotide sequence as a template. The capture oligonucleotide can be provided in solid form, e.g., lyophilized, in solution, or immobilized on a support surface, e.g., on a particle (e.g., microparticle, bead) or in an array. Two or more capture oligonucleotides can be provided together. Examples of two or more capture oligonucleotides provided together include a set or subset (also referred to herein as a set) of the parent capture oligonucleotides described herein.

[0099] "Probe" or "primer" refers to a reagent containing an oligonucleotide sequence capable of hybridizing to a target nucleotide sequence. A probe can contain a single-stranded sequence that is complementary or substantially complementary to a portion of the target nucleotide sequence. A probe can also contain a tag sequence (sometimes referred to herein as a directional sequence) that is complementary to a capture oligonucleotide. The sequences complementary to the target nucleotide sequence and the tag sequence can be on the same nucleic acid strand within the probe, or they can be on different strands within the probe. For example, a probe can contain a first strand having a sequence complementary to the target sequence and a bridging sequence, and a second strand having a sequence complementary to the tag sequence and a bridging sequence on the first strand, where the first and second strands can hybridize or hybridize via the bridging sequence. A probe can be DNA or RNA, contain modified nucleobase analogs, or be modified with a label or a linker suitable for attaching a label. Probes must be long enough to allow hybridization of the probe to the target nucleotide sequence, typically about 5 to about 100, about 10 to about 50, about 20 to about 30, or at least about 5, 6, 7, 8, 9, 10, 15, 20, or 25, and up to about 30, 35, 40, 45, 50, 75, or 100 nucleotides in length. Probes can be prepared by any suitable method known in the art, including chemical or enzymatic synthesis, or by cleavage of a larger nucleic acid using nonspecific nucleic acid-cleaving chemicals or enzymes, or by using site-specific restriction endonucleases. In some applications, probes that hybridize to complementary regions of the target sequence can prime extension of the probe by a polymerase, serving as initiation points for replication of adjacent single-stranded regions of the target sequence.

[0100] A "linker" (also referred to herein as a "spacer") refers to one or more atoms that connect one chemical moiety to another, e.g., one or more atoms that connect a reactive functional group or label to an oligonucleotide. A linker can be a nucleotide or non-nucleotide compound containing one or more atoms, e.g., from about 2, 3, 4, 5, 6, 7, 8, 9, or 10 atoms to about 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms, and can include atoms such as carbon, oxygen, sulfur, nitrogen, and phosphorus, as well as combinations thereof. Examples of linkers include low-molecular-weight groups such as amide, ester, carbonate, and ether groups, as well as high-molecular-weight linking groups such as polyethylene glycol (PEG) and alkyl chains. Thus, a linker can contain one or more atoms, units, or molecules.

[0101] A "label" refers to a chemical group or moiety that has a detectable physical property or is capable of exhibiting a detectable physical property to a chemical group or moiety, including, for example, an enzyme that catalyzes the conversion of a substrate to a detectable product. Labels can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other methods. Examples of labels include, but are not limited to, radioisotopes, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, electrochemiluminescent moieties, magnetic particles, and bioluminescent moieties. In another embodiment, a label is a compound that is a member of a binding pair, where the first member of the binding pair (which can be referred to as the "primary binding reagent") is attached to, for example, a substrate and oligonucleotide, and the other member of the binding pair (which can be referred to as the "secondary binding reagent") has a detectable physical property. Non-limiting examples of binding pairs include biotin and streptavidin, or avidin, complementary oligonucleotides, haptens and hapten-binding partners, and antibody / antigen binding pairs.

[0102] "Detection" refers to detecting, observing, or quantifying the presence of a substance, such as an oligonucleotide, based on the presence or absence of a label.

[0103] "Complementary" refers to nucleic acid molecules or sequences of nucleic acid molecules that interact with each other through the formation of hydrogen bonds, for example, according to the Watson-Crick base pairing model. For example, hybridization can occur between two complementary DNA molecules (DNA-DNA hybridization), between two RNA molecules (RNA-RNA hybridization), or between complementary DNA and RNA molecules (DNA-RNA hybridization). Hybridization can occur between short nucleotide sequences that are complementary to a portion of a longer nucleotide sequence. Hybridization can occur between sequences that do not have 100% "sequence complementarity" (i.e., sequences in which less than 100% of the nucleotides are aligned based on a base pairing model, such as the Watson-Crick base pairing model), but sequences with low sequence complementarity are less stable and less likely to hybridize than sequences with high sequence complementarity. In one embodiment, the nucleotides of complementary sequences have 100% sequence complementarity based on the Watson-Crick model. In another embodiment, the nucleotides of the complementary sequences have at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity based on the Watson-Crick model.

[0104] Whether two complementary sequences hybridize can depend on the stringency of the hybridization conditions, which can vary depending on conditions such as temperature, solvent, ionic strength, and other parameters. The stringency of hybridization conditions can be selected to provide for the selective formation or maintenance of a desired hybridization product of two complementary nucleic acid sequences in the presence of other potentially cross-reacting or interfering sequences. Stringent conditions are sequence-dependent; typically, longer complementary sequences hybridize specifically at higher temperatures than shorter complementary sequences. Generally, stringent hybridization conditions are those that achieve the thermal melting point (T) of a particular nucleotide sequence at a defined ionic strength, concentration of chemical denaturant, pH, and concentration of hybridization partner. m) (i.e., the temperature at which 50% of the sequence hybridizes to a substantially complementary sequence). Generally, nucleotide sequences with a higher percentage of G and C bases hybridize under more stringent conditions than nucleotide sequences with a lower percentage of G and C bases. Generally, stringency can be increased by increasing the temperature, increasing the pH, decreasing the ionic strength, or increasing the concentration of chemical nucleic acid denaturants (such as formamide, dimethylformamide, dimethyl sulfoxide, ethylene glycol, propylene glycol, and ethylene carbonate). Stringent hybridization conditions typically include salt concentrations of less than about 1 M, 500 mM, or 200 mM; hybridization temperatures of greater than about 20°C, 30°C, 40°C, 60°C, or 80°C; and chemical denaturant concentrations of greater than about 10%, 20%, 30%, 40%, or 50%. Because many factors can affect the stringency of hybridization, the combination of parameters may be more important than the absolute value of any parameter alone.

[0105] The term "complement" or "complementary" refers to two oligonucleotides whose bases form complementary base pairs, for example, A pairs with T or U, and C pairs with G. Full complementarity or 100% complementarity refers to a situation in which each nucleotide of one oligonucleotide sequence or region can hydrogen bond with each nucleotide of a second oligonucleotide sequence or region. "Substantial complementarity" refers to sequences that are partially complementary and can hybridize under stringent hybridization conditions. Substantially complementary sequences do not need to hybridize along their entire length.

[0106] "Corresponding" can be used to refer to the relationship between a capture oligonucleotide and an oligonucleotide tag, where the oligonucleotide tag is designed to specifically bind to a particular capture oligonucleotide sequence under stringent hybridization conditions. In one embodiment, the oligonucleotide tag specifically binds to its corresponding capture molecule and does not bind or cross-react with other capture molecules under stringent conditions. In one embodiment, the oligonucleotide tag specifically binds to its corresponding capture molecule and does not bind or cross-react with other capture molecules in the array under stringent conditions. In one embodiment, the oligonucleotide tag is a single-stranded oligonucleotide having a sequence that is complementary to at least a portion of the sequence of its "corresponding" capture oligonucleotide. In one embodiment, the nucleotides of the "corresponding" oligonucleotide tag and the capture oligonucleotide sequence have 100% sequence complementarity based on the Watson-Crick model. In another embodiment, the nucleotides of the corresponding sequence have at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity based on the Watson-Crick model.

[0107] A single-stranded polynucleotide has a "direction" or "directivity" because adjacent nucleotides are joined by phosphodiester bonds between their 3' and 5' carbon atoms, resulting in the terminal 5' and 3' carbon atoms being exposed at either end of the polynucleotide (sometimes referred to as the 5'-(phosphoryl) and 3'-(hydroxyl) ends of the molecule). A "reverse" oligonucleotide has a reverse sequence to the reference oligonucleotide when read in the 5'-to-3' direction. For example, if the reference oligonucleotide sequence is 5'-ACCGATCATG-3' (SEQ ID NO: 1649), the "reverse" oligonucleotide sequence is 5'-GTACTAGCCA-3' (SEQ ID NO: 1650).

[0108] According to the rules defined by Watson-Crick base pairing and the antiparallelism of DNA-DNA, RNA-RNA, and RNA-DNA double helices, the complement of a sequence contains, in 3' to 5' order, a string of bases that are (or substantially are) Watson-Crick partners of the bases of the original sequence. An example of the complement of the sequence 5'-ACCGATCATG-3' (SEQ ID NO: 1649) is 5'-CATGATCGGT-3' (SEQ ID NO: 1651). When the term reverse complement is used in reference to a sequence herein, it is used to refer to the reverse complement of the original sequence. An example of the reverse complement of the sequence 5'-ACCGATCATG-3' (SEQ ID NO: 1649) is 5'-TGGCTAGTAC-3' (SEQ ID NO: 1652).

[0109] "Cross-react" or "cross-reactivity" refers to the ability of an oligonucleotide sequence to hybridize to two or more other oligonucleotide sequences in a sample. In one embodiment, the term "cross-react" refers to the ability of a first oligonucleotide sequence to hybridize to a second oligonucleotide sequence in a sample, where the second oligonucleotide sequence is not complementary or substantially complementary to the first oligonucleotide sequence. In one embodiment, the term "cross-react" or "cross-reactivity" refers to the ability of a capture oligonucleotide to hybridize to two or more oligonucleotide tags or two or more tagged target nucleotide sequences in a sample. In one embodiment, a cross-reactive capture oligonucleotide hybridizes to one or more oligonucleotide tags in a sample under stringent capture hybridization conditions. In one embodiment, stringent capture hybridization conditions include a temperature of 27°C to 47°C, a formamide concentration of 21% to 41%, a salt concentration of 300 mM to 500 mM, and a pH of 7.5 to 8.5. In one embodiment, stringent capture hybridization conditions comprise a temperature of about 37° C., a formamide concentration of about 31%, a salt concentration of about 400 mM, and a pH of 8.0.

[0110] " Non-cross-reactive " or " non-cross-reacting " refers to the ability of a first oligonucleotide sequence to only hybridize with a specific oligonucleotide sequence in a sample, for example, to only hybridize with its corresponding complementary sequence in a sample.In one embodiment, the term " non-cross-reactive " refers to the ability of a capture oligonucleotide to only hybridize with one oligonucleotide tag in a sample that contains two or more oligonucleotide tags or two or more tagged target nucleotide sequences.In one embodiment, a non-cross-reactive oligonucleotide probe only hybridizes with one oligonucleotide tag in a sample under stringent hybridization conditions.In one embodiment, non-cross-reactive means that the ratio of the first oligonucleotide binding to sequences other than its complementary sequence in a sample is less than 0.05% under stringent capture hybridization conditions.

[0111] "Ligase" refers to a class of enzymes that can join nucleotide sequences together by catalyzing the formation of a phosphodiester bond between the 3' hydroxyl of one nucleotide sequence with the 5' phosphate of a second nucleotide sequence. Ligases include E. coli DNA ligase, T4 DNA ligase, T4 RNA ligase, T. aquaticus (Taq) ligase, T. thermophilus DNA ligase (e.g., HiFi ligase), or Pyrococcus DNA ligase. In one embodiment, the ligase is a thermostable ligase. "Ligation" refers to the process of joining two nucleotide sequences together by the formation of a phosphodiester bond between the 3' hydroxyl of one nucleotide sequence and the 5' phosphate of a second nucleotide sequence.

[0112] "Array" refers to one or more support surfaces having two or more spatially distinct (i.e., non-overlapping) addressable locations, referred to herein as binding domains or array elements. In one aspect, each addressable location contains assay reagents, including, for example, capture molecules.

[0113] "Support surface" refers to a surface material onto which various substances, such as oligonucleotides or polypeptides, can be immobilized. A "support surface" can be planar or non-planar. In one embodiment, the support surface comprises a flat surface. In one embodiment, the support surface is a plate with multiple wells, i.e., a "multiwell plate." A multiwell plate can contain any number of wells of any size or shape, arranged in any pattern or configuration. In another embodiment, the support surface has a curved surface. In one embodiment, the support surface is provided by one or more particles, beads, or microspheres. The terms particle, bead, or microsphere can be used interchangeably unless otherwise specified. In one embodiment, the support surface comprises color-coded particles, beads, or microspheres. In one embodiment, the support surface comprises an assay module, such as an assay plate, slide, cartridge, bead, or chip. In one embodiment, the support surface comprises an assay flow cell or assay fluidics.

[0114] In one embodiment, the support surface comprises a plurality of addressable locations (sometimes referred to as "spots"), e.g., as is common in "gene chip" devices. In another embodiment, the array comprises a plurality of support surfaces, each having one addressable location, such as in a "bead array" approach in which each bead in a suspension of beads represents an addressable location (e.g., can be addressed using flow cytometry or microscopic detection techniques). In another embodiment, the array comprises a plurality of support surfaces, each having one or more, or two or more, addressable locations per surface. The addressable locations on the support surface can be arranged in uniform rows and columns, or can form other patterns. The number of addressable locations on an array can vary, e.g., from fewer than 10 to more than 50, 100, 200, 500, or 1000. "Multiplexing" refers to the simultaneous analysis of two or more assay targets in a single assay.

[0115] "Carbon-based" refers to a material containing elemental carbon (C) as the primary component. Examples of carbon-containing or carbon-based materials include, but are not limited to, carbon, carbon black, graphitic carbon, glassy carbon, carbon nanotubes, carbon fibrils, graphite, carbon fiber, and mixtures thereof. Carbon-based materials can include, for example, elemental carbon with graphite, carbon black, or carbon nanotubes. In one embodiment, carbon-based materials include conductive carbon-polymer composites, conductive polymers, or conductive particles dispersed in a matrix, such as carbon ink, carbon paste, or metal ink. Conductive carbon particles can include, for example, carbon fibrils, carbon black, or graphitic carbon dispersed in a polymer matrix, such as ethylene vinyl acetate (EVA), polystyrene, polyethylene, polyvinyl alcohol, polyvinyl acetate, polyvinyl chloride, or acrylonitrile butadiene styrene (ABS). Such polymer matrices can also include copolymers with two or more types of component monomers, which may include monomers selected from vinyl acetate, ethylene, vinyl alcohol, vinyl chloride, acrylonitrile, butadiene, styrene, or other monomers.

[0116] An "allele" refers to a genomic variant of a target nucleotide sequence that, when translated, may result in a functional or dysfunctional gene product. The two allelic forms are sometimes referred to as the "wild-type allele" and the "mutant" or "variant" allele. "Wild-type" refers to a nucleotide sequence that is predominant in a population. "Mutant" or "variant" refers to a nucleotide sequence that is less frequent in a population. A mutant or variant nucleotide sequence may or may not have functional consequences.

[0117] A "polymorphism" or "polymorphic site" refers to a variant in a group of two or more nucleic acids. A "single nucleotide variant" (also called a "single nucleotide polymorphism," "SNP," or "single base change") refers to a variant that involves only a single base. A single nucleotide variant can involve a substitution of one nucleotide for another at the polymorphic site, or a deletion of a nucleotide from a reference nucleotide sequence, or an insertion of a nucleotide into a reference nucleotide sequence. A single nucleotide variant can be common (e.g., present in at least 1% of a population) or rare (e.g., present in less than 1% of a population).

[0118] A "kit" refers to a set of components provided or collected to be used together, for example, to create a composition, manufacture a device, or perform a method. A kit can include one or more components. The components of a kit can be provided in one package or multiple packages, each containing one or more of the components. The listed components of a kit may be provided as a single physical part or multiple parts that are combined for use in the kit. For example, instrument components of a kit may be provided fully assembled or as multiple instrument parts that are assembled before use. Similarly, liquid reagent components of a kit may be provided as a complete liquid formulation in a container, as one or more dry reagents and one or more liquid diluents that are combined to provide a complete liquid formulation, or as two or more liquid solutions that must be combined to provide a complete liquid formulation. As is known in the art, kit components for assays are often shipped and stored separately due to different storage needs, such as storage temperatures of 4°C versus -70°C.

[0119] B. Overview

[0003] Described herein are kits for identifying, detecting, or quantifying one or more target analytes in a sample, as well as methods for making and using the same. In one embodiment, the method or kit includes one or more capture molecules that are immobilized or can be immobilized to individual binding domains on a support surface. In one embodiment, the capture molecules are single-stranded capture oligonucleotides having a nucleotide sequence that is complementary to the nucleotide sequence of a single-stranded oligonucleotide tag attached to a probe or reaction product. In one embodiment, the probe including the oligonucleotide tag associates with the target analyte and guides the target analyte to the capture molecule. In one embodiment, the target analyte is associated with a first probe including the oligonucleotide tag and a second probe including a label. In one embodiment, a reaction product is generated using the target nucleotide sequence as a template. In one embodiment, the reaction product includes the oligonucleotide tag and a label. In one embodiment, the method or kit includes one or more oligonucleotide tags. In one aspect, hybridization between the capture oligonucleotide and the complementary nucleotide sequence of the tag on the reaction product immobilizes the reaction product to a support surface so that the captured reaction product can be identified, detected, or quantified based on the attached label.

[0120] In one aspect, a method for immobilizing one or more oligonucleotides on a support surface is provided. In one aspect, the method includes immobilizing one or more oligonucleotides containing a thiol-reactive group on the support surface. In one aspect, one or more capture oligonucleotides are immobilized on the support surface in one or more binding domains. In one aspect, the method includes washing the support surface with a thiol-containing wash solution (also referred to herein as a blocking solution or blocker) to remove unbound oligonucleotides. In one aspect, each binding domain contains less than about 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of contaminating capture oligonucleotides.

[0121] In one embodiment, the methods and kits for identifying, detecting, or quantifying one or more target analytes in a sample described herein provide greater sensitivity than conventional methods. In one embodiment, the disclosed methods and kits can detect nanomolar, suitably picomolar, or more suitably femtomolar concentrations of target analytes in a sample. In one embodiment, the disclosed methods and kits can detect at least about 0.1 fM, 1 fM, 25 fM, 50 fM, 75 fM, or 100 fM and up to about 500 fM, 1 pM, 10 pM, 100 pM, 500 pM, or 1 nM of target analyte in a sample, or about 0.1 fM to about 1 nM, about 1 fM to about 100 pM, about 10 fM to about 10 pM, about 50 fM to about 1 pM, or about 100 fM to about 500 fM of target analyte. In one embodiment, the methods and kits of the disclosure are capable of detecting about 0.1 fM, about 1 fM, about 2.5 fM, about 5 fM, about 10 fM, about 25 fM, about 50 fM, about 100 fM, about 250 fM, about 500 fM, about 1 pM, about 2.5 pM, about 5 pM, about 10 pM, about 25 pM, about 50 pM, about 100 pM, or about 1 nM of target analyte in a sample.

[0122] In certain aspects, the methods and kits provided herein are capable of detecting femtomolar concentrations of polynucleotides, e.g., therapeutic oligonucleotides or RNA such as mRNA, in a sample, which advantageously allows for detection, identification, and / or quantification without the need to amplify the polynucleotide.

[0123] In one embodiment, the methods and kits provided herein can reduce the time required to identify, detect, or quantify one or more target analytes in a sample compared to conventional methods. In one embodiment, the methods and kits disclosed herein can identify, detect, or quantify one or more target analytes in a sample in about 1 hour to about 48 hours, about 1.5 hours to about 24 hours, about 2 hours to about 18 hours, about 2.5 hours to about 12 hours, about 3 hours to about 10 hours, about 3.5 hours to about 8 hours, about 4 hours to about 6 hours, or about 4.5 hours to about 5 hours. In one embodiment, the methods and kits of the present disclosure are capable of identifying, detecting or quantifying one or more target analytes in a sample in less than about 48 hours, less than about 36 hours, less than about 24 hours, less than about 18 hours, less than about 12 hours, less than about 10 hours, less than about 9 hours, less than about 8 hours, less than about 7 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour.

[0124] C. Capture molecules In one embodiment, the method or kit includes one or more capture molecules that are immobilized or can be immobilized to individual binding domains on a support surface. In one embodiment, the capture molecules are not naturally occurring sequences. In another embodiment, the capture molecules are recombinantly produced. In one embodiment, the sequences of the set of non-cross-reactive capture molecules are generated using a mathematical algorithm.

[0125] In one embodiment, the capture molecule is a single-stranded capture oligonucleotide having a nucleotide sequence that is complementary to the nucleotide sequence of the single-stranded oligonucleotide tag. In one embodiment, the oligonucleotide tag is attached to the target analyte. In one embodiment, the oligonucleotide tag is attached to a probe associated with the target analyte. In one embodiment, the oligonucleotide tag is attached to a reaction product generated using the target nucleotide sequence as a template. In one embodiment, hybridization between the capture oligonucleotide and the complementary nucleotide sequence of the oligonucleotide tag immobilizes the target or reaction product of interest on a support surface. The captured target or reaction product can then be identified, detected, or quantified based on the attached label.

[0126] In one embodiment, this method or kit comprises a different capture oligonucleotide for each target nucleotide sequence to be identified, detected or measured.In one embodiment, the hybridization between multiple capture oligonucleotides and their complementary oligonucleotide tags occurs simultaneously in parallel across the entire array of capture oligonucleotides.The array can comprise or consist of two or more capture oligonucleotides as described herein. Thus, an array can contain from 2 to 150 or more capture oligonucleotides, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or up to 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 capture oligonucleotides. The oligonucleotides in the array may comprise or consist of a "parent set" or "subset" (also referred to herein as a "set") of oligonucleotides described herein.

[0127] In one embodiment, one or more capture oligonucleotides comprise single-stranded nucleic acid sequences, including, for example, nucleic acid sequences comprising deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or structural analogs containing non-naturally occurring chemical structures that may also participate in hybridization reactions.

[0128] In one embodiment, the capture oligonucleotides used in a particular array have similar binding energies or melting temperatures (Tm), e.g., at least about 0.5°C, 1°C, 2°C, 3°C, 4°C, or within 5°C of each other, and the melting temperatures (T m ) refers to the temperature at which 50% of the oligonucleotides are hybridized to their complements and 50% are free in solution. m can be determined using known methods, for example, by measuring the change in absorbance of an oligonucleotide with its complement as a function of temperature. In one embodiment, the capture oligonucleotide has a melting temperature (T) in 50 mM NaCl of about 50°C to about 70°C, 55°C to about 65°C, or at least about 50°C, 55°C, or 60°C up to about 60°C, 65°C, or 70°C. m In one embodiment, the capture oligonucleotide has a GC content of about 40% to about 60%, or about 40% to about 50%.

[0129] In one embodiment, the capture oligonucleotide is about 20 to about 100, about 30 to about 50, or about 35 to about 40 nucleotides in length, e.g., at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 to a maximum of about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 75, or 100 nucleotides in length. In one embodiment, the capture oligonucleotide comprises at least 20, 24, 30, or 36 nucleotides. Capture oligonucleotides that are at least about 20, 24, 30, or 36 nucleotides in length can bind to tagged targets or reaction products and remain bound at higher temperatures, resulting in improved specificity (i.e., less nonspecific binding) compared to shorter capture oligonucleotides. In one embodiment, one or more capture oligonucleotides in the array are not identical in length to the nucleic acid sequence of their complementary oligonucleotide tag.In fact, it may be desirable to include a capture oligonucleotide that has a sequence that is longer than its complementary single-stranded oligonucleotide tag, for example, by up to 5, 10, 15, 20, or 25 bases.In one embodiment, the tagged target or reaction product and the capture oligonucleotide are included in a ratio of about 1:1.In another embodiment, the tagged target or reaction product is present in excess to increase the likelihood that the tagged target or reaction product will bind to the capture oligonucleotide.In one embodiment, the tagged reaction product and the capture oligonucleotide are included in a ratio of about 2:1, 3:1, 4:1, or 5:1.

[0130] In one embodiment, one or more capture oligonucleotides are covalently or non-covalently immobilized on a support surface. In one embodiment, one or more capture oligonucleotides are covalently or non-covalently immobilized to one or more binding domains on the support surface. In one embodiment, the capture oligonucleotides are adsorbed to the support surface, for example, via electrostatic interactions between negatively charged phosphate groups on the oligonucleotides and positive charges on the support surface. In one embodiment, one or more capture oligonucleotides are immobilized on the support surface via binding of a first binding partner attached to the capture oligonucleotide (directly or via a linker moiety) to a second binding partner immobilized on the surface. In one embodiment, one or more capture oligonucleotides are covalently immobilized on the support surface. In one embodiment, one or more capture oligonucleotides are directly immobilized on the support surface. In another embodiment, the capture oligonucleotides are immobilized on the support surface via a linker.

[0131] In one embodiment, one or more capture oligonucleotides comprise a reactive functional group. In one embodiment, the functional group comprises a thiol (-SH) or amine (-NH) group. In one embodiment, one or more capture oligonucleotides are immobilized to a support surface via a reactive functional group. In one embodiment, one or more capture oligonucleotides are immobilized to a support surface via a reactive functional group attached to the capture oligonucleotide via a linker. In one embodiment, the capture oligonucleotides are immobilized to a support surface via a thiol or amine group. In one embodiment, the capture oligonucleotides are immobilized to a support surface via a thiol or amine group attached to the capture oligonucleotide via a linker (also referred to herein as a "spacer"). In one embodiment, the linker comprises from about 3 to about 20 atoms or molecules or units, or at least about 3, 4, 5, 6, 7, 8, 9, 10 to up to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms or molecules or units. In one embodiment, the linker is a carbon atom linker. In one embodiment, the linker is an ethylene glycol linker or a polyethylene glycol (PEG) linker. In one embodiment, the linker comprises up to 3, 4, 5, or 6 consecutive PEG units. In another embodiment, the linker comprises 3 consecutive PEG units. In another embodiment, the linker comprises 6 consecutive PEG units. The linker may have the structure shown in Example 2.

[0132] In one embodiment, one or more capture oligonucleotides are immobilized on a support surface pretreated with a protein such as bovine serum albumin (BSA). In another embodiment, the capture oligonucleotides are immobilized on the support surface via a crosslinker. Suitable homobifunctional and heterobifunctional crosslinkers for connecting proteins and nucleic acids to each other or to other materials are well known in the art; see, for example, Thermo Scientific Crosslinking Technical Handbook, published by Thermo Fisher Scientific in 2012. In one embodiment, the crosslinker is a heterobifunctional crosslinker containing an amine-reactive moiety (such as N-hydroxysuccinimide or N-hydroxysulfosuccinimide ester) and a thiol-reactive moiety such as maleimide, iodosuccinimide, or activated disulfide (such as pyridyl disulfide). Examples of such heterobifunctional cross-functional crosslinkers include, for example, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). In one embodiment, an amine-reactive moiety (e.g., the N-hydroxysuccinimide (NHS) moiety of SMCC) reacts with a protein to introduce a thiol-reactive moiety (e.g., the maleimide moiety of SMCC) into the protein. The thiol-reactive moiety then reacts with a thiol-modified capture oligonucleotide to form a protein-oligonucleotide conjugate linked via a stable thioether bond. Arrays of protein-oligonucleotide conjugates can be formed by printing a pattern of reagents onto a surface that adsorbs or reacts with the protein to produce a patterned array. In one embodiment, the array is formed by printing the protein-oligonucleotide conjugate onto a graphitic carbon surface, such as a screen-printed carbon ink electrode. See, e.g., U.S. Patent Publication No. 2016 / 0069872 and U.S. Patents 6,977,722 and 7,842,246, the disclosures of which are incorporated herein by reference in their entireties.In one embodiment, one or more capture oligonucleotides are immobilized on a support surface that has not been pretreated with a protein. In one embodiment, as described above, the protein component of the protein-oligonucleotide used to immobilize the oligonucleotide is BSA.

[0133] In one approach, a computer algorithm is used to identify oligonucleotides that meet the following requirements: (a) a GC content of about 40% to about 50%, (b) an AG content of about 30% to about 70%, (c) a CT content of about 30% to about 70%, (d) a maximum string of three or fewer base repeats in the sequence, (e) no undesired oligonucleotide-oligonucleotide interactions with strings of more than seven consecutive complementary base pair matches, (f) no undesired oligonucleotide-oligonucleotide interactions with strings of 18 or fewer consecutive bases where (i) the terminal bases on both ends are complementary matches and (ii) the sum of the complementary base pair matches minus the sum of the mismatches is greater than 7, and (g) a match to a sequence (or the complement of the sequence, or both) in a given genome, e.g., the human genome or a sequence in nature. A set of capture oligonucleotides of the above lengths (e.g., 24-, 30-, or 36-mer) is generated that satisfies one or more of the following: (i) there are no predicted hairpin loops with four or more consecutive matches within the stem; (j) there are no predicted hairpin loops with four or more consecutive matches within the stem and a loop size greater than six bases. In one embodiment, at least criteria (a) through (h) are considered. In this context, an undesired oligonucleotide-oligonucleotide interaction refers to an interaction between an oligonucleotide and itself, another sequence in the set, or the complement of another sequence in the set. The free energy of hybridization (ΔG) is generally calculated for a specified ionic strength, temperature, and pH, for example, physiological ionic strength and pH (about 150 mM NaCl, about pH 7.2) at room temperature (about 25°C) or about 200 mM monovalent cations, about pH 7.0 at about 23°C, or other relevant conditions.Alternatively or additionally, one or more of the following configurations can be avoided: the formation of a single nucleotide loop or a single nucleotide mismatch positioned between the G / C-rich sequences when paired with other capture oligonucleotides used in the assay.

[0134] In one embodiment, the capture molecule comprises an oligonucleotide sequence set forth in any of SEQ ID NOs: 1-774 (Tables 1-12). In one embodiment, the capture oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in any of SEQ ID NOs: 1-774. In another embodiment, the capture oligonucleotide has a nucleotide sequence that comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of the sequence set forth in any of SEQ ID NOs: 1-774. In another embodiment, the capture oligonucleotide has a nucleotide sequence that comprises at least 20 consecutive nucleotides of the sequence set forth in any of SEQ ID NOs: 1-774.

[0135] In another embodiment, the capture oligonucleotide has a nucleotide sequence set forth in any of SEQ ID NOs: 1-64. In one embodiment, the capture oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in any of SEQ ID NOs: 1-64. In another embodiment, the capture oligonucleotide has a nucleotide sequence that comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of the sequence set forth in any of SEQ ID NOs: 1-64. In another embodiment, the capture oligonucleotide has a nucleotide sequence that comprises at least 20 consecutive nucleotides of the sequence set forth in any of SEQ ID NOs: 1-64.

[0136] In another embodiment, the capture oligonucleotide has a nucleotide sequence set forth in any of SEQ ID NOs: 1-10, 11-13, 25-26, 33-37, 42, 44-46, 54, and 59-62. In one embodiment, the capture oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in any of SEQ ID NOs: 1-10, 11-13, 25-26, 33-37, 42, 44-46, 54, and 59-62. In another embodiment, the capture oligonucleotide has a nucleotide sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of the sequence set forth in any of SEQ ID NOs: 1-10, 11-13, 25-26, 33-37, 42, 44-46, 54, and 59-62. In another embodiment, the capture oligonucleotide has a nucleotide sequence comprising at least 20 consecutive nucleotides of the sequence set forth in any of SEQ ID NOs: 1-10, 11-13, 25-26, 33-37, 42, 44-46, 54, and 59-62.

[0137] In another embodiment, the capture oligonucleotide has a nucleotide sequence set forth in any of SEQ ID NOs: 1-10. In one embodiment, the capture oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in any of SEQ ID NOs: 1-10. In another embodiment, the capture oligonucleotide has a nucleotide sequence that comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of the sequence set forth in any of SEQ ID NOs: 1-10. In another embodiment, the capture oligonucleotide has a nucleotide sequence that comprises at least 20 consecutive nucleotides of the sequence set forth in any of SEQ ID NOs: 1-10. In another embodiment, the capture oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides of a sequence set forth in any of SEQ ID NOs: 1-10. In another embodiment, the capture oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a sequence comprising at least 20 contiguous nucleotides of a sequence set forth in any of SEQ ID NOs: 1-10.

[0138] In one embodiment, the base sequence is used to generate a set of non-cross-reactive capture oligonucleotides using an algorithm. In one embodiment, up to four sets of non-cross-reactive capture oligonucleotides are generated, where (a) a first set of non-cross-reactive capture oligonucleotides is generated using the base sequence, (b) a second set of non-cross-reactive capture oligonucleotides having sequences complementary to the capture oligonucleotide sequences of the first set can be generated, (c) a third set of non-cross-reactive capture oligonucleotides having reverse sequences of the capture oligonucleotide sequences of the first set can be generated, and (d) a fourth set of non-cross-reactive capture oligonucleotides having sequences that are the reverse complement of the capture oligonucleotide sequences of the first set can be generated.

[0139] In one embodiment, each set of non-cross-reactive capture oligonucleotides generated using the base sequences is referred to as a "parent set." Two or more oligonucleotides from a parent set can be selected to form a "subset" (also referred to herein as a "set") of non-cross-reactive capture oligonucleotides, where each oligonucleotide in a subset is a member of the same parent set (i.e., a subset cannot include capture oligonucleotides from more than one parent set).

[0140] For example, the base sequences can be used to generate (a) a first set of parent non-cross-reactive capture oligonucleotides, (b) a second set of parent non-cross-reactive capture oligonucleotides having sequences that are complementary to the capture oligonucleotide sequences of the first set, (c) a third set of parent non-cross-reactive capture oligonucleotides having reverse sequences of the capture oligonucleotide sequences of the first set, and (d) a fourth set of parent non-cross-reactive capture oligonucleotides having sequences that are the reverse complement of the capture oligonucleotide sequences of the first set.

[0141] The subset (or set) can include (a) two or more non-cross-reactive capture oligonucleotides from a first parent set, (b) two or more non-cross-reactive capture oligonucleotides from a second parent set, (c) two or more non-cross-reactive capture oligonucleotides from a third parent set, or (d) two or more non-cross-reactive capture oligonucleotides from a fourth parent set. In one embodiment, the set or subset of non-cross-reactive capture oligonucleotides includes about 50 to about 150, about 50 to about 100, about 60 to about 75, or about 60 to about 65 non-cross-reactive capture oligonucleotides selected from the set of parent non-cross-reactive oligonucleotides. In one embodiment, the set or subset of non-cross-reactive capture oligonucleotides comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 and up to about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 70, 75, 80, 85, 90, 95, 100, 125, or 150 non-cross-reactive oligonucleotides selected from the set of parent non-cross-reactive oligonucleotides.

[0142] In one embodiment, a first base sequence is used to generate a first set of non-cross-reactive capture oligonucleotides shown in Table 1 (SEQ ID NOS: 1-64). The complementary sequence of this first set of non-cross-reactive capture oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 4 (SEQ ID NOS: 187-250). The reverse sequence of this first set of non-cross-reactive capture oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 7 (SEQ ID NOS: 373-436). The reverse complementary sequence of this first set of non-cross-reactive capture oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 10 (SEQ ID NOS: 559-622).

[0143] In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 1 (SEQ ID NOS: 1-64). In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 4 (SEQ ID NOS: 187-250). In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 7 (SEQ ID NOS: 373-436). In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 10 (SEQ ID NOS: 559-622). In one embodiment, the set of non-cross-reactive capture oligonucleotides is a subset of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, and up to 64, non-cross-reactive sequences selected from the parent set shown in Table 1 (SEQ ID NOs: 1-64), Table 4 (SEQ ID NOs: 187-250), Table 7 (SEQ ID NOs: 373-436), or Table 10 (SEQ ID NOs: 559-622).

[0144] In one embodiment, the set of non-cross-reactive capture oligonucleotides includes one or more capture oligonucleotides having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence set forth in Table 1 (SEQ ID NOs: 1-64), Table 4 (SEQ ID NOs: 187-250), Table 7 (SEQ ID NOs: 373-436), or Table 10 (SEQ ID NOs: 559-622). In another embodiment, the set of non-cross-reactive capture oligonucleotides includes one or more capture oligonucleotides having a nucleotide sequence that includes at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence set forth in Table 1 (SEQ ID NOs: 1-64), Table 4 (SEQ ID NOs: 187-250), Table 7 (SEQ ID NOs: 373-436), or Table 10 (SEQ ID NOs: 559-622). In one embodiment, the set of non-cross-reactive capture oligonucleotides includes one or more capture oligonucleotides having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 consecutive nucleotides of a sequence set forth in Table 1 (SEQ ID NOs: 1-64), Table 4 (SEQ ID NOs: 187-250), Table 7 (SEQ ID NOs: 373-436), or Table 10 (SEQ ID NOs: 559-622).

[0145] In one embodiment, the second base sequence is used to generate a second set of non-cross-reactive capture oligonucleotides shown in Table 2 (SEQ ID NOS: 65-122). The complementary sequences of this second set of non-cross-reactive capture oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 5 (SEQ ID NOS: 251-308). The reverse sequences of this second set of non-cross-reactive capture oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 8 (SEQ ID NOS: 437-494). The reverse complementary sequences of this second set of non-cross-reactive capture oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 11 (SEQ ID NOS: 623-680).

[0146] In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 2 (SEQ ID NOS: 65-122). In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 5 (SEQ ID NOS: 251-308). In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 8 (SEQ ID NOS: 437-494). In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 11 (SEQ ID NOS: 623-680). In one embodiment, the set of non-cross-reactive capture oligonucleotides is a subset of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, and up to 64, non-cross-reactive sequences selected from the parent set shown in Table 2 (SEQ ID NOs: 65-122), Table 5 (SEQ ID NOs: 251-308), Table 8 (SEQ ID NOs: 437-494), or Table 11 (SEQ ID NOs: 623-680).

[0147] In one embodiment, the set of non-cross-reactive capture oligonucleotides includes one or more capture oligonucleotides having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence set forth in Table 2 (SEQ ID NOs:65-122), Table 5 (SEQ ID NOs:251-308), Table 8 (SEQ ID NOs:437-494), or Table 11 (SEQ ID NOs:623-680). In another embodiment, the set of non-cross-reactive capture oligonucleotides includes one or more capture oligonucleotides having a nucleotide sequence that includes at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence set forth in Table 2 (SEQ ID NOs:65-122), Table 5 (SEQ ID NOs:251-308), Table 8 (SEQ ID NOs:437-494), or Table 11 (SEQ ID NOs:623-680). In one embodiment, the set of non-cross-reactive capture oligonucleotides includes one or more capture oligonucleotides having a nucleotide sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides that are at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence set forth in Table 2 (SEQ ID NOs: 65-122), Table 5 (SEQ ID NOs: 251-308), Table 8 (SEQ ID NOs: 437-494), or Table 11 (SEQ ID NOs: 623-680).

[0148] In one embodiment, the third base sequence is used to generate a third set of non-cross-reactive capture oligonucleotides shown in Table 3 (SEQ ID NOS: 123-186). The complementary sequences of this third set of non-cross-reactive capture oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 6 (SEQ ID NOS: 309-372). The reverse sequences of this third set of non-cross-reactive capture oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 9 (SEQ ID NOS: 495-558). The reverse complementary sequences of this third set of non-cross-reactive capture oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 12 (SEQ ID NOS: 681-744).

[0149] In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 3 (SEQ ID NOS: 123-186). In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 6 (SEQ ID NOS: 309-372). In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 9 (SEQ ID NOS: 495-558). In one embodiment, the set of non-cross-reactive capture oligonucleotides comprises two or more sequences from the parent set shown in Table 12 (SEQ ID NOS: 681-744). In one embodiment, the set of non-cross-reactive capture oligonucleotides is a subset of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, and up to 64, non-cross-reactive sequences selected from the parent set shown in Table 3 (SEQ ID NOs: 123-186), Table 6 (SEQ ID NOs: 309-372), Table 9 (SEQ ID NOs: 495-558), or Table 12 (SEQ ID NOs: 681-744).

[0150] In one embodiment, the set of non-cross-reactive capture oligonucleotides includes one or more capture oligonucleotides having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence set forth in Table 3 (SEQ ID NOs:123-186), Table 6 (SEQ ID NOs:309-372), Table 9 (SEQ ID NOs:495-558), or Table 12 (SEQ ID NOs:681-744). In another embodiment, the set of non-cross-reactive capture oligonucleotides includes one or more capture oligonucleotides having a nucleotide sequence that includes at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence set forth in Table 3 (SEQ ID NOs:123-186), Table 6 (SEQ ID NOs:309-372), Table 9 (SEQ ID NOs:495-558), or Table 12 (SEQ ID NOs:681-744). In one embodiment, the set of non-cross-reactive capture oligonucleotides includes one or more capture oligonucleotides having a nucleotide sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides that are at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence set forth in Table 3 (SEQ ID NOs:123-186), Table 6 (SEQ ID NOs:309-372), Table 9 (SEQ ID NOs:495-558), or Table 12 (SEQ ID NOs:681-744).

[0151] In one embodiment, the set of non-cross-reactive capture oligonucleotides includes capture oligonucleotides having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-64, capture oligonucleotides having a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 1-64, and capture oligonucleotides having a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 1-64. The capture oligonucleotides include one or more capture oligonucleotides selected from: a capture oligonucleotide having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-64; a capture oligonucleotide having a sequence selected from SEQ ID NOs: 1-64; and combinations thereof.

[0152] In one embodiment, the set of non-cross-reactive capture oligonucleotides includes capture oligonucleotides having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-10, capture oligonucleotides having a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 1-10, and capture oligonucleotides having a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 1-10. The capture oligonucleotides include one or more capture oligonucleotides selected from: a capture oligonucleotide having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-10; a capture oligonucleotide having a sequence selected from SEQ ID NOs: 1-10; and combinations thereof.

[0153] In one embodiment, the capture oligonucleotide is covalently bound to a protein, and immobilization to the support surface is achieved through protein adsorption to the support surface. Examples of proteins that can be used include albumins, such as bovine serum albumin (BSA), immunoglobulins, or other proteins selected for their ability to adsorb to the support surface. In another embodiment, the capture oligonucleotide is attached (directly or via a linker) to a first binding partner from a binding partner pair, and immobilization is achieved by binding of this first binding partner to a second binding partner from the binding partner pair immobilized on the support surface. Binding partner pairs suitable for use in immobilizing the capture oligonucleotide include those known in the art, such as biotin-streptavidin, biotin-avidin, antibody-hapten, antibody-epitope tag (e.g., antibody-FLAG), nickel-NTA, and receptor-ligand pairs. In one embodiment, the capture oligonucleotide is covalently bound to the protein or first binding partner via a thiol (-SH) or amine (-NH) group. This attachment can be direct or via a linking group (e.g., a bifunctional linking group such as those described in the Thermo Scientific Crosslinking Technical Handbook, published by Thermo Fisher Scientific in 2012). In one embodiment, the thiol or amine group is at the 5' or 3' end of the capture oligonucleotide. In one embodiment, the capture oligonucleotide is a 5'-terminally thiolated oligonucleotide. In one embodiment, the capture oligonucleotide is a 3'-terminally thiolated oligonucleotide. In one embodiment, the thiol group is incorporated at an internal position of the capture oligonucleotide. In one embodiment, the capture oligonucleotide has a nucleotide sequence comprising a sequence set forth in any of SEQ ID NOs: 1489-1498 (Table 25). In one embodiment, the capture oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in any of SEQ ID NOs: 1489-1498.In another embodiment, the capture oligonucleotide has a nucleotide sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides of a sequence set forth in SEQ ID NOs: 1489-1498. In another embodiment, the capture oligonucleotide has a nucleotide sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a sequence set forth in any of SEQ ID NOs: 1489-1498.

[0154] In one embodiment, the capture oligonucleotide is covalently attached to the support surface via a thiol (-SH) or amine (-NH2) group. In one embodiment, the thiol or amine group is at the 5' or 3' end of the capture oligonucleotide. In one embodiment, the capture oligonucleotide is a 5'-terminally thiolated oligonucleotide. In one embodiment, the capture oligonucleotide is a 3'-terminally thiolated oligonucleotide. In one embodiment, the thiol group is incorporated at an internal position of the capture oligonucleotide. In one embodiment, the capture oligonucleotide has a nucleotide sequence comprising a sequence set forth in any of SEQ ID NOs: 1489-1498 (Table 25). In one embodiment, the capture oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in any of SEQ ID NOs: 1489-1498. In another embodiment, the capture oligonucleotide has a nucleotide sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides of a sequence set forth in SEQ ID NOs: 1489-1498. In another embodiment, the capture oligonucleotide has a nucleotide sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a sequence set forth in any of SEQ ID NOs: 1489-1498.

[0155] In one embodiment, the capture oligonucleotide has a nucleotide sequence that is the complement, reverse complement, or reverse complement of the nucleotide sequence set forth in SEQ ID NOs: 1489-1498. In one embodiment, the capture oligonucleotide has a nucleotide sequence that is the complement, reverse complement, or reverse complement of a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NOs: 1489-1498. In another embodiment, the capture oligonucleotide has a sequence that is the complement, reverse complement, or reverse complement of a sequence having at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides of the sequence set forth in SEQ ID NOs: 1489-1498. In another embodiment, the capture oligonucleotide has a nucleotide sequence that is the complement, reverse complement, or inverted complement of a sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a sequence set forth in any of SEQ ID NOs:1489-1498.

[0156] In one embodiment, one or more capture oligonucleotides contain only three bases (TAG) to reduce hybridization with native sequences, similar to Luminex x-TAG technology.

[0157] D.Support surface In one embodiment, one or more capture oligonucleotides are immobilized on a support surface. The capture oligonucleotides can be immobilized on a variety of support surfaces, including support surfaces used in conventional binding assays. In one embodiment, the support surface has a flat surface. In another embodiment, the support surface has a curved surface. In one embodiment, the support surface comprises an assay module such as an assay plate, slide, cartridge, bead, or chip. In one embodiment, the support surface comprises color-coded microspheres. See, e.g., Yang et al. (2001) BADGE, BeadsArray for the Detection of Gene Expression, a High-Throughput Diagnostic Bioassay. Genome Res. 11(11):1888-1898. In one embodiment, the support surface comprises one or more beads to which one or more capture oligonucleotides are immobilized.

[0158] The support surface can be made from a variety of suitable materials, including polymers such as polystyrene and polypropylene, ceramics, glasses, and composite materials, including carbon-polymer composites such as carbon-based inks. In one aspect, the support surface is a carbon-based support surface.

[0159] In one embodiment, the support surface is provided by one or more particles or "beads." In one embodiment, the beads can have a diameter of up to about 1 cm (or 10,000 μm), 5,000 μm, 1,000 μm, 500 μm, or 100 μm. In one embodiment, the beads have a diameter of about 10 nm to about 100 μm, about 100 nm to about 10 μm, or about 0.5 μm to about 5 μm. In one embodiment, the beads are paramagnetic, providing the ability to capture the beads using a magnetic field. In one embodiment, the support surface is provided by streptavidin- or avidin-coated magnetic beads, and biotin-labeled capture oligonucleotides are immobilized on the beads.

[0160] In one embodiment, the support surface is a plate with multiple wells, i.e., a "multiwell plate." A multiwell plate can contain any number of wells of any size or shape, arranged in any pattern or configuration. In one embodiment, the multiwell plate contains from about 1 to about 10,000 wells. In one embodiment, the multiwell assay plate uses an industry-standard format for the number, size, shape, and configuration of plates and wells. Examples of standard formats include 96-, 384-, 1536-, and 9600-well plates, where the wells are arranged in a two-dimensional array. Other multiwell formats include single-well, 2-well, 6-well, 24-well, and 6144-well plates. In one embodiment, the support surface comprises a 96-well plate.

[0161] In one embodiment, the support surface comprises a two-dimensional patterned array in which capture molecules are printed at known locations, called binding domains. In one embodiment, the support surface comprises a patterned array of distinct, non-overlapping, addressable binding domains to which capture oligonucleotides are immobilized, and the sequence of the capture oligonucleotides in each binding domain is known and can be correlated with the appropriate target analyte or target reaction product. In one embodiment, all capture oligonucleotides within a particular binding domain have the same sequence, and capture oligonucleotides in one binding domain have sequences that differ from those in other binding domains. In one embodiment, multiple binding domains are arranged in ordered rows and columns on the support surface, and the exact location and sequence of each binding domain is recorded in a computer database. In one embodiment, the array is arranged in a symmetrical grid pattern. In other embodiments, the array is arranged in other patterns, including, but not limited to, radially distributed lines, spiral lines, or ordered clusters. In another embodiment, each binding domain is positioned on the surface of one or more microparticles or beads, which are coded to enable discrimination between different binding domains.

[0162] In one embodiment, the support surface is a multi-well plate containing one or more individual addressable binding domains in each well corresponding to one or more capture oligonucleotides. In one embodiment, the support surface contains at least one binding domain for detecting wild-type nucleotide sequences and a separate binding domain for detecting mutant nucleotide sequences. In one embodiment, each well contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 binding domains. In one embodiment, each well contains at least 7, 10, 16, or 25 binding domains.

[0163] In one embodiment, the support surface is a multiwell plate containing at least 24, 96, or 384 wells, each well containing an array of up to 10 binding domains, with a different capture oligonucleotide immobilized on each binding domain. In a more specific embodiment, the support surface is a 96-well plate, each well containing an array with up to 10 binding domains. In one embodiment, each well of the 96-well plate contains up to 10 binding domains, with up to 10 individual capture oligonucleotides immobilized thereon. In one embodiment, each well contains the same patterned array with the same capture oligonucleotide. In another embodiment, different wells may contain different patterned arrays of capture oligonucleotides.

[0164] E. Electrode In one embodiment, target analytes, including, for example, polypeptides or nucleic acid sequences, are identified, detected, or quantified using electrochemiluminescence. Multiplexed measurement of analytes using electrochemiluminescence is described in U.S. Patent Nos. 7,842,246 and 6,977,722, the disclosures of which are incorporated herein by reference in their entireties.

[0165] In one embodiment, the support surface comprises one or more electrodes. In one embodiment, the support surface comprises one or more working electrodes and one or more counter electrodes. In one embodiment, the support surface comprises one or more binding domains formed on the one or more electrodes for use in electrochemical or electrochemiluminescent assays.

[0166] In one aspect, the binding domains are formed by collecting beads coated with capture oligonucleotides onto an electrode surface, hi one aspect, the beads are paramagnetic and are collected onto the electrode by using a magnetic field.

[0167] In one embodiment, one or more capture oligonucleotides are covalently or non-covalently immobilized to one or more binding domains on one or more electrodes on a support surface, hi one embodiment, multiple individual binding domains are present on one or more electrodes for multiplexed measurement of target analytes in a sample.

[0168] In one embodiment, the electrodes are provided in an assay module that provides an assay vessel, an assay flow cell, assay fluidics, or other components useful for performing an assay. Examples of assay modules for performing electrochemiluminescence assays include, for example, a multi-array case, an assay plate case, a cartridge case, and the like. In one embodiment, the electrodes are provided in an assay module that provides an assay vessel, an assay flow cell, assay fluidics, or other components useful for performing an assay. Examples of assay modules for performing electrochemiluminescence assays are described in U.S. Patent Nos. 6,673,533, 7,842,246, 9,731,297, and 8,298,834. In one embodiment, the support surface is a multiwell plate that includes at least one electrode. In one embodiment, each well of the multiwell assay plate includes at least one electrode. In one embodiment, at least one well of the multiwell assay plate includes a working electrode. In another embodiment, at least one well of the multiwell assay plate includes a working electrode and a counter electrode. In another aspect, each well of the multi-well assay plate comprises a working electrode and a counter electrode. In one aspect, the working electrode is adjacent to, but not in electrical contact with, the counter electrode.

[0169] In one embodiment, the electrodes are constructed from conductive materials, including metals such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, conductive alloys, or combinations thereof. In another embodiment, the electrodes include semiconductor materials such as silicon and germanium, or semiconductor films such as indium tin oxide (ITO) and antimony tin oxide (ATO). In another embodiment, the electrodes include metals coated with oxides, such as aluminum coated with aluminum oxide. In one embodiment, the electrodes include carbon-based materials. In one embodiment, the electrodes include conductive composites, inks, pastes, polymer blends, and mixtures of materials containing metal / non-metal composites, including, for example, mixtures of conductive or semiconductive and non-conductive materials. In one embodiment, the electrodes include carbon-based materials, such as carbon, glassy carbon, carbon black, graphitic carbon, carbon nanotubes, carbon fibrils, graphite, carbon fibers, and mixtures thereof. In one embodiment, the electrode includes a conductive carbon-polymer composite, a conductive polymer, or conductive particles dispersed in a matrix, such as carbon ink, carbon paste, or metal ink. In one embodiment, the working electrode is comprised of a carbon-polymer composite including conductive carbon particles, such as carbon fibrils, carbon black, or graphitic carbon, dispersed in a polymer matrix, such as ethylene vinyl acetate (EVA), polystyrene, polyethylene, polyvinyl acetate, polyvinyl chloride, polyvinyl alcohol, acrylonitrile butadiene styrene (ABS), or a copolymer of one or more of these polymers.

[0170] In one embodiment, the working electrode is made of a continuous conductive sheet or film of one or more conductive materials, which can be extruded, pressed, or molded. In another embodiment, the working electrode is made of a conductive material deposited or patterned on a substrate by, for example, printing, painting, coating, spin coating, vapor deposition, chemical vapor deposition, electrodeposition, electroless deposition, photolithography, or other electronics microfabrication techniques. In one embodiment, the working electrode includes a conductive carbon ink printed on a polymer support by, for example, inkjet printing, laser printing, or screen printing. Carbon inks are known and include materials produced by Acheson Colloids Co. (e.g., Acheson 440B, 423ss, PF407A, PF407C, PM-003A, 30D071, 435A, Electrodag 505SS, and Aquadag™), EI Du Pont de Nemours and Co. (e.g., DuPont 7105, 7101, 7102, 7103, 7144, 7082, 7861D, and CB050), Conductive Compounds Inc. (e.g., C-100), and Ercon Inc. (e.g., G-451).

[0171] In one embodiment, the working electrode is a continuous film. In another embodiment, the working electrode includes one or more discrete regions or a pattern of discrete regions. Alternatively, the working electrode may include multiple connected regions. One or more regions of exposed electrode surface on the working electrode may be defined by a patterned insulating layer covering the working electrode, for example, by screen printing a patterned dielectric ink layer on the working electrode or by adhering a die-cut insulating film. The exposed regions may define array elements of an array of reagents printed on the working electrode, and may take the shape and pattern of an array as described above. In one embodiment, the insulating layer defines a series of circular regions (or "spots") of exposed working electrode surface.

[0172] The counter electrode may generally have one or more of the properties described above for the working electrode. In one embodiment, the working electrode and the counter electrode are constructed of the same material. In another embodiment, the working electrode and the counter electrode are not constructed of the same material; for example, the working electrode may be a carbon electrode and the counter electrode may be a metal electrode.

[0173] In one embodiment, one or more capture oligonucleotides are immobilized on one or more electrodes by passive adsorption. In another embodiment, one or more capture oligonucleotides are covalently immobilized on the electrode. In one embodiment, the electrode is derivatized or modified, for example, to immobilize reagents, such as capture oligonucleotides, on the surface of the electrode. In one embodiment, the electrode is modified by chemical or mechanical treatment to improve reagent immobilization, for example, to introduce functional groups for reagent immobilization or to enhance its adsorption properties. Examples of functional groups that can be introduced include carboxylic acid (COOH), hydroxy (OH), amino (NH), activated carboxyl (e.g., N-hydroxysuccinimide (NHS)-ester), poly-(ethylene glycol), thiol, alkyl ((CH) n) groups, or combinations thereof. In one embodiment, one or more reagents, e.g., capture oligonucleotides, are immobilized to a carbon-containing electrode, e.g., carbon black, fibrils, or carbon dispersed in another material, by either covalent or non-covalent means. It has been discovered that capture molecules bearing thiol groups can be covalently attached to a carbon-containing electrode, e.g., a screen-printed carbon ink electrode, without the need to first deposit an additional thiol-reactive layer, such as a protein layer or a chemical crosslinking layer. In one embodiment, a method is provided for directly attaching capture molecules bearing thiol groups, such as thiol-modified oligonucleotides, to an electrode, providing a simple, robust, efficient, and reproducible process for forming capture surfaces and arrays on the electrode. In one embodiment, one or more capture oligonucleotides bearing thiol groups are immobilized directly to a carbon-containing electrode, such as a screen-printed carbon ink electrode, through reaction of the thiol with the electrode, without first adding a thiol-reactive layer to the electrode.

[0174] In one embodiment, the electrode is treated with a plasma, e.g., a low-temperature plasma such as a glow discharge plasma, to alter the physical properties, chemical composition, or surface chemistry of the electrode, for example, to aid in the immobilization of reagents such as capture oligonucleotides, or to reduce contaminants, improve adhesion to other materials, alter surface wettability, promote material deposition, create patterns, or improve uniformity. Examples of useful plasmas include oxygen, nitrogen, argon, ammonia, hydrogen, fluorocarbons, water, and combinations thereof. In one embodiment, an oxygen plasma is used to treat the electrode with carbon particles in a carbon-polymer composite. In another embodiment, oxygen is used to introduce carboxylic acid or other oxidized carbon functional groups into carbon or organic materials (e.g., active esters or acyl chlorides) to facilitate reagent coupling. In another embodiment, an ammonia-containing plasma can be used to introduce amino groups for use in coupling assay reagents. In one embodiment, the electrode is not pretreated to aid in the immobilization of one or more capture oligonucleotides.

[0175] In one embodiment, the support surface comprises an assay module such as a multiwell plate having one or more working or counter electrodes in each well. In one embodiment, the multiwell plate comprises multiple working or counter electrodes in each well. In one embodiment, the working or counter electrode of the multiwell plate comprises a screen-printed layer of carbon, e.g., carbon ink. In one embodiment, one or more capture oligonucleotides are immobilized on the screen-printed carbon ink via thiol moieties on the capture oligonucleotides. In one embodiment, the working electrode is used to induce an electrochemiluminescent signal from a label attached to the reaction product. In one embodiment, the electrochemiluminescent signal is released from ruthenium-tris-bipyridine in the presence of a co-reactant such as a tertiary alkylamine, e.g., tripropylamine or butyldiethanolamine.

[0176] In one embodiment, the electrode contains binding domains as described above, defined by a dielectric ink (i.e., an electrically insulating ink). The electrode is a working electrode with a dielectric printed thereon in a pattern defining the binding domains. In one embodiment, the binding domains are approximately circular areas of exposed working electrodes (or "spots"). The electrodes are in a 96-well plate formed by adhering the top of an injection-molded 96-well plate to a Mylar sheet that defines the bottom of the wells. The top surface of the Mylar sheet is printed with screen-printed carbon ink electrodes, with each well containing a carbon ink working electrode approximately in the center of the well and two carbon ink counter electrodes approximately toward the two ends of the well. Electrodes printed on the bottom of the Mylar sheet and connected to the top of the sheet via conductive through-holes provide contacts for applying voltages to the working and counter electrodes.

[0177] F. Methods for Immobilizing Capture Molecules In one embodiment, a method for immobilizing one or more capture molecules on a support surface is provided, in one embodiment, the one or more capture molecules comprise one or more single-stranded capture oligonucleotide molecules as described herein.

[0178] In one embodiment, the method comprises immobilizing one or more capture molecules on a support surface comprising a carbon-based support surface. In one embodiment, the method comprises immobilizing one or more capture molecules on a support surface comprising one or more electrodes. In one embodiment, the method comprises immobilizing one or more capture molecules on a support surface comprising one or more carbon-based electrodes.

[0179] In one embodiment, the support surface is a multi-well plate comprising one or more electrodes. In one embodiment, the support surface is a multi-well plate comprising one or more electrodes in each well. In one embodiment, one or more capture molecules are immobilized on the support surface in an array.

[0180] In one embodiment, the method includes spotting or printing two or more capture oligonucleotides in an array onto a first electrode of a first well of a multiwell plate, followed by printing one or more capture oligonucleotides in an array onto electrodes of one or more additional wells of the multiwell plate. In one embodiment, at least some of the printed arrays in each well are the same. In another embodiment, at least some of the printed arrays in each well are different.

[0181] In one embodiment, one or more capture molecules are spotted or printed in one or more known positions in the array, which are called binding domains.In one embodiment, one or more capture oligonucleotides are immobilized in individual, non-overlapping, addressable binding domains, and the sequence of the capture oligonucleotide in each binding domain is known and can be correlated with target analyte.In one embodiment, all capture oligonucleotides in a specific binding domain have the same sequence, and the capture oligonucleotide in one binding domain has a different sequence from the capture oligonucleotide in other binding domains.

[0182] In one embodiment, one or more capture molecules are spotted or printed onto individual binding domains on the support surface. In one embodiment, an array of capture oligonucleotides is spotted or printed onto individual binding domains on the support surface. In one embodiment, the capture molecules are spotted or printed by contact printing, including, for example, contact pin printing or microstamping, or by non-contact printing, including, for example, photolithography, laser writing, electrospray deposition, and inkjet printing. Generally, the spotting or printing method involves applying one or more droplets containing one or more capture molecules to individual binding domains on the support surface and allowing the droplets to dry. In one embodiment, the droplets can spread to cover an area of ​​the support surface. In one embodiment, the support surface comprises one or more areas of higher wettability and one or more areas of lower wettability, where the higher wettability areas define the binding domains or array elements. Wettability refers to the interaction between a liquid and a solid surface, more specifically, the phenomenon in which an aqueous solution does not spread on a solid surface but shrinks to form droplets. In one embodiment, the solid support has surface properties that promote droplet formation when a small amount of aqueous solution is dispensed onto one or more individual binding domains. A solution of capture molecules printed onto the highly wettable regions spreads to the boundary with the less wettable regions, precisely controlling the shape and location of the binding domains. In one embodiment, the binding domains are regions of the exposed electrode surface on the working electrode, and a patterned insulating layer on the working electrode (e.g., screen-printed dielectric ink on a screen-printed carbon ink electrode) defines the lower wettable boundary of the exposed electrode region.

[0183] Methods for immobilizing oligonucleotides on support surfaces are known (see, e.g., Balasheb Nimse et al. (2014) Immobilization Techniques for Microarray: Challenges and Applications Sensors. 14(2):22208-22229) and generally rely on one or more of the following mechanisms: (1) physical adsorption, e.g., via charge-charge or hydrophobic interactions; (2) covalent immobilization, e.g., via chemical bonds; and (3) non-covalent protein-ligand interactions, such as streptavidin-biotin immobilization. In one embodiment, one or more oligonucleotides are immobilized on a functionalized support surface. In one embodiment, one or more oligonucleotides are immobilized on a support surface that has not been modified to include one or more functional groups. In one embodiment, one or more oligonucleotides are immobilized by physical absorption onto a support surface comprising one or more of the following moieties: amine, nitrocellulose, poly(l-lysine), PAAH, and diazonium. In one embodiment, one or more oligonucleotides are immobilized on the support surface by covalent interaction, for example, via a thiol (-SH) or amine (-NH) group, or a hydrazide group. In one embodiment, the support surface contains or is modified to contain a reactive functional group, for example, including carboxyl (-COOH), aldehyde (-CHO), epoxy (-CHCHO), isothiocyanate (-N=C=S), maleimide (-HC(CO)NH), or mercaptosilane (-Si-R-SH). In one embodiment, the oligonucleotide contains or is modified to contain a reactive functional group, for example, including a thiol, amine, or hydrazide group. In one embodiment, one or more oligonucleotides are immobilized on the support surface via a nucleophilic or electrophilic functional group present on the support surface.

[0184] In one embodiment, one or more capture molecules comprise a thiol group. In one embodiment, one or more capture molecules are immobilized on a support surface via a thiol group present on the capture molecule. In one embodiment, the method comprises spotting or printing one or more capture molecules comprising a thiol group onto a carbon-based support surface, and incubating the printed support surface to immobilize one or more capture molecules on the support surface via the thiol group. In one embodiment, one or more capture molecules are covalently attached to the support surface via the thiol group.

[0185] In one embodiment, one or more capture molecules are immobilized on a support surface by printing droplets (e.g., 50 nL) containing the capture molecules onto the support surface, spreading the droplets, drying the droplets, and incubating the dried droplets for a time sufficient to immobilize the capture molecules on the support surface (e.g., overnight). In one embodiment, one or more capture molecules comprising thiol groups are immobilized on a carbon-based support surface by printing droplets containing the capture molecules onto the support surface, spreading the droplets, drying the droplets, and incubating the dried droplets for a time sufficient to immobilize the capture molecules on the support surface via the thiol groups. In one embodiment, the droplets are printed in an array. In one embodiment, the droplets are printed with one or more binding domains. In one embodiment, the carbon-based support surface comprises one or more carbon-based electrodes. In one embodiment, the one or more capture molecules are covalently attached to the carbon-based electrodes via the thiol groups. In one embodiment, a patterned insulating layer is included on the carbon-based support surface to define the spreading of droplets printed on the support surface.

[0186] In one embodiment, the carbon-based support surface is pretreated, for example, to introduce one or more functional groups onto the support surface, for example, to increase the reactivity between the thiol groups on the capture molecules and the support surface. In one embodiment, the carbon-based support surface is pretreated with a protein such as bovine serum albumin (BSA). In another embodiment, the carbon-based support surface is not pretreated to introduce functional groups onto the support surface before immobilizing one or more capture oligonucleotides onto the support surface via the thiol groups. In one embodiment, the support surface is not modified with a protein to increase the reactivity between the capture molecules and the thiol groups on the support surface.

[0187] In one embodiment, after one or more capture oligonucleotides are spotted or printed onto a surface to remove free capture oligonucleotides (i.e., capture oligonucleotides not immobilized on the support surface), the support surface is washed with a washing (or blocking) solution (also referred to herein as the "blocking" step; see, e.g., Example 3). In one embodiment, the support surface is washed with a washing solution after printing and drying. In one embodiment, the support surface is washed before being packaged in a dry package. In another embodiment, the support surface is washed after being packaged in a dry package.

[0188] In one aspect, the washing or blocking step involves adding a washing or blocking solution to the surface (e.g., 50 uL of solution per well for a 96-well assay plate) and incubating for 30-60 minutes. The incubation temperature can be any convenient temperature, e.g., room temperature or 37°C. The incubation can be performed while shaking the surface. The washing or blocking step can include removing the washing or blocking solution and rinsing the surface with a buffer such as PBS.

[0189] In one embodiment, the wash solution contains a thiol-containing compound. During the wash step, excess thiol-containing capture molecules from one binding domain on the carbon-based electrode may migrate to another binding domain and become permanently immobilized. This migration of capture molecules and the resulting cross-contamination of binding domains can be reduced by including a thiol-containing compound in the wash solution. Without wishing to be bound by theory, it is believed that the thiol-containing compound in the wash solution competes with free (unbound) capture oligonucleotides, preventing cross-contamination of binding domains due to the binding of excess capture oligonucleotides removed from different binding domains. In one embodiment, the wash solution contains a water-soluble thiol-containing compound. In one embodiment, the wash solution contains a water-soluble thiol-containing compound having a molecular weight of less than about 200 g / mol, about 175 g / mol, about 150 g / mol, or about 125 g / mol. In one embodiment, the water-soluble thiol-containing compound contains a zwitterion.

[0190] In one embodiment, the cleaning solution comprises a water-soluble thiol selected from cysteine ​​(e.g., L-cysteine), cysteamine, dithiothreitol, 3-mercaptoproprionate, and 3-mercapto-1-propanesulfonic acid. In one embodiment, the water-soluble thiol-containing compound comprises cysteine.

[0191] In one embodiment, the wash solution comprises a pH buffering component. In one embodiment, the pH buffering component comprises Tris. In one embodiment, the wash solution comprises a surfactant. In one embodiment, the surfactant comprises Triton X-100. In one embodiment, the wash solution comprises a metal chelator.

[0192] In one embodiment, the wash solution contains about 5 mM to about 750 mM, about 10 mM to about 500 mM, about 25 mM and about 75 mM, or about 50 mM of a thiol-containing compound. In one embodiment, the wash solution contains about 5 mM to about 750 mM, about 10 mM to about 500 mM, about 25 mM and about 75 mM, or about 50 mM of cysteine. In one embodiment, the wash solution contains about 10 mM to about 30 mM, or about 15 mM to about 25 mM, or about 20 mM of a buffer such as Tris. In one embodiment, the wash solution contains about 0.05% to about 0.5%, or about 0.05% to 0.2%, or about 0.1% of a surfactant such as Triton X-100. In one embodiment, the wash solution has a pH of about 7 to about 9, about 7.5 to about 8.5, or about 8.0.

[0193] In one embodiment, the wash or blocking solution may contain the following reagents: (i) known polymers useful for reducing background signals in hybridization assays, including, but not limited to, PS20, polyvinyl alcohol (PVA), polyvinylpyrrolidone (about 1,000 kD or about 360 kD), Ficoll, and polyethylene glycol (about 3 kD and about 10 kD); and (ii) nucleic acids or their derivatives, including, but not limited to, salmon sperm DNA, herring DNA, calf thymus DNA, sheared polyA, and yeast tRNA. and heparin; (iii) monomeric and polymeric protein blocking agents, including but not limited to BSA and polyBSA; (iv) surfactants, including but not limited to sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Triton-100, and Tween-20; and (v) hydrogen bond destabilizing agents, including but not limited to formamide and propylene glycol.

[0194] In one embodiment, the method includes immobilizing one or more capture oligonucleotides on a support surface and then washing excess non-immobilized capture oligonucleotides from the support surface with a wash solution. In one embodiment, the washing includes washing the immobilized capture oligonucleotides under stringent wash conditions. In one embodiment, stringent wash conditions include a temperature of about 27°C to about 47°C, a formamide concentration of about 21% to about 41%, a salt concentration of about 300 mM to about 500 mM, and a pH of about 7.5 to about 8.5. In one embodiment, high stringency conditions include a temperature of about 37°C, a formamide concentration of about 31%, a salt concentration of about 400 mM, and a pH of 8.0. In one embodiment, the immobilized oligonucleotides are exposed to the high stringency conditions for at least 5, 10, 30, or 60 minutes. In another embodiment, high stringency conditions comprise low salt conditions, e.g., a buffer having a salt concentration of less than about 40 mM, 20 mM, 15 mM, or 10 mM. In one embodiment, high stringency conditions comprise low salt conditions, such as 0.1x PBS at 37°C.

[0195] In one embodiment, one or more capture oligonucleotides are immobilized on the support surface in an array.In one embodiment, one or more capture oligonucleotides are immobilized on the support surface in one or more binding domains.In one embodiment, the capture oligonucleotides printed on one binding domain of the array have a different sequence from the capture oligonucleotides printed on other binding domains of the array.

[0196] Without wishing to be bound by theory, it is believed that the wash solution brings loosely bound capture oligonucleotides into solution, from which they may be redeposited on the surface via either SH covalent bonds or other mechanisms. If a capture oligonucleotide is redeposited on a binding domain having a capture oligonucleotide with a different nucleotide sequence, it is considered a contaminating capture molecule. The presence of contaminating capture molecules may interfere with the results of the assay. In one embodiment, the binding domains of the array prepared by the methods described herein contain less than about 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% contaminating capture molecules.

[0197] In one embodiment, the cross-reactivity between the binding partners (i.e., oligonucleotide tags) of a set of capture molecules is less than about 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01%. In one embodiment, the specificity of the assay (including cross-reactivity from either binding of non-complementary sequences or cross-contamination of capture oligonucleotides) is determined. In one embodiment, the specificity is determined by adding one or more samples containing one or more labeled QC probes to one or more replicate plates under conditions in which the QC probes hybridize to the corresponding complementary capture molecules immobilized on the plate surface. The plate is then washed to remove excess QC probes, and the presence of bound QC probes is detected by either detecting the primary label or adding a secondary binding partner. Cross-reactivity can be calculated for each array, e.g., for each well of a multi-well plate, as the percentage of the signal detected from the probe binding to a spot with a non-specific capture nucleotide relative to the signal from the probe binding to a spot with the corresponding complementary capture nucleotide. In one embodiment, the calculation includes correction for the non-specific background signal detected in the absence of the QC probe.

[0198] In one embodiment, assay specificity is determined using a series of quality control (QC) oligonucleotide probes. In one embodiment, the QC probes comprise nucleotide sequences complementary to at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleic acids of corresponding capture molecules in a set of non-cross-reactive capture molecules immobilized on a surface. In one embodiment, the set includes QC probes having nucleotide sequences complementary to at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleic acids of corresponding capture molecules in a set of non-cross-reactive capture molecules having a sequence set forth in any of SEQ ID NOs: 1-774. In one embodiment, the set includes QC probes having nucleotide sequences complementary to at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleic acids of corresponding capture molecules in the set of non-cross-reactive capture molecules set forth in SEQ ID NOs: 1-64. In one embodiment, the set includes QC probes having nucleotide sequences complementary to at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleic acids of corresponding capture molecules in the set of non-cross-reactive capture molecules set forth in SEQ ID NOs: 1-10.

[0199] In one embodiment, the QC probe comprises a label. In one embodiment, the label is directly attached to the QC probe. In another embodiment, the label is attached to the QC probe via a linker. In one embodiment, the label is a compound that is a member of a binding pair, where the first member of the binding pair (which can be referred to as the "primary binding reagent") is attached, for example, to a substrate or oligonucleotide, and the other member of the binding pair (which can be referred to as the "secondary binding reagent") has a detectable physical property. Examples or primary labels include, but are not limited to, electrochemiluminescent labels and organometallic complexes containing transition metals, such as ruthenium. In one embodiment, the primary label comprises streptavidin. In one embodiment, the primary label comprises MSD SULFO-TAG-labeled streptavidin.

[0200] In one embodiment, the label comprises a secondary binding reagent that binds to the primary binding reagent. In one embodiment, the primary binding reagent includes biotin, a hapten, streptavidin, avidin, or an antibody or antigen. In one embodiment, the secondary binding reagent includes biotin, a hapten, streptavidin, avidin, or an antibody or antigen. In one embodiment, the secondary binding reagent includes an electrochemiluminescent label. In one embodiment, the secondary binding reagent includes an organometallic complex containing a transition metal, e.g., ruthenium. In one embodiment, the QC probe comprises biotin and the secondary binding reagent comprises MSD SULFO-TAG-labeled streptavidin. In one embodiment, the QC probe is modified at the 3' end with biotin, as shown in the following structure: [ka]

[0201] In one embodiment, the percent of contaminated capture molecules is measured by the method of Example 4.

[0202] In one embodiment, the uniformity of one or more binding domains on a plate (intra-plate) or across two or more plates (inter-plate) can be determined using known methods for determining the coefficient of variation (CV). In one embodiment, the intra-plate or inter-plate binding domains have a CV of less than about 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, or 1%. In one embodiment, the average intra-plate or inter-plate CV is about 3% to about 6%, or less than about 5%. In one embodiment, the uniformity of the binding domains is measured by the method of Example 5.

[0203] G. Aptamer Immobilization In one embodiment, a method for immobilizing one or more aptamers on a support surface is provided. In one embodiment, one or more aptamers are immobilized on a support surface by binding to one or more single-stranded capture molecules that are immobilized on the support surface as described herein. In one embodiment, the aptamer is an oligonucleotide capable of specifically binding to a target molecule, and can include, for example, DNA, RNA, or XNA aptamers that bind to molecular targets including small molecules, proteins, nucleic acids, cells, tissues, and organisms through non-covalent interactions (such as electrostatic interactions and hydrophobic interactions). In another embodiment, the aptamer is a peptide capable of specifically binding to a target molecule comprising at least one or more variable peptide domains displayed by a protein scaffold. In one embodiment, the immobilized aptamer is used as a probe for one or more target analytes. In one embodiment, the immobilized aptamer is used in a microarray.

[0204] H. Oligonucleotide Probes In one embodiment, the method or kit comprises one or more probe reagents capable of specifically binding to a target analyte in a sample, hi one embodiment, the oligonucleotide probe comprises a binding partner capable of specifically binding to a target analyte in a sample.

[0205] As used herein, the term "binding partner" refers to a member of a pair of moieties that specifically bind to each other under a particular set of conditions; i.e., the binding pair binds to each other to the substantial exclusion of other moieties present in the environment. A binding partner can be any molecule, such as a polypeptide, lipid, glycolipid, nucleic acid molecule, carbohydrate, or other molecule, with which another molecule specifically interacts, e.g., through a covalent or non-covalent interaction, including, for example, an antibody with a cognate antigen, an interaction between two complementary nucleotide sequences, or an interaction between biotin and streptavidin or avidin. The term "corresponding" refers to the relationship between two specific binding partners, such that one member of a binding partner pair "corresponds" to the other member of the pair.

[0206] In one embodiment, the binding partner comprises an antibody that specifically binds to the target analyte, hi another embodiment, the binding partner comprises an oligonucleotide sequence that is complementary to an oligonucleotide sequence of the target analyte such that the oligonucleotide probe can hybridize to the target nucleotide sequence.

[0207] In one embodiment, the oligonucleotide probe comprises an oligonucleotide tag and a binding partner. In one embodiment, the binding partner comprises a single-stranded sequence that is complementary or substantially complementary to a portion of the target nucleotide sequence. In one embodiment, the probe comprises an oligonucleotide tag having a sequence that is complementary to the sequence of the capture oligonucleotide. In one embodiment, the oligonucleotide tag and the binding partner are different regions of a single oligonucleotide strand.

[0208] In one embodiment, the probe is a single-stranded nucleic acid sequence, including, for example, a nucleic acid sequence comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), peptide nucleic acid (PNA) or locked nucleic acid (LNA). In one embodiment, the probe comprises one or more modified nitrogenous base analogs, or bases modified to include a label or a reactive functional group or linker suitable for attaching a label.

[0209] In one embodiment, the probe is about 5 to about 100, about 10 to about 50, about 20 to about 30, or at least about 5, 6, 7, 8, 9, 10, 15, 20, or 25, and up to about 30, 35, 40, 45, 50, 75, or 100 nucleotides in length. Probes can be prepared by any suitable method known in the art, including chemical or enzymatic synthesis, or by cleavage of a larger nucleic acid using nonspecific nucleic acid-cleaving chemicals or enzymes, or by using site-specific restriction endonucleases. In some applications, a probe that hybridizes to a complementary region of a target sequence can prime extension of the probe by a polymerase, serving as an initiation point for replication of adjacent single-stranded regions on the target sequence.

[0210] In one embodiment, the probe comprises a label. In one embodiment, the label is directly attached to the probe. In another embodiment, the label is attached to the probe via a linker. In one embodiment, the label is a compound that is a member of a binding pair, where the first member of the binding pair (which can be referred to as the "primary binding reagent") is attached to, for example, a substrate, an oligonucleotide, and the other member of the binding pair (which can be referred to as the "secondary binding reagent") has a detectable physical property. Examples or primary labels include, but are not limited to, electrochemiluminescent labels, organometallic complexes containing transition metals, such as ruthenium. In one embodiment, the primary label is an MSD SULFO-TAG label.

[0211] In one embodiment, the secondary binding reagent binds to the primary binding reagent. In one embodiment, the primary binding reagent includes biotin, a hapten, streptavidin, avidin, or an antibody or antigen. In one embodiment, the secondary binding reagent includes biotin, a hapten, streptavidin, avidin, or an antibody or antigen. In one embodiment, the secondary binding reagent includes an electrochemiluminescent label. In one embodiment, the secondary binding reagent includes an organometallic complex containing a transition metal, e.g., ruthenium. In one embodiment, the secondary binding reagent includes an MSD SULFO-TAG label.

[0212] In one embodiment, the kit comprises one or more probe reagents. In one embodiment, the end user prepares the one or more probe reagents.

[0213] I. Oligonucleotide Tags In one embodiment, the probe comprises an oligonucleotide tag having a sequence that specifically binds to the oligonucleotide sequence of the capture molecule. In one embodiment, the tag comprises a single-stranded oligonucleotide that is complementary to at least a portion of the nucleotide sequence of the single-stranded capture oligonucleotide. In one embodiment, the oligonucleotide tag is recombinantly produced. In one embodiment, the oligonucleotide tag is not a naturally occurring sequence. In one embodiment, one or more capture oligonucleotides comprise a single-stranded nucleic acid sequence, for example, a nucleic acid sequence comprising deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a structural analogue comprising a non-naturally occurring chemical structure that can also participate in a hybridization reaction.

[0214] In one embodiment, the tag is attached to the 5' end of the probe. In another embodiment, the tag is attached to the 3' end of the probe. In one embodiment, the tag is not complementary to the target nucleotide sequence and does not hybridize with the target nucleotide sequence.

[0215] In one embodiment, the sequence that is complementary to target nucleotide sequence and oligonucleotide tag sequence exists on one nucleic acid strand in probe.In another embodiment, the sequence that is complementary to target nucleotide sequence and oligonucleotide tag sequence exists on different nucleic acid strands.In one embodiment, probe comprises a first strand that has the sequence that is complementary to target sequence and first bridging sequence, and a second strand that has the second bridging sequence that is complementary to oligonucleotide tag sequence and first bridging sequence, and the first and second strands can hybridize or hybridize through the first and second bridging sequence.

[0216] In one embodiment, the oligonucleotide tag comprises a label. In one embodiment, the label is attached directly to the oligonucleotide tag. In another embodiment, the label is attached to the oligonucleotide tag via a linker. In one embodiment, the label is attached to the 5'-terminal nucleotide of the oligonucleotide tag. In another embodiment, the label is attached to the 3'-terminal nucleotide of the oligonucleotide tag. In one embodiment, the label is attached along the length of the oligonucleotide tag.

[0217] In one embodiment, the label includes radioactive, fluorescent, chemiluminescent, electrochemiluminescent, light absorption, light scattering, electrochemical, magnetic, and enzymatic labels. In one embodiment, the label comprises an electrochemiluminescent label. In one embodiment, the label comprises a hapten. In one embodiment, the label is biotin, fluorescein, or digoxigenin. In one embodiment, the label comprises an organometallic complex comprising a transition metal. In one embodiment, the transition metal comprises ruthenium. In one embodiment, the label is an MSD SULFO-TAG™ label.

[0218] In one aspect, the oligonucleotide tag comprises a primary binding reagent as a label, and the primary binding reagent is a binding partner of the secondary binding reagent. In one aspect, the primary binding reagent includes biotin, streptavidin, avidin, or an antigen. In one aspect, the secondary binding reagent includes biotin, streptavidin, avidin, or an antibody. In one aspect, the primary binding reagent comprises an oligonucleotide, and the secondary binding reagent is an oligonucleotide having a sequence complementary to that of the primary binding reagent.

[0219] In one embodiment, the tag has a nucleotide sequence that is at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, and up to about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or about 15 to about 40, or about 20 to about 30 nucleotides in length. In one embodiment, the tag comprises a nucleotide sequence that is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and up to about 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or about 1 to about 20, or about 10 to about 15, or about 12 to about 13 nucleotides shorter than the complementary capture oligonucleotide sequence. In one embodiment, the tag has a nucleotide sequence that is at least about 24, 30, or 36 nucleotides in length.

[0220] In one embodiment, the oligonucleotide tag has a sequence that hybridizes to a capture molecule having a sequence set forth in any of SEQ ID NOs: 1-774 (Tables 1-12). In one embodiment, the oligonucleotide tag has a sequence that hybridizes to a complementary capture molecule having a sequence set forth in any of SEQ ID NOs: 1-744 (Tables 1-12). In one embodiment, the tag has a nucleotide sequence that is complementary to a sequence that is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence set forth in any of SEQ ID NOs: 1-744. In one embodiment, the tag has a nucleotide sequence that is complementary to a sequence that is at least about 24, 30, or 36 consecutive nucleotides of a sequence set forth in any of SEQ ID NOs: 1-744. In one embodiment, the oligonucleotide tag has a nucleic acid sequence set forth in any of SEQ ID NOs: 745-1488 (Tables 13-24).

[0221] In one embodiment, the oligonucleotide tag has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence set forth in any of SEQ ID NOs: 745-1488 (Tables 13-24). In another embodiment, the oligonucleotide tag has a nucleotide sequence that comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 consecutive nucleotides of a sequence set forth in any of SEQ ID NOs: 745-1488 (Tables 13-24). In another embodiment, the oligonucleotide tag has a nucleotide sequence that comprises at least 20 consecutive nucleotides of a sequence set forth in any of SEQ ID NOs: 745-1488 (Tables 13-24). In another embodiment, the oligonucleotide tag has a nucleotide sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any of SEQ ID NOs: 745-1488 (Tables 13-24). In another embodiment, the oligonucleotide tag has a nucleotide sequence comprising at least 20 consecutive nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any of SEQ ID NOs: 745-1488 (Tables 13-24).

[0222] In one embodiment, the oligonucleotide tag has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to any of the sequences set forth in any of SEQ ID NOs: 745-754, 755-757, 769-770, 777-781, 786, 788-790, 798, and 803-806. In another embodiment, the oligonucleotide tag has a nucleotide sequence that includes at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 consecutive nucleotides of any of the sequences set forth in any of SEQ ID NOs: 745-754, 755-757, 769-770, 777-781, 786, 788-790, 798, and 803-806. In another embodiment, the oligonucleotide tag has a nucleotide sequence comprising at least 20 consecutive nucleotides of a sequence set forth in any of SEQ ID NOs: 745-754, 755-757, 769-770, 777-781, 786, 788-790, 798, and 803-806. In another embodiment, the oligonucleotide tag has a nucleotide sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any of SEQ ID NOs: 745-754, 755-757, 769-770, 777-781, 786, 788-790, 798, and 803-806. In one embodiment, the oligonucleotide tag has a nucleotide sequence set forth in any of SEQ ID NOs: 745-754, 755-757, 769-770, 777-781, 786, 788-790, 798, and 803-806.

[0223] In one embodiment, the oligonucleotide tag has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in any of SEQ ID NOs: 745-754. In another embodiment, the oligonucleotide tag has a nucleotide sequence that comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 consecutive nucleotides of the sequence set forth in any of SEQ ID NOs: 745-754. In another embodiment, the oligonucleotide tag has a nucleotide sequence that comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 consecutive nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any of SEQ ID NOs: 745-754. In one embodiment, the oligonucleotide tag has a nucleotide sequence set forth in any of SEQ ID NOs: 745-754.

[0224] In one embodiment, the method or kit includes a set of non-cross-reactive oligonucleotide tags selected from a "parent set" of non-cross-reactive oligonucleotide tags. In one embodiment, the set of non-cross-reactive oligonucleotide tags is complementary to a set of non-cross-reactive capture oligonucleotides. In one embodiment, the non-cross-reactive oligonucleotide tags in a set are configured to hybridize to their corresponding complementary sequences in a corresponding set of capture oligonucleotides. In one embodiment, the oligonucleotide tags in a set hybridize to less than 0.05% of the non-complementary sequences in a corresponding set of capture oligonucleotides compared to the complementary sequences.

[0225] Two or more oligonucleotides from parent set can be selected to form a "subset" of non-cross-reactive oligonucleotide tags, and each oligonucleotide in the subset is a member of the original parent set.Subset cannot include oligonucleotide tags from two or more parent sets.In one embodiment, the set or subset of non-cross-reactive oligonucleotide tags comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, and up to 64 non-cross-reactive sequences selected from the set of parent non-cross-reactive sequences.

[0226] In one embodiment, a first set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 1 (SEQ ID NOS: 1-64). In one embodiment, the first set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 13 (SEQ ID NOS: 745-808).

[0227] In one embodiment, a second set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 2 (SEQ ID NOS: 65-122). In one embodiment, the second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 14 (SEQ ID NOS: 809-866).

[0228] In one embodiment, a third set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 3 (SEQ ID NOS: 123-186). In one embodiment, the third set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 15 (SEQ ID NOS: 867-930).

[0229] In one embodiment, a fourth set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 4 (SEQ ID NOs: 187-250). In one embodiment, the fourth set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 16 (SEQ ID NOs: 931-994).

[0230] In one embodiment, a fifth set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 5 (SEQ ID NOS:251-308). In one embodiment, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 17 (SEQ ID NOS:995-1052).

[0231] In one embodiment, a sixth set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 6 (SEQ ID NOs: 309-372). In one embodiment, the second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 18 (SEQ ID NOs: 1053-1116).

[0232] In one embodiment, a seventh set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 7 (SEQ ID NOS: 373-436). In one embodiment, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 19 (SEQ ID NOS: 1117-1180).

[0233] In one embodiment, an eighth set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 8 (SEQ ID NOS: 437-494). In one embodiment, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 20 (SEQ ID NOS: 1181-1238).

[0234] In one embodiment, a ninth set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 9 (SEQ ID NOS:495-558). In one embodiment, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 21 (SEQ ID NOS:1239-1302).

[0235] In one embodiment, a tenth set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 10 (SEQ ID NOS: 559-622). In one embodiment, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 22 (SEQ ID NOS: 1303-1366).

[0236] In one embodiment, an eleventh set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 11 (SEQ ID NOs:623-680). In one embodiment, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 23 (SEQ ID NOs:1367-1424).

[0237] In one embodiment, a twelfth set of non-cross-reactive oligonucleotide tags is generated that are complementary to one or more capture sequences set forth in Table 12 (SEQ ID NOs:681-744). In one embodiment, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set set forth in Table 24 (SEQ ID NOs:1425-1488).

[0238] In one embodiment, the set of non-cross-reactive oligonucleotide tags includes one or more tags having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence that is complementary to a sequence of a capture oligonucleotide in Table 1 (SEQ ID NOs: 1-64), Table 2 (SEQ ID NOs: 65-122), Table 3 (SEQ ID NOs: 123-186), Table 4 (SEQ ID NOs: 187-250), Table 5 (SEQ ID NOs: 251-308), Table 6 (SEQ ID NOs: 309-372), Table 7 (SEQ ID NOs: 373-436), Table 8 (SEQ ID NOs: 437-494), Table 9 (SEQ ID NOs: 495-558), Table 10 (SEQ ID NOs: 559-622), Table 11 (SEQ ID NOs: 623-680), or Table 12 (SEQ ID NOs: 681-744). In one embodiment, the set of non-cross-reactive oligonucleotide tags includes one or more tags having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence set forth in Table 13 (SEQ ID NOs:745-808), Table 14 (SEQ ID NOs:809-866), Table 15 (SEQ ID NOs:867-930), Table 16 (SEQ ID NOs:931-994), Table 17 (SEQ ID NOs:995-1052), Table 18 (SEQ ID NOs:1053-1116), Table 19 (SEQ ID NOs:1117-1180), Table 20 (SEQ ID NOs:1181-1238), Table 21 (SEQ ID NOs:1239-1302), Table 22 (SEQ ID NOs:1303-1366), Table 23 (SEQ ID NOs:1367-1424), or Table 24 (SEQ ID NOs:1425-1488).

[0239] In another embodiment, the set of non-cross-reactive oligonucleotide tags includes one or more tags having a nucleotide sequence comprising at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence complementary to the sequence of a capture oligonucleotide in Table 1 (SEQ ID NOs: 1-64), Table 2 (SEQ ID NOs: 65-122), Table 3 (SEQ ID NOs: 123-186), Table 4 (SEQ ID NOs: 187-250), Table 5 (SEQ ID NOs: 251-308), Table 6 (SEQ ID NOs: 309-372), Table 7 (SEQ ID NOs: 373-436), Table 8 (SEQ ID NOs: 437-494), Table 9 (SEQ ID NOs: 495-558), Table 10 (SEQ ID NOs: 559-622), Table 11 (SEQ ID NOs: 623-680), or Table 12 (SEQ ID NOs: 681-744). In another embodiment, the set of non-cross-reactive oligonucleotide tags includes one or more tags having a nucleotide sequence comprising at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence set forth in Table 13 (SEQ ID NOs:745-808), Table 14 (SEQ ID NOs:809-866), Table 15 (SEQ ID NOs:867-930), Table 16 (SEQ ID NOs:931-994), Table 17 (SEQ ID NOs:995-1052), Table 18 (SEQ ID NOs:1053-1116), Table 19 (SEQ ID NOs:1117-1180), Table 20 (SEQ ID NOs:1181-1238), Table 21 (SEQ ID NOs:1239-1302), Table 22 (SEQ ID NOs:1303-1366), Table 23 (SEQ ID NOs:1367-1424), or Table 24 (SEQ ID NOs:1425-1488).

[0240] In another embodiment, the set of non-cross-reactive oligonucleotide tags includes one or more tags having a nucleotide sequence comprising at least 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence complementary to a sequence of a capture oligonucleotide in Table 1 (SEQ ID NOs: 1-64), Table 2 (SEQ ID NOs: 65-122), Table 3 (SEQ ID NOs: 123-186), Table 4 (SEQ ID NOs: 187-250), Table 5 (SEQ ID NOs: 251-308), Table 6 (SEQ ID NOs: 309-372), Table 7 (SEQ ID NOs: 373-436), Table 8 (SEQ ID NOs: 437-494), Table 9 (SEQ ID NOs: 495-558), Table 10 (SEQ ID NOs: 559-622), Table 11 (SEQ ID NOs: 623-680), or Table 12 (SEQ ID NOs: 681-744). In one aspect, the set of non-cross-reactive oligonucleotide tags is selected from the group consisting of Table 13 (SEQ ID NOs: 745-808), Table 14 (SEQ ID NOs: 809-866), Table 15 (SEQ ID NOs: 867-930), Table 16 (SEQ ID NOs: 931-994), Table 17 (SEQ ID NOs: 995-1052), Table 18 (SEQ ID NOs: 1053-1116), Table 19 (SEQ ID NOs: 1117-1180), Table 20 (SEQ ID NOs: 1181-1238), Table 21 (SEQ ID NOs: 1239-1300), Table 22 (SEQ ID NOs: 1241-1245), Table 23 (SEQ ID NOs: 1251-1252), Table 24 (SEQ ID NOs: 1253-1254), Table 25 (SEQ ID NOs: 1255-1256), Table 26 (SEQ ID NOs: 1257-1258), Table 27 (SEQ ID NOs: 1261-1262), Table 28 (SEQ ID NOs: 1263-1264), Table 29 (SEQ ID NOs: 1265-1266), Table 30 (SEQ ID NOs: 1266-1268), Table 31 (SEQ ID NOs: 1271-1272), Table 32 (SEQ ID NOs: 1273-1274), Table 33 (SEQ ID NOs: 1275-1276), Table 34 (SEQ ID NOs: 1276-1278), Table 35 (SEQ ID NOs: 1 and one or more oligonucleotide tags having a nucleotide sequence comprising at least 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence set forth in Table 22 (SEQ ID NOs: 1303-1366), Table 23 (SEQ ID NOs: 1367-1424), or Table 24 (SEQ ID NOs: 1425-1488).

[0241] In one embodiment, the non-cross-reactive oligonucleotide tags in the set are selected from: oligonucleotide tags having a sequence having at least 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 745-808; oligonucleotide tags having a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 745-808; oligonucleotide tags having a sequence having at least 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 745-808; oligonucleotide tags having a sequence selected from SEQ ID NOs: 745-808; and combinations thereof.

[0242] In one embodiment, the non-cross-reactive oligonucleotide tags in the set are selected from: oligonucleotide tags having a sequence having at least 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 745-754; oligonucleotide tags having a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 745-754; oligonucleotide tags having a sequence having at least 20, 21, 22, 23, or 24 contiguous nucleotides of a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 745-754; oligonucleotide tags having a sequence selected from SEQ ID NOs: 745-754; and combinations thereof.

[0243] J. Detection of Labeled Oligonucleotide Products In one embodiment, a method and kit are provided for labeling and detecting one or more target analytes in a sample. In one embodiment, the presence of one or more target analytes in a sample is determined by generating a reaction product comprising an oligonucleotide tag. In one embodiment, the reaction product comprises a label. Various methods can be used to generate the reaction product. In one embodiment, the reaction product is generated by a method described herein, including, but not limited to, a sandwich assay, an oligonucleotide ligation assay (OLA), a primer extension assay (PEA), a direct hybridization assay, a polymerase chain reaction (PCR)-based assay or other target amplification assay, and a nuclease protection assay.

[0244] 1. Sandwich Assay In one embodiment, a method and kit are provided for detecting, identifying, or quantifying one or more target analytes in a sample using a sandwich assay. In one embodiment, the method or kit includes a set of one or more probes, including a targeting probe and a detection probe. In one embodiment, the targeting probe includes a single-stranded oligonucleotide tag that is complementary to at least a portion of a capture oligonucleotide immobilized on a support surface and a first binding partner. In one embodiment, the first binding partner includes a first nucleic acid sequence. In one embodiment, the first nucleic acid sequence of the first binding partner is complementary to a first region of a target nucleotide sequence in the sample. In one embodiment, the first nucleic acid sequence of the first binding partner includes a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide includes an RNA oligonucleotide sequence. In one embodiment, the therapeutic oligonucleotide is selected from miRNA, therapeutic RNA, mRNA, RNA virus, antisense oligonucleotide (ASO), or a combination thereof. In one embodiment, the first nucleic acid sequence of the first binding partner is specifically bound by an anti-drug antibody (ADA) in the sample. In another embodiment, the first binding partner comprises an antibody that specifically binds to a target analyte in the sample.In one embodiment, the detection probe comprises a label and a second binding partner.

[0245] In one embodiment, the second binding partner comprises a second nucleic acid sequence. In one embodiment, the second nucleic acid sequence of the second binding partner is complementary to a second region of the target nucleotide sequence. In one embodiment, the second nucleic acid sequence of the second binding partner comprises a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide comprises an RNA oligonucleotide sequence. In one embodiment, the therapeutic oligonucleotide is selected from miRNA, therapeutic RNA, mRNA, RNA viruses, antisense oligonucleotides (ASOs), or combinations thereof. In one embodiment, the second nucleic acid sequence of the second binding partner is specifically bound by an anti-drug antibody (ADA) in the sample. In one embodiment, the first ASO of the first binding partner and the second ASO of the second binding partner are specifically bound by the same anti-drug antibody.

[0246] In one embodiment, the nucleotide sequence of the first ASO of the first binding partner and the nucleotide sequence of the second ASO of the second binding partner are at least about 95%, 96%, 97%, 98%, 99% or 100% identical. In one embodiment, the second binding partner comprises an antibody that specifically binds to a target analyte in a sample. In one embodiment, the targeting probe and the detection probe can simultaneously bind to the same target analyte in a sample to form a reaction product. In one embodiment, the reaction product is a sandwich complex.

[0247] In one embodiment, the method or kit includes multiple sets of probes that can be used in a multiplexed array to detect, identify, or quantify multiple target analytes in parallel. In one embodiment, each set of probes includes a targeting probe having a first binding partner that specifically binds to a first target analyte that is different from the targeting probes in another set, and an oligonucleotide tag having a sequence complementary to a capture oligonucleotide sequence that is different from the targeting probes in the other set. In one embodiment, each set of probes includes a detection probe having a second binding partner that specifically binds to the first target analyte and a label. In one embodiment, the method or kit includes multiple sets of oligonucleotide probes. In one embodiment, each set of probes includes a targeting probe whose first binding partner includes a nucleic acid sequence complementary to a first target nucleotide sequence, and an oligonucleotide tag having a sequence complementary to a capture oligonucleotide sequence, and the target nucleotide sequence of one set of targeting probes is different from the target nucleotide sequence of another set of targeting probes. In one embodiment, the sequence of the oligonucleotide tag of one set of targeting probes is complementary to a capture oligonucleotide sequence that is different from the sequence of the oligonucleotide tag of another set of targeting probes. In one embodiment, the method or kit includes a detection probe having a second binding partner that is a second target nucleotide sequence that is complementary to a second target nucleotide sequence in the one or more target nucleotides.

[0248] In one embodiment, the method includes providing an array comprising one or more carbon-based electrodes having one or more surfaces and one or more non-cross-reactive capture oligonucleotides described herein, wherein the one or more non-cross-reactive capture oligonucleotides are immobilized to one or more binding domains on one or more surfaces of the one or more carbon-based electrodes. In one embodiment, the method includes associating one or more target analytes with oligonucleotide tags complementary to at least a portion of the capture oligonucleotides immobilized on the support surface and a label, and then contacting the array with a composition comprising one or more tagged and labeled target analytes or reaction products. As used herein, "associated" or "associated" means that the oligonucleotide tag or label is either covalently or non-covalently bound to the target analyte. In one embodiment, the one or more target analytes are associated with the oligonucleotide tag and label in a sandwich complex. In one embodiment, the target analyte is used to generate a reaction product comprising the oligonucleotide tag and label. In one aspect, the method includes incubating the sandwich complexes or reaction products with a support surface under conditions in which the oligonucleotide tags of the sandwich complexes or reaction products hybridize to their corresponding complementary capture oligonucleotides, thereby identifying, detecting, or quantifying target analytes based on the presence or absence of the label at the array location.

[0249] 2. Oligonucleotide Ligation Assay (OLA) In one aspect, the array is contacted with a composition comprising a plurality of target analytes, each target analyte associated with an oligonucleotide tag that is complementary to a different capture oligonucleotide, and the target analyte can be identified, detected, or quantified based on binding of the oligonucleotide tag at the array location. In one aspect, the array is contacted with a composition comprising a plurality of tags and labeled reaction products, and each target analyte is used to generate a reaction product that includes an oligonucleotide tag that is complementary to a different capture oligonucleotide, and the target analyte in the sample can be identified, detected, or quantified based on binding of the reaction product at the array location.

[0250] In one embodiment, the tag and labeled reaction product are prepared by oligonucleotide ligation assay (OLA), and can be captured and detected to identify, detect, or quantify one or more target nucleotide sequences. In one embodiment, the ligation assay is used to detect, identify, or quantify single nucleotide polymorphisms (SNPs) in one or more target nucleotide sequences. In one embodiment, the ligation assay is performed following amplification of one or more target nucleotide sequences in a sample. In another embodiment, the ligation assay is performed on a sample in which one or more target nucleotide sequences have not been amplified. In one embodiment, the reaction product from the ligation assay is amplified before capture and detection. In another embodiment, the reaction product from the ligation assay is not amplified before capture and detection. The reaction product of the ligation assay can be amplified using known methods.

[0251] Methods for performing oligonucleotide ligation reactions are known and generally include the following steps: A sample containing or capable of containing one or more nucleotide sequences of interest is contacted with a pair of single-stranded oligonucleotide probes complementary to one or more target nucleotide sequences, allowing them to hybridize to the target nucleotide sequences; Probes that hybridize to adjacent regions of the target nucleotide sequence are ligated to form a reaction product; In one embodiment, these steps can be repeated to obtain multiple copies of the reaction product; In one embodiment, the nucleotide sequences in the ligation reaction mixture are denatured before the annealing step; The target nucleotide sequence can be detected, identified, or quantified based on the presence, absence, or amount of the reaction product in the sample.

[0252] Probe ligation by DNA ligase depends on three events: (1) hybridization of the oligonucleotide probe to a complementary sequence within the target nucleotide sequence, (2) the oligonucleotide probes being adjacent to each other in the 5' to 3' direction with no intervening nucleotides, and (3) the oligonucleotide probes having perfect base-pair complementarity with the target nucleotide sequence at their binding sites. A single-nucleotide mismatch between primer and target can inhibit ligation.

[0253] In one embodiment, the probes are generated by identifying nucleic acid sequences comprising approximately 40 base pairs on either side of the SNP site in the target nucleotide sequence (a total of approximately 80 base pairs), and generating probes having complementary sequences upstream and downstream of the SNP spanning approximately 18 to 28 nucleotides. In one embodiment, two targeting probes are generated that differ at the SNP position. Typically, only one detection probe is required to detect the wild-type and variant alleles.

[0254] In another embodiment, the target nucleotide sequence is a small nucleic acid, e.g., at least about 15 base pairs, at least about 16 base pairs, at least about 17 base pairs, at least about 18 base pairs, at least about 19 base pairs, or at least about 20 base pairs in length, and up to about 20 base pairs in length, up to about 25 base pairs in length, up to about 30 base pairs in length, up to about 40 base pairs in length, or up to about 50 base pairs in length. In one embodiment, a probe for detecting such a small nucleic acid target comprises at least about 8 base pairs, at least about 9 base pairs, at least about 10 base pairs, at least about 11 base pairs, or at least about 12 base pairs in length, and up to about 20 base pairs in length, up to about 25 base pairs in length, up to about 30 base pairs in length, up to about 40 base pairs in length, or up to about 50 base pairs in length, and the probe and small nucleic acid target are ligated after hybridizing to each other as described herein.

[0255] The length of the oligonucleotide probe sequence can be varied based on the ligation temperature requirements of the OLA reaction (e.g., about 62°C to about 64°C). Bases can be added to or removed from the targeting or detection probe until the probe length is appropriate for a given reaction temperature.

[0256] After the sequence and length of the targeting and detection probes are determined, oligonucleotide tags can be added to the targeting probe.In one embodiment, the oligonucleotide tags are added to the 5' end of the upstream targeting probe.In one embodiment, each oligonucleotide tag is complementary to a different capture oligonucleotide immobilized on the support surface.In one embodiment, the detection probe comprises a label.In one embodiment, the detection probe comprises a 5' phosphate group and a 3' label.In one embodiment, the detection probe comprises a 5' phosphate group and a 3' biotin label.

[0257] In one embodiment, this method involves the use of two or more pairs of probes. In one embodiment, a pair of probes is provided for each target sequence in a sample. In one embodiment, three probes are prepared for detecting SNP pairs: two targeting probes that vary with the single nucleotide polymorphism, and one detection probe. In one embodiment, the two targeting probes comprise a 5' oligonucleotide tag and a 3' nucleic acid that is complementary to either the wild-type or variant single nucleotide polymorphism in the target nucleic acid of interest, and the detection probe comprises a 3' label. In one embodiment, the 3' label is a primary binding reagent that binds to a detectable secondary binding reagent. In one embodiment, the 3' label comprises biotin, and the secondary binding reagent comprises MSD SULFO-TAG streptavidin. In one embodiment, a pair of probes can be prepared for each allele of the polymorphic site, for example, two probes can be prepared, one for the wild-type allele and one for the mutant allele. In one embodiment, a ligation reaction is performed for each target nucleotide sequence. In another embodiment, a multiplexed ligation reaction is performed on two or more target nucleotide sequences. In one embodiment, a multiplexed ligation reaction is performed on about 1 to about 100, or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, or 100 target nucleotide sequences. In one embodiment, a multiplexed ligation reaction is performed to detect, identify, or quantify up to 10 target nucleotide sequences in each well. In one embodiment, multiple allele pairs are detected, identified, or quantified. In one embodiment, up to five allele pairs (i.e., wild-type and mutant SNP pairs) are detected, identified, or quantified in each well. In one embodiment, detecting, identifying, or quantitating includes determining whether the sample is homozygous, heterozygous, or null for a variant allele.

[0258] In one embodiment, the probes are attached using a template-dependent ligase, such as E. coli DNA ligase, T4 DNA ligase, T. aquaticus (Taq) ligase, T. Thermophilus DNA ligase, or Pyrococcus DNA ligase. In one embodiment, the ligase is a thermostable ligase. In another embodiment, the probes are attached by chemical ligation. In one embodiment, hybridization and ligation are performed in combined steps, for example, using multiple thermocycles and a thermostable ligase. In one embodiment, the reaction mixture contains at least about 100 U / mL, 500 U / mL, or 1000 U / mL and up to about 1500 U / mL or 2000 U / mL ligase.

[0259] In one embodiment, the ligation assay is performed by combining a sample with one or more probe pairs and a ligase in a ligation buffer. In one embodiment, the sample, probe, and ligase are combined with the ligation buffer to form a ligation reaction mixture having a volume of at least about 10 μL, 15 μL, or 20 μL and up to about 20 μL, 25 μL, or 50 μL.

[0260] In one embodiment, each pair of probes comprises a targeting probe and a detection probe. In one embodiment, the targeting probe comprises a nucleotide sequence complementary to a first region of the target nucleotide sequence and a single-stranded oligonucleotide tag complementary to at least a portion of the capture oligonucleotide immobilized on the support surface. In one embodiment, the detection probe comprises a label and a nucleotide sequence complementary to a second region of the target nucleotide sequence adjacent to the first region to which the first nucleic acid sequence of the targeting probe sequence is complementary. In one embodiment, the 5' end of the targeting probe is phosphorylated, and when the pair of probes is annealed to the target nucleotide sequence, it is adjacent to the 3'-hydroxyl of the detection probe, so that the ends of the two probes can be ligated by forming a phosphodiester bond. In one embodiment, the 5' end of the detection probe is phosphorylated, and when the pair of probes is annealed to the target nucleotide sequence, it is adjacent to the 3'-hydroxyl of the targeting probe, so that the ends of the two probes can be ligated by forming a phosphodiester bond.

[0261] In one embodiment, the targeting probe comprises about 5 to about 100, about 10 to about 50, about 20 to about 30, or at least about 5, 6, 7, 8, 9, 10, 15, 20, or 25, and up to about 30, 35, 40, 45, 50, 75, or 100 nucleotides. In one embodiment, at least about 1 nM, 2 nM, 3 nM, 4 nM, or 5 nM, and up to about 5 nM, 10 nM, 25 nM, or 50 nM, of targeting probe is included in the reaction mixture.

[0262] In one embodiment, the entire length of the targeting probe is complementary to the target nucleotide sequence. In another embodiment, only a portion of the targeting probe is complementary to the target nucleotide sequence. In one embodiment, the targeting probe is complementary to the target nucleotide sequence downstream of the polymorphic site. In one embodiment, the targeting probe is an allele-specific probe comprising a nucleic acid sequence that is complementary to a region of the target nucleotide sequence that contains a single nucleotide variant. In one embodiment, the targeting probe is an allele-specific probe comprising a nucleic acid sequence that is complementary to a region of the target nucleotide sequence that contains a single nucleotide polymorphism. In one embodiment, the 3'-terminal nucleic acid of the targeting probe is complementary to the polymorphic nucleic acid of the target nucleotide sequence. In another embodiment, the 3'-terminal nucleic acid of the targeting probe is complementary to the nucleotide 3' of the polymorphic nucleic acid of the target nucleotide sequence.

[0263] In one embodiment, the targeting probe comprises a tag that specifically binds to the capture molecule. In one embodiment, the tag comprises a single-stranded oligonucleotide sequence that is complementary to at least a portion of the nucleotide sequence of the single-stranded capture oligonucleotide. In one embodiment, the tag is attached to the 5' end of the targeting probe. In another embodiment, the tag is attached to the 3' end of the targeting probe. In one embodiment, the tag is not complementary to the target nucleotide sequence and does not hybridize with the target nucleotide sequence.

[0264] In one embodiment, the tag comprises a nucleotide sequence that is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and up to about 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or about 1 to about 20, or about 10 to about 15, or about 12 to about 13 nucleotides shorter than the complementary capture oligonucleotide sequence. In one embodiment, the tag has a nucleotide sequence that is at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, and up to about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or about 15 to about 40, or about 20 to about 30 nucleotides in length.

[0265] In one embodiment, the tag comprises a nucleotide sequence that is complementary to at least a portion of the nucleotide sequence of the capture oligonucleotide set forth in SEQ ID NOs: 1 to 10. In one embodiment, the tag comprises a nucleotide sequence that is complementary to about 20 to about 25, or about 24, consecutive nucleotides of the sequence of the capture oligonucleotide set forth in SEQ ID NOs: 1 to 10. In one embodiment, the single-stranded oligonucleotide tag is prepared using known methods based on the sequence of the capture oligonucleotide.

[0266] In one embodiment, each pair of oligonucleotide probes includes detection probes having about 5 to about 100, about 10 to about 50, about 20 to about 30, or at least about 5, 6, 7, 8, 9, 10, 15, 20, or 25, and up to about 30, 35, 40, 45, 50, 75, or 100 nucleotides.

[0267] In one embodiment, the targeting and detection probes have melting temperatures of about 60° C. to about 65° C., or about 62° C. to about 64° C. In one embodiment, the targeting and detection probes have similar melting temperatures (i.e., within about 1° C., 2° C., 3° ​​C., 4° C., or 5° C.).

[0268] In one embodiment, the targeting and detection probes for the target nucleotide sequence are included in the ligation reaction mixture in a 1:1 ratio. In another embodiment, the detection probe is included in excess, e.g., the ligation reaction mixture can include at least about 5, 10, or 20 times more detection probe than the targeting probe. In one embodiment, at least about 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, or 200 nM of detection probe is included in the reaction mixture.

[0269] In one embodiment, the entire length of the detection probe is complementary to the target nucleotide sequence. In another embodiment, only a portion of the detection probe is complementary to the target nucleotide sequence. In one embodiment, the detection probe is complementary to the target nucleotide sequence upstream of the polymorphic site. In one embodiment, the detection probe comprises a nucleic acid sequence that is complementary to a region of the target nucleotide sequence that contains a single nucleotide variant. In one embodiment, the detection probe comprises a nucleic acid sequence that is complementary to a region of the target nucleotide sequence that contains a single nucleotide polymorphism. In one embodiment, the 5'-terminal nucleic acid of the detection probe is complementary to the polymorphic nucleic acid of the target nucleotide sequence. In another embodiment, the 5'-terminal nucleic acid of the detection probe hybridizes to a nucleic acid that is 5' to the polymorphic nucleic acid of the target nucleotide sequence.

[0270] In one embodiment, the detection probe comprises a label. In one embodiment, the label is attached to the 3' end of the detection probe. In one embodiment, the label is attached to the 3' end of the detection probe, and the 5' end has a nucleic acid sequence that is complementary to the sequence of the target nucleotide immediately adjacent to the sequence of the target nucleotide to which the 3' end of the targeting probe hybridizes. In one embodiment, the label is attached to the 5' end of the detection probe, and the 3' end has a nucleic acid sequence that is complementary to the sequence of the target nucleotide immediately adjacent to the sequence of the target nucleotide to which the 5' end of the targeting probe hybridizes.

[0271] In one embodiment, the targeting probe hybridizes to the target nucleotide sequence, so that the 3' end of the targeting probe is located directly above the polymorphic nucleotide of the target nucleotide sequence, and the detection probe hybridizes to the target nucleotide sequence adjacent to the polymorphic site, and provides the 5' end of the ligation reaction.If the targeting probe is complementary to the polymorphic nucleotide of the target nucleotide sequence, the first oligonucleotide can hybridize to the target nucleotide sequence at the polymorphic site, and ligation can occur.If the targeting probe is not complementary to the polymorphic nucleotide of the nucleotide sequence, the first oligonucleotide does not hybridize to the target nucleotide sequence at the polymorphic site, and ligation does not occur.

[0272] In another embodiment, the targeting probe hybridizes to the target nucleotide sequence such that the terminal 5'-base of the targeting probe is located directly above the polymorphic nucleotide of the target nucleotide sequence and the detection probe hybridizes to the target nucleotide sequence adjacent to the polymorphic site to provide the 3' end of the ligation reaction.

[0273] In another embodiment, the detection probe hybridizes to the target nucleotide sequence such that the terminal 5'-base of the detection probe is positioned directly above the polymorphic nucleotide of the target nucleotide sequence and the targeting probe hybridizes to the target nucleotide sequence adjacent to the polymorphic site to provide the 3' end of the ligation reaction.

[0274] In another embodiment, the detection probe hybridizes to the target nucleotide sequence such that the terminal 3'-base of the detection probe is positioned directly above the polymorphic nucleotide of the target nucleotide sequence and the targeting probe hybridizes to the target nucleotide sequence adjacent to the polymorphic site to provide the 5' end of the ligation reaction.

[0275] In one aspect, the method includes: (i) contacting a sample containing one or more target nucleotides with a pair of oligonucleotide probes and DNA ligase to form a ligation reaction mixture; (ii) hybridizing a pair of probes to the target nucleotide sequence, the pair including a capture or detection probe having a terminal 3' or 5' base located directly above a polymorphic nucleotide in the target nucleotide sequence; (iii) ligating the targeting and detection probes together to form labeled and tagged reaction products; (iv) contacting one or more capture oligonucleotides with a support surface on which the labeled and tagged reaction products are immobilized; (v) hybridizing the tag to the capture oligonucleotide; and (vi) detecting the presence of the tag and labeled reaction products.

[0276] In one embodiment, the probe used in the ligation assay is contained beyond the target nucleotide sequence (i.e., at nM level), and therefore, in some cases, non-specific binding of oligonucleotide and target can be detected on the plate as a positive signal.Without wishing to be bound by theory, it is believed that non-specific hybridization may be the result of the probe hybridizing to the target nucleotide sequence and remaining hybridized without ligation, resulting in a signal that is not due to the ligation reaction product but is a non-specific signal called bridging background.

[0277] In one embodiment, the method includes providing one or more blocking probes in the ligation reaction mixture. In one embodiment, including one or more blocking probes in the ligation reaction mixture reduces nonspecific bridging background. As used herein, the term "blocking probe" refers to a single-stranded nucleotide sequence that is complementary to a target nucleotide sequence and spans the probe ligation site but does not contain a tag or label, or a single-stranded nucleotide sequence that is complementary to a probe designed to hybridize to a target nucleotide sequence. In one embodiment, the blocking probe is approximately collinear with the probe sequence. In one embodiment, the blocking probe contains at least about 20, 25, 30, 35, 40, 45, or 50, and up to about 50, 75, 100, 150, or 200, or about 20 to about 200, or about 50 to about 100, nucleotides that are complementary to either the target nucleotide sequence or a probe directed against the target nucleotide sequence. In one embodiment, a pair of blocking probes is included in the ligation reaction mixture, where the first blocking probe has a sequence identical to the connection probe but does not have a complementary oligonucleotide tag, and the second blocking probe has a sequence identical to the detection probe but does not have a biotin label. In one embodiment, up to 2, 3, 4, or 5 additional nucleotides can be added to the 5' and 3' ends of the blocking probe that are complementary to the target nucleotide sequence adjacent to the probe sequence.

[0278] Without wishing to be bound by theory, it is believed that the presence of blocking probe reduces the formation of a complex in which the target nucleotide sequence acts as a bridge for the probe that is annealed to the target sequence but not ligated, and this complex may generate false signals.In one embodiment, a pair of blocking probes is included in the ligation reaction mixture.In another embodiment, one or more blocking probes are included in the ligation reaction mixture in excess of corresponding OLA probes.In one embodiment, one or more blocking probes are included in at least about 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times molar excess of corresponding OLA probes.

[0279] One embodiment of an oligonucleotide ligation assay is depicted schematically in Figure 1. Briefly, a target nucleotide sequence 1 containing a polymorphic site 2 is contacted with a pair of oligonucleotide probes, including a targeting probe 3 bearing an oligonucleotide tag 4 and a detection probe 5 bearing a nucleotide complementary to the polymorphic site and a label 6. The oligonucleotide probes 3 and 5 are hybridized to the target nucleotide sequence (Figure 1A). The oligonucleotide probes 3 and 5 that hybridize with perfect complementarity at the polymorphic site are ligated to form a tagged 4 and labeled 6 reaction product 11 (Figure 1B). A reaction mixture containing the tagged 4 and labeled 6 ligation product 11 is introduced to a support surface having one or more capture oligonucleotides 7 immobilized to one or more binding domains 9. A signal 10 is detected when the tagged 4 and labeled 6 ligation product 11 is immobilized to the support surface due to hybridization between the complementary nucleotide contained in the tagged oligonucleotide 4 and the capture oligonucleotide 7 (Figure 1C).

[0280] In one embodiment, a multiplex ligase detection reaction is provided. In one embodiment, a sample is contacted with one or more allele-specific probes and one or more general probes. In one embodiment, the one or more allele-specific probes include an upstream probe that includes a sequence complementary to a capture oligonucleotide sequence and a 5' oligonucleotide tag with a 3' sequence corresponding to the polymorphism of interest. In one embodiment, the one or more general probes are 5'-phosphorylated and 3'-biotinylated downstream probes. In one embodiment, the multiplex ligation probes are contacted with a sample containing one or more target analytes, hybridized, and adjacent probes are ligated with DNA ligase to form ligation products. In one embodiment, one or more immobilized capture oligonucleotides are contacted with the ligation products, and the oligonucleotide tags can hybridize with their corresponding capture oligonucleotides. The immobilized ligation products can be detected, for example, using labeled streptavidin, for example, SULFO-TAG labeled streptavidin.

[0281] In one embodiment, oligonucleotide ligation assays (OLAs) are used to detect, identify, and / or quantify a target nucleotide sequence in a sample, which may contain degradation products of the target nucleotide sequence, also known as oligonucleotide metabolites. In one embodiment, the sample containing the target nucleotide sequence further contains one or more oligonucleotide metabolites. In one embodiment, OLA is used to measure the amount of the target nucleotide sequence in a sample relative to the oligonucleotide metabolites. In one embodiment, OLA is used to determine the pharmacokinetic parameters of the target nucleotide sequence. In one embodiment, the pharmacokinetic parameters measured are clearance, volume distribution, plasma concentration, half-life, peak time, peak concentration, availability, or a combination thereof. Measurement and interpretation of pharmacokinetic parameters are described herein. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one embodiment, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. Therapeutic oligonucleotides, ASOs, and their metabolism and pharmacology are described herein.

[0282] In one embodiment, the oligonucleotide metabolite is shorter than the target nucleotide sequence by 1 nucleotide or more, 2 nucleotides or more, 3 nucleotides or more, 4 nucleotides or more, 5 nucleotides or more, 6 nucleotides or more, 7 nucleotides or more, 8 nucleotides or more, 9 nucleotides or more, 10 nucleotides or more, 15 nucleotides or more, or 20 nucleotides or more. In one embodiment, the oligonucleotide metabolite is shorter than the target nucleotide sequence by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one embodiment, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite.

[0283] An exemplary embodiment is shown in Figure 15. In Figure 15, a sample containing a target nucleotide sequence is contacted with a template oligonucleotide. The template oligonucleotide comprises a first sequence complementary to the target nucleotide sequence and a second sequence adjacent to the first sequence and complementary to a ligation partner of the target nucleotide sequence. In one aspect, the target nucleotide sequence hybridizes to the first sequence of the template oligonucleotide, and the ligation partner of the target nucleotide sequence hybridizes to the second sequence of the template oligonucleotide. In one aspect, the target nucleotide sequence and the ligation partner hybridize over the entire length of the template oligonucleotide. In one aspect, the target nucleotide sequence and the ligation partner hybridize with the template oligonucleotide to form a double-stranded complex. The target nucleotide sequence and the ligation partner are ligated together using the methods described herein to form a target nucleotide sequence ligation product. The target nucleotide sequence ligation product is then contacted with a pair of single-stranded oligonucleotide probes that are complementary to the target nucleotide sequence ligation product and can hybridize to the target nucleotide sequence ligation product. In one embodiment, probes that can hybridize to adjacent regions of the target nucleotide sequence ligation product are added to the target nucleotide sequence ligation product. In one embodiment, two adjacent probes that each hybridize to adjacent regions of the target nucleotide sequence ligation product are ligated to form a reaction product. In one embodiment, the probes include a targeting probe and a detection probe as described herein. In one embodiment, the targeting probe and the detection probe hybridize over the entire length of the target nucleotide sequence ligation product. In one embodiment, the targeting probe includes an oligonucleotide tag. The targeting probe and the oligonucleotide tag are further described herein. In one embodiment, the oligonucleotide tag is complementary to at least a portion of the capture oligonucleotide immobilized on the support surface.In one embodiment, the detection probe comprises a label. Detection probes and labels are further described herein. In one embodiment, the label comprises biotin, and the detection reagent is linked to streptavidin. In another embodiment, the label comprises a hapten, and the detection reagent is linked to a hapten-binding partner, such as an antibody. The label, the detection reagent, and the binding mode between the label and the detection reagent are further described herein. In one embodiment, the surface is contacted with a detection reagent for binding to the label. In one embodiment, the detection reagent is an electrochemiluminescent reagent. In one embodiment, the detection reagent comprises MSD SULFO-TAG. In one embodiment, electrochemiluminescence is measured as described herein to detect, identify, and / or quantify a target nucleotide sequence. In one embodiment, the amount of the target nucleotide sequence in a sample is measured to determine pharmacokinetic parameters of the target nucleotide sequence. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide. In one embodiment, the therapeutic oligonucleotide is detected without amplifying the therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotides in the sample are detected without a nucleic acid extraction step.

[0284] In one embodiment, a sample containing a target nucleotide sequence also contains one or more oligonucleotide metabolites. In one embodiment, the oligonucleotide metabolites interfere with the detection, identification, and / or quantification of the target nucleotide sequence. Therefore, it may be desirable to remove the oligonucleotide metabolites from the sample. Thus, in one embodiment, a nuclease specific to single-stranded oligonucleotides (i.e., a "single-strand-specific nuclease") is added to the sample, and the target nucleotide sequence ligation product and the template oligonucleotide are hybridized before being added to the probe as outlined in Figure 15. The single-strand-specific nuclease specifically removes the single-stranded oligonucleotide metabolites while being substantially unreactive with the hybridized target nucleotide sequence ligation product and the template oligonucleotide. In one embodiment, the single-strand-specific nuclease also removes excess unhybridized template oligonucleotides. Non-limiting examples of single-strand-specific nucleases include nuclease S1 (e.g., isolated from Aspergillus oryzae), nuclease P1 (e.g., isolated from Penicillium citrinum), nuclease MB (e.g., isolated from mung bean Vigna radiata), and nucleases isolated from Alteromonas espejiana, Neurospora crassa, and Ustilago maydis. Single-strand-specific nucleases can also include RNases such as, for example, RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V, PNPase, RNase PH, RNase R, RNase D, RNase T, RNaseONE, oligoribonuclease, exoribonuclease I, and exoribonuclease II. Additional nucleases that may be suitable for the methods of the present invention include certain DNases.Additional nucleases, including single-strand-specific nucleases, are provided, for example, in Yang, Q Rev Biophys 44(1):1-93 (2011) and Desai et al., FEMS Microbiol Rev 26:457-491 (2003). In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide. In one embodiment, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. In one embodiment, the target nucleotide sequence comprises RNA. In one embodiment, the target nucleotide sequence comprises miRNA, therapeutic RNA, mRNA, RNA virus, or a combination thereof.

[0285] 3. Primer Extension Assay (PEA) In another embodiment, one or more target nucleotide sequences in a sample are detected, identified, or quantified using primer extension assay (PEA). In one embodiment, the target nucleotide sequence comprises one or more single nucleotide variants (SNVs). In another embodiment, the nucleotide sequence comprises one or more single nucleotide polymorphisms (SNPs). In one embodiment, primer extension is performed following amplification of the target nucleotide sequence in the sample. In another embodiment, primer extension is performed on an unamplified sample.

[0286] Methods for performing primer extension assays are known and generally include the following steps: contacting a sample with a probe having a nucleotide sequence complementary to a target nucleotide sequence. In one embodiment, the entire length of the probe is complementary to the target nucleotide sequence. In another embodiment, only a portion of the probe is complementary to the target nucleotide sequence. In one embodiment, the probe comprises a nucleic acid sequence complementary to the nucleic acid sequence of the target nucleotide sequence immediately adjacent to the 3' end of the polymorphism such that the probe hybridizes to the target nucleotide sequence downstream of the polymorphic nucleotide. In one embodiment, the probe comprises a length of about 5 to about 100, about 10 to about 50, about 20 to about 30, or at least about 5, 6, 7, 8, 9, 10, 15, 20, or 25, and up to about 30, 35, 40, 45, 50, 75, or 100 nucleotides.

[0287] In one embodiment, the probe is a targeting probe that comprises a tag that specifically binds to the capture molecule. In one embodiment, the tag comprises a single-stranded oligonucleotide sequence that is complementary to the nucleotide sequence of the single-stranded capture oligonucleotide. In one embodiment, the tag is attached to the 5'-end of the targeting probe. In one embodiment, the tag is attached to the 5'-end of the targeting probe, and the 3'-end nucleic acid of the targeting probe is complementary to the nucleic acid immediately downstream of the polymorphic site of the target nucleotide sequence. In one embodiment, the single-stranded oligonucleotide tag is prepared by the end user using a known method based on the sequence of the capture oligonucleotide.

[0288] In one embodiment, the tag comprises a nucleotide sequence that is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and up to about 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or about 1 to about 20, or about 10 to about 15, nucleotides shorter than the capture oligonucleotide sequence. In one embodiment, the tag has a nucleotide sequence that is at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, and up to about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or about 15 to about 40, or about 20 to about 30 nucleotides in length. In one embodiment, the tag comprises a nucleotide sequence that is complementary to at least a portion of the nucleotide sequence of the capture oligonucleotide set forth in SEQ ID NOs: 1-64. In one embodiment, the tag comprises a nucleotide sequence that is complementary to at least a portion of the nucleotide sequence of the capture oligonucleotide set forth in SEQ ID NOs: 1-10.

[0289] In one embodiment, one or more tag oligonucleotides comprise a sequence that is complementary to the complete sequence of their corresponding capture oligonucleotides. In one embodiment, one or more tag oligonucleotides comprise a sequence that is complementary to only a portion of the sequence of their corresponding capture oligonucleotides. For example, but not by way of limitation, a capture oligonucleotide may comprise a linker as described herein, which may consist of or comprise an oligonucleotide sequence that is not complementary to the tag oligonucleotide sequence (e.g., beginning with a thiol-modified terminal nucleotide) proximal to the surface to which it is attached. The region of complementarity between tag oligonucleotides and capture oligonucleotides may also vary in length. In some embodiments of the present invention, the region of complementarity between oligonucleotides is at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides in length.

[0290] In one embodiment, this method comprises: contacting a sample containing one or more target nucleotide sequences with a targeting probe; and hybridizing the targeting probe to the target oligonucleotide in the presence of a primer extension reaction mixture containing a polymerase and one or more 2'3'-dideoxynucleotide triphosphates (ddNTPs), including ddA, ddT, ddC, and ddG. In one embodiment, the ddNTP is complementary to the polymorphic site and is labeled. In one embodiment, the ddNTP that is not complementary to the polymorphic site is unlabeled. In one embodiment, the ddNTP that is complementary to the wild-type polymorphic nucleotide is labeled. In another embodiment, the ddNTP is complementary to the mutant polymorphic nucleotide and is labeled. In one embodiment, the 3' end of the targeting probe is extended by a single ddNTP. In one embodiment, if the labeled ddNTP is complementary to the polymorphic nucleotide, the primer is extended by one labeled ddNTP to form a tag and a labeled reaction product. If the labeled ddNTP is not complementary to the polymorphic nucleotide, the primer is extended by unlabeled ddNTP and is not detected.

[0291] Suitable polymerase enzymes include, but are not limited to, DNA polymerase, RNA polymerase, DNA-dependent RNA polymerase (reverse transcriptase), and their active subunits (including, for example, the Klenow fragment of DNA polymerase). In one embodiment, the polymerase is a DNA polymerase. In one embodiment, the polymerase is a thermostable polymerase such as Taq polymerase.

[0292] One embodiment of a primer extension assay is depicted schematically in Figure 2. Briefly, a target nucleotide sequence 21 containing a polymorphic site 22 is contacted with a targeting probe 23 bearing an oligonucleotide tag 25 in the presence of a primer extension reaction mixture containing a DNA polymerase and 2'3'-dideoxynucleotide triphosphates (ddNTPs) (i.e., ddA, ddT, ddC, ddG), where the ddNTP 25 complementary to the polymorphic site is labeled 26. The 3' end of the targeting probe is extended by a single ddNTP. As shown in Figure 2A, if the labeled ddNTP is complementary to the polymorphic nucleotide, the primer is extended by the single labeled ddNTP to form a tag and a labeled reaction product. As shown in Figure 2B, if the polymorphic nucleotide is not complementary to the labeled ddNTP, the primer is extended by an unlabeled ddNTP, resulting in an undetectable unlabeled reaction product.

[0293] 4. Direct Hybridization In one embodiment, a method or kit is provided for detecting, identifying, or quantifying one or more target analytes in a sample using a direct hybridization method. In one embodiment, the method or kit includes one or more capture oligonucleotides (referred to herein as "target-specific capture oligonucleotides") that contain one or more nucleic acid sequences that are complementary to the sequences of one or more target nucleic acids in the sample. In one embodiment, the method or kit includes multiple target-specific capture oligonucleotides that can be used in a multiplexed array to detect, identify, or quantify multiple target analytes in parallel.

[0294] In one aspect, the method includes providing a support surface on which one or more target-specific capture molecules are immobilized. In one aspect, the support surface has a flat surface. In one aspect, the support surface is a plate with multiple wells, i.e., a "multiwell plate." A multiwell plate can contain any number of wells of any size or shape, arranged in any pattern or configuration. In another aspect, the support surface has a curved surface. In one aspect, the support surface comprises an assay module, such as an assay plate, slide, cartridge, bead, or chip. In one aspect, the support surface is provided by one or more particles or "beads." In one aspect, the support surface comprises color-coded microspheres. See, e.g., Yang et al. (2001) BADGE, BeadsArray for the Detection of Gene Expression, a High-Throughput Diagnostic Bioassay. Genome Res. 11(11):1888-1898. In one aspect, the support surface comprises one or more beads on which one or more target-specific capture oligonucleotides are immobilized.

[0295] In one embodiment, one or more target-specific capture molecules are immobilized to binding domains within the array. In one embodiment, the support surface comprises one or more carbon-based electrodes having one or more surfaces, and one or more target-specific capture oligonucleotides immobilized to one or more binding domains on one or more surfaces of the one or more carbon-based electrodes.

[0296] In one embodiment, a sample containing or suspected to contain one or more target analytes is contacted with one or more oligonucleotide probes comprising one or more sequences complementary to the sequences on one or more target nucleic acids, and labeled primers comprising sequences complementary to one or more target analytes under conditions in which the labeled primers hybridize to the target analytes.The target analytes can then be amplified using known techniques, such as PCR amplification, to form labeled reaction products.

[0297] In one embodiment, the support surface on which one or more target-specific capture oligonucleotide sequences are immobilized is contacted with the labeled reaction products under conditions in which the labeled reaction products hybridize to their corresponding complementary capture oligonucleotide sequences, allowing the target analyte to be identified, detected, or quantified based on the presence or absence of the label at the array location.

[0298] In one embodiment, direct hybridization is used to detect, identify, or quantify the presence of a virus in a sample. In one embodiment, direct hybridization can be used for human papillomavirus (HPV) genotyping. Human papillomavirus (HPV) infection is the primary cause of cervical cancer. More than 200 HPV genotypes have been identified, with approximately 40 responsible for genital tract infections. HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 are considered to be carcinogenic. Munoz et al. (2003) Epidemiologic classification of human papillomavirus types associated with cervical cancer. N. Engl. J. Med. 3(48):518.

[0299] In one embodiment, direct hybridization is used to detect, identify, or quantify the presence of bacteria in a sample. In one embodiment, direct hybridization is used to detect, identify, or quantify Chlamydia trachomatis (C. trachomatis) in a sample. In one embodiment, direct hybridization is used to detect, identify, or quantify one or more of the three major serovars of C. trachomatis (serovars A-C).

[0300] In one embodiment, direct hybridization is used to detect, identify, or quantify the presence of Salmonella enterica in a sample. Over 2,600 different serotypes have been identified, which can be divided into typhoidal and non-typhoidal serovars. Gal-mor et al. (2014) Same species, different diseases: how and why typhoidal and non-typhoidal Salmonella enterica serovars differ. Front. Microbiol. 5(391) doi:10.3389 / fmicb.2014.00391.

[0301] In one embodiment, direct hybridization is used to detect, identify, and / or quantify a target nucleotide sequence, e.g., a therapeutic oligonucleotide, in a sample that may contain oligonucleotide metabolites. In one embodiment, the sample containing the target nucleotide sequence further comprises one or more oligonucleotide metabolites. In one embodiment, direct hybridization is used to measure the amount of the target nucleotide sequence in a sample relative to the oligonucleotide metabolites. In one embodiment, direct hybridization is used to determine the pharmacokinetic parameters of the target nucleotide sequence. In one embodiment, the pharmacokinetic parameters measured are clearance, volume distribution, plasma concentration, half-life, peak time, peak concentration, availability, or a combination thereof. Measurement and interpretation of pharmacokinetic parameters are described herein. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). Therapeutic oligonucleotides, ASOs, and their metabolism and pharmacology are described herein.

[0302] In one embodiment, the oligonucleotide metabolite is shorter than the target nucleotide sequence by 1 nucleotide or more, 2 nucleotides or more, 3 nucleotides or more, 4 nucleotides or more, 5 nucleotides or more, 6 nucleotides or more, 7 nucleotides or more, 8 nucleotides or more, 9 nucleotides or more, 10 nucleotides or more, 15 nucleotides or more, or 20 nucleotides or more. In one embodiment, the oligonucleotide metabolite is shorter than the target nucleotide sequence by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one embodiment, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite.

[0303] An exemplary embodiment of the method is shown in FIG. 16. In FIG. 16, a sample containing a target nucleotide sequence is contacted with a target nucleotide sequence complement comprising a complementary sequence to the target nucleotide sequence under conditions in which the target nucleotide sequence and the target nucleotide sequence complement hybridize. In one aspect, the target nucleotide sequence and the target nucleotide sequence complement hybridize over their entire length. In one aspect, the target nucleotide sequence analyte and the target nucleotide sequence complement hybridize to form a double-stranded complex. In one aspect, the sample containing the target nucleotide sequence further comprises one or more oligonucleotide metabolites. Metabolites of the target nucleotide sequence, e.g., therapeutic oligonucleotides such as ASOs, are described herein. In one aspect, the method includes removing the oligonucleotide metabolites. In one aspect, a single-strand-specific nuclease is added to the sample while the target nucleotide sequence and the target nucleotide sequence complement are hybridized. In one aspect, the single-strand-specific nuclease is substantially unreactive with the hybridized target nucleotide sequence and the target nucleotide sequence complement while specifically removing the single-stranded oligonucleotide metabolites. In one aspect, the single-strand-specific nuclease further removes excess unhybridized target nucleotide sequence complements. Examples of suitable nucleases, including single-strand-specific nucleases, are provided herein. In one aspect, the target nucleotide sequence is a therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide is an antisense oligonucleotide. In one aspect, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite.

[0304] In one embodiment, after removal of oligonucleotide metabolites and / or unhybridized target nucleotide sequence complements by single-strand-specific nucleases, probes capable of hybridizing to adjacent regions of the target nucleotide sequence are added. In one embodiment, two adjacent probes, each hybridizing to adjacent regions of the target nucleotide sequence, are ligated to form a reaction product. In one embodiment, the probes comprise a targeting probe and a detection probe as described herein. In one embodiment, the targeting probe and the detection probe hybridize over the entire length of the target nucleotide sequence. In one embodiment, the targeting probe comprises an oligonucleotide tag. The targeting probe and the oligonucleotide tag are further described herein. In one embodiment, the oligonucleotide tag is complementary to at least a portion of the capture oligonucleotide immobilized on the support surface. In one embodiment, the detection probe comprises a label. The detection probe and the label are further described herein. In one embodiment, the detection probe can be bound to a detection reagent. In one embodiment, the detection probe comprises a biotin label. In one embodiment, the label comprises biotin and the detection reagent is linked to streptavidin. In another embodiment, the label comprises a hapten and the detection reagent is linked to a hapten-binding partner, such as an antibody. The label, the detection reagent, and the binding mode between the label and the detection reagent are further described herein. In one embodiment, the surface is contacted with a detection reagent for binding to the label. In one embodiment, the detection reagent is an electrochemiluminescent reagent. In one embodiment, the detection reagent comprises MSD SULFO-TAG. In one embodiment, electrochemiluminescence is measured as described herein to detect, identify, and / or quantify the target nucleotide sequence. In one embodiment, the amount of the target nucleotide sequence in a sample is measured to determine pharmacokinetic parameters of the target nucleotide sequence. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide. In one embodiment, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. In one embodiment, the target nucleotide sequence comprises RNA.In one embodiment, the target nucleotide sequence comprises an miRNA, a therapeutic RNA, an mRNA, an RNA virus, or a combination thereof.

[0305] 5. Polymerase Chain Reaction (PCR) In one embodiment, a method or kit is provided for detecting, identifying, or quantifying one or more target analytes in a sample using polymerase chain reaction (PCR). In one embodiment, the target nucleic acid is amplified using PCR. In one embodiment, a method or kit is provided that includes one or more sets of PCR primers, each set of primers including an upstream primer and a downstream primer. In one embodiment, the target nucleotide sequence in the sample is amplified using one or more upstream and downstream PCR primers.

[0306] In one embodiment, one or more target nucleotide analytes in a sample are amplified using one or more modified upstream or downstream primers.In one embodiment, one or more target nucleotide analytes are amplified using one or more upstream primers comprising an oligonucleotide tag sequence configured to hybridize with a capture oligonucleotide having a complementary sequence.In one embodiment, one or more target nucleotide analytes are amplified using one or more downstream primers comprising a label.In one embodiment, one or more target nucleotide analytes are amplified using one or more downstream primers comprising an oligonucleotide tag sequence configured to hybridize with a capture oligonucleotide having a complementary sequence.In one embodiment, one or more target nucleotide analytes are amplified using one or more upstream primers comprising a label.

[0307] In one embodiment, a target nucleotide sequence is amplified using one or more modified PCR primers to form a PCR reaction product comprising an oligonucleotide tag configured to hybridize to a capture oligonucleotide immobilized on a support surface. In one embodiment, a target nucleotide sequence is amplified using one or more modified PCR primers to form a PCR reaction product comprising a label. In one embodiment, a target nucleotide sequence is amplified using one or more modified PCR primers to form a PCR reaction product comprising an oligonucleotide tag configured to hybridize to a capture oligonucleotide immobilized on a support surface and a label. Methods for labeling PCR reaction products are known, including, for example, labeled deoxynucleotide triphosphates (dNTPs) or modified upstream or downstream primers comprising a label.

[0308] In one embodiment, one or more capture oligonucleotides are immobilized to binding domains in an array on the support surface, hi one embodiment, PCR reaction products are captured on the support surface by hybridization of oligonucleotide tags to their corresponding capture oligonucleotides.

[0309] In one embodiment, one or more target analytes are detected, identified, or quantified using ligation-mediated amplification (LM PCR). In one embodiment, one or more target analytes are detected, identified, or quantified using multiplex "ligation-mediated amplification" in combination with the methods described herein. In one embodiment, one or more target nucleotide analytes are reverse transcribed using upstream and downstream probes. In one embodiment, the upstream probe comprises a nucleotide sequence complementary to a universal primer site such as T7, an oligonucleotide tag sequence, and a gene-specific sequence, and the downstream probe comprises a gene-specific fragment that is in contact with the gene-specific fragment of the upstream probe and the universal primer site such as T3. In one embodiment, the downstream probe is 5'-phosphorylated. In one embodiment, the probes are annealed to their targets, free probes are removed, and the annealed probes are ligated using a ligase to obtain an amplification template. In one embodiment, PCR is performed using T3 and 5'-biotinylated T7 primers. In one embodiment, capture oligonucleotides immobilized on a support surface are contacted with biotinylated amplicons under conditions in which the oligonucleotide tags hybridize to their corresponding capture oligonucleotides. In one embodiment, the captured labeled amplicons are incubated with labeled streptavidin, such as SULFO-TAG labeled streptavidin, to detect, identify, or quantify the captured labeled amplicons. See, for example, Peck et al. (2006) A method for high-throughput gene expression signature analysis Genome Biol. 7(7):R61.

[0310] In one embodiment, the target analyte is cDNA. In one embodiment, the target analyte is mRNA. In one embodiment, cDNA is synthesized from poly-A tailed mRNA using oligo-dT primers. In one embodiment, cDNA can be generated from mRNA using random primed cDNA synthesis.

[0311] 6. Nuclease Protection Assay (NPA) In one embodiment, a method or kit is provided for detecting, identifying, or quantifying one or more target analytes in a sample using a nuclease protection assay. In one embodiment, a nuclease protection assay is used to detect, identify, or quantify a target analyte in a sample containing or suspected of containing the target analyte. In one embodiment, the target analyte comprises a single-stranded nucleic acid, including, for example, single-stranded RNA. In one embodiment, the target analyte comprises a microRNA (miRNA). In one embodiment, the sample is contacted with one or more single-stranded probes comprising a sequence complementary to the sequence of the target analyte and an oligonucleotide tag sequence under conditions in which the target analyte hybridizes to the probe to form a tagged reaction product. In one embodiment, the probe is a DNA / RNA hybrid probe comprising a single-stranded DNA tag sequence and a single-stranded RNA sequence complementary to the nucleic acid sequence of the target analyte. In one embodiment, the hybrid probe comprises a biotin label.

[0312] In one embodiment, the support surface on which one or more capture oligonucleotides are immobilized is contacted with a mixture containing tagged reaction products under the condition that one or more oligonucleotide tag sequences are hybridized with the corresponding capture oligonucleotide sequences immobilized on the support surface.After the oligonucleotide tags are hybridized with the corresponding capture oligonucleotides on the support surface, the support surface is washed, and contacted with a single-stranded RNA-specific RNase, such as RNase A or RNase I, under the condition that the RNase can digest single-stranded RNA molecules, remove excess probes that are bound to spots that do not hybridize with target RNA, and cleave the mismatched sites between probes and target RNA.

[0313] In one embodiment, the miRNA analysis involves a step-down probe hybridization step in which a DNA / RNA chimeric probe hybridizes to a target miRNA during a gradual decrease in annealing temperature.

[0314] In one embodiment, a direct surface-coated nuclease protection assay (NPA) is used to detect, identify, and / or quantify a target nucleotide sequence in a sample that may contain degradation products of the target nucleotide sequence, also known as oligonucleotide metabolites. In one embodiment, the sample containing the target nucleotide sequence further comprises one or more oligonucleotide metabolites. In one embodiment, the direct surface-coated NPA is used to measure the amount of the target nucleotide sequence in the sample relative to the oligonucleotide metabolites. In one embodiment, the direct surface-coated NPA is used to determine the pharmacokinetic parameters of a therapeutic oligonucleotide. In one embodiment, the pharmacokinetic parameters measured are clearance, volume distribution, plasma concentration, half-life, peak time, peak concentration, availability, or a combination thereof. Measurement and interpretation of pharmacokinetic parameters are described herein. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one embodiment, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. Therapeutic oligonucleotides, antisense oligonucleotides, and their metabolism and pharmacology are described herein.

[0315] In one embodiment, the oligonucleotide metabolite is 1 or more nucleotides, 2 or more nucleotides, 3 or more nucleotides, 4 or more nucleotides, 5 or more nucleotides, 6 or more nucleotides, 7 or more nucleotides, 8 or more nucleotides, 9 or more nucleotides, 10 or more nucleotides, 15 or more nucleotides, or 20 or more nucleotides shorter than the target nucleotide sequence. In one embodiment, the oligonucleotide metabolite is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% shorter than the target nucleotide sequence. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one embodiment, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. In one embodiment, the therapeutic oligonucleotide is detected without amplifying the therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide in the sample is detected without a nucleic acid extraction step.

[0316] An exemplary embodiment is shown in Figure 17. In Figure 17, a target nucleotide sequence complement comprising a complementary sequence to the target nucleotide sequence is used as a capture oligonucleotide. The target nucleotide sequence complement comprises a label at one end and a surface-attached moiety at the other end. Methods for immobilizing the capture oligonucleotide to a surface are described herein and include, for example, electrostatic interactions, complementary binding partners, complementary reactive functional groups, linkers (e.g., cross-linkers comprising reactive functional groups), and the like. In one aspect, a surface is coated with the target nucleotide sequence complement via the surface-attached moiety of the target nucleotide sequence complement. In one aspect, the surface-attached moiety comprises a thiol. In one aspect, the surface-attached moiety comprises biotin.

[0317] In one embodiment, a surface coated with a target nucleotide sequence complement is contacted with a sample containing the target nucleotide sequence under conditions in which the target nucleotide sequence complement and the target nucleotide sequence hybridize. In one embodiment, the target nucleotide sequence and the target nucleotide sequence complement hybridize over their entire length. In one embodiment, the target nucleotide sequence and the target nucleotide sequence complement hybridize to form a double-stranded complex. In one embodiment, the sample containing the target nucleotide sequence further comprises one or more oligonucleotide metabolites. Metabolites of the target nucleotide sequence, such as therapeutic oligonucleotides such as ASOs, are described herein. In one embodiment, the method includes removing the oligonucleotide metabolites. In one embodiment, a single-strand-specific nuclease is added to the sample while the target nucleotide sequence and the target nucleotide sequence complement are hybridized. In one embodiment, the single-strand-specific nuclease is substantially unreactive with the hybridized target nucleotide sequence-target nucleotide sequence complement while specifically removing the single-stranded oligonucleotide metabolites. Examples of suitable nucleases, including single-strand-specific nucleases, are provided herein.

[0318] In one embodiment, after removal of the oligonucleotide metabolites by the single-strand-specific nuclease, the surface is contacted with a detection reagent capable of binding to the label on the target nucleotide sequence complement. In one embodiment, the label comprises biotin, and the detection reagent is linked to streptavidin. In another embodiment, the label comprises a hapten, and the detection reagent is linked to a hapten-binding partner, such as an antibody. The label, the detection reagent, and the binding mode between the label and the detection reagent are further described herein. In one embodiment, the detection reagent is an electrochemiluminescent reagent. In one embodiment, the detection reagent comprises MSD SULFO-TAG. In one embodiment, electrochemiluminescence is measured as described herein to detect, identify, and / or quantify the target nucleotide sequence. In one embodiment, the amount of the target nucleotide sequence in the sample is measured to determine the pharmacokinetic parameters of the target nucleotide sequence. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide. In one embodiment, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. In one embodiment, the target nucleotide sequence comprises RNA. In one embodiment, the target nucleotide sequence comprises miRNA, therapeutic RNA, mRNA, an RNA virus, or a combination thereof.

[0319] In another embodiment, the target nucleotide sequence is a small nucleic acid, e.g., at least about 15 base pairs, at least about 16 base pairs, at least about 17 base pairs, at least about 18 base pairs, at least about 19 base pairs, or at least about 20 base pairs in length, and up to about 20 base pairs in length, up to about 25 base pairs in length, up to about 30 base pairs in length, up to about 40 base pairs in length, or up to about 50 base pairs in length. In one embodiment, a probe for detecting such a small nucleic acid target comprises at least about 8 base pairs, at least about 9 base pairs, at least about 10 base pairs, at least about 11 base pairs, or at least about 12 base pairs in length, and up to about 20 base pairs in length, up to about 25 base pairs in length, up to about 30 base pairs in length, up to about 40 base pairs in length, or up to about 50 base pairs in length, and the probe and small nucleic acid target are ligated after hybridizing to each other as described herein.

[0320] 7. Hybridization / Protection Assay In one embodiment, a hybridization / protection assay is used to detect, identify, and / or quantify a target nucleotide sequence, e.g., a therapeutic oligonucleotide, in a sample that may contain oligonucleotide metabolites. In one embodiment, the sample containing the target nucleotide sequence further comprises one or more oligonucleotide metabolites. In one embodiment, a hybridization / protection assay is used to measure the amount of a target nucleotide sequence in a sample relative to the oligonucleotide metabolites. In one embodiment, a hybridization / protection assay is used to determine the pharmacokinetic parameters of a therapeutic oligonucleotide. In one embodiment, the pharmacokinetic parameters measured are clearance, volume distribution, plasma concentration, half-life, peak time, peak concentration, availability, or a combination thereof. Measurement and interpretation of pharmacokinetic parameters are described herein. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). Therapeutic oligonucleotides, ASOs, and their metabolism and pharmacology are described herein.

[0321] In one embodiment, the oligonucleotide metabolite is 1 or more nucleotides, 2 or more nucleotides, 3 or more nucleotides, 4 or more nucleotides, 5 or more nucleotides, 6 or more nucleotides, 7 or more nucleotides, 8 or more nucleotides, 9 or more nucleotides, 10 or more nucleotides, 15 or more nucleotides, or 20 or more nucleotides shorter than the target nucleotide sequence. In one embodiment, the oligonucleotide metabolite is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% shorter than the target nucleotide sequence. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one embodiment, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. In one embodiment, the therapeutic oligonucleotide is detected without amplifying the therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide in the sample is detected without a nucleic acid extraction step.

[0322] An exemplary embodiment is shown in Figure 18. In Figure 18, a sample containing a target nucleotide sequence is contacted with a target nucleotide sequence complement probe comprising (i) a target nucleotide sequence complement sequence complementary to the target nucleotide sequence, (ii) an oligonucleotide tag, and (iii) a label. In one aspect, the oligonucleotide tag of the target nucleotide sequence complement probe is complementary to at least a portion of a capture oligonucleotide immobilized on a support surface. In one aspect, the oligonucleotide tag of the target nucleotide sequence is double-stranded, and one strand of the oligonucleotide tag is complementary to at least a portion of a capture oligonucleotide immobilized on a support surface. The oligonucleotide tag and the capture oligonucleotide are further described herein. In one aspect, the label of the target nucleotide sequence complement probe can be bound to a detection reagent. In one aspect, the label comprises biotin, and the detection reagent is linked to streptavidin. In another aspect, the label comprises a hapten, and the detection reagent is linked to a hapten-binding partner, such as an antibody. The label, the detection reagent, and the binding mode between the label and the detection reagent are further described herein.

[0323] In one embodiment, the target nucleotide sequence hybridizes with the target nucleotide sequence complement probe. In one embodiment, the target nucleotide sequence and the target nucleotide sequence complement probe hybridize over the entire length of the target nucleotide sequence and the target nucleotide sequence complement sequence. In one embodiment, the oligonucleotide tag is double-stranded, and the target nucleotide sequence and the target nucleotide sequence complement sequence hybridize to form a double-stranded complex. In one embodiment, the sample containing the target nucleotide sequence further comprises one or more oligonucleotide metabolites. Metabolites of the target nucleotide sequence, for example, therapeutic oligonucleotides such as ASOs, are described herein. In one embodiment, the method includes removing the oligonucleotide metabolites. In one embodiment, while the target nucleotide sequence and the target nucleotide sequence complement are hybridized, a single-strand-specific nuclease is added to the sample. In one embodiment, the single-strand-specific nuclease is substantially non-reactive with the hybridized target nucleotide sequence-target nucleotide sequence complement, while specifically removing the single-stranded oligonucleotide metabolites. In one aspect, the single-strand-specific nuclease further removes excess unhybridized target nucleotide sequence complement probes. Examples of suitable nucleases, including single-strand-specific nucleases, are provided herein.

[0324] In one embodiment, after removal of the oligonucleotide metabolites and / or unhybridized target nucleotide sequence complement probes, the hybridized target nucleotide sequence-target nucleotide sequence complement probes are immobilized on a support surface via binding of an oligonucleotide tag on the target nucleotide sequence complement probe to a capture oligonucleotide on the surface. In one embodiment, the oligonucleotide metabolites and / or unhybridized target nucleotide sequence complement probes are removed prior to immobilization of the hybridized target nucleotide sequence-target nucleotide sequence complement probes, providing improved sensitivity compared to simultaneous removal / immobilization or immobilization followed by removal formats. In one embodiment, a detection reagent is added to the surface, and the detection reagent binds to a label on the target nucleotide sequence complement probe. In one embodiment, the detection reagent is an electrochemiluminescent reagent. In one embodiment, the detection reagent comprises MSD SULFO-TAG. In one embodiment, electrochemiluminescence is measured as described herein to detect, identify, and / or quantify the target nucleotide sequence. In one embodiment, the amount of the target nucleotide sequence in a sample is measured to determine pharmacokinetic parameters of the target nucleotide sequence. In one embodiment, the target nucleotide sequence is a therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide. In one embodiment, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. In one embodiment, the target nucleotide sequence comprises RNA. In one embodiment, the target nucleotide sequence comprises miRNA, therapeutic RNA, mRNA, RNA virus, or a combination thereof.

[0325] K. Sample The methods or kits described herein are suitable for detecting one or more target analytes in a sample containing or suspected of containing one or more target analytes. In one embodiment, the target analyte comprises a target nucleotide sequence. In another embodiment, the target analyte comprises a target protein. In one embodiment, the sample contains or is suspected of containing one or more prokaryotic or eukaryotic DNA or RNA sequences of interest. In one embodiment, the sample is a biological sample obtained from an organism such as a human or other mammal, including, but not limited to, a non-human primate, dog, cat, cow, sheep, poultry, or horse, or another organism such as a plant, bacterium, fungus, protist, or virus. In one embodiment, the biological sample includes tissue, cells, cell extracts, or biopsies, or biological fluids such as urine, blood, saliva, amniotic fluid, exudates from an area of ​​infection or inflammation, mouthwash containing buccal cells, cerebrospinal fluid, or synovial fluid. In one embodiment, the sample is isolated from an individual. In another embodiment, the sample is derived from a group of individuals. In one embodiment, the sample comprises one or more, or multiple, individual or pooled samples.

[0326] In one embodiment, the sample contains one or more target DNA sequences, including, but not limited to, single-stranded or double-stranded DNA, including, but not limited to, genomic DNA, mitochondrial DNA, cDNA, whole genome amplified DNA, or combinations thereof. In another embodiment, the sample contains one or more target RNA sequences, including, but not limited to, single-stranded or double-stranded RNA, including, but not limited to, ribosomal RNA, mRNA, miRNA, siRNA, RNAi, or combinations thereof. In another embodiment, the sample contains or is suspected to contain one or more target nucleotide sequences that are amplicons, such as PCR products, plasmids, cosmids, DNA libraries, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), synthetic oligonucleotides, restriction fragments, DNA / RNA hybrids, PNAs (peptide nucleic acids), or DNA / RNA mosaic nucleic acids. In the case of double-stranded nucleic acids, the target nucleotide sequences can be present on either strand. In one embodiment, the sample does not contain ethylenediaminetetraacetic acid (EDTA).

[0327] In one embodiment, the sample contains one or more target nucleotide sequences, e.g., therapeutic oligonucleotides, and the sample may also contain oligonucleotide metabolites. As used herein, "therapeutic oligonucleotide" refers to an oligonucleotide that can interact with a biomolecule to provide a therapeutic effect. In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). ASOs are single-stranded oligonucleotides that are typically about 5, 10, 15, 20, or 25 nucleotides to about 30, 35, 40, 45, or 50 nucleotides in length. ASOs can affect RNA processing and / or regulate protein expression. ASOs are single-stranded oligonucleotides that bind to and inactivate single-stranded RNA. In one embodiment, the ASO binds to the messenger RNA (mRNA) of a gene, thereby inactivating the gene. In one embodiment, the gene is a disease gene. Thus, the ASO can inactivate the mRNA of the disease gene to prevent or ameliorate the production of a protein that causes a particular disease. In one embodiment, the ASO comprises DNA, RNA, or a combination thereof.

[0328] Oligonucleotides in a sample, such as therapeutic oligonucleotides such as ASOs, can degrade over time due to various factors, such as the presence of nucleases, temperature, pH, and salt concentration. In certain embodiments, degradation of therapeutic oligonucleotides in a sample indicates a pharmacodynamic response to the therapeutic oligonucleotide. Degraded or truncated therapeutic oligonucleotides, also referred to herein as therapeutic oligonucleotide metabolites, can lose therapeutic efficacy. In one embodiment, a sample contains a therapeutic oligonucleotide and one or more therapeutic oligonucleotide metabolites. In one embodiment, the therapeutic oligonucleotide metabolite is 1 or more nucleotides, 2 or more nucleotides, 3 or more nucleotides, 4 or more nucleotides, 5 or more nucleotides, 6 or more nucleotides, 7 or more nucleotides, 8 or more nucleotides, 9 or more nucleotides, 10 or more nucleotides, 15 or more nucleotides, or 20 or more nucleotides shorter than the therapeutic oligonucleotide. In one embodiment, the therapeutic oligonucleotide metabolite is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% shorter than the therapeutic oligonucleotide.

[0329] In one embodiment, the method provided herein is used to measure the amount of therapeutic oligonucleotide in a sample compared to therapeutic oligonucleotide metabolites. In one embodiment, the pharmacokinetic parameters of a therapeutic oligonucleotide are determined by measuring the rate and / or amount of degradation of the therapeutic oligonucleotide in a biological environment, such as a patient. Thus, in one embodiment, the method provided herein is used to determine the pharmacokinetic parameters of a therapeutic oligonucleotide. In one embodiment, the pharmacokinetic parameters measured are clearance, volume distribution, plasma concentration, half-life, peak time, peak concentration, availability, or a combination thereof. The measurement and interpretation of pharmacokinetic parameters are further described herein.

[0330] In one embodiment, the sample contains one or more anti-drug antibodies (ADAs). In one embodiment, the ADAs bind to therapeutic polypeptides, including but not limited to therapeutic proteins or therapeutic antibodies. In one embodiment, the ADAs bind to therapeutic oligonucleotides, including but not limited to antisense oligonucleotides (ASOs), short interfering RNAs, microRNAs, and synthetic guide strands of CRISPR / Cas. In one embodiment, the ADAs can bind to biopharmaceutical products. In one embodiment, the ADAs can inhibit the functional activity of therapeutic products.

[0331] In one embodiment, the sample contains one or more unamplified target nucleotide sequences. In another embodiment, the sample contains one or more target nucleotide sequences obtained by amplifying or cloning sequences from a biological sample. Amplification can be achieved by methods including, but not limited to, polymerase chain reaction (PCR), whole genome amplification (WGA), reverse transcription followed by polymerase chain reaction (RT-PCR), strand displacement amplification (SDA), or rolling circle amplification (RCA).

[0332] In one embodiment, the sample contains or is suspected to contain one or more target proteins.In one embodiment, the target protein comprises a DNA binding protein, for example, a protein with a DNA binding domain that can bind to single-stranded or double-stranded DNA.Examples of DNA binding proteins include, but are not limited to, transcription factors, polymerases, nucleases, and histones.In one embodiment, the DNA binding protein binds to a specific DNA sequence, for example, a transcription factor.

[0333] In one embodiment, one or more target analytes are purified from a biological sample. Methods for purifying target analytes from a sample are known. Methods for purifying nucleotide sequences from biological samples are known, including, for example, high performance liquid chromatography (HPLC), such as reverse-phase high performance liquid chromatography (RP-HPLC) or anion exchange high pressure liquid chromatography (AEX HPLC) or polyacrylamide gel electrophoresis (PAGE). Methods for purifying proteins from biological samples are known, including, for example, chromatography such as size exclusion chromatography, high performance liquid chromatography (HPLC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, affinity chromatography, and electrophoresis.

[0334] In one embodiment, the sample comprises at least about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, or 10 μg, and up to about 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, or 50 μg, or between about 1 μg and about 50 μg, or between about 5 μg and about 20 μg of one or more target analytes, e.g., genomic DNA purified from a cell line or whole genome amplified DNA. In one embodiment, the sample contains at least about 0.1 μL, 0.5 μL, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and up to about 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 15 μL, 20 μL, or 25 μL, or from about 1 μL to about 25 μL, or from about 0.1 μL to about 5 μL of one or more target analytes, e.g., a sample containing one or more amplification products, e.g., PCR amplicons generated from cell line DNA. In one embodiment, the sample has an analyte concentration of at least about 1 ng / μL, 5 ng / μL, or 10 ng / μL, and up to about 25 ng / μL, 50 ng / μL, or 100 ng / μL.

[0335] In one embodiment, the sample contains at least one copy of the target analyte. 7 , 10 6 , 105 , 10 4 , 10 3 , 10 2 or 10 1 In one embodiment, the copies are present in a sample of about 0.001 mL to about 1 mL, or less than about 1 mL, 0.1 mL, 0.01 mL, or 0.001 mL.

[0336] L. Sample Amplification Although the methods or kits described herein can be used in connection with samples in which one or more target nucleotide sequences are not amplified, it may be desirable to include an amplification step to increase the amount of target nucleotides in the sample. For example, it may be desirable to amplify the target nucleotide sequence if the target nucleotide sequence contains one or more rare mutations, such as one or more rare or low allele fraction mutations associated with cancer.

[0337] In one embodiment, the target nucleotide sequence is amplified by polymerase chain reaction (PCR). Methods of PCR amplification are known. See, for example, Saiki et al., "Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase," Science, 239:487-491. Briefly, in PCR amplification, the target nucleotide sequence is contacted with two oligonucleotide primers that flank the specific nucleotide sequence to be amplified. Repeated cycles of heat denaturation, annealing of the primers to their complementary sequences, and extension of the annealed primers by DNA polymerase result in approximately 2 n of target fragments accumulate exponentially, where n is the number of cycles.

[0338] In another embodiment, the target nucleotide sequence is amplified using rolling circle amplification (RCA), an isothermal nucleic acid (e.g., DNA or RNA) amplification technique in which a polymerase sequentially adds single nucleotides to a primer annealed to a circular template, resulting in a long single-stranded DNA or RNA sequence containing multiple, e.g., tens to hundreds, tandem repeats that are complementary to the circular template.

[0339] In another embodiment, whole genome amplification (WGA) is used to amplify genomic DNA samples. Whole genome amplification methods are known, including, for example, multiple displacement amplification (MDA), degenerate oligonucleotide PCR (DOP-PCR) and primer extension preamplification (PEP). DOP-PCR and PEP are based on standard PCR technology, while MDA uses an isothermal reaction setup.

[0340] In some embodiments, amplification involves the use of one or more oligonucleotide primers used by a polymerase to initiate DNA or RNA synthesis. Primers can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), DNA containing phosphorothioate linkages, or combinations thereof, and include nucleotide analogs or modified nucleotides. Generally, primers are single-stranded oligonucleotides about 10 to about 100, or about 15 to about 30, or at least about 10, 15, or 20 to a maximum of about 25, 30, 35, 40, 45, or 50 nucleotides in length. In some embodiments, the oligonucleotide primers are specific primers complementary to specific regions of the target nucleotide sequence, such that the region of the template to be amplified is defined by the primer. Methods for preparing oligonucleotide primers are known. In one embodiment, commercially available amplification primers can be used. In one embodiment, the primers are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0341] In one embodiment, the sample contains PCR products. In one embodiment, the PCR products are about 25 bp to about 500 bp, or about 50 bp to about 300 bp, or about 75 bp to about 200 bp in length. In one embodiment, the PCR primers have a melting temperature similar to (i.e., within about 5°C or 1°C of) other primers used in the multiplex PCR assay.

[0342] M. Detection In one embodiment, target analytes are detected, identified, or quantified in an array. In one embodiment, reaction products, including, for example, PCR reaction products, OLA reaction products, PEA reaction products, sandwich complexes, or NPA reaction products described herein, can be detected, identified, or quantified in an array. In one embodiment, the array is a multiplexed array, and target analytes are detected, identified, or quantified by detecting labels attached to immobilized target molecules on the array. In one embodiment, a support surface is contacted with a hybridization mixture containing tagged reaction products, and the reaction products are immobilized on the support surface by hybridization of the single-stranded oligonucleotide tag with its corresponding complementary capture oligonucleotide. In one embodiment, the reaction products are amplified before contacting the solid support with the reaction products. In one embodiment, the reaction products are amplified at least about 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold. In one embodiment, the hybridization mixture includes a hybridization buffer. In one aspect, the presence or amount of the reaction product can be detected, identified, or quantified based on a label attached to the reaction product. In one aspect, the support surface is washed with a wash buffer after the reaction product is immobilized thereon.

[0343] In one embodiment, the presence of one or more target nucleotide sequences is detected, identified, or quantified based on the detection of reaction products immobilized on a support surface. In one embodiment, the presence of the immobilized reaction products is detected by monitoring light emission from a label on the reaction products, including, but not limited to, fluorescence, time-resolved fluorescence, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), luminescence, chemiluminescence, bioluminescence, fluorescence, light scattering, or electrode-induced luminescence. In another embodiment, the label comprises an enzyme or other chemically reactive species having chemical activity that produces a measurable signal, such as light scattering, absorbance, or fluorescence. Examples of enzymatic labels include, but are not limited to, horseradish peroxidase or alkaline phosphatase. In one embodiment, the label is a detectable hapten, including, but not limited to, biotin, fluorescein, or digoxigenin. In one embodiment, the reaction product comprises a biotin label.

[0344] In one embodiment, the reaction product is immobilized on one or more binding domains located on a support surface. In one embodiment, the one or more binding domains are located on one or more electrodes, and detecting, identifying, or quantifying includes applying a voltage waveform to one or more electrodes to stimulate the label of the captured reaction product to generate an electrochemical or luminescent signal. In one embodiment, detecting, identifying, or quantifying includes measuring an electrochemiluminescent signal and correlating the signal with the presence or amount of target nucleotide sequence in the sample. In one embodiment, the intensity of the emitted light is proportional to the amount of target in the sample, so that the emitted light can provide a quantitative determination of the amount of target nucleotide in the sample.

[0345] In one embodiment, the support surface is contacted with a detection mixture after the reaction product is immobilized thereon. In one embodiment, the detection mixture includes an electrochemiluminescent label. Examples of electrochemiluminescent labels include: i) organometallic compounds in which the metal is derived from a Group VIII noble metal, including, for example, Ru- and Os-containing organometallic compounds such as tris-bipyridyl-ruthenium (RuBpy) moieties; and ii) luminol and related compounds. In one embodiment, the detection mixture also includes one or more electrochemiluminescent coreactants and one or more additional components, such as a pH buffer, a surfactant, an antifoaming agent, an antifoaming agent, a salt, a metal ion, or a metal chelator. The term "electrochemiluminescent coreactant" refers to the species involved in the electrochemiluminescent label and includes, but is not limited to, tertiary amines such as tripropylamine (TPA), oxalate ions, ascorbic acid, and persulfate for RuBpy and hydrogen peroxide for luminol. Methods for measuring electrochemiluminescence are known, and instruments for performing the measurements are commercially available. For example, multiplexed measurement of analytes using electrochemiluminescence is used in Meso Scale Diagnostics, LLC, MULTI-ARRAY® and SECTOR® Imager lines or products (see, e.g., U.S. Pat. Nos. 7,842,246 and 6,977,722, the disclosures of which are incorporated herein by reference in their entireties).

[0346] In one embodiment, biotin is covalently attached to the reaction product, and the detection mixture includes a streptavidin-conjugated label that binds to the immobilized reaction product via an avidin moiety. In one embodiment, the streptavidin-conjugated label is an electrochemiluminescent (ECL) label. In one embodiment, the electrochemiluminescent label is an n-hydroxysuccinimide ester, such as Sulfo-TAG NHS-ester (Meso Scale Diagnostics).

[0347] In one embodiment, the kit or method is used to detect, identify or quantify one or more single nucleotide polymorphisms (SNPs) in one or more target nucleotide sequences. In one embodiment, the presence of the SNP of interest is detected by determining the ratio between wild-type alleles and variant alleles in a sample. In one embodiment, the ratio is determined by determining the ratio of the detectable labels of wild-type and variant alleles present in a sample. In one embodiment, the ratio of the electrochemiluminescent labels of wild-type and variant alleles is determined. The ratio of wild-type or variant alleles present in a sample can be determined using the following formula: ECL ratio WT=(SignalWT-) / (SignalWT-Bkg+SignalMUT-Bkg) ECL ratio MUT=(SignalMUT-Bkg) / (SignalWT-Bkg+SignalMUT-Bkg) where SignalWT is the ECL signal detected by the wild-type allele, SignalMUT is the signal detected by the variant allele, and Bkg is the background signal. In one embodiment, the background signal may vary between binding domains, so the background signal is specific to the binding domain corresponding to the wild-type or variant allele. In one embodiment, the background value is the average value of duplicate spots in two wells of the "no ligase control" sample.

[0348] This ratio estimates the proportion of wild-type and variant sequences present in the sample. In one embodiment, the likelihood of a sample is homozygous wild-type, heterozygous, or homozygous variant. In one embodiment, the homozygous wild-type or variant ratio should be greater than about 0.8, heterozygous should be about 0.3 to about 0.7, and the absence of variants (or wild-type) should be less than about 0.2. The ratio of homozygous alleles may be greater than 1.0 due to signal variability. Similarly, if an allele is absent, the ratio may be less than zero due to background subtraction.

[0349] In one embodiment, the kit or method is used to detect one or more rare or low allele fractions of cancer mutations. In one embodiment, the frequency of the rare or low allele fraction mutations present in a sample is determined by generating a calibration curve from the ECL signal using the following formula: ECL ratio MUT=(SignalMUT) / (SignalWT+SignalMUT).

[0350] Background subtraction is not required to create a standard curve, as all signals are compared to the standard curve and the background is taken into account in the goodness of fit. The standard curve establishes the lowest percent variant allele detectable for a particular allele, and fitting sample data to the curve allows for the determination of the percent variant present in each sample.

[0351] In one embodiment, the assay comprises approximately 1 x 10 per well. 5 ~10×10 5 , or approximately 10 × 10 5 , 9×10 5 , 8×10 5 , 7×10 5 , 6×10 5 , 5×10 5 , 4×10 5 , 3×10 5...

Claims

(i) a set of two or more non-cross-reactive capture oligonucleotides, the set of two or more non-cross-reactive capture oligonucleotides being a subset of a set of parent capture oligonucleotides, the set of parent capture oligonucleotides comprising: (a) a parent capture set 1 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 1-64; (d) a parent capture set 4 comprising a capture oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 187-250; A set of two or more non-cross-reactive capture oligonucleotides selected from: (ii) a corresponding set of two or more non-cross-reactive oligonucleotide tags, said set of two or more non-cross-reactive oligonucleotide tags being a subset of a set of parent oligonucleotide tags, said set of parent oligonucleotide tags comprising: (a) a parent tag set 1 comprising an oligonucleotide tag containing a nucleotide sequence selected from SEQ ID NOs: 745-808; (d) a parent tag set 4 comprising an oligonucleotide tag containing a nucleotide sequence selected from SEQ ID NOs: 931-994; and is selected from said set of two or more non-cross-reactive oligonucleotide tags hybridize to their corresponding complementary set of two or more non-cross-reactive capture oligonucleotides; A set of two or more corresponding non-cross-reactive oligonucleotide tags Including the kit.

2. 2. The kit of claim 1, wherein the set of two or more non-cross-reactive capture oligonucleotides is selected from capture oligonucleotides comprising a sequence selected from SEQ ID NOs: 1-64.

3. The kit of claim 1 or 2, wherein the set of two or more non-cross-reactive capture oligonucleotides is selected from capture oligonucleotides comprising a sequence selected from SEQ ID NOs: 1-10.

4. The kit of any one of claims 1 to 3, wherein the set of two or more non-cross-reactive capture oligonucleotides comprises a reactive functional group.

5. the reactive functional group is attached to the capture oligonucleotide via a linker; The kit according to claim 4.

6. The kit of claim 4 or 5, wherein the reactive functional group comprises a thiol group.

7. The kit of any one of claims 4 to 6, wherein the set of two or more non-cross-reactive capture oligonucleotides is immobilized on a surface via the reactive functional groups.

8. The kit of claim 7 , wherein the surface comprises an electrode surface.

9. The kit of claim 8 , wherein the electrode comprises a carbon-based electrode.

10. The kit of claim 9 , wherein the electrodes comprise carbon ink electrodes.

11. The kit of claim 1, wherein the set of two or more non-cross-reactive capture oligonucleotides is at least 24 nucleotides in length.

12. The kit of claim 1, wherein the set of two or more non-cross-reactive capture oligonucleotides is at least 36 nucleotides in length.

13. (a) one or more support surfaces; (b) a set of two or more non-cross-reactive capture oligonucleotides according to any one of claims 1 to 12, wherein the set of two or more non-cross-reactive capture oligonucleotides is immobilized on one or more support surfaces; Including the kit.

14. (a) one or more carbon-based electrodes having one or more surfaces; (b) a set of two or more non-cross-reactive capture oligonucleotides according to any one of claims 1 to 12, wherein the set of two or more non-cross-reactive capture oligonucleotides is immobilized on one or more surfaces of the carbon-based electrode; Including the kit.

15. 1. A method for identifying, detecting, or quantifying a target analyte in a sample, the method comprising: (a) (i) one or more carbon-based electrodes having one or more surfaces; (ii) a set of two or more non-cross-reactive capture oligonucleotides according to any one of claims 1 to 12, wherein the set of two or more non-cross-reactive capture oligonucleotides is immobilised on one or more surfaces of the carbon-based electrode; (iii) a set of two or more corresponding non-cross-reactive oligonucleotide tags according to any one of claims 1 to 12; contacting the array with a composition comprising one or more target analytes, contacting, wherein the one or more target analytes are associated with a label and an oligonucleotide tag that is complementary to at least a portion of the capture oligonucleotide; (b) incubating said composition of one or more target analytes under conditions in which said oligonucleotide tags hybridize to their corresponding complementary capture oligonucleotides to form hybridization complexes; (c) identifying, detecting, or quantifying the target analyte based on the presence or absence of the label at the array location; and A method comprising:

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