Kits for detecting one or more target analytes in a sample and methods of making and using the same

Abstract

This invention provides oligonucleotides, methods, and kits for detecting, identifying, or quantifying one or more target analytes in a sample, as well as methods for immobilizing oligonucleotides onto a carrier surface.

Description

[0001] This application is a divisional application of the same invention patent application 202080045391.2 filed on April 30, 2020. Technical Field

[0002] This disclosure relates to a kit for detecting one or more target analytes in a sample, and methods for preparing and using the kit.

[0003] [Sequence List]

[0004] This application contains a sequence list that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on April 17, 2020, and is named 0076-0006WO1_SL.txt, with a size of 431,364 bytes. Background Technology

[0005] Single nucleotide polymorphisms (SNPs) are single nucleotide variations in the genome of an organism, characterized by two or more distinct nucleotide residues (alleles) that appear in a significant portion (>1%) of the population. SNPs are the most frequent form of sequence variation in an individual and are involved in the pathogenesis of various genetic diseases. Wang et al. (1998), "Large-scale identification, localization, and genotyping of single nucleotide polymorphisms in the human genome" (… Large-Scale Identification, Mapping, and Genotyping of Single- Nucleotide Polymorphisms in the Human Genome ), Science, 280:1077-1082. An estimated 10 million SNPs exist in the human genome, which can occur in both coding and non-coding regions. Kruglyak et al. (2001), "Change for Life," [reference needed]. Variation is the Spice of Life ( ), Nature Genetics, 27:234-236. Many SNPs have no effect on cell function, but others have been associated with hereditary traits, hereditary diseases, age-related diseases, and responses to drugs and environmental factors.

[0006] Genotyping analysis is a gene test used to detect the presence of nucleotide sequences in a sample and can be used to detect the presence of SNPs or other sequence variants in the sample, including (but not limited to) deletions and insertions, duplications, and translocations. High-density oligonucleotide arrays use hundreds of thousands of probes arranged on a chip to study multiple nucleotide sequences simultaneously.

[0007] Because large-scale analysis of nucleotide sequences in samples is required to associate sequences with diseases or susceptibility to diseases, with individual variability in drug response, or for population studies, kits for identifying nucleotide sequences in samples are still needed. Summary of the Invention

[0008] This invention relates to kits for identifying, detecting, or quantifying one or more target analytes in a sample, and methods for their preparation and use. In one aspect, the target analyte comprises a nucleic acid sequence. In another aspect, the target analyte comprises a polypeptide sequence. In one aspect, the method or kit comprises one or more capture molecules in a dispersed binding domain immobilized or immobilizable on a carrier surface. In one aspect, a collection of two or more non-cross-reactive capture oligonucleotides is provided.

[0009] In one aspect, the capturing oligonucleotide set is a subset of the parent set of capturing oligonucleotides, and one or more capturing 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 having one or more nucleotide sequences from the parent set, wherein the parent set of capturing oligonucleotides is selected from the following:

[0010] (a) Capture set 1, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 1-64;

[0011] (b) Capture set 2, which comprises capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 65-122;

[0012] (c) Capture set 3, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 123-186;

[0013] (d) Capture set 4, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 187-250;

[0014] (e) Capture set 5, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 251-308;

[0015] (f) Capture set 6, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 309-372;

[0016] (g) Capture set 7, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 373-436;

[0017] (h) Capture set 8, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 437-494;

[0018] (i) Capture set 9, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 495-558;

[0019] (j) Capture set 10, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 559-662;

[0020] (k) Capture set 11, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 623-680; and

[0021] (l) Capture set 12, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 681-744.

[0022] In another aspect, a set of two or more non-cross-reactive capturing oligonucleotides is provided, wherein the set of capturing oligonucleotides is a subset of a parent set of capturing oligonucleotides, and one or more capturing oligonucleotides in the set comprise a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more nucleotide sequences from the parent set, wherein the parent set of capturing oligonucleotides is selected from the following:

[0023] (a) Capture set 1, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 1-64;

[0024] (b) Capture set 2, which comprises capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 65-122;

[0025] (c) Capture set 3, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 123-186;

[0026] (d) Capture set 4, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 187-250;

[0027] (e) Capture set 5, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 251-308;

[0028] (f) Capture set 6, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 309-372;

[0029] (g) Capture set 7, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 373-436;

[0030] (h) Capture set 8, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 437-494;

[0031] (i) Capture set 9, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 495-558;

[0032] (j) Capture set 10, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 559-662;

[0033] (k) Capture set 11, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 623-680; and

[0034] (l) Capture set 12, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 681-744.

[0035] In another aspect, a set of two or more non-cross-reactive capturing oligonucleotides is provided, wherein the set of capturing oligonucleotides is a subset of a parent set of capturing oligonucleotides, wherein the parent set of capturing oligonucleotides is selected from the following:

[0036] (a) Capture set 1, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 1-64;

[0037] (b) Capture set 2, which comprises capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 65-122;

[0038] (c) Capture set 3, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 123-186;

[0039] (d) Capture set 4, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 187-250;

[0040] (e) Capture set 5, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 251-308;

[0041] (f) Capture set 6, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 309-372;

[0042] (g) Capture set 7, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 373-436;

[0043] (h) Capture set 8, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 437-494;

[0044] (i) Capture set 9, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 495-558;

[0045] (j) Capture set 10, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 559-662;

[0046] (k) Capture set 11, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 623-680; and

[0047] (l) Capture set 12, which contains capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 681-744.

[0048] In one aspect, the set of two or more non-cross-reactive capturing oligonucleotides comprises one or more capturing oligonucleotides selected from the following:

[0049] (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 selected from the sequence of SEQ ID No: 1-64;

[0050] (b) Capture oligonucleotides comprising a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID No: 1-64;

[0051] (c) 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 having at least 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID No: 1-64;

[0052] (d) Capture oligonucleotides comprising sequences selected from SEQ ID No: 1-64; and

[0053] (e) Capture oligonucleotides, which are selected from any one of (a)-(d).

[0054] In one aspect, the set of two or more non-cross-reactive capturing oligonucleotides comprises one or more capturing oligonucleotides selected from the following:

[0055] (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 selected from the sequences of SEQ ID No: 1-10;

[0056] (b) Capture oligonucleotides comprising a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID No: 1-10;

[0057] (c) 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 having at least 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID No: 1-10;

[0058] (d) Capture oligonucleotides comprising sequences selected from SEQ ID No: 1-10; and

[0059] (e) Capture oligonucleotides, which are selected from any one of (a)-(d).

[0060] In one aspect, a set of non-cross-reactive capture oligonucleotides immobilized in an array is provided, wherein the set of non-cross-reactive oligonucleotides satisfies one or more of the following requirements:

[0061] (a) GC content is between approximately 40% and approximately 50%;

[0062] (b) AG content is between approximately 30% and approximately 70%;

[0063] (c) CT content is between approximately 30% and approximately 70%;

[0064] (d) A chain of sequences with no more than three maximum base repeats;

[0065] (e) No unwanted oligonucleotide-oligonucleotide interactions with strands that match more than 7 consecutive complementary base pairs;

[0066] (f) No unwanted oligonucleotide-oligonucleotide interactions with chains of 18 consecutive bases or less, wherein:

[0067] (i) The terminal bases at each end are complementary; and

[0068] (ii) The sum of the complementary base pair matches minus the sum of the mismatches is greater than 7;

[0069] (g) No strands of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 base pairs or longer, said strands matching sequences in the genome or in nature or complementary sequences of sequences or both;

[0070] (h) The difference in hybridization free energy between the sequence and its complementary sequence is less than about 1 kCal / mol, about 2 kCal / mol, about 3 kCal / mol, or about 4 kCal / mol;

[0071] (i) There are no predicted hairpin loops with 4 or more consecutive matches in the stem; and

[0072] (j) There are no predicted hairpin loops with 4 or more consecutive matching in the stem, and the loop size is greater than 6 bases.

[0073] In one aspect, the collection of two or more non-cross-reactive capturing oligonucleotides comprises one or more capturing oligonucleotides having a reactive functional group. In one aspect, the reactive functional group is linked to the capturing oligonucleotide via a linker. In one aspect, the reactive functional group comprises a thiol group.

[0074] In one aspect, one or more oligonucleotides are immobilized on a surface by reactive functional groups. In one aspect, the surface comprises an electrode surface. In one aspect, the electrode comprises a carbon-based electrode. In one aspect, the electrode comprises a carbon-ink electrode.

[0075] In one aspect, one or more non-reactive capturing oligonucleotides are at least 20 nucleotides long. In one aspect, one or more non-reactive capturing oligonucleotides are at least 24 nucleotides long. In one aspect, one or more non-reactive capturing oligonucleotides are at least 36 nucleotides long.

[0076] In one aspect, a kit is provided comprising a collection of two or more non-cross-reactive capture oligonucleotides. In another aspect, the kit comprises a collection 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 capture oligonucleotides. In another aspect, the kit comprises at least 10 non-cross-reactive capture oligonucleotides.

[0077] 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 parent set of oligonucleotide tags, and one or more oligonucleotide tags in the set comprise a nucleotide sequence having at least 20, 21, 22, 23, or 24 consecutive nucleotides having one or more nucleotide sequences from the parent set, wherein the parent set is selected from:

[0078] (a) Tag set 1, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 745-808;

[0079] (b) Tag set 2, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 809-866;

[0080] (c) Tag set 3, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 867-930;

[0081] (d) Tag set 4, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 931-994;

[0082] (e) Tag set 5, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 995-1052;

[0083] (f) Tag set 6, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1053-1116;

[0084] (g) Tag set 7, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1117-1180;

[0085] (h) Tag set 8, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1181-1238;

[0086] (i) Tag set 9, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1239-1302;

[0087] (j) Tag set 10, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1303-1366;

[0088] (k) Tag set 11, comprising oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1367-1424; and

[0089] (l) Tag set 12, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1425-1488.

[0090] 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 parent set of oligonucleotide tags, and one or more oligonucleotides in the set comprise a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more nucleotide sequences from the parent set, wherein the parent set is selected from:

[0091] (a) Tag set 1, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 745-808;

[0092] (b) Tag set 2, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 809-866;

[0093] (c) Tag set 3, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 867-930;

[0094] (d) Tag set 4, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 931-994;

[0095] (e) Tag set 5, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 995-1052;

[0096] (f) Tag set 6, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1053-1116;

[0097] (g) Tag set 7, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1117-1180;

[0098] (h) Tag set 8, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1181-1238;

[0099] (i) Tag set 9, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1239-1302;

[0100] (j) Tag set 10, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1303-1366;

[0101] (k) Tag set 11, comprising oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1367-1424; and

[0102] (l) Tag set 12, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1425-1488.

[0103] 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 parent set of oligonucleotide tags, wherein the parent set is selected from:

[0104] (a) Tag set 1, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 745-808;

[0105] (b) Tag set 2, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 809-866;

[0106] (c) Tag set 3, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 867-930;

[0107] (d) Tag set 4, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 931-994;

[0108] (e) Tag set 5, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 995-1052;

[0109] (f) Tag set 6, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1053-1116;

[0110] (g) Tag set 7, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1117-1180;

[0111] (h) Tag set 8, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1181-1238;

[0112] (i) Tag set 9, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1239-1302;

[0113] (j) Tag set 10, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1303-1366;

[0114] (k) Tag set 11, comprising oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1367-1424; and

[0115] (l) Tag set 12, which contains oligonucleotide tags having nucleotide sequences selected from SEQ ID No: 1425-1488.

[0116] In one aspect, a set of two or more non-cross-reactive oligonucleotide tags contains one or more oligonucleotide tags selected from the following:

[0117] (a) An oligonucleotide tag comprising a sequence having at least 20, 21, 22, 23 or 24 consecutive nucleotides selected from the sequence of SEQ ID No: 745-808;

[0118] (b) An oligonucleotide tag comprising a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID No: 745-808;

[0119] (c) An oligonucleotide tag having at least 20, 21, 22, 23 or 24 consecutive nucleotides having at least 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID No: 745-808;

[0120] (d) An oligonucleotide tag comprising a sequence selected from SEQ ID No: 745-808; and

[0121] (e) An oligonucleotide tag, selected from any one of (a)-(d).

[0122] In one aspect, a set of two or more non-cross-reactive oligonucleotide tags contains one or more oligonucleotide tags selected from the following:

[0123] (a) An oligonucleotide tag comprising a sequence having at least 20, 21, 22, 23 or 24 consecutive nucleotides selected from the sequence of SEQ ID No: 745-754;

[0124] (b) An oligonucleotide tag comprising a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID No: 745-754;

[0125] (c) An oligonucleotide tag having at least 20, 21, 22, 23 or 24 consecutive nucleotides having at least 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID No: 745-754;

[0126] (d) An oligonucleotide tag comprising a sequence selected from SEQ ID No: 745-754; and

[0127] (e) An oligonucleotide tag, selected from any one of (a)-(d).

[0128] In one aspect, the set comprises two or more non-cross-reactive oligonucleotide tags, wherein one or more oligonucleotide tags in the set hybridize with less than 0.05% of the non-complementary capturing oligonucleotide relative to the complementary capturing oligonucleotide.

[0129] In one aspect, one or more oligonucleotide tags in the set comprise a marker. In one aspect, the marker is linked to the oligonucleotide tag via a linker. In one aspect, the marker is selected from radioactive, fluorescent, chemiluminescent, electrochemiluminescent, light-absorbing, light-scattering, electrochemical, magnetic, and enzyme-labeled markers. In one aspect, the marker comprises an electrochemiluminescent marker. In one aspect, the marker is selected from radioactive, fluorescent, colorimetric, antigenic, and enzyme-labeled markers. In one aspect, the marker comprises biotin or a hapten. In one aspect, the marker comprises biotin, fluorescein, or digoxigenin. In one aspect, the marker comprises an organometallic complex containing a transition metal. In one aspect, the transition metal comprises ruthenium. In one aspect, the marker comprises an MSD SULFO-TAG. TM A label. In one aspect, the label comprises a primary binding agent, said primary binding agent being a conjugate of a secondary binding agent. In one aspect, the secondary binding agent comprises biotin, streptavidin, avidin, or an antibody. In one aspect, the primary binding agent comprises an oligonucleotide, and the secondary binding agent comprises an oligonucleotide complementary to the primary binding agent.

[0130] In one aspect, a kit is provided comprising a set of two or more non-cross-reactive oligonucleotide tags. In another aspect, the kit comprises a set 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 oligonucleotide tags. In another aspect, the kit comprises a set of at least 10 non-cross-reactive oligonucleotide tags.

[0131] In one aspect, a method is provided for immobilizing an oligonucleotide on a carbon-based support surface. In one aspect, the oligonucleotide comprises a thiol group. In one aspect, the method comprises:

[0132] (a) Printing one or more droplets containing the oligonucleotide onto the surface of the carbon-based support;

[0133] (b) Allow the droplets to diffuse on the surface;

[0134] (c) Dry the droplets to form dry droplets;

[0135] (d) Immobilizing the oligonucleotide onto the surface of the carbon-based support via the thiol group; and

[0136] (e) Wash the dried droplets with a washing solution containing a thiol compound to remove unfixed oligonucleotides.

[0137] In one aspect, a method is provided for manufacturing a carbon-based support surface having one or more immobilized oligonucleotides. In one aspect, the method comprises:

[0138] (a) Printing one or more droplets comprising thiol-containing oligonucleotides onto the surface of the carbon-based support;

[0139] (b) Allow the droplets to diffuse on the surface;

[0140] (c) Dry the droplets to form dry droplets;

[0141] (d) Immobilizing the oligonucleotide onto the surface of the carbon-based support via the thiol group; and

[0142] (e) Wash the dried droplets with a washing solution containing thiol compounds to remove unfixed oligonucleotides.

[0143] In one aspect, the carbon-based support surface includes a carbon-based electrode. In another aspect, an oligonucleotide is covalently linked to the carbon-based support surface via a thiol group. In yet another aspect, the method comprises printing an array of droplets containing the captured oligonucleotide onto a plurality of binding domains on the carbon-based support surface.

[0144] In one aspect, a perforated plate is provided, comprising:

[0145] (a) having one or more holes with one or more carbon-based electrodes; and

[0146] (b) A collection of one or more non-cross-reactive capture oligonucleotides selected from the parent set of non-cross-reactive capture oligonucleotides as described herein, wherein one or more non-cross-reactive capture oligonucleotides are immobilized on one or more carbon-based electrodes.

[0147] In one aspect, a method for manufacturing a porous plate is provided, wherein the method comprises:

[0148] (a) Printing one or more droplets comprising a thiol-containing oligonucleotide onto the carbon-based electrode;

[0149] (b) Allow the droplets to diffuse on the surface;

[0150] (c) Dry the droplets to form dry droplets;

[0151] (d) Immobilizing the oligonucleotide to the carbon-based electrode via the thiol group; and

[0152] (e) Wash the dried droplets with a washing solution containing thiol compounds to remove unfixed oligonucleotides.

[0153] In one aspect, the carbon-based support surface includes a carbon-based electrode. In another aspect, an oligonucleotide is covalently linked to the carbon-based support surface via a thiol group. In yet another aspect, the method comprises printing an array of droplets containing the captured oligonucleotide onto a plurality of binding domains on the support surface.

[0154] In one aspect, a method is provided for immobilizing a capture oligonucleotide array on the surface of one or more carbon-based electrodes. In one aspect, the method comprises:

[0155] (a) Printing an array of droplets containing 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 contain thiol groups;

[0156] (b) Allow the droplets to diffuse on the surface;

[0157] (c) Dry the droplets to form dry droplets;

[0158] (d) Cultivating the dried droplets for a sufficient amount of time to immobilize one or more oligonucleotides onto the surface of the carbon-based electrode via the thiol groups; and

[0159] (e) Wash the dried droplets with a washing solution containing a thiol compound to remove excess unfixed oligonucleotides.

[0160] In one aspect, the capturing oligonucleotide printed on one binding domain of the array has a sequence different from that printed on the capturing oligonucleotides on other binding domains of the array. 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 capturing oligonucleotides. In one aspect, each binding domain contains less than about 0.05% of contaminating capturing oligonucleotides.

[0161] In one aspect, the method includes printing an array on a plurality of carbon-based electrodes. In one aspect, one or more carbon-based electrodes comprise a plurality of binding domains. In one aspect, the carbon-based electrodes comprise carbon ink electrodes. In one aspect, one or more carbon-based electrodes are in a porous plate.

[0162] In one aspect, the droplet contains a surfactant. In one aspect, 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 aspect, the thiol-containing compound is selected from cysteine, cysteamine, dithiothreitol, 3-mercaptopropionate, 3-mercapto-1-propanesulfonic acid, and combinations thereof. In one aspect, the thiol-containing compound contains cysteine. In one aspect, the washing solution contains cysteine ​​between about 5 mM and about 750 mM, cysteine ​​between about 10 mM and about 500 mM, or cysteine ​​between about 25 mM and about 75 mM. In one aspect, the washing solution contains a pH buffer component, a surfactant, or a combination thereof, and has a pH between about 7 and 9. In one aspect, the pH buffer component contains Tris, the surfactant contains Triton X-100, and the pH is about 8.0.

[0163] 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 prior to a washing step. In one aspect, the carbon-based electrode is packaged in a dry package after a washing step.

[0164] In one aspect, a kit is provided comprising:

[0165] (a) One or more carrier surfaces; and

[0166] (b) A collection of one or more non-cross-reactive capture oligonucleotides selected from a parent set of non-cross-reactive capture oligonucleotides, wherein one or more non-cross-reactive capture oligonucleotides are immobilized on one or more carrier surfaces.

[0167] In one aspect, one or more support surfaces provided in the kit comprise a carbon-based support surface. In another aspect, one or more support surfaces comprise a carbon-based electrode.

[0168] In one aspect, a kit is provided comprising:

[0169] (a) One or more carbon-based electrodes having one or more surfaces; and

[0170] (b) A collection of one or more non-cross-reactive capture oligonucleotides selected from a parent set of non-cross-reactive capture oligonucleotides, wherein one or more non-cross-reactive capture oligonucleotides are immobilized on one or more surfaces of a carbon-based electrode.

[0171] In one aspect, the kit contains one or more carbon ink electrodes.

[0172] In one aspect, the kit comprises one or more non-cross-reactive capturing oligonucleotides in an array immobilized on one or more carrier surfaces. In one aspect, one or more non-cross-reactive capturing oligonucleotides are immobilized in one or more binding domains. In one aspect, two or more non-cross-reactive capturing oligonucleotides are immobilized in two or more unique binding domains, wherein the capturing oligonucleotides immobilized in each unique binding domain have the same sequence.

[0173] In one aspect, the kit contains one or more of the following components:

[0174] (a) A labeled oligonucleotide probe containing a sequence complementary to the target sequence in the nucleic acid of interest.

[0175] (b) One or more end-capping probes,

[0176] (c) One or more nucleoside triphosphates,

[0177] (d) One or more labeled nucleoside triphosphates,

[0178] (e) One or more labeled dideoxynucleotide triphosphates;

[0179] (f) One or more conjugases, and

[0180] (g) One or more polymerases.

[0181] In one aspect, the kit comprises a plurality of labeled oligonucleotide probes, the plurality of labeled oligonucleotide probes comprising a first sequence complementary to a target sequence in the nucleic acid of interest and an oligonucleotide tag having a sequence complementary to the sequence of the captured oligonucleotide.

[0182] 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 polymorphic nucleotides, and the kit comprises at least one pair of oligonucleotide probes containing:

[0183] (i) a targeting probe comprising a single-stranded oligonucleotide tag complementary to at least a portion of a capture oligonucleotide immobilized on a carrier surface and a first nucleic acid sequence complementary to a first region of a target nucleotide sequence in a sample; and

[0184] (ii) A detection probe comprising a label and a second nucleic acid sequence complementary to a second region of a target nucleotide sequence, the second region being adjacent to a first region complementary to the first nucleic acid sequence of the same target probe sequence, wherein the target probe or the detection probe comprises a terminal 3' or 5' nucleotide of a polymorphic nucleotide located in the target nucleotide sequence.

[0185] In one aspect, the targeting probe has a terminal 3' nucleotide complementary to a region of the target nucleotide sequence, said region being adjacent to a region complementary to the 5' terminal nucleotide of the detection probe. In another aspect, the terminal 3' nucleotide of the targeting probe is complementary to a polymorphic nucleotide of the target nucleotide sequence.

[0186] In one aspect, the kit comprises first and second targeting probes that bind to a target nucleotide sequence, wherein the first and second targeting probes differ only in their terminal 3' nucleotides. In another aspect, the first targeting probe is complementary to a wild-type sequence, and the second targeting probe is complementary to a mutant sequence.

[0187] In one aspect, the kit contains multiple oligonucleotide probe pairs for multiple target nucleotide sequences.

[0188] In one aspect, the kit contains a detection probe with a label attached to its 3' end.

[0189] 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 contain polymorphic nucleotides, and the kit comprises one or more targeting probes containing:

[0190] (a) A single-stranded oligonucleotide tag complementary to at least a portion of the capture oligonucleotide immobilized on the surface of the carrier;

[0191] (b) A target nucleic acid sequence complementary to the target nucleotide sequence in the sample; and

[0192] (c) Marking.

[0193] In one aspect, the kit contains one or more of the following:

[0194] (a) polymerase; and

[0195] (b) One or more dideoxynucleotide triphosphates (ddNTPs).

[0196] In one aspect, the kit includes an oligonucleotide tag attached to the 5' end of a targeting probe, and the targeting nucleic acid sequence includes a 3' end complementary to a nucleotide adjacent to a polymorphic nucleotide in one or more target nucleotide sequences in the sample. In another aspect, the oligonucleotide tag is attached to the 5' end of a targeting probe, and the targeting nucleic acid sequence includes a terminal 3' nucleotide complementary to a polymorphic nucleotide in one or more target nucleotide sequences in the sample.

[0197] In one aspect, the kit contains multiple probes containing a target nucleic acid sequence complementary to multiple target nucleotide sequences in the sample.

[0198] In one aspect, the kit comprises a labeled nucleoside triphosphate. In one aspect, the kit comprises a labeled nucleoside triphosphate and a secondary binding reagent. In one aspect, the kit comprises a labeled nucleoside triphosphate containing a binding complex with a secondary binding reagent. In one aspect, the kit comprises: a secondary binding reagent comprising avidin, streptavidin, or an antibody; and a labeled nucleoside triphosphate comprising biotin or a hapten label.

[0199] In one aspect, the kit comprises nucleoside triphosphates labeled with radioactive, fluorescent, chemiluminescent, electrochemiluminescent, light-absorbing, light-scattering, electrochemical, magnetic, or enzyme-labeled substances. In another aspect, the nucleoside triphosphates are labeled with electrochemiluminescent labels.

[0200] In one aspect, the kit contains a labeled dideoxynucleotide triphosphate complementary to the polymorphic nucleotide of the target nucleotide sequence.

[0201] In one aspect, the kit contains one or more of the following components:

[0202] (a) Hybridization buffer,

[0203] (b) Binding buffer,

[0204] (c) Marking,

[0205] (d) Secondary binding reagent,

[0206] (d) Reading buffer; and

[0207] (e) Unique reagent kit identifier.

[0208] In one aspect, the hybridization buffer contains a nucleic acid denaturing agent. In one aspect, the nucleic acid denaturing agent contains formamide. In one aspect, the hybridization buffer is provided as two separate components that can be combined to form a hybridization buffer. In one aspect, the binding buffer contains a surfactant. In one aspect, the reading buffer contains an electrochemiluminescent reading buffer. In one aspect, the electrochemiluminescent reading buffer contains one or more electrochemiluminescent co-reactants selected from tertiary amines, tripropylamine, and N-butyldiethanolamine.

[0209] In one aspect, the kit comprises one or more non-cross-reactive capturing oligonucleotides in one or more binding domains immobilized on the surface of one or more carbon-based electrodes. In one aspect, the one or more capturing oligonucleotides comprise a thiol group and are covalently bound to the carbon-based surface via the thiol group. In one aspect, the capturing oligonucleotide is linked to the thiol group via a linker. In one aspect, the capturing oligonucleotide is covalently linked to a carrier surface. In one aspect, the non-cross-reactive capturing oligonucleotide is immobilized in an array. In one aspect, the non-cross-reactive capturing oligonucleotide is immobilized on a bead array.

[0210] In one aspect, the kit contains a set of two or more non-cross-reactive capture oligonucleotides selected from the following:

[0211] (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 selected from the sequence of SEQ ID No: 1-64;

[0212] (b) Capture oligonucleotides comprising a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID No: 1-64;

[0213] (c) Capture oligonucleotides comprising sequences selected from SEQ ID No: 1-64; and

[0214] (d) Capture oligonucleotides selected from any of the aforementioned sets.

[0215] In one aspect, the kit contains a set of two or more non-cross-reactive capture oligonucleotides selected from the following:

[0216] (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 selected from the sequences of SEQ ID No: 1-10;

[0217] (b) Capture oligonucleotides comprising a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID No: 1-10;

[0218] (c) Capture oligonucleotides comprising sequences selected from SEQ ID No: 1-10; and

[0219] (d) Capture oligonucleotides selected from any of the aforementioned sets.

[0220] In one aspect, the oligonucleotide tag in the kit contains 24 or more nucleotides. In another aspect, the oligonucleotide tag contains 36 or more nucleotides.

[0221] In one aspect, oligonucleotide tags bind to less than 0.05% of non-complementary capturing oligonucleotides, relative to complementary capturing oligonucleotides.

[0222] In one aspect, the kit contains more than one electrode having one or more binding domains.

[0223] In one aspect, the kit comprises two or more capture oligonucleotides immobilized in two or more unique binding domains, wherein the capture oligonucleotides immobilized in each unique binding domain have identical sequences. In one aspect, the carrier surface comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 different capture oligonucleotides immobilized in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 unique binding domains. In one aspect, one or more binding domains comprise at least some capture oligonucleotides that are not covalently bound to the electrode surface via thiol groups. In one aspect, one or more binding domains comprise greater than 10%, 15%, 20%, 25%, 50%, or 75% of the capture oligonucleotides that are not covalently bound to the carrier surface via thiol groups. In one aspect, the 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.

[0224] In one aspect, the kit includes a carrier surface containing a multiwell plate. In one aspect, one or more wells of the multiwell plate contain one or more electrodes. In one aspect, the one or more electrodes contain carbon-based electrodes. In one aspect, the one or more electrodes contain carbon ink. In one aspect, one or more electrodes in one or more wells of the multiwell plate contain one or more binding domains. In one aspect, one or more capture oligonucleotides are immobilized onto one or more binding domains on one or more electrodes.

[0225] In one aspect, the kit comprises a wash solution containing a thiol-containing compound. In one aspect, 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 aspect, the thiol-containing compound is selected from the following: cysteine, cysteamine, dithiothreitol, 3-mercaptopropionate, 3-mercapto-1-propanesulfonic acid, and combinations thereof. In one aspect, the thiol-containing compound contains cysteine. In one aspect, the thiol-containing compound contains zwitterions. In one aspect, the wash solution comprises an aqueous solution. In one aspect, the aqueous wash solution contains cysteine ​​between about 5 mM and 750 mM, cysteine ​​between about 10 mM and about 500 mM, or cysteine ​​between about 25 mM and about 75 mM. In one aspect, the wash solution comprises a pH buffer component, a surfactant, or a combination thereof. In one aspect, the pH buffer contains Tris, and the surfactant contains Triton X-100. In one aspect, the wash solution has a pH between 7 and 9. In another aspect, the wash solution has a pH of approximately 8. In one aspect, the wash solution contains between approximately 15 mM and approximately 25 mM Tris, between approximately 0.05% and approximately 0.15% Triton X-100, between approximately 5 mM and approximately 750 mM cysteine, and has a pH of approximately 8.0. In one aspect, the wash solution contains between approximately 15 mM and approximately 25 mM Tris, between approximately 0.05% and approximately 0.15% Triton X-100, between approximately 25 mM and approximately 75 mM cysteine, and has a pH of approximately 8.0. In one aspect, the wash solution contains approximately 20 mM Tris, approximately 0.1% Triton X-100, approximately 50 mM cysteine, and has a pH of approximately 8.0. In one aspect, one or more components of the wash solution are provided in anhydrous form. In one aspect, the kit includes a liquid diluent for reconstituted components of the wash solution.

[0226] In one aspect, the kit comprises one or more capping probes. In another aspect, the kit comprises one or more pairs of capping probes, wherein each pair of capping probes comprises:

[0227] (a) A first capping probe containing a sequence identical to that of the target probe, but without the single-stranded oligonucleotide tag; and

[0228] (b) A second end-capping probe having a sequence consistent with that of the detection probe, but without the marker.

[0229] In one aspect, the kit contains at least one pair of capping probes for each pair of oligonucleotide probes.

[0230] In one aspect, the kit comprises a label selected from: radioactive, fluorescent, chemiluminescent, electrochemiluminescent, light-absorbing, light-scattering, electrochemical, magnetic, and enzyme-labeled. In one aspect, the label comprises an electrochemiluminescent label. In one aspect, the label comprises an organometallic complex containing a transition metal. In one aspect, the transition metal comprises ruthenium. In one aspect, the label comprises MSD SULFO-TAG. TM A label. In one aspect, the label comprises a binding complex of a secondary binding agent. In one aspect, the secondary binding agent comprises biotin, streptavidin, avidin, or an antibody. In one aspect, the label comprises a hapten selected from biotin, fluorescein, and digoxigenin. In one aspect, the label comprises a primary binding agent containing a first oligonucleotide sequence, and the secondary binding agent comprises a second oligonucleotide sequence complementary to the first oligonucleotide sequence of the primary binding agent.

[0231] In one aspect, a method is provided for identifying, detecting, or quantifying a target analyte. In one aspect, the method comprises providing an array of one or more carbon-based electrodes having one or more surfaces; and one or more non-cross-reactive capturing oligonucleotides immobilized on one or more surfaces of the one or more carbon-based electrodes in one or more binding domains; contacting the array with a composition comprising one or more target analytes, wherein the one or more target analytes are linked to an oligonucleotide tag and label complementary to at least a portion of the capturing oligonucleotides immobilized on the carrier surface; cultivating the composition of one or more target analytes under conditions in which the oligonucleotide tag hybridizes with its complementary capturing oligonucleotide to form a hybridization complex; and identifying, detecting, or quantifying the target analyte based on the presence or absence of a label at a location in the array.

[0232] In one aspect, the method includes contacting an array with a composition comprising a plurality of target analytes, wherein each target analyte is linked to an oligonucleotide tag complementary to a different capturing oligonucleotide, and the target analyte can be identified, detected, or quantified based on the binding of the oligonucleotide tag at the array location. In one aspect, less than about 0.05% of the oligonucleotide tags bind to non-complementary capturing oligonucleotides on the array relative to their corresponding complementary capturing oligonucleotides. In one aspect, the oligonucleotide tag comprises at least 12, 24, or 36 nucleotides. In one aspect, one or more electrodes comprise carbon ink electrodes. In one aspect, one or more electrodes are contained in a multiwell plate. In one aspect, each well of the multiwell plate comprises an electrode. In one aspect, the electrodes in each well of the multiwell plate comprise an array of capturing oligonucleotides immobilized in a plurality of binding domains. In one aspect, one or more target analytes are linked to oligonucleotide tags via a binding complex. In one aspect, the binding complex comprises an antibody that specifically binds to the target analyte. In one aspect, the binding complex comprises an oligonucleotide sequence complementary to the oligonucleotide sequence of the target analyte. In one aspect, the oligonucleotide tag and the binding covariate are different regions of an oligonucleotide single strand. In another aspect, one or more target analytes comprise nucleic acid sequences, and the binding covariate comprises an oligonucleotide sequence complementary to a target sequence in the nucleic acid sequence of interest.

[0233] In one aspect, the method comprises cultivating a composition of one or more target analytes at a temperature between about 27°C and about 47°C, a formamide concentration between about 21% and about 41%, a salt concentration between about 300 mM and about 500 mM, and a pH between about 7.5 and about 8.5, such that an oligonucleotide tag hybridizes with its complementary capturing oligonucleotide to form a hybridization complex. In one aspect, cultivation 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.

[0234] In one aspect, one or more target analytes are labeled with a primary binding reagent. In another aspect, one or more target analytes are incubated with one or more pairs of oligonucleotide probes comprising the following:

[0235] (a) A targeting probe comprising a single-stranded oligonucleotide tag complementary to at least a portion of a capture oligonucleotide immobilized on a carrier surface and a first nucleic acid sequence complementary to a first region of a target nucleotide sequence in a sample; and

[0236] (b) A detection probe comprising a label and a second nucleic acid sequence complementary to the second region of the target nucleotide sequence.

[0237] The cultivation process involves cultivating the probe under conditions in which it binds to its corresponding target analyte.

[0238] In one aspect, the targeting probe and the detection probe can simultaneously bind to the target analyte to form a sandwich complex. In another aspect, the targeting probe and the detection probe have nucleotide sequences complementary to adjacent sequences of the target nucleotide sequence. In another aspect, the targeting probe has a 3' nucleic acid sequence containing a 3' end that hybridizes to the target nucleotide sequence adjacent to the 5' end of the same detection probe. In another aspect, the 3' nucleotide of the targeting probe is complementary to a polymorphic nucleotide in the target nucleotide sequence.

[0239] In one aspect, the method includes culturing a hybridization complex in the presence of a nuclease, wherein the nuclease conjugates a target probe and a detection probe to form a reaction product. In one aspect, the method includes exposing the reaction product to denaturing conditions that dissociate the reaction product from a target nucleotide sequence. In one aspect, the method includes adding one or more capping probes to the reaction product. In one aspect, the method includes culturing one or more target analytes together with a target probe comprising:

[0240] (a) A single-stranded oligonucleotide tag complementary to at least a portion of the capture oligonucleotide immobilized on the surface of the carrier;

[0241] (b) A target nucleic acid sequence complementary to the target nucleotide sequence in the sample; and

[0242] (c) Marking.

[0243] In one aspect, the method includes incubating one or more target analytes with a target probe under conditions in which a target nucleic acid sequence of a target probe hybridizes with a complementary sequence on a target nucleotide sequence to form a hybridization complex. In one aspect, the method includes incubating the hybridization complex in the presence of a nucleic acid polymerase, wherein the polymerase extends a first probe sequence to form an extended sequence. In one aspect, the method includes incubating the polymerase with one or more labeled nucleoside triphosphates, wherein the extended sequence comprises a labeled nucleoside triphosphate.

[0244] In one aspect, the method comprises incubating a polymerase with one or more unlabeled nucleoside triphosphates, wherein the length of the extended sequence is longer than one nucleotide and it contains an unlabeled nucleoside triphosphate. In one aspect, the labeled nucleoside triphosphate contains a chain-terminating nucleoside triphosphate, and the length of the extended sequence is one nucleotide. In one aspect, the labeled nucleoside triphosphate contains a labeled dideoxynucleoside triphosphate. In one aspect, the first nucleic acid sequence of the targeting probe has a 3' end complementary to a nucleotide adjacent to a polymorphic nucleotide in the same nucleic acid of interest, and the polymerase adds a nucleotide to the 3' end of the targeting nucleic acid sequence.

[0245] In one aspect, the method includes washing the array with a wash buffer after the incubation step. In one aspect, washing includes immersing the array in a wash buffer under high-tightness conditions. In one aspect, the high-tightness conditions include a temperature between about 27°C and about 47°C, a formamide concentration between about 21% and about 41%, a salt concentration between about 300 mM and about 500 mM, and a pH between 7.5 and 8.5. In one aspect, the high-tightness 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 aspect, the method includes immersing the array under high-tightness conditions for at least 5, 10, 30, or 60 minutes. In another aspect, the high-tightness conditions include low-salt conditions, such as a buffer with a salt concentration of less than about 40 mM, 20 mM, 15 mM, or 10 mM. In one aspect, the high-tightness conditions include low-salt conditions, such as 0.1X PBS at 37°C.

[0246] In one aspect, the label comprises an electrochemiluminescent label, and the method comprises the steps of generating an analytical signal by contacting an electrode with an electrochemiluminescent reading buffer containing an electrochemiluminescent co-reactant and applying a potential to the electrode. In one aspect, the co-reactant is selected from tertiary amines, tripropylamine, N-butyldiethanolamine, and combinations thereof. In one aspect, the method comprises imaging the analytical signal to determine the analytical signal associated with each captured oligonucleotide. Attached Figure Description

[0247] Figure 1A This is a schematic diagram of the hybridization steps in an oligonucleotide ligation assay (OLA). Figure 1B This is a schematic diagram of the OLA bonding process; Figure 1C This is a schematic diagram of the OLA detection steps; Figure 1D This is a schematic diagram of mismatched OLA probes that do not hybridize.

[0248] Figure 2A This is a schematic diagram of a primer extension assay (PEA), in which labeled ddNTPs are added to the 3' end of the probe. Figure 2B This is a schematic diagram of PEA, in which unlabeled ddNTPs are added to the 3' end of the probe.

[0249] Figure 3 A graph illustrating the effect of varying the length of the linker (or spacer) between the captured oligonucleotide and the electrode on the hybridization of the probe with the captured oligonucleotide and detection using electrochemiluminescence.

[0250] Figure 4A graph showing the effect of different washing conditions for the captured oligonucleotide array on the measured cross-reactivity of oligonucleotide probes specific to a component of the array.

[0251] Figure 5 To compare the analytical signals of electrochemiluminescent OLA of BRAF mutants with varying nucleic acid template concentrations containing the target BRAF gene region; and to compare the signals generated by mutant sequences with wild-type sequences.

[0252] Figure 6 A graph showing the analytical signals generated by a group of electrochemiluminescent OLAs as the concentration of their specific target sequences varies.

[0253] Figure 7 A graph showing how the bridging background signal of a set of electrochemiluminescent OLAs can be reduced by including capped oligonucleotides.

[0254] Figure 8 The graph shows that the increased background of electrochemiluminescent OLA, attributed to the nonspecific binding of the probe to the capturing oligonucleotide, can be reduced by including a capped oligonucleotide or an additional high-rigidity thermal soaking step.

[0255] Figure 9 Electrochemiluminescence (OLA) results of PCR-amplified genomic DNA extracted from a mixture of mutant and wild-type cells are shown, with the predicted percentage of mutant BRAF and NRAS sequences compared to the actual percentage of mutant sequences.

[0256] Figure 10 The analysis signal of electrochemiluminescent PEA for the BRAF 1799T>A mutation is shown as the concentration of template nucleic acid representing mutant and wild-type sequences varies, demonstrating that the analysis is specific for mutant sequences.

[0257] Figure 11 A graph showing that a set of electrochemiluminescent PEAs of BRAF and NRAS SNPs have a linear response to input DNA concentration.

[0258] Figure 12 The image shows the predicted percentage of mutant BRAF 1799T>A sequences versus the actual percentage of mutant sequences using electrochemiluminescence BRAF 1799T>A OLA analysis of PCR-amplified genomic DNA extracted from a mixture of mutant and wild-type cells.

[0259] Figure 13The image shows the percentage of predicted mutant NRAS 181C>A sequences versus the actual percentage of mutant sequences using electrochemiluminescence NRAS 181C>A OLA analysis of PCR-amplified genomic DNA extracted from a mixture of mutant and wild-type cells.

[0260] Figure 14 The image shows the predicted percentage of mutant 182A>T sequences versus the actual percentage of mutant sequences using electrochemiluminescence 182A>T OLA analysis of PCR-amplified genomic DNA extracted from a mixture of mutant and wild-type cells.

[0261] Figure 15 Oligonucleotide conjugation amplification (OLA) analysis is shown to detect, identify, and / or quantify target nucleotide sequences (e.g., therapeutic oligonucleotides) in samples that may contain oligonucleotide metabolites, as described in the examples herein.

[0262] Figure 16 This paper demonstrates a direct hybridization method for detecting, identifying, and / or quantifying target nucleotide sequences (e.g., therapeutic oligonucleotides) in samples that may contain oligonucleotide metabolites, as described in the examples herein.

[0263] Figure 17 Nuclease protection assay (NPA) with direct surface coating, as described in the examples herein, is used to detect, identify, and / or quantify target nucleotide sequences (e.g., therapeutic oligonucleotides) in samples that may contain oligonucleotide metabolites.

[0264] Figure 18 Hybridization / protection analyses are shown for detecting, identifying, and / or quantifying target nucleotide sequences (e.g., therapeutic oligonucleotides) in samples that may contain oligonucleotide metabolites, as described in the examples herein.

[0265] Figure 19 This demonstrates sandwich analysis used to detect, identify, and / or quantify antibodies (e.g., anti-drug antibodies (ADA)) in a sample.

[0266] Figure 20 This diagram illustrates the targeting probe and detection probe bridged by a positive control oligonucleotide containing a nucleotide sequence complementary to the ASO sequence.

[0267] Figure 21 show Figure 19 The improvement shown is a sandwich analysis for detecting, identifying, and / or quantifying antibodies (e.g., anti-drug antibodies (ADA)) in a sample. Detailed Implementation

[0268]

A. Definition

[0269] Unless otherwise defined, the scientific and technical terms used herein shall have the meanings commonly understood by one of ordinary skill in the art to which they pertain. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms, such as “a / an” including multiples, such as “one or more” or “at least one”, and the term “or” may mean “and / or” unless explicitly indicated to refer only to substitutes or substitutes being mutually exclusive. The term “including / includes / included” is not restrictive. Any type of range provided herein includes all values ​​within the particular range described and values ​​near the endpoints of the particular range.

[0270] As used herein, the term "about" is used to modify, for example, the quantity of an ingredient in a composition, the concentration, volume, process temperature, process time, yield, flow rate, pressure, and range thereof used in the invention. The term "about" refers to numerical quantities that can vary, for example, through variations in the procedures typically used to measure and prepare compounds, compositions, concentrates, or formulations; through unintentional errors in these procedures; through differences in the manufacture, origin, or purity of the starting materials or ingredients used to carry out the method; and similar considerations. The term "about" also covers amounts that differ from a particular initial concentration or mixture due to aging of the formulation, and amounts that differ from a particular initial concentration or mixture due to mixing or processing of the formulation. When modified by the term "about," the appended claims include such equivalents.

[0271] As used in this article, the range described by the phrase "between..." includes the endpoints of the range. Therefore, for example, the range between 50°C and 70°C includes 50°C to 70°C, that is, it includes the endpoints 50°C and 70°C.

[0272] Generally, the nomenclature and techniques described herein in conjunction with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization are those well-known and commonly used in the art. Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their generally accepted single-letter codes.

[0273] "Target analyte" may comprise any molecule of interest that can be detected and analyzed by the methods and kits described herein, and may include biomolecules such as nucleic acids, proteins, carbohydrates, sugars, and lipids. In one aspect, the target analyte is a target nucleotide sequence. In another aspect, the target analyte is a protein. In one aspect, the target analyte is a DNA-binding protein. The term "target analyte" may refer to the entire molecule of interest or a segment or portion of the molecule of interest. In one aspect, the target analyte comprises a modified molecule, such as a labeled, cleaved, or chemically or enzymatically treated form of the molecule of interest.

[0274] The “target nucleotide sequence” may include any nucleotide sequence of interest, including (but not limited to) sequences found in DNA or RNA of prokaryotic or eukaryotic DNA organisms. These may include single-stranded or double-stranded DNA, single-stranded or double-stranded RNA, DNA / RNA hybrids, or DNA / RNA chimeras. The target nucleotide sequence may include miRNA, therapeutic RNA, mRNA, RNA viruses, or combinations thereof. For double-stranded nucleotide sequences, the target nucleotide sequence may be identified in either strand. The target nucleotide sequence may require extraction, for example, nuclear DNA or viral genomic DNA or RNA, or may be manipulated directly in a sample, such as cell-free fetal DNA or cell-free tumor DNA in serum or plasma, or circulating therapeutic oligonucleotides. The target nucleotide sequence may be directly isolated from a biological sample or may include amplified sequences from a biological sample. The amplification methods are known and include (but are not limited to) polymerase chain reaction (PCR), whole genome amplification (WGA), reverse transcription followed by the polymerase chain reaction (RT-PCR), strand displacement amplification (SDA), or rolling circle amplification (RCA). Polymerases suitable for the amplification methods described herein include, for example, Taq, Phi, Bst, and Vent-exo for DNA amplification; and, for example, T7 RNA polymerase for RNA amplification.

[0275] The target nucleotide sequence may be an oligonucleotide, such as a therapeutic oligonucleotide. As used herein, a "therapeutic oligonucleotide" refers to an oligonucleotide capable of interacting with a biomolecule to provide a therapeutic effect. In one aspect, a therapeutic oligonucleotide is an antisense oligonucleotide (ASO). ASOs can influence RNA processing and / or regulate protein expression. An ASO is a single-stranded oligonucleotide that binds to single-stranded RNA to inactivate it. ASOs are single-stranded oligonucleotides typically ranging in length from about 5, 10, 15, 20, or 25 nucleotides to about 30, 35, 40, 45, or 50 nucleotides. In one aspect, an ASO binds to the messenger RNA (mRNA) of a gene, thereby inactivating the gene. In one aspect, the gene is a disease gene. Thus, an ASO can inactivate the mRNA of a disease gene to prevent or improve the production of a specific disease-causing protein. In one aspect, an ASO comprises DNA, RNA, or a combination thereof. Therapeutic oligonucleotides and ASOs are further described, for example, in Goodchild, *Methods in Molecular Biology*. Methods Mol Biol )》 764:1-15 (2011); Smith et al., Annual Review of Pharmacology and Toxicology ( Ann Rev Pharmacol Toxicol )》 59:605-630 (2019); and Stein et al., Molecular Therapy ( Mol Ther )》 25(5):1069-1075 (2017)

[0276] In one aspect, the target analyte is an antidrug antibody (ADA). As used herein, "antidrug antibody" or "ADA" refers to an antibody that induces a response to a biopharmaceutical product in vivo. ADAs can induce responses to biopharmaceutical products, such as therapeutic peptides comprising (but not limited to) proteins and antibodies; and therapeutic oligonucleotides comprising (but not limited to) antisense oligonucleotides (ASOs), short interfering RNA, microRNAs, and CRISPR / Cas synthesis guide chains. ADAs may contain any antibody isotype capable of binding to the biopharmaceutical product, referred to as binding antibodies, and may also contain a subset of binding antibodies capable of inhibiting the functional activity of the therapeutic product, referred to as neutralizing antibodies. Detection of ADAs can be an important measure of immunogenicity, which can affect both the safety and efficacy of the biopharmaceutical product.

[0277] The target nucleotide sequence in the sample, such as a therapeutic oligonucleotide, may be degraded over time, i.e. shortened, due to various factors such as the presence of nucleases, temperature, pH, salt concentration, etc. The degradation products of the target nucleotide sequence are also referred to as oligonucleotide metabolites. In one aspect, the oligonucleotide metabolite is shorter than the target nucleotide sequence by one or more nucleotides, two or more nucleotides, three or more nucleotides, four or more nucleotides, five or more nucleotides, six or more nucleotides, seven or more nucleotides, eight or more nucleotides, nine or more nucleotides, ten or more nucleotides, fifteen or more nucleotides, or twenty or more nucleotides. In one aspect, 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 aspect, the sample of this disclosure comprises: a target nucleotide sequence, such as a therapeutic oligonucleotide; and one or more oligonucleotide metabolites, such as therapeutic oligonucleotide metabolites.

[0278] In one aspect, the target nucleotide sequence is a therapeutic oligonucleotide. In some aspects, degradation of the therapeutic oligonucleotide in a sample indicates a pharmacokinetic response to the therapeutic oligonucleotide. Degraded or shortened therapeutic oligonucleotides, also referred to herein as therapeutic oligonucleotide metabolites, may lose their therapeutic efficacy. The methods of this disclosure can be used to measure the amount of a target nucleotide sequence (e.g., a therapeutic oligonucleotide) relative to an oligonucleotide metabolite (e.g., a therapeutic oligonucleotide metabolite). In one aspect, the methods of this disclosure are used to determine pharmacokinetic parameters of a target nucleotide sequence (e.g., a therapeutic oligonucleotide). In one aspect, the pharmacokinetic parameters of a target nucleotide sequence (e.g., a therapeutic oligonucleotide) are determined by measuring the degradation rate and / or amount of the target nucleotide sequence (e.g., a therapeutic oligonucleotide) in a biological environment (e.g., a patient). In one aspect, the measured pharmacokinetic parameters are clearance, volume distribution, plasma concentration, half-life, peak time, peak concentration, available rate, or combinations thereof. Further discussion of the measurement and interpretation of pharmacokinetic parameters can be found, for example, in Benet, *European Journal of Respiratory Diseases Supplement*. Eur J Respir Dis Suppl References include Le et al., “Overview of Pharmacokinetics”, Merck Handbook Professional Edition, revised May 2019. Oligonucleotide metabolites present in a sample can also interfere with the detection, identification, and / or quantification of the target nucleotide sequence in the sample. Therefore, it may be necessary to remove oligonucleotide metabolites from the sample. Consequently, the methods of this 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 the target nucleotide sequence.

[0279] "Polymerase chain reaction" or "PCR" refers to a technique used to amplify a target nucleotide sequence, which involves a repetitive cycle of the following 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 of the target DNA sequence on their sides; and (3) extension, in which a DNA polymerase extends the 3' ends of each primer along the template strand. PCR can use a thermostable DNA polymerase, such as Taq polymerase.

[0280] A nucleotide is a monomeric unit comprising a nitrogenous base, a pentose sugar (ribose or deoxyribose), and at least one phosphate group. Nucleotides include: nucleoside triphosphates, such as ATP, UTP, CTG, and GTP found in RNA; deoxynucleoside triphosphates, most commonly found in DNA, such as dATP, dCTP, dGTP, and dTTP; and dideoxyribonucleoside triphosphates (ddNTPs), which lack the 3'-OH required for polymerase-mediated elongation, including, for example, ddATP, ddCTP, ddGTP, and ddTTP.

[0281] “Oligonucleotide (oligonucleotide / oligo)” refers to a nucleic acid having a nucleotide sequence of length between about 5 and about 100, about 10 and about 50, or about 10 and about 25 nucleotides, or a length of at least about 10, 15, 20, 25, 30, 35, 40, 45, or 50 and at most about 50, 75, or 100 nucleotides. Oligonucleotides, including (but not limited to) the probes, primers, tags, or capture oligonucleotides described herein, can be synthesized using known methods, including, for example, those described by Beaucage and Carruthers (1981) in "Deoxynucleoside aminophosphates—Key Intermediates in the Synthesis of a New Class of Deoxypolynucleotides" (…). Deoxynucleoside phosphoramidites- a new class of key intermediates for deoxypolynucleotide synthesis The aminophosphate method described in Tetrahedron Letters, 22(2):1859-1862, or according to Matteucci and Caruthers (1981), Synthesis of deoxynucleotides on polymer supports. Synthesis of deoxynucleotides on a polymer support The preparation was carried out by the triester method. (J. Am. Chem. Soc., 103(11):3185-3191)

[0282] Nucleotides and nucleic acids of this disclosure, including, for example, those in the target sequence or oligonucleotide reagents of this disclosure, may contain structural analogs with non-naturally occurring chemical structures that can also participate in hybridization reactions. In one instance, the nucleotide or nucleic acid may contain chemical modifications that link it to a label or provide a reactive functional group that can be linked to the label, for example, by using a nucleotide base modified with an amine or thiol group, a phosphate group, or a sugar group. The term "reactive functional group" refers to an atom or group of related atoms that can undergo another chemical reaction, such as forming a covalent bond with another functional group. Examples of reactive functional groups include (but are not limited to) amino, thiol, hydroxyl, and carbonyl groups. In one aspect, the reactive functional group includes a thiol group. Labels that can be linked to nucleotides or nucleic acids through these chemical modifications may contain (but are not limited to) detectable moieties, such as biotin, haptens, fluorophores, and electrochemiluminescent (ECL) labels.

[0283] In another respect, nucleotides can be modified to prevent enzymatic or chemical elongation of the nucleic acid chain into which they are incorporated, for example by replacing the ribose or deoxyribose group with a dideoxyribose. In another instance, the backbone component linking nucleotide bases (e.g., sugar or phosphate groups) together can be modified or substituted, for example by using peptide nucleic acids (PNAs) or by incorporating ribosomes, such as those found in 2'-O-methyl-substituted RNA, locked nucleic acids, bridging nucleic acids, and morpholinonucleotides. These "backbone" analogs can be present in one, some, or all of the backbone links of nucleic acids or oligonucleotides and can provide certain advantages, such as improved binding stability or hybridization stability with nucleases. In another instance of nucleotide and nucleic acid structural analogs, non-natural nucleotide bases can be included. Non-natural (also called "atypical") bases can hybridize with natural (typical) bases or can hybridize with another non-natural base.

[0284] "Separated" refers to a target analyte that is substantially or essentially free from other sequences or components that normally accompany or interact with it in its natural environment, such as polypeptide or protein, or oligonucleotide or nucleic acid sequences. In one aspect, a separated nucleotide sequence includes components or sequences that are not found in their natural environment along with the nucleic acid sequence. The term "separated" also includes oligonucleotide or protein sequences that are not naturally occurring or recombinantly generated, as such sequences do not exist in nature. Specifically, oligonucleotides that are not naturally occurring or recombinantly generated may have closely spaced sequences that are not found to exist naturally.

[0285] 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 or contains at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a reference polypeptide or oligonucleotide sequence.

[0286] The term "identity" refers to the fact that two polynucleotide or polypeptide sequences contain the same nucleic acid base or amino acid residue at the same position in a comparison window. The term "percentage sequence identity" can be determined by comparing two aligned sequences through a comparison window; determining the number of positions in the two sequences where the same nucleic acid base or amino acid residue is present, obtaining the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window; and multiplying the result by 100 to obtain the percentage of sequence identity. The comparison window may contain the full-length sequence or a sub-section of a larger sequence. Various methods and algorithms are known for determining the percentage identity between two or more sequences, including (but not limited to) MEGALIGN (DNASTAR, Inc., Madison, Wisconsin), FASTA, BLAST, or ENTREZ.

[0287] "Capture oligonucleotide" refers to an oligonucleotide reagent that can be immobilized on a carrier surface and designed to hybridize (and thus be captured on the surface) with complementary oligonucleotides. In one aspect, the capture oligonucleotide is a single-stranded sequence that can selectively hybridize, for example, with a single-stranded oligonucleotide tag present on a target reaction product under stringent hybridization conditions. In one aspect, the target reaction product is generated by using the target nucleotide sequence as a template via oligonucleotide conjugation. In another aspect, the target reaction product is generated by using the target nucleotide sequence as a template via primer extension. Capture oligonucleotides may be provided in solid form (e.g., lyophilized), in solution form, or immobilized on a carrier surface, such as on or in an array of particles (e.g., microparticles, beads). Two or more capture oligonucleotides may be provided together. Examples of two or more capture oligonucleotides provided together comprise a set or subset (also referred to herein as a set) of parent capture oligonucleotides as described herein.

[0288] A "probe" or "primer" is a reagent containing an oligonucleotide sequence capable of hybridizing with a target nucleotide sequence. A probe may contain a single-stranded sequence complementary to a portion or substantially complementary to the target nucleotide sequence. The probe may also contain a tag sequence complementary to the capturing oligonucleotide (which may also be referred to herein as a guide sequence). The sequence complementary to the target nucleotide sequence and the tag sequence may be present on the same nucleic acid strand within the probe, or they may be present on different strands within the probe. For example, a probe may contain a first strand having a sequence complementary to the target sequence and a bridging sequence, and a second strand having a tag sequence and a sequence complementary to the bridging sequence on the first strand, wherein the first and second strands hybridize or hybridize via the bridging sequence. The probe may be DNA or RNA and may contain modified nitrogenous base analogs, or other labeled or suitable linker modifications for linking tags. The probe should be long enough to allow hybridization with the target nucleotide sequence, typically between about 5 and about 100, about 10 and about 50, about 20 and about 30, or at least about 5, 6, 7, 8, 9, 10, 15, 20, or 25 and at most about 30, 35, 40, 45, 50, 75, or 100 nucleotides. The probe can be prepared by any suitable method known in the art, involving chemical or enzymatic synthesis, or by cleaving larger nucleic acids using nonspecific nucleic acid-cleavage chemicals or enzymes, or by cleaving with site-specific restriction endonucleases. In some applications, the probe hybridizing with a complementary region in the target sequence can initiate probe extension via polymerase, acting as the starting point for replication of adjacent single-stranded regions on the target sequence.

[0289] A linker (also referred to herein as a "spacer") is one or more atoms that join one chemical part to another, such as one or more atoms that join a reactive functional group or label to an oligonucleotide. Linkers can be nucleotide or non-nucleotide compounds containing one or more atoms, for example, 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 contain atoms such as carbon, oxygen, sulfur, nitrogen, and phosphorus, and combinations thereof. Examples of linkers include: low molecular weight groups, such as amides, esters, carbonates, and diethyl ethers; and higher molecular weight linking groups, such as polyethylene glycol (PEG) and alkyl chains. Therefore, a linker can contain one or more atoms, units, or molecules.

[0290] "Label" refers to a chemical group or part that has detectable physical properties or enables a chemical group or part to exhibit detectable physical properties, including, for example, enzymes that catalyze the conversion of a substrate into 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) radioactive isotopes, enzymes, substrates, fluorescent molecules, chemiluminescent components, electrochemiluminescent components, magnetic particles, and bioluminescent components. In another aspect, a label is a compound that is a member of a binding pair, wherein the first member of the binding pair (which may be referred to as the "primary binding agent") is linked to a substrate (e.g., an oligonucleotide), and the other member of the binding pair (which may be referred to as the "secondary binding agent") has detectable physical properties. Non-limiting examples of binding pairs include biotin and streptavidin or avidin; complementary oligonucleotides; haptens and hapten conjugates; and antibody / antigen binding pairs.

[0291] "Detection" refers to the detection, observation, or quantification of a substance (such as an oligonucleotide) based on the presence or absence of a marker.

[0292] "Complementarity" refers to nucleic acid molecules or sequences of nucleic acid molecules that interact by forming 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 with less than 100% nucleotide alignment based on, for example, the Watson-Crick base pairing model), but sequences with less sequence complementarity are less stable and less likely to hybridize compared to sequences with greater sequence complementarity. In one aspect, based on the Watson-Crick model, the nucleotides of the complementary sequence have 100% sequence complementarity. In another aspect, based on the Watson-Crick model, the nucleotides of the complementary sequence have at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity.

[0293] Whether two complementary sequences hybridize depends on the stringency of the hybridization conditions, which can vary depending on factors such as temperature, solvent, ionic strength, and other parameters. The stringency of hybridization conditions can be chosen to selectively form or maintain the desired hybridization product of two complementary nucleic acid sequences in the presence of other potential cross-reacting or interfering sequences. Stringent conditions are sequence-dependent; typically, longer complementary sequences hybridize specifically at higher temperatures than shorter complementary sequences. Generally, for a specific nucleotide sequence at defined ionic strengths, chemical denaturant concentrations, pH, and hybridization compound concentrations, stringent hybridization conditions are more effective than thermal melting points (T0). m (That is, the temperature at which 50% of the sequence hybridizes to a substantially complementary sequence) approximately 5°C to approximately 10°C lower. Generally, nucleotide sequences with a higher percentage of G and C bases hybridize under more stringent conditions compared to nucleotide sequences with a lower percentage of G and C bases. Generally, stringency can be increased by increasing temperature, increasing pH, decreasing ionic strength, or increasing the concentration of chemical nucleic acid denaturants (e.g., formamide, dimethylformamide, dimethyl sulfoxide, ethylene glycol, propylene glycol, and ethyl carbonate). Stringent hybridization conditions typically involve salt concentrations less than approximately 1 M, 500 mM, or 200 mM; hybridization temperatures above approximately 20°C, 30°C, 40°C, 60°C, or 80°C; and chemical denaturant concentrations above approximately 10%, 20%, 30%, 40%, or 50%. Because many factors can affect hybridization stringency, the combination of parameters may be more important than the absolute value of any single parameter.

[0294] The term "complementary sequence" refers to a sequence in which each base forms a complementary base pair, such as two oligonucleotides where A pairs with T or U and C pairs with G. Perfect complementarity, or 100% complementarity, means that each nucleotide in one polynucleotide sequence or region can form hydrogen bonds with each nucleotide in a second polynucleotide chain or region. "Largely complementary" refers to a partially complementary sequence that can hybridize under strict hybridization conditions. Largely complementary sequences do not necessarily hybridize along their entire length.

[0295] The term "corresponding" can be used to refer to the relationship between a capturing oligonucleotide and an oligonucleotide tag, wherein the oligonucleotide tag is designed to specifically bind to a specific capturing oligonucleotide sequence under strict hybridization conditions. In one aspect, under strict conditions, the oligonucleotide tag specifically binds to its corresponding capturing molecule and does not bind to or cross-react with other capturing molecules. In another aspect, under strict conditions, the oligonucleotide tag specifically binds to its corresponding capturing molecule and does not bind to or cross-react with other capturing molecules in the array. In one aspect, the oligonucleotide tag is a single-stranded oligonucleotide having a sequence complementary to at least a portion of the sequence of its "corresponding" capturing oligonucleotide. In one aspect, based on the Watson-Crick model, the nucleotide of the "corresponding" oligonucleotide tag has 100% sequence complementarity with the capturing oligonucleotide sequence. In another aspect, based on the Watson-Crick model, the nucleotide of the corresponding sequence has at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity.

[0296] Single-stranded polynucleotides have a “direction” or “directionality” because adjacent nucleotides are linked by a phosphodiester bond between their 3' and 5' carbon atoms, resulting in terminal 5' and 3' carbons at either end of the polynucleotide, which may be referred to as the 5'-(phosphoryl) and 3'-(hydroxyl) ends of the molecule. “Reverse” oligonucleotides have a sequence that is reversed when read in the 5'-to-3' direction compared to a reference oligonucleotide. For example, for the reference oligonucleotide sequence 5'-ACCGATCATG-3' (SEQ ID NO: 1649), the “reverse” oligonucleotide sequence should be 5'-GTACTAGCCA-3' (SEQ ID NO: 1650).

[0297] Based on the rules defined by Watson-Crick base pairing and the antiparallel nature of DNA-DNA, RNA-RNA, and RNA-DNA double helices, the complementary sequence of a sequence comprises (or substantially comprises) a Watson-Crick pair of bases from the initial sequence but sequenced from 3' to 5'. An example of the complementary sequence of sequence 5'-ACCGATCATG-3' (SEQ ID NO: 1649) is 5'-CATGATCGGT-3' (SEQ ID NO: 1651). When the term reverse complement is used herein, it refers to the complementary sequence of the reverse of the initial sequence. An example of the reverse complement of sequence 5'-ACCGATCATG-3' (SEQ ID NO: 1649) is 5'-TGGCTAGTAC-3' (SEQ ID NO: 1652).

[0298] "Cross-reactivity" or "cross-reactivity" refers to the ability of an oligonucleotide sequence to hybridize with more than one other oligonucleotide sequence in a sample. In one aspect, the term "cross-reactivity" refers to the ability of a first oligonucleotide sequence to hybridize with a second oligonucleotide sequence in a sample, wherein the second oligonucleotide sequence is not complementary to or substantially complementary to the first oligonucleotide sequence. In one aspect, the term "cross-reactivity" or "cross-reactivity" refers to the ability of a capturing oligonucleotide to hybridize with more than one oligonucleotide tag or more than one tagged target nucleotide sequence in a sample. In one aspect, a cross-reactive capturing oligonucleotide hybridizes with one or more oligonucleotide tags in a sample under stringent capture hybridization conditions. In one aspect, stringent capture hybridization conditions include a temperature between 127°C and 47°C, a formamide concentration between 21% and 41%, a salt concentration between 300 mM and 500 mM, and a pH between 7.5 and 8.5. In one aspect, stringent capture hybridization conditions include a temperature of approximately 37°C, a formamide concentration of approximately 31%, a salt concentration of approximately 400 mM, and a pH of 8.0.

[0299] "Non-cross-reactive" refers to a first oligonucleotide sequence that hybridizes only with a specific oligonucleotide sequence in a sample, such as the ability of a first oligonucleotide sequence to hybridize only with its corresponding complementary sequence in the sample. In one aspect, the term "non-cross-reactive" refers to the ability of a capturing oligonucleotide to hybridize only with one oligonucleotide tag in a sample containing more than one oligonucleotide tag or more than one tagged target nucleotide sequence. In one aspect, a non-cross-reactive oligonucleotide probe hybridizes only with one oligonucleotide tag in the sample under stringent hybridization conditions. In one aspect, non-cross-reactive means that, under stringent capture hybridization conditions, the binding rate of the first oligonucleotide to sequences other than its complementary sequence in the sample is less than 0.05%.

[0300] "Conjugases" are a class of enzymes that catalyze the formation of a phosphodiester bond between the 3' hydroxyl group of one nucleotide sequence and the 5' phosphate group of a second nucleotide sequence, thereby joining nucleotide sequences together. Conjugases include those found in *Escherichia coli* (E. coli). E. coli DNA conjugase, T4 DNA conjugase, T4 RNA conjugase, and *Thermophyton aquatilis* ( T. aquaticus Taq) conjugase, thermophilic bacteria ( T . Thermophilus DNA conjugase (e.g., HiFi conjugase) or *Pyrococcus* spp. ( PyrococcusDNA conjugase. In one aspect, the conjugase is a thermostable conjugase. "Conjugation" refers to the process of joining two nucleotide sequences together by forming a phosphodiester bond between the 3' hydroxyl group of one nucleotide sequence and the 5' phosphate group of the second nucleotide sequence.

[0301] "Array" refers to one or more carrier surfaces having more than one spatially distinct (i.e., non-overlapping) addressable site, referred herein as a binding domain or array assembly. In one aspect, each addressable site contains an analytical reagent, including, for example, a trapping molecule.

[0302] A "carrier surface" refers to a surface material on which various substances, such as oligonucleotides or peptides, can be immobilized. A "carrier surface" can be planar or non-planar. In one aspect, the carrier surface comprises a flat surface. In one aspect, the carrier surface is a plate with multiple pores, i.e., a "porous plate." A porous plate can contain any number of pores of any size or shape arranged in any pattern or configuration. In another aspect, the carrier surface has a curved surface. In one aspect, the carrier surface is provided by one or more particles, beads, or microspheres. Unless otherwise indicated, the terms particles, beads, or microspheres are used interchangeably. In one aspect, the carrier surface comprises color-coded particles, beads, or microspheres. In one aspect, the carrier surface comprises an analytical module, such as an analytical plate, slide, cylinder, bead, or chip. In one aspect, the carrier surface comprises an analytical flow cell or analytical fluid.

[0303] In one aspect, the carrier surface contains multiple addressable sites (which may be referred to as "spots"), as is commonly seen in "gene chip" devices. In another aspect, the array comprises multiple carrier surfaces, each having one addressable site, as in the "bead array" method, where each bead in the bead suspension represents an addressable site (which can be addressed, for example, using flow cytometry or microscopic detection techniques). In yet another aspect, the array comprises multiple carrier surfaces, each with one or more, or two or more, addressable sites. The addressable sites on the carrier surface may be arranged in rows and columns, or may form other patterns. The number of addressable sites on the array can vary, for example, from less than 10 to greater than 50, 100, 200, 500, or 1000. "Multiplexing" refers to the simultaneous analysis of more than one analytical target in a single analysis.

[0304] "Carbon-based" refers to materials containing elemental carbon (C) as a main component. Examples of carbon-containing or carbon-based materials include (but are not limited to) carbon, carbon black, graphite carbon, glassy carbon, carbon nanotubes, carbon filaments, graphite, carbon fibers, and mixtures thereof. Carbon-based materials may contain elemental carbon, including, for example, graphite, carbon black, or carbon nanotubes. In one aspect, the carbon-based material comprises a conductive carbon-polymer composite, a conductive polymer, or conductive particles, such as carbon ink, carbon paste, or metallic ink, dispersed in a matrix. The conductive particles comprise, for example, carbon filaments, carbon black, or graphite carbon dispersed in a matrix, such as a polymer matrix, for example, ethylene vinyl acetate (EVA), polystyrene, polyethylene, polyvinyl alcohol, polyvinyl acetate, polyvinyl chloride, or acrylonitrile butadiene styrene (ABS). Such polymer matrices may also comprise copolymers having more than one type of component monomers, which may include monomers selected from: vinyl acetate, ethylene, vinyl alcohol, vinyl chloride, acrylonitrile, butadiene, styrene, or other monomers.

[0305] An "allele" is a genomic variant of the target nucleotide sequence that, when translated, produces a functional or abnormal gene product. Two allele forms can be referred to as a "wild-type allele" and a "mutant" or "variant" allele. The "wild-type" allele is the dominant nucleotide sequence in the population. The "mutant" or "variant" allele is the less common nucleotide sequence in the population. Mutant or variant nucleotide sequences may or may not have a functional outcome.

[0306] A "polymorphism" or "polymorphic site" refers to a variant of one of two or more nucleic acids. A "single nucleotide variant" (sometimes also called a "single nucleotide polymorphism," "SNP," or "single nucleotide variation") is a variant involving only a single nucleotide. A single nucleotide variant may involve the substitution of one nucleotide by another at the polymorphic site, the deletion of a nucleotide from a reference nucleotide sequence, or the insertion of a nucleotide into a reference nucleotide sequence. Single nucleotide variants can be common (e.g., present in at least 1% of the population) or rare (e.g., present in less than 1% of the population).

[0307] A “kit” refers to a collection of components provided or assembled for use, for example, to generate a composition, manufacture an apparatus, or perform a method. A kit may contain one or more components. The components of a kit may be provided in one or more packages, each containing one or more of the components. The listed components of a kit may also be provided as a single physical component or as multiple components used in combination with the kit. For example, the instrument components of a kit may be provided fully assembled or as multiple instrument parts to be assembled prior to use. Similarly, the liquid reagent components of a kit may be provided as a complete liquid formulation in a container, as one or more drying reagents and one or more liquid diluents to be combined to provide a complete liquid formulation, or as two or more liquid solutions to be combined to provide a complete liquid formulation. As is known in the art, kit components for analysis are typically shipped and stored separately due to different storage requirements, such as storage temperatures of 4°C and -70°C.

[0308]

B. Overview

[0309] This document describes kits for identifying, detecting, or quantifying one or more target analytes in a sample, and methods for their preparation and use. In one aspect, the method or kit comprises one or more capture molecules in a dispersed binding domain immobilized or immobilizable on a carrier surface. In one aspect, the capture molecule is a single-stranded capture oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence of a single-stranded oligonucleotide tag linked to the same probe or reaction product. In one aspect, a probe containing an oligonucleotide tag binds to a target analyte to direct the target analyte to the capture molecule. In one aspect, the target analyte binds to a first probe containing an oligonucleotide tag and a second probe containing a label. In one aspect, a target nucleotide sequence is used as a template to generate a reaction product. In one aspect, the reaction product comprises an oligonucleotide tag and a label. In one aspect, the method or kit comprises 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 the carrier surface, such that the captured reaction product can subsequently be identified, detected, or quantified based on the attached label.

[0310] In one aspect, a method is provided to immobilize one or more oligonucleotides on a carrier surface. In one aspect, the method comprises immobilizing one or more oligonucleotides comprising a thiol-reactive group on the carrier surface. In one aspect, one or more capturing oligonucleotides are immobilized in one or more binding domains on the carrier surface. In one aspect, the method comprises washing the carrier surface with a thiol-containing washing solution (also referred to herein as a blocking solution or capping agent) to remove unbound oligonucleotides. In one aspect, each 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 capturing oligonucleotides.

[0311] In one aspect, the methods and kits described herein for identifying, detecting, or quantifying one or more target analytes in a sample provide increased sensitivity relative to conventional methods. In one aspect, the methods and kits of this disclosure are capable of detecting target analytes in nanomolar, appropriate picomolar, or more appropriate femtomolar concentrations in a sample. In one aspect, the methods and kits of this disclosure are capable of detecting target analytes in samples at concentrations of at least about 0.1 fM, 1 fM, 25 fM, 50 fM, 75 fM, or 100 fM and at most about 500 fM, 1 pM, 10 pM, 100 pM, 500 pM, or 1 nM, or from 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. In one aspect, the methods and kits of this disclosure are capable of detecting target analytes in samples at concentrations of 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.

[0312] In one particular aspect, the methods and kits provided herein are capable of detecting femtomolar concentrations of polynucleotides, such as therapeutic oligonucleotides or RNA (e.g., mRNA), in a sample, which preferably allow for identification, detection, and / or quantification without amplifying the polynucleotides.

[0313] In one aspect, the methods and kits provided herein reduce the amount of time required to identify, detect, or quantify one or more target analytes in a sample compared to conventional methods. In one aspect, the methods and kits of this disclosure are capable of identifying, detecting, or quantifying one or more target analytes in a sample within the following timeframes: 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 aspect, the methods and kits of this disclosure are capable of identifying, detecting, or quantifying one or more target analytes in a sample within the following timeframes: 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.

[0314]

C. Capture molecules

[0315] In one aspect, the method or kit comprises one or more capture molecules in a dispersed binding domain immobilized or immobilizable on a carrier surface. In one aspect, the capture molecules are not naturally occurring sequences. In another aspect, the capture molecules are generated via recombination. In one aspect, a mathematical algorithm is used to generate a sequence of a set of non-cross-reactive capture molecules.

[0316] In one aspect, the capture molecule is a single-stranded capture oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence of the single-stranded oligonucleotide tag. In one aspect, the oligonucleotide tag is linked to a target analyte. In one aspect, the oligonucleotide tag is linked to a probe that binds to the target analyte. In one aspect, the oligonucleotide tag is linked to a reaction product generated using the target nucleotide sequence as a template. In one aspect, hybridization between the capture oligonucleotide and the complementary nucleotide sequence of the oligonucleotide tag immobilizes the target of interest or the reaction product onto the carrier surface. The captured target or reaction product can then be identified, detected, or quantified based on the linked tag.

[0317] In one aspect, the method or kit comprises different capture oligonucleotides targeting individual target nucleotide sequences to be identified, detected, or measured. In another aspect, hybridization between multiple capture oligonucleotides and their complementary oligonucleotide tags occurs simultaneously and in parallel throughout the capture oligonucleotide array. The array may include or consist of two or more capture oligonucleotides as described herein. Therefore, the array may contain 2 to 150 or more capturing oligonucleotides, such as 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 capturing oligonucleotides. The oligonucleotides in the array may include or consist of a “parent set” or “subset” (also referred to herein as a “set”) of oligonucleotides as described herein.

[0318] In one aspect, one or more capture oligonucleotides comprise a single-stranded nucleic acid sequence, including, for example, a nucleic acid sequence comprising deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a structural analog comprising a non-naturally occurring chemical structure that may also participate in hybridization reactions.

[0319] In one aspect, the capturing oligonucleotides used in a particular array have similar binding energies or melting temperatures (Tm), for example, within at least about 0.5°C, 1°C, 2°C, 3°C, 4°C, or 5°C, wherein the melting temperature (Tm) of the oligonucleotides is... m The temperature at which 50% of the oligonucleotide hybridizes to its complementary sequence and 50% remains free in solution is called T. Known methods can be used to determine T, such as by measuring the change in absorbance of the oligonucleotide with its complementary sequence as a function of temperature. m In one aspect, the captured oligonucleotide has a melting temperature (T0) at 50 mM NaCl between about 50°C and about 70°C, between 55°C and about 65°C, or at least about 50°C, 55°C, or 60°C and at most about 60°C, 65°C, or 70°C. m In one aspect, the captured oligonucleotide has a GC content between approximately 40% and approximately 60% or between approximately 40% and approximately 50%.

[0320] In one aspect, the capturing oligonucleotide is between about 20 and about 100 nucleotides, about 30 and about 50 nucleotides, or about 35 and about 40 nucleotides in length, for example, at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides in length and at most about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 75, or 100 nucleotides in length. In another aspect, the capturing oligonucleotide comprises at least 20, 24, 30, or 36 nucleotides. Capturing oligonucleotides with a length of at least about 20, 24, 30, or 36 nucleotides are capable of binding to the tagged target or reaction product and, compared to shorter capturing oligonucleotides, bind at higher temperatures and exhibit improved specificity (i.e., less nonspecific binding). In one aspect, the length of one or more capture oligonucleotides in the array is inconsistent with the nucleic acid sequence of their complementary oligonucleotide tag. In practice, it may be necessary to include capture oligonucleotides with sequences that are, for example, up to 5, 10, 15, 20, or 25 bases longer than their complementary single-stranded oligonucleotide tag. In one aspect, the tagged target or reaction product and the capture oligonucleotide are contained in a ratio of approximately 1:1. In another aspect, the tagged target or reaction product is present in excess to increase the likelihood of the tagged target or reaction product binding to the capture oligonucleotide. In one aspect, the tagged reaction product and the capture oligonucleotide are contained in a ratio of approximately 2:1, 3:1, 4:1, or 5:1.

[0321] In one aspect, one or more capturing oligonucleotides are covalently or non-covalently immobilized to a support surface. In another aspect, one or more capturing oligonucleotides are covalently or non-covalently immobilized to one or more binding domains on a support surface. In one aspect, the capturing oligonucleotide is adsorbed onto the support surface via, for example, electrostatic interaction between a negatively charged phosphate group on the oligonucleotide and a positive charge on the support surface. In one aspect, one or more capturing oligonucleotides are immobilized to the support surface via the binding of a first binding complex linked to the capturing oligonucleotide (directly or through a linker portion) to a second binding complex immobilized on the surface. In one aspect, one or more capturing oligonucleotides are covalently immobilized to the support surface. In one aspect, one or more capturing oligonucleotides are directly immobilized to the support surface. In another aspect, the capturing oligonucleotide is immobilized to the support surface via a linker.

[0322] In one aspect, one or more capturing oligonucleotides comprise a reactive functional group. In one aspect, the functional group comprises a thiol group (-SH) or an amino group (-NH2). In one aspect, one or more capturing oligonucleotides are immobilized to a support surface via a reactive functional group. In one aspect, one or more capturing oligonucleotides are immobilized to a support surface linked to a reactive functional group, said reactive functional group being linked to the capturing oligonucleotide via a linker. In one aspect, the capturing oligonucleotide is immobilized to a support surface via a thiol group or an amino group. In one aspect, the capturing oligonucleotide is immobilized to a support surface via a thiol group or an amino group, said thiol group or amino group being linked to the capturing oligonucleotide via a linker (also referred to herein as a "spacer"). In one aspect, the linker comprises between about 3 and about 20 atoms or molecules or units, or at least about 3, 4, 5, 6, 7, 8, 9, 10 and at most about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 atoms or molecules or units. In one aspect, the linker is a carbon linker. In one aspect, the linker is an ethylene glycol linker or a polyethylene glycol (PEG) linker. In one aspect, the linker contains up to 3, 4, 5, or 6 consecutive PEG units. In another aspect, the linker contains three consecutive PEG units. In yet another aspect, the linker contains six consecutive PEG units. The linker may have the structure shown in Example 2.

[0323] In one aspect, one or more capture oligonucleotides are immobilized onto the surface of a carrier pretreated with, for example, bovine serum albumin (BSA). In another aspect, the capture oligonucleotides are immobilized onto the carrier surface using a crosslinking agent. Hybrid and heterobifunctional crosslinking agents suitable for linking proteins and nucleic acids to each other or to other materials are well known in the art; see, for example, the Thermo Scientific Crosslinking Technical Handbook published by Thermo Fisher Scientific, 2012. In one aspect, the crosslinking agent is a heterobifunctional crosslinking agent comprising an amine-reactive moiety (e.g., N-hydroxysuccinimide or N-hydroxysulfosuccinimide ester) and a thiol-reactive moiety (e.g., maleic animide, iodosuccinimide, or an activated disulfide (e.g., pyridyl disulfide)); such heterobifunctional crosslinking agents contain, for example, sulfosuccinimide-4-(N-cis-butenyliminomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). In one aspect, the amine-reactive moiety (e.g., the N-hydroxysuccinimide (NHS) moiety of SMCC) reacts with a protein to introduce the thiol-reactive moiety (e.g., the maleic animide moiety of SMCC) into the protein. The thiol-reactive moiety then reacts with a thiol-modified trapping oligonucleotide to form a protein-oligonucleotide conjugate linked by a stable thioether bond. Arrays of protein-oligonucleotide conjugates can be formed by printing a reagent pattern onto a surface that adsorbs or reacts with a protein to produce a patterned array. In one aspect, the array is formed by printing the protein-oligonucleotide conjugate onto a graphite carbon surface, such as a screen-printed carbon ink electrode. See, for example, U.S. Patent Publication No. 2016 / 0069872, U.S. Patents 6,977,722 and 7,842,246, the disclosure of which is incorporated herein by reference in its entirety. In one aspect, one or more capture oligonucleotides are immobilized onto a carrier surface that has not been pretreated with a protein. In one aspect, the protein component of the protein-oligonucleotide conjugate used for immobilization as described above is BSA.

[0324] In one approach, a computer algorithm is used to generate a collection of capture oligonucleotides of lengths (e.g., 24, 30, or 36-mers) that meet one or more of the following requirements: (a) GC content between approximately 40% and approximately 50%; (b) AG content between approximately 30% and approximately 70%; (c) CT content between approximately 30% and approximately 70%; (d) the maximum number of repeating bases in the sequence is no more than three; (e) there are no unwanted oligonucleotide-oligonucleotide interactions with strands matching more than seven consecutive complementary base pairs; (f) The oligonucleotide-oligonucleotide interaction is not required with a chain of 18 consecutive bases or less, wherein (i) the terminal bases at each end are complementary and (ii) the sum of the complementary base pairs minus the sum of the mismatches is greater than 7; (g) there is no matching of 20 base pairs or longer (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36) in a given genome (e.g., the human genome) or in nature (or the complementary sequence of the sequence or both). (a) a chain of (b) bp; (h) the difference in the free energy of hybridization between the sequence and its complementary sequence (or the first 24 oligonucleotides from the 5' end and their complementary sequence) is less than about 1 kCal / mol, about 2 kCal / mol, about 3 kCal / mol, or about 4 kCal / mol; (i) an unpredictable hairpin loop with 4 or more consecutive matching rings in the stem; (j) an unpredictable hairpin loop with 4 or more consecutive matching rings in the stem and a ring size greater than 6 bases. In one aspect, at least criteria (a) to (h) are considered. In this case, the unwanted oligonucleotide-oligonucleotide interaction refers to the interaction of the oligonucleotide itself, the oligonucleotide with another sequence within the set, or the oligonucleotide with the complementary sequence of another sequence within the set. Hybridization free energy (DG) is typically calculated for specified ionic strength, temperature, and pH, such as physiological ionic strength and pH at room temperature (approximately 25°C) (approximately 150 mM NaCl, approximately pH 7.2); or approximately 200 mM monovalent cation at approximately 23°C, approximately pH 7.0; 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 between G / C-rich sequences when paired with other capturing oligonucleotides used in the analysis.

[0325] In one aspect, the capturing molecule comprises the oligonucleotide sequence shown in any one of SEQ ID Nos: 1-774 (Tables 1-12). In one aspect, the capturing oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in any one of SEQ ID Nos: 1-774. In another aspect, the capturing 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 shown in any one of SEQ ID Nos: 1-774. In yet another aspect, the capturing oligonucleotide has a nucleotide sequence comprising at least 20 consecutive nucleotides of the sequence shown in any one of SEQ ID Nos: 1-774.

[0326] In another aspect, the capturing oligonucleotide has the nucleotide sequence shown in any one of SEQ ID Nos: 1-64. In one aspect, the capturing oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in any one of SEQ ID Nos: 1-64. In another aspect, the capturing 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 shown in any one of SEQ ID Nos: 1-64. In another aspect, the capturing oligonucleotide has a nucleotide sequence comprising at least 20 consecutive nucleotides of the sequence shown in any one of SEQ ID Nos: 1-64.

[0327] In another aspect, the capturing oligonucleotide has the nucleotide sequence shown in any one of SEQ ID Nos: 1 to 10, 11 to 13, 25 to 26, 33 to 37, 42, 44 to 46, 54, and 59 to 62. In one aspect, the capturing oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in any one of SEQ ID Nos: 1 to 10, 11 to 13, 25 to 26, 33 to 37, 42, 44 to 46, 54, and 59 to 62. In another aspect, the capturing 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 shown in any one of SEQ ID Nos: 1 to 10, 11 to 13, 25 to 26, 33 to 37, 42, 44 to 46, 54, and 59 to 62. In yet another aspect, the capturing oligonucleotide has a nucleotide sequence comprising at least 20 consecutive nucleotides of the sequence shown in any one of SEQ ID Nos: 1 to 10, 11 to 13, 25 to 26, 33 to 37, 42, 44 to 46, 54, and 59 to 62.

[0328] In another aspect, the capturing oligonucleotide has the nucleotide sequence shown in any one of SEQ ID Nos: 1-10. In one aspect, the capturing oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in any one of SEQ ID Nos: 1-10. In another aspect, the capturing 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 shown in any one of SEQ ID Nos: 1-10. In another aspect, the capturing oligonucleotide has a nucleotide sequence comprising at least 20 consecutive nucleotides of the sequence shown in any one of SEQ ID Nos: 1-10. In another aspect, the capturing oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in any one of SEQ ID Nos: 1-10, consisting of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides. Alternatively, the capturing oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in any one of SEQ ID Nos: 1-10, consisting of at least 20 consecutive nucleotides.

[0329] In one aspect, an algorithm is used to generate a set of non-cross-reactive capturing oligonucleotides. In one aspect, up to four sets of non-cross-reactive capturing oligonucleotides are generated: (a) a first set of non-cross-reactive capturing oligonucleotides is generated using a base sequence; (b) a second set of non-cross-reactive capturing oligonucleotides may be generated having a sequence complementary to the capturing oligonucleotide sequence in the first set; (c) a third set of non-cross-reactive capturing oligonucleotides may be generated having a reverse sequence of the capturing oligonucleotide sequence in the first set; and (d) a fourth set of non-cross-reactive capturing oligonucleotides may be generated having a sequence having a reverse complementary sequence of the capturing oligonucleotide sequence in the first set.

[0330] In one aspect, the sets of non-cross-reactive capturing oligonucleotides generated using base sequences are referred to as “parent sets”. Two or more oligonucleotides from the parent set may be selected to form a “subset” (also referred to herein as a “set”) of non-cross-reactive capturing oligonucleotides, wherein each oligonucleotide in the subset is a member of the same parent set (i.e., a subset cannot contain capturing oligonucleotides from more than one parent set).

[0331] For example, the base sequence can be used to generate: (a) a first parent set of non-cross-reactive capturing oligonucleotides; (b) a second parent set of non-cross-reactive capturing oligonucleotides having a sequence complementary to the capturing oligonucleotide sequence in the first set; (c) a third parent set of non-cross-reactive capturing oligonucleotides having an inverse sequence of the capturing oligonucleotide sequence in the first set; and (d) a fourth parent set of non-cross-reactive capturing oligonucleotides having a sequence having an inverse complementary sequence of the capturing oligonucleotide sequence in the first set.

[0332] A subset (or set) may contain: (a) two or more non-cross-reactive capturing oligonucleotides from a first parent set; (b) two or more non-cross-reactive capturing oligonucleotides from a second parent set; (c) two or more non-cross-reactive capturing oligonucleotides from a third parent set; or (d) two or more non-cross-reactive capturing oligonucleotides from a fourth parent set. In one aspect, a set or subset of non-cross-reactive capturing oligonucleotides comprises between about 50 and about 150, about 50 and about 100, about 60 and about 75, or about 60 and about 65 non-cross-reactive capturing oligonucleotides selected from the parent sets of non-cross-reactive oligonucleotides. In one aspect, 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 at most 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 parent set of non-cross-reactive oligonucleotides.

[0333] In one aspect, the first base sequence is used to generate a first set of non-cross-reactive capturing oligonucleotides shown in Table 1 (SEQ ID NO: 1-64). The complementary sequence of this first set of non-cross-reactive capturing oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 4 (SEQ ID NO: 187-250). The reverse sequence of this first set of non-cross-reactive capturing oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 7 (SEQ ID NO: 373-436). The reverse complementary sequence of this first set of non-cross-reactive capturing oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 10 (SEQ ID NO: 559-622).

[0334] In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 1-64) shown in Table 1. In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 187-250) shown in Table 4. In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 373-436) shown in Table 7. In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 559-622) shown in Table 10. In one aspect, 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 sets shown below: Table 1 (SEQ ID NO: 1-64), Table 4 (SEQ ID NO: 187-250), Table 7 (SEQ ID NO: 373-436), or Table 10 (SEQ ID NO: 559-622).

[0335] In one aspect, the collection of non-cross-reactive capture oligonucleotides comprises one or more capture oligonucleotides having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% consistent with the sequences shown below: Table 1 (SEQ ID NO: 1-64), Table 4 (SEQ ID NO: 187-250), Table 7 (SEQ ID NO: 373-436), or Table 10 (SEQ ID NO: 559-622). In another respect, the collection of non-cross-reactive capture oligonucleotides comprises 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 of the sequence shown in Table 1 (SEQ ID NO: 1-64), Table 4 (SEQ ID NO: 187-250), Table 7 (SEQ ID NO: 373-436), or Table 10 (SEQ ID NO: 559-622). In one aspect, the collection of non-cross-reactive capture oligonucleotides comprises one or more capture oligonucleotides having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% consistent with 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 as shown in Table 1 (SEQ ID NO: 1-64), Table 4 (SEQ ID NO: 187-250), Table 7 (SEQ ID NO: 373-436), or Table 10 (SEQ ID NO: 559-622).

[0336] In one aspect, the second base sequence is used to generate a second set of non-cross-reactive capturing oligonucleotides shown in Table 2 (SEQ ID NO: 65-122). The complementary sequence of this second set of non-cross-reactive capturing oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 5 (SEQ ID NO: 251-308). The reverse sequence of this second set of non-cross-reactive capturing oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 8 (SEQ ID NO: 437-494). The reverse complementary sequence of this second set of non-cross-reactive capturing oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 11 (SEQ ID NO: 623-680).

[0337] In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 65-122) shown in Table 2. In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 251-308) shown in Table 5. In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 437-494) shown in Table 8. In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 623-680) shown in Table 11. In one aspect, 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 sets shown below: Table 2 (SEQ ID NO: 65-122), Table 5 (SEQ ID NO: 251-308), Table 8 (SEQ ID NO: 437-494), or Table 11 (SEQ ID NO: 623-680).

[0338] In one aspect, the collection of non-cross-reactive capture oligonucleotides comprises one or more capture oligonucleotides having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% consistent with the sequences shown below: Table 2 (SEQ ID NO: 65-122), Table 5 (SEQ ID NO: 251-308), Table 8 (SEQ ID NO: 437-494), or Table 11 (SEQ ID NO: 623-680). In another aspect, the collection of non-cross-reactive capture oligonucleotides comprises 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 of the sequence shown in Table 2 (SEQ ID NO: 65-122), Table 5 (SEQ ID NO: 251-308), Table 8 (SEQ ID NO: 437-494), or Table 11 (SEQ ID NO: 623-680). In one aspect, the collection of non-cross-reactive capture oligonucleotides comprises 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% consistent with the sequence shown below: Table 2 (SEQ ID NO: 65-122), Table 5 (SEQ ID NO: 251-308), Table 8 (SEQ ID NO: 437-494), or Table 11 (SEQ ID NO: 623-680).

[0339] In one aspect, the third base sequence is used to generate a third set of non-cross-reactive capturing oligonucleotides shown in Table 3 (SEQ ID NO: 123-186). The complementary sequence of this third set of non-cross-reactive capturing oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 6 (SEQ ID NO: 309-372). The reverse sequence of this third set of non-cross-reactive capturing oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 9 (SEQ ID NO: 495-558). The reverse complementary sequence of this third set of non-cross-reactive capturing oligonucleotides can be used to generate another set of non-cross-reactive sequences shown in Table 12 (SEQ ID NO: 681-744).

[0340] In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 123-186) shown in Table 3. In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 309-372) shown in Table 6. In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 495-558) shown in Table 9. In one aspect, the set of non-cross-reactive capturing oligonucleotides comprises two or more sequences from the parental set (SEQ ID NO: 681-744) shown in Table 12. In one aspect, 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 sets shown below: Table 3 (SEQ ID NO: 123-186), Table 6 (SEQ ID NO: 309-372), Table 9 (SEQ ID NO: 495-558), or Table 12 (SEQ ID NO: 681-744).

[0341] In one aspect, the collection of non-cross-reactive capture oligonucleotides comprises one or more capture oligonucleotides having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% consistent with the sequences shown below: Table 3 (SEQ ID NO: 123-186), Table 6 (SEQ ID NO: 309-372), Table 9 (SEQ ID NO: 495-558), or Table 12 (SEQ ID NO: 681-744). In another respect, the collection of non-cross-reactive capture oligonucleotides comprises 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 of the sequence shown in Table 3 (SEQ ID NO: 123-186), Table 6 (SEQ ID NO: 309-372), Table 9 (SEQ ID NO: 495-558), or Table 12 (SEQ ID NO: 681-744). In one aspect, the collection of non-cross-reactive capture oligonucleotides comprises 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% consistent with the sequence shown below: Table 3 (SEQ ID NO: 123-186), Table 6 (SEQ ID NO: 309-372), Table 9 (SEQ ID NO: 495-558), or Table 12 (SEQ ID NO: 681-744).

[0342] In one aspect, the collection of non-cross-reactive capturing oligonucleotides comprises one or more capturing oligonucleotides selected from the following: capturing 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 No: 1-64; capturing oligonucleotides having a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID No: 1-64; capturing 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 ... selected from SEQ ID No: 1-64; capturing oligonucleotides having a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID No: 1-64; capturing oligonucleotides having a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID No: 1-64; Capture oligonucleotides of sequences No:1-64; and their combinations.

[0343] In one aspect, the collection of non-cross-reactive capturing oligonucleotides comprises one or more capturing oligonucleotides selected from the following: capturing 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 No: 1-10; capturing oligonucleotides having a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID No: 1-10; ... a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID No: 1-10; capturing oligonucleotides having a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID No: 1-10; capturing oligonucleotides having a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID No: 1-10; capturing oligonucleotides having a sequence that is at least 95%, 96%, 9 Capture oligonucleotides of sequences 1-10; and their combinations.

[0344] In one aspect, the capturing oligonucleotide is covalently bound to a protein, and immobilization on the carrier surface is achieved by adsorbing the protein onto the carrier surface. Examples of proteins that can be used include: albumins, such as bovine serum albumin (BSA); immunoglobulins; or another protein selected for its ability to adsorb onto the carrier surface. In another aspect, the capturing oligonucleotide is linked (directly or via a linker) to a first conjugate from a conjugate pair, and immobilization is achieved by the binding of this first conjugate to a second conjugate from a conjugate pair immobilized on the carrier surface. Conjugate pairs suitable for immobilizing the capturing oligonucleotide include those known in the art, such as biotin-antibiotic streptavidin, biotin-antibiotin protein, antibody-hapten, antibody-antigenic determinant tag (e.g., antibody-FLAG), nickel-NTA, and receptor-ligand pairs. In one aspect, the capturing oligonucleotide is covalently bound to the protein or the first conjugate via a thiol group (-SH) or an amino group (-NH2). This binding can be direct or via a linker group (e.g., a bifunctional linker group, such as those described in Thermo Scientific Crosslinking Technical Handbook, 2012, published by Thermo Fisher Scientific). In one aspect, the thiol group or amino group is at the 5' or 3' end of the capturing oligonucleotide. In one aspect, the capturing oligonucleotide is a 5'-terminally thiolized oligonucleotide. In one aspect, the capturing oligonucleotide is a 3'-terminally thiolized oligonucleotide. In one aspect, the thiol group is incorporated at an internal position within the capturing oligonucleotide. In one aspect, the capturing oligonucleotide has a nucleotide sequence comprising the sequence shown in any one of SEQ ID NO: 1489-1498 (Table 25). In one aspect, the capturing oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in any one of SEQ ID NO: 1489-1498. In another aspect, the capturing 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 comprising the sequence shown in SEQ ID No: 1489-1498. In yet another aspect, the capturing 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 comprising a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in any one of SEQ ID NO: 1489-1498.

[0345] In one aspect, the capturing oligonucleotide is covalently bound to the carrier surface via a thiol group (-SH) or an amino group (-NH2). In one aspect, the thiol group or amino group is at the 5' or 3' end of the capturing oligonucleotide. In one aspect, the capturing oligonucleotide is a 5'-terminally thiolized oligonucleotide. In one aspect, the capturing oligonucleotide is a 3'-terminally thiolized oligonucleotide. In one aspect, the thiol group is incorporated at an internal position of the capturing oligonucleotide. In one aspect, the capturing oligonucleotide has a nucleotide sequence comprising the sequence shown in any one of SEQ ID NO: 1489-1498 (Table 25). In one aspect, the capturing oligonucleotide has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in any one of SEQ ID NO: 1489-1498. In another aspect, the capturing 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 comprising the sequence shown in SEQ ID No: 1489-1498. In yet another aspect, the capturing 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 comprising a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in any one of SEQ ID NO: 1489-1498.

[0346] In one aspect, the capturing oligonucleotide has a nucleotide sequence having a complementary, reverse, or reverse complementary sequence to the nucleotide sequence shown in SEQ ID NO: 1489-1498. In another aspect, the capturing oligonucleotide has a nucleotide sequence having a complementary, reverse, or reverse complementary sequence to the nucleotide sequence shown in SEQ ID NO: 1489-1498, comprising at least 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence. In yet another aspect, the capturing oligonucleotide has a complementary, reverse, or reverse complementary sequence to the 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 the sequence shown in SEQ ID No: 1489-1498. In another aspect, the captured oligonucleotide has a nucleotide sequence comprising a complementary, reverse, or reverse complementary sequence of 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%, or 99% identical to the sequence shown in any one of SEQ ID NO: 1489-1498.

[0347] In one aspect, one or more capture oligonucleotides contain only three bases (TAG) to reduce hybridization with the native sequence, similar to Luminex x-TAG technology.

[0348] [D. Carrier surface]

[0349] In one aspect, one or more capture oligonucleotides are immobilized on a carrier surface. The capture oligonucleotides can be immobilized on a variety of carrier surfaces, including those used in conventional binding assays. In one aspect, the carrier surface has a flat surface. In another aspect, the carrier surface has a curved surface. In one aspect, the carrier surface contains analytical modules, such as analytical plates, slides, tubes, beads, or chips. In one aspect, the carrier surface contains color-coded microspheres. See, for example, Yang et al. (2001), “BADGE, BeadsArray for the Detection of Gene Expression, a High-Throughput Diagnostic Bioassay.” *Genome Research* Genome Res )》. 11(11):1888-1898. In one aspect, the carrier surface contains one or more beads on which one or more capture oligonucleotides are immobilized.

[0350] The carrier surface can be made of a variety of suitable materials, including polymers such as polystyrene and polypropylene; ceramics; glass; and composite materials, including, for example, carbon-polymer composites, such as carbon-based inks. In one aspect, the carrier surface is a carbon-based carrier surface.

[0351] In one aspect, the carrier surface is provided by one or more particles or "beads". In one aspect, the beads may 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 aspect, the beads have a diameter between about 10 nm and about 100 µm, between about 100 nm and about 10 µm, or between about 0.5 µm and about 5 µm. In one aspect, the beads are paramagnetic, providing the ability to capture the beads using a magnetic field. In one aspect, the carrier surface is provided by magnetic beads coated with streptavidin or avidin, and biotin-labeled capturing oligonucleotides are immobilized on the beads.

[0352] In one aspect, the carrier surface is a plate with multiple wells, i.e., a "well plate". A well plate can contain any number of wells of any size or shape arranged in any pattern or configuration. In one aspect, the well plate contains between about 1 and about 10,000 wells. In one aspect, the number, size, shape, and configuration of the plate and wells in the well plate use industry-standard types. Examples of standard types include 96, 384, 1536, and 9600-well plates, where the wells are arranged in a two-dimensional array. Other well types include single-well, two-well, six-well, twenty-four-well, and 6144-well plates. In one aspect, the carrier surface comprises a 96-well plate.

[0353] In one aspect, the carrier surface comprises a two-dimensional patterned array in which capture molecules are printed at known locations (referred to as binding domains). In another aspect, the carrier surface comprises a patterned array of dispersed, non-overlapping, addressable binding domains in which capture oligonucleotides are immobilized, wherein the sequences of the capture oligonucleotides in each binding domain are known and can be associated with a suitable target analyte or target reaction product. In one aspect, all capture oligonucleotides in a particular binding domain have the same sequence, and the capture oligonucleotides in one binding domain have sequences different from those in other binding domains. In one aspect, multiple binding domains are arranged in ordered rows and columns on the carrier surface, and the precise locations and sequences of each binding domain are recorded in a computer database. In one aspect, the array is arranged in a symmetrical grid pattern. In other aspects, the array is arranged in another pattern, including (but not limited to) radial distribution lines, helices, or ordered clusters. In yet another aspect, each binding domain is located on the surface of one or more microparticles or beads, wherein the microparticles or beads are encoded to allow differentiation between different binding domains.

[0354] In one aspect, the vector surface is a multi-well plate containing one or more dispersed addressable binding domains corresponding to one or more capture oligonucleotides within each well. In one aspect, the vector 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 aspect, 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 aspect, each well contains at least 7, 10, 16, or 25 binding domains.

[0355] In one aspect, the carrier surface is a multi-well plate comprising an array of at least 24, 96, or 384 wells, each well containing an array of up to 10 binding domains, wherein different capturing oligonucleotides are immobilized in dispersed binding domains. In a more particular aspect, the carrier surface is a 96-well plate, wherein each well contains an array having up to 10 binding domains. In one aspect, each well of the 96-well plate contains up to 10 binding domains on which up to 10 different capturing oligonucleotides are immobilized. In one aspect, each well contains an identical patterned array having the same capturing oligonucleotide. In another aspect, different wells may contain different patterned arrays of capturing oligonucleotides.

[0356]

E. Electrode

[0357] In one aspect, electrochemiluminescence is used to identify, detect, or quantify target analytes, comprising, for example, peptide or nucleic acid sequences. The use of electrochemiluminescence for multitasking analyte measurement 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 entirety.

[0358] In one aspect, the support surface includes one or more electrodes. In another aspect, the support surface includes one or more working electrodes and one or more opposing electrodes. In yet another aspect, the support surface includes one or more binding domains formed on one or more electrodes for electrochemical or electrochemiluminescence analysis.

[0359] In one aspect, binding domains are formed by assembling beads coated with trapping oligonucleotides onto an electrode surface. In another aspect, the beads are paramagnetic, and the beads are assembled onto the electrode using a magnetic field.

[0360] In one aspect, one or more capture oligonucleotides are covalently or non-covalently immobilized on one or more electrodes on a carrier surface and onto one or more binding domains. In another aspect, multiple different binding domains are present on one or more electrodes for multi-task measurement of a target analyte in a sample.

[0361] In one aspect, the electrode is provided within an analytical module that provides an analytical container, an analytical flow cell, an analytical fluid, or other components suitable for analysis. Examples of analytical modules for performing electrochemiluminescence analysis include, for example, multi-array configurations, analytical plate configurations, and barrel configurations. In one aspect, the electrode is provided within an analytical module that provides an analytical container, an analytical flow cell, an analytical fluid, or other components suitable for analysis. Examples of analytical modules for performing electrochemiluminescence analysis can be found in U.S. Patents 6,673,533, 7,842,246, 9,731,297, and 8,298,834. In one aspect, the carrier surface is a porous plate containing at least one electrode. In one aspect, each well of the porous analytical plate contains at least one electrode. In one aspect, at least one well of the porous analytical plate contains a working electrode. In another aspect, at least one well of the porous analytical plate contains a working electrode and a counter electrode. In another aspect, each well of the porous analytical plate contains a working electrode and a counter electrode. In one aspect, the working electrodes are adjacent but not in electrical contact with the opposing electrodes.

[0362] In one aspect, the electrode is constructed of a conductive material, which includes, for example, metals such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, conductive alloys, or combinations thereof. In another aspect, the electrode comprises: a semiconductive material, such as silicon and germanium; or a semiconductive film, such as indium tin oxide (ITO) and antimony tin oxide (ATO). In another aspect, the electrode comprises an oxide-coated metal, such as aluminum oxide-coated aluminum. In one aspect, the electrode comprises a carbon-based material. In one aspect, the electrode comprises a mixture of materials containing conductive composites, inks, pastes, polymer blends, and metal / non-metal composites, comprising, for example, a mixture of conductive or semiconductive materials with nonconductive materials. In one aspect, the electrode comprises a carbon-based material, such as carbon, glassy carbon, carbon black, graphitic carbon, carbon nanotubes, carbon filaments, graphite, carbon fibers, and mixtures thereof. In one aspect, the electrode comprises a conductive carbon-polymer composite, a conductive polymer, or conductive particles, such as carbon ink, carbon paste, or metallic ink, dispersed in a matrix. In one aspect, the working electrode is made of a carbon-polymer composite material comprising, for example, conductive carbon particles dispersed in a matrix, such as carbon filaments, carbon black, or graphite carbon, the matrix being a polymer matrix, such as ethylene vinyl acetate (EVA), polystyrene, polyethylene, polyvinyl acetate, polyvinyl chloride, polyvinyl alcohol, acrylonitrile butadiene styrene (ABS), or copolymers of one or more of these polymers.

[0363] In one aspect, the working electrode is made of a continuous conductive sheet or a film of one or more conductive materials, which may be extruded, pressed, or molded. In another aspect, the working electrode is made of a conductive material deposited or patterned on a substrate, for example by printing, brushing, coating, spin coating, evaporation, chemical vapor deposition, electrolytic deposition, electrodeposition, photolithography, or other electronic microfabrication techniques. In one aspect, the working electrode comprises conductive carbon ink printed on a polymer carrier, for example by inkjet printing, laser printing, or screen printing. Carbon ink is known to contain materials manufactured 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).

[0364] In one aspect, the working electrode is a continuous film. In another aspect, the working electrode comprises one or more dispersed regions or a pattern of dispersed regions. Alternatively, the working electrode may comprise multiple connected regions. One or more regions of the exposed electrode surface on the working electrode may be defined by a patterned insulating layer covering the working electrode, for example by printing a patterned dielectric ink layer onto the working electrode, or by attaching a die-cut insulating film. The exposed regions may define array components of a reagent array printed on the working electrode, and may be in the array shape and pattern described above. In one aspect, the insulating layer defines a series of annular regions (or “spots”) on the exposed working electrode surface.

[0365] The opposing electrode may have one or more of the characteristics generally described above for the working electrode. In one aspect, the working electrode and the opposing electrode are constructed of the same material. In another aspect, the working electrode and the opposing electrode are not constructed of the same material; for example, the working electrode may be a carbon electrode and the opposing electrode may be a metal electrode.

[0366] In one aspect, one or more capturing oligonucleotides are immobilized onto one or more electrodes via passive adsorption. In another aspect, one or more capturing oligonucleotides are covalently immobilized onto the electrode. In one aspect, the electrode is derivatized or modified, for example, to immobilize a reagent, such as a capturing oligonucleotide, onto the electrode surface. In one aspect, the electrode is modified by chemical or mechanical treatment to improve reagent immobilization (e.g., by introducing functional groups for reagent immobilization) or to enhance its adsorption properties. Examples of functional groups that can be introduced include (but are not limited to) carboxylic acids (COOH), hydroxyl groups (OH), amino groups (NH2), activated carboxyl groups (e.g., N-hydroxysuccinimide (NHS)-ester), poly(ethylene glycol), thiols, and alkyl groups ((CH2)). n (or combinations thereof). In one aspect, one or more reagents, such as capturing oligonucleotides, are immobilized onto a carbon-containing electrode, such as carbon black, filaments, or carbon dispersed in another material, by covalent or non-covalent means. It has been found that capturing molecules with thiol groups can be covalently bound to carbon-containing electrodes (e.g., screen-printed carbon ink electrodes) without first depositing an additional thiol-reactive layer, such as a protein layer or a chemically cross-linked layer. In one aspect, a method is provided for guiding capturing molecules with thiol groups (e.g., thiol-modified oligonucleotides) to an electrode, providing a simple, robust, efficient, and reproducible process for forming capturing surfaces and arrays on the electrode. In one aspect, one or more capturing oligonucleotides with thiol groups are directly immobilized onto a carbon-containing electrode (e.g., a screen-printed carbon ink electrode) by a reaction of the thiol groups with the electrode, without first adding a thiol-reactive layer to the electrode.

[0367] In one aspect, the electrode is treated with plasma, such as cryogenic plasma or glow discharge plasma, to alter its physical properties, chemical composition, or surface chemistry, for example, to aid in the fixation of reagents that capture oligonucleotides or reduce impurities, improve adhesion to other materials, alter surface wettability, promote material deposition, generate patterns, or improve uniformity. Examples of suitable plasmas include oxygen, nitrogen, argon, ammonia, hydrogen, fluorocarbons, water, and combinations thereof. In one aspect, oxygen plasma is used to treat electrodes of carbon-polymer composite materials containing carbon particles. In another aspect, oxygen is used to introduce carboxylic acids or other oxidized carbon functional groups into carbon or organic materials (e.g., activated esters or acyl chlorides) to facilitate reagent coupling. In another aspect, ammonia-containing plasma can be used to introduce amino groups for coupling analytical reagents. In one aspect, the electrode is not pretreated to aid in the fixation of one or more captured oligonucleotides.

[0368] In one aspect, the carrier surface includes an analytical module, such as a porous plate with one or more working or opposing electrodes in each well. In another aspect, the porous plate includes multiple working or opposing electrodes in each well. In another aspect, the working or opposing electrodes of the porous plate comprise carbon, such as a screen-printed carbon ink layer. In another aspect, one or more capture oligonucleotides are immobilized on screen-printed carbon ink by capturing thiol moieties on the oligonucleotides. In another aspect, the working electrode is used to induce an electrochemiluminescence signal from a label linked to a reaction product. In another aspect, the electrochemiluminescence signal is emitted from ruthenium terpyridine in the presence of a co-reactant (e.g., a tertiary alkylamine, such as tripropylamine or butyldiethanolamine).

[0369] In one aspect, the electrode contains a bonding structure domain defined by dielectric ink (i.e., electrically insulating ink) as described above. The electrode is a working electrode on which a dielectric material is printed within a pattern defining the bonding structure domain described above. In another aspect, the bonding structure domain is an approximately annular region (or “spot”) of the exposed working electrode. The electrode is in a 96-well plate formed by attaching the top of an injection-molded 96-well plate to the bottom of a polyester film sheet defining the wells. The top surface of the polyester film sheet has screen-printed carbon ink electrodes printed thereon, such that each well contains a carbon ink working electrode approximately at the center of the well and two carbon ink opposing electrodes approximately facing the two edges of the well. Electrodes printed on the bottom of the polyester film sheet and connected to the top of the sheet via conductive perforations provide contact for applying voltage to the working electrode and the opposing electrode.

[0370] [F. Methods for immobilizing captured molecules]

[0371] In one aspect, a method is provided for immobilizing one or more capture molecules on a carrier surface. In another aspect, the one or more capture molecules comprise one or more single-stranded capture oligonucleotide molecules as described herein.

[0372] In one aspect, the method includes immobilizing one or more trapping molecules on a carrier surface comprising a carbon-based support surface. In another aspect, the method includes immobilizing one or more trapping molecules on a carrier surface comprising one or more electrodes. In yet another aspect, the method includes immobilizing one or more trapping molecules on a carrier surface comprising one or more carbon-based electrodes.

[0373] In one aspect, the support surface is a porous plate containing one or more electrodes. In another aspect, the support surface is a porous plate where each pore contains one or more electrodes. In yet another aspect, one or more trapping molecules are immobilized in an array on the support surface.

[0374] In one aspect, the method includes spotting or printing two or more capture oligonucleotides onto an array of electrodes in a first well of a multiwell plate, and subsequently printing one or more capture oligonucleotides onto an array of electrodes in one or more additional wells of the multiwell plate. In one aspect, at least some of the printed arrays in each well are identical. In another aspect, at least some of the printed arrays in each well are different.

[0375] In one aspect, one or more capture molecules are spotted or printed at one or more known locations (referred to as binding domains) within an array. In another aspect, one or more capture oligonucleotides are immobilized in dispersed, non-overlapping, addressable binding domains, wherein the sequences of the capture oligonucleotides in each binding domain are known and relevance to the target analyte. In yet another aspect, all capture oligonucleotides in a particular binding domain have the same sequence, and the capture oligonucleotides in one binding domain have sequences different from those in the other binding domains.

[0376] In one aspect, one or more capture molecules are spotted or printed onto dispersed binding domains on a carrier surface. In another aspect, an array of capture oligonucleotides is spotted or printed onto dispersed binding domains on a carrier surface. In one aspect, the capture molecules are spotted or printed by contact printing, including, for example, stylus printing or microstamping, or by non-contact printing, including, for example, photolithography, laser writing, electrojet deposition, and inkjet printing. Generally, the spotting or printing method involves applying one or more droplets containing one or more capture molecules onto dispersed binding domains on a carrier surface and allowing the droplets to dry. In one aspect, the droplets are diffused to cover a region of the carrier surface. In one aspect, the carrier surface includes one or more highly wettable regions and one or more less wettable regions, wherein the more wettable regions define binding domains or array assemblies. Wetness refers to the interaction between a liquid and a solid surface, and more specifically, the phenomenon in which an aqueous solution does not diffuse onto a solid surface but actually contracts to form droplets. In one aspect, the solid carrier has surface properties that encourage droplet formation when a small volume of aqueous solution is applied to one or more dispersed binding domains. The solution of the trapped molecules printed on the higher wettability region diffuses to the boundary with the lower wettability region, thereby providing precise control over the shape and position of the binding domain. In one aspect, the binding domain is a region on the exposed electrode surface of 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 wettability boundary of the exposed electrode region.

[0377] Methods for immobilizing oligonucleotides onto the surface of a carrier are known (see, for example, Balasheb Nimse et al. (2014), "Immobilization Techniques for Microarray: Challenges and Applications"). [The last sentence appears to be a fragment and doesn't translate directly. It likely refers to a separate document or article.] Sensors(14(2): 22208-22229), and is generally based on one or more of the following mechanisms: (1) physisorption, e.g., through charge-charge or hydrophobic interactions; (2) covalent fixation, e.g., through chemical bonding; and (3) non-covalent protein-ligand interactions, e.g., streptavidin-biotin fixation. In one aspect, one or more oligonucleotides are fixed to a functionalized carrier surface. In one aspect, one or more oligonucleotides are fixed to a carrier surface that has not been modified to include one or more functional groups. In one aspect, one or more oligonucleotides are fixed to a carrier surface containing one or more of the following components by physisorption: amine, nitrocellulose, poly(l-lysine), PAAH, and diazo. In one aspect, one or more oligonucleotides are fixed to a carrier surface by covalent interaction, e.g., through a thiol group (-SH), an amino group (-NH2), or an acylhydrazide group. In one aspect, the support surface includes or is modified to include reactive functional groups, such as carboxyl (-COOH), aldehyde (-CHO), epoxy (-CHCH2O), isothiocyanate (-N=C=S), maleic anhydride (-HC2(CO)2NH), or mercaptosilane (-Si-R-SH). In one aspect, the oligonucleotide includes or is modified to include reactive functional groups, such as thiol, amine, or hydrazide groups. In one aspect, one or more oligonucleotides are immobilized to the support surface by means of nucleophilic or electrophilic functional groups present on the support surface.

[0378] In one aspect, one or more capturing molecules comprise a thiol group. In one aspect, one or more capturing molecules are immobilized on a support surface by the thiol group present on the capturing molecule. In one aspect, the method comprises: spotting or printing one or more capturing molecules comprising a thiol group onto a carbon-based support surface, and cultivating the printed support surface to immobilize one or more capturing molecules on the support surface by the thiol group. In one aspect, one or more capturing molecules are covalently linked to the support surface by the thiol group.

[0379] In one aspect, one or more trapping molecules are immobilized onto a support surface by: printing droplets containing trapping molecules (e.g., 50 nL) onto the support surface, allowing the droplets to diffuse, allowing the droplets to dry, and cultivating the dried droplets for a sufficient time (e.g., overnight) to immobilize the trapping molecules onto the support surface. In one aspect, one or more trapping molecules containing thiol groups are immobilized onto a carbon-based support surface by: printing droplets containing trapping molecules onto the support surface, allowing the droplets to diffuse, allowing the droplets to dry, and cultivating the dried droplets for a sufficient time to immobilize the trapping molecules onto the support surface via the thiol groups. In one aspect, droplets are printed in an array. In one aspect, droplets are printed in one or more binding domains. In one aspect, the carbon-based support surface includes one or more carbon-based electrodes. In one aspect, one or more trapping molecules are covalently linked to the carbon-based electrodes via thiol groups. In one aspect, a patterned insulating layer is included on the carbon-based support surface to delineate the diffusion of droplets printed on the support surface.

[0380] In one aspect, the carbon-based support surface is pretreated, for example, to introduce one or more functional groups onto the support surface, such as to increase the reactivity between the thiol groups on the capture molecule and the support surface. In one aspect, the carbon-based support surface is pretreated with a protein, such as bovine serum albumin (BSA). In another aspect, the carbon-based support surface is not pretreated to introduce any functional groups onto the support surface before one or more capture oligonucleotides are immobilized onto the support surface via thiol groups. In one aspect, the support surface is not modified with a protein to increase the reactivity between the thiol groups on the capture molecule and the support surface.

[0381] In one aspect, after spotting or printing one or more capture oligonucleotides onto the surface, the carrier surface is washed with a washing (or capping) solution to remove free capture oligonucleotides (i.e., capture oligonucleotides not immobilized to the carrier surface) (also referred to herein as the “capping” step; see, for example, Example 3). In one aspect, the carrier surface is washed with a washing solution after printing and drying. In one aspect, the carrier surface is washed before being packaged in a dried package. In another aspect, the carrier surface is washed after being packaged in a dried package.

[0382] In one aspect, the washing or capping step includes adding a washing or capping solution (e.g., 50 μL per well for a 96-well analytical plate) to the surface and incubating for 30 to 60 minutes. The incubation temperature can be any convenient temperature, such as room temperature or 37°C. Incubation can occur while the surface is shaken. The washing or capping step may include removing the washing or capping solution and rinsing the surface with a buffer such as PBS.

[0383] In one aspect, the washing solution contains a thiol-containing compound. During the washing step, excess thiol-containing capture molecules can be transferred from one binding domain on the carbon-based electrode to another binding domain and become permanently attached. This transfer of capture molecules and cross-contamination of the resulting binding domains can be reduced by including the thiol-containing compound in the washing solution. While not wishing to be bound by theory, it is believed that the thiol-containing compound in the washing solution competes with free (unbound) capture oligonucleotides and prevents cross-contamination of the binding domains from binding excess capture oligonucleotides removed from different binding domains. In one aspect, the washing solution contains a water-soluble thiol-containing compound. In one aspect, the washing 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 aspect, the water-soluble thiol-containing compound contains a zwitterion.

[0384] In one aspect, the washing solution comprises a water-soluble thiol group selected from: cysteine ​​(e.g., L-cysteine), cysteamine, dithiothreitol, 3-mercaptopropionate, and 3-mercapto-1-propanesulfonic acid. In another aspect, the water-soluble thiol compound comprises cysteine.

[0385] In one aspect, the washing solution contains a pH buffer component. In one aspect, the pH buffer component contains Tris. In one aspect, the washing solution contains a surfactant. In one aspect, the surfactant contains Triton X-100. In one aspect, the washing solution contains a metal chelating agent.

[0386] In one aspect, the washing solution contains a thiol-containing compound between about 5 mM and about 750 mM, between about 10 mM and about 500 mM, between about 25 mM and about 75 mM, or about 50 mM. In one aspect, the washing solution contains cysteine ​​between about 5 mM and about 750 mM, between about 10 mM and about 500 mM, between about 25 mM and about 75 mM, or about 50 mM. In one aspect, the washing solution contains a buffer solution, such as Tris, between about 10 mM and about 30 mM, between about 15 mM and about 25 mM, or about 20 mM. In one aspect, the washing solution contains a surfactant, such as Triton X-100, between about 0.05% and about 0.5%, between about 0.05% and about 0.2%, or about 0.1%. In one aspect, the washing solution has a pH between about 7 and about 9, between about 7.5 and about 8.5, or about 8.0.

[0387] In one aspect, the washing or capping solution contains one or more of the following reagents: (i) known polymers suitable for reducing background signals in hybridization analysis, including (but not limited to) PS20, polyvinyl alcohol (PVA), polyvinylpyrrolidone (~1,000 kD or ~360 kD), Ficoll, and polyethylene glycol (~3 kD and ~10 kD); (ii) nucleic acids or other multivalent anions, including (but not limited to) salmon sperm DNA, herring DNA, calf thymus DNA, cleaved PolyA, yeast tRNA, and heparin; (iii) monomeric and polymeric protein capping agents, including (but not limited to) BSA and polyBSA; (iv) surfactants, including (but not limited to) sodium dodecyl sulfate (SDS), 3-[(3-choleylaminopropyl)dimethylammonium]-1-propanesulfonate (CHAPS), triton-100, and tween-20; and (v) hydrogen bond destabilizers, including (but not limited to) formamide and propylene glycol.

[0388] In one aspect, the method comprises the steps of immobilizing one or more capture oligonucleotides on a carrier surface and subsequently washing away excess unimmobilized capture oligonucleotides from the carrier surface with a washing solution. In one aspect, the washing comprises washing the immobilized capture oligonucleotides under stringent washing conditions. In one aspect, stringent washing conditions comprise a temperature between about 27°C and about 47°C, a formamide concentration between about 21% and about 41%, a salt concentration between about 300 mM and about 500 mM, and a pH between about 7.5 and about 8.5. In one aspect, high stringent 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. In one aspect, the immobilized oligonucleotides are exposed to high stringent conditions for at least 5, 10, 30, or 60 minutes. In another aspect, high stringent conditions comprise low-salt conditions, such as buffer solutions with salt concentrations less than about 40 mM, 20 mM, 15 mM, or 10 mM. In one aspect, high stringency conditions include low-salt conditions, such as 0.1X PBS at 37°C.

[0389] In one aspect, one or more capture oligonucleotides are immobilized in an array on a carrier surface. In another aspect, one or more capture oligonucleotides are immobilized in one or more binding domains on a carrier surface. In another aspect, the capture oligonucleotide printed on one binding domain of the array has a different sequence from the capture oligonucleotides printed on other binding domains in the array.

[0390] While not wishing to be bound by theory, it is believed that the washing solution can introduce loosely bound capture oligonucleotides into the solution, which may potentially redeposit onto the surface via SH covalent binding or other mechanisms. If the capture oligonucleotides are redeposited onto binding domains containing capture oligonucleotides with different nucleotide sequences, then contaminating capture molecules should be considered. The presence of contaminating capture molecules can interfere with analytical results. In one aspect, the binding domains of arrays 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% of contaminating capture molecules.

[0391] In one aspect, the cross-reactivity between binding coordinators (i.e., oligonucleotide tags) of the capture molecule set 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 aspect, analytical specificity (including cross-reactivity of binding or capture oligonucleotide cross-contaminants of non-complementary sequences) is determined. In one aspect, 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 with their corresponding complementary capture molecules immobilized on the plate surface. Subsequently, the plates are washed to remove excess QC probes, and the presence of bound QC probes is detected by detecting primary labels or by adding secondary binding coordinators. The cross-reactivity of each well in each array, such as a multi-well plate, can be calculated as the percentage of the signal detected from the binding of the probe to a spot with a non-specific capture nucleotide. In one aspect, the calculation includes correction for nonspecific background signals detected in the absence of any QC probes.

[0392] In one aspect, a quality control (QC) oligonucleotide probe set is used to determine analytical specificity. In one aspect, the QC probe comprises a nucleotide sequence 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 a corresponding capture molecule in a set of non-cross-reactive capture molecules immobilized on a surface. In one aspect, the set comprises a QC probe having a nucleotide sequence 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 a corresponding capture molecule in a set of non-cross-reactive capture molecules having the sequence shown in any one of SEQ ID NO: 1-774. In one aspect, the collection comprises a QC probe having a nucleotide sequence 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 a corresponding capture molecule in the set of non-cross-reactive capture molecules shown in SEQ ID NO: 1-10. In another aspect, the collection comprises a QC probe having a nucleotide sequence 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 a corresponding capture molecule in the set of non-cross-reactive capture molecules having the sequence shown in SEQ ID NO: 1-10.

[0393] In one aspect, the QC probe comprises a label. In another aspect, the label is directly linked to the QC probe. In yet another aspect, the label is linked to the QC probe via a linker. In one aspect, the label is a compound that is a member of a binding pair, wherein the first member of the binding pair (which may be referred to as the “primary binding agent”) is linked to a substrate (e.g., an oligonucleotide), and the other member of the binding pair (which may be referred to as the “secondary binding agent”) has a detectable physical property. Examples or primary labels include (but are not limited to) electrochemiluminescent labels and organometallic complexes containing transition metals (e.g., ruthenium). In one aspect, the primary label comprises streptomycin. In one aspect, the primary label comprises streptomycin labeled with MSD SULFO-TAG.

[0394] In one aspect, the label comprises a secondary binding agent that binds to a primary binding agent. In one aspect, the primary binding agent comprises biotin, a hapten, streptavidin, avidin, or an antibody or antigen. In one aspect, the secondary binding agent comprises biotin, a hapten, streptavidin, avidin, or an antibody or antigen. In one aspect, the secondary binding agent comprises an electrochemiluminescent label. In one aspect, the secondary binding agent comprises an organometallic complex containing a transition metal (e.g., ruthenium). In one aspect, the QC probe comprises biotin, and the secondary binding agent comprises streptavidin labeled with MSD SULFO-TAG. In one aspect, the QC probe is modified with biotin at its 3' end, as shown in the following structure:

[0395]

[0396] In one aspect, the percentage of contaminating trapped molecules is measured using a method such as in Example 4.

[0397] In one aspect, the uniformity of one or more bonded structural domains on a plate (intra-plate) or between two or more plates (inter-plate) can be determined using known methods for determining the coefficient of variation (CV). In one aspect, the intra-plate or inter-plate bonded structural domains have CVs 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 aspect, the average intra-plate or inter-plate CV is between about 3% and about 6%, or less than about 5%. In one aspect, the uniformity of the bonded structural domains is measured by a method as described in Example 5.

[0398] G. Adaptor fixation

[0399] In one aspect, a method is provided for immobilizing one or more aptamers on a carrier surface. In one aspect, one or more aptamers are immobilized on a carrier surface by binding to one or more single-stranded capture molecules immobilized to a carrier surface as described herein. In one aspect, the aptamer is an oligonucleotide capable of specifically binding to a target molecule and may comprise, for example, a DNA, RNA, or XNA aptamer binding to a molecular target, said molecular target comprising, for example, small molecules, proteins, nucleic acids, cells, tissues, and organisms with non-covalent interactions, such as electrostatic and hydrophobic interactions. In another aspect, the aptamer is a peptide capable of specifically binding to a target molecule comprising at least one or more variable peptide domains displayed through a protein backbone. In one aspect, the immobilized aptamer is used as a probe for one or more target analytes. In one aspect, the immobilized aptamer is used in a microarray.

[0400]

H. Oligonucleotide probe

[0401] In one aspect, the method or kit comprises one or more probe reagents capable of specifically binding to a target analyte in a sample. In another aspect, the oligonucleotide probe comprises a binding complex capable of specifically binding to a target analyte in a sample.

[0402] As used herein, the term "conjugate" refers to members of a pair of molecules that bind specifically to each other under specific set of conditions, under which the conjugate pairs bind to each other to substantially exclude other molecules present in the environment. A conjugate can be any molecule that specifically interacts with another molecule, such as polypeptides, lipids, glycolipids, nucleic acid molecules, carbohydrates, or other molecules, for example through covalent or non-covalent interactions, including, for example, the interaction between an antibody and its homologous antigen, the interaction between two complementary nucleotide sequences, or the interaction between biotin and streptavidin or avidin. The term "correspondence" refers to a relationship between two specific conjugates such that one member of a conjugate pair "corresponds" to the other member of the pair.

[0403] In one aspect, the conjugate contains an antibody that specifically binds to the target analyte. In another aspect, the conjugate contains an oligonucleotide sequence complementary to the oligonucleotide sequence of the target analyte, enabling the oligonucleotide probe to hybridize with the target nucleotide sequence.

[0404] In one aspect, the oligonucleotide probe comprises an oligonucleotide tag and a binding conjugate. In one aspect, the binding conjugate comprises a single-stranded sequence complementary to or substantially complementary to a portion of the target nucleotide sequence. In one aspect, the probe comprises an oligonucleotide tag having a sequence complementary to the sequence of the captured oligonucleotide. In one aspect, the oligonucleotide tag and the binding conjugate are different regions of an oligonucleotide single strand.

[0405] In one aspect, the probe is a single-stranded nucleic acid sequence comprising, for example, a nucleic acid sequence comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), peptide nucleic acid (PNA) or locked nucleic acid (LNA). In another aspect, the probe comprises one or more modified nitrogenous base analogs or bases modified to contain labeled or reactive functional groups or linkers suitable for linking labels.

[0406] In one aspect, the probe length is between about 5 and about 100, between about 10 and about 50, between about 20 and about 30, or at least about 5, 6, 7, 8, 9, 10, 15, 20, or 25 and at most about 30, 35, 40, 45, 50, 75, or 100 nucleotides. The probe can be prepared by any suitable method known in the art, including chemical or enzymatic synthesis, or by cleaving larger nucleic acids using nonspecific nucleic acid-cleavage chemicals or enzymes or by cleaving them with site-specific restriction endonucleases. In some applications, a probe hybridizing to a complementary region in a target sequence can initiate probe extension via polymerase, acting as the starting point for replication of adjacent single-stranded regions on the target sequence.

[0407] In one aspect, the probe contains a label. In another aspect, the label is directly attached to the probe. In yet another aspect, the label is attached to the probe via a linker. In one aspect, the label is a compound that is a member of a binding pair, wherein the first member of the binding pair (which may be referred to as the “primary binding agent”) is attached to a substrate (e.g., an oligonucleotide), and the other member of the binding pair (which may be referred to as the “secondary binding agent”) has detectable physical properties. Examples or primary labels include (but are not limited to) electrochemiluminescent labels and organometallic complexes containing transition metals (e.g., ruthenium). In one aspect, the primary label is an MSD SULFO-TAG label.

[0408] In one aspect, the secondary binding agent binds to the primary binding agent. In one aspect, the primary binding agent comprises biotin, a hapten, streptavidin, avidin, or an antibody or antigen. In one aspect, the secondary binding agent comprises biotin, a hapten, streptavidin, avidin, or an antibody or antigen. In one aspect, the secondary binding agent comprises an electrochemiluminescent label. In one aspect, the secondary binding agent comprises an organometallic complex containing a transition metal (e.g., ruthenium). In one aspect, the secondary binding agent comprises an MSD SULFO-TAG label.

[0409] In one aspect, the kit contains one or more probe reagents. In another aspect, the end user prepares one or more probe reagents.

[0410]

I. Oligonucleotide Tag

[0411] In one aspect, the probe comprises an oligonucleotide tag having a sequence complementary to the oligonucleotide sequence of the capture molecule. In one aspect, the tag comprises a single-stranded oligonucleotide complementary to at least a portion of the nucleotide sequence of the single-stranded capture oligonucleotide. In one aspect, the oligonucleotide tag is generated by recombination. In one aspect, the oligonucleotide tag is not a naturally occurring sequence. In one aspect, one or more capture oligonucleotides comprise a single-stranded nucleic acid sequence, comprising, for example, a nucleic acid sequence comprising deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a structural analog comprising a non-naturally occurring chemical structure that may also participate in hybridization reactions.

[0412] In one aspect, the tag is attached to the 5' end of the probe. In another aspect, the tag is attached to the 3' end of the probe. In one aspect, the tag is not complementary to the target nucleotide sequence and does not hybridize with it.

[0413] In one aspect, a sequence complementary to the target nucleotide sequence and an oligonucleotide tag sequence are present on one nucleic acid strand within the probe. In another aspect, a sequence complementary to the target nucleotide sequence and an oligonucleotide tag sequence are present on different nucleic acid strands. In one aspect, the probe comprises: a first strand having a sequence complementary to the target sequence and a first bridging sequence; and a second strand having an oligonucleotide tag sequence and a second bridging sequence complementary to the first bridging sequence, wherein the first and second strands hybridize or can hybridize via the first and second bridging sequences.

[0414] In one aspect, the oligonucleotide tag contains a marker. In another aspect, the marker is directly linked to the oligonucleotide tag. In yet another aspect, the marker is linked to the oligonucleotide tag via a linker. In one aspect, the marker is linked to the 5' terminal nucleotide of the oligonucleotide tag. In yet another aspect, the marker is linked to the 3' terminal nucleotide of the oligonucleotide tag. In one aspect, the marker is linked along the length of the oligonucleotide tag.

[0415] In one aspect, the label includes radioactive, fluorescent, chemiluminescent, electrochemiluminescent, light-absorbing, light-scattering, electrochemical, magnetic, or enzyme-based labeling. In one aspect, the label includes an electrochemiluminescent label. In one aspect, the label includes a hapten. In one aspect, the label is biotin, fluorescein, or digoxigenin. In one aspect, the label includes an organometallic complex containing a transition metal. In one aspect, the transition metal includes ruthenium. In one aspect, the label is MSD SULFO-TAG. TM mark.

[0416] In one aspect, the oligonucleotide tag includes a primary binding agent as a tag, wherein the primary binding agent is a conjugate of a secondary binding agent. In one aspect, the primary binding agent includes biotin, streptavidin, avidin, or an antigen. In one aspect, the secondary binding agent includes biotin, streptavidin, avidin, or an antibody. In one aspect, the primary binding agent includes an oligonucleotide, and the secondary binding agent is an oligonucleotide having a sequence complementary to the sequence of the primary binding agent.

[0417] In one aspect, the tag has a nucleotide sequence of the following length: at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 and at most about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, or between about 15 and about 40 or between about 20 and about 30 nucleotides. In another aspect, the tag contains a nucleotide sequence shorter than the complementary capture oligonucleotide sequence: at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and at most about 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or between about 1 and about 20, or between about 10 and about 15 or between about 12 and about 13 nucleotides. In one aspect, the tag has a nucleotide sequence of at least about 24, 30, or 36 nucleotides in length.

[0418] In one aspect, the oligonucleotide tag has a sequence that hybridizes with a capture molecule having the sequence shown in any one of SEQ ID NO: 1-774 (Tables 1-12). In one aspect, the oligonucleotide tag has a sequence that hybridizes with a complementary capture molecule having the sequence shown in any one of SEQ ID NO: 1-744 (Tables 1-12). In one aspect, the tag has a nucleotide sequence complementary to a sequence of at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of the sequence shown in any one of SEQ ID NO: 1-744. In one aspect, the tag has a nucleotide sequence complementary to a sequence of at least about 24, 30, or 36 consecutive nucleotides of the sequence shown in any one of SEQ ID NO: 1-744. In one aspect, the oligonucleotide tag has the nucleic acid sequence shown in any of SEQ ID NO: 745-1488 (Tables 13-24).

[0419] In one aspect, the oligonucleotide tag has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any one of SEQ ID NO: 745-1488 (Tables 13-24). In another aspect, 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 the sequence shown in any one of SEQ ID NO: 745-1488 (Tables 13-24). In yet another aspect, the oligonucleotide tag has a nucleotide sequence comprising at least 20 consecutive nucleotides of the sequence shown in any one of SEQ ID NO: 745-1488 (Tables 13-24). In another aspect, 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 comprising a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence in any of SEQ ID NO: 745-1488 (Tables 13-24). In yet another aspect, the oligonucleotide tag has a nucleotide sequence comprising at least 20 consecutive nucleotides comprising a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence in any of SEQ ID NO: 745-1488 (Tables 13-24).

[0420] In one aspect, the oligonucleotide tag has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of the following: SEQ ID NO: 745-754, 755-757, 769-770, 777-781, 786, 788-790, 798, and 803-806. In another aspect, 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 the sequence shown in any of the following: SEQ ID NO: 745-754, 755-757, 769-770, 777-781, 786, 788-790, 798, and 803-806. In another respect, the oligonucleotide tag has a nucleotide sequence comprising at least 20 consecutive nucleotides of the sequence shown in any of the following: SEQ ID NO: 745-754, 755-757, 769-770, 777-781, 786, 788-790, 798 and 803-806. In another aspect, 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 comprising a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence in any of the following: SEQ ID NO: 745-754, 755-757, 769-770, 777-781, 786, 788-790, 798, and 803-806. In one aspect, the oligonucleotide tag has a nucleotide sequence shown in any of the following: SEQ ID NO: 745-754, 755-757, 769-770, 777-781, 786, 788-790, 798, and 803-806.

[0421] In one aspect, the oligonucleotide tag has a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any one of SEQ ID NO: 745-754. In another aspect, 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 the sequence shown in any one of SEQ ID NO: 745-754. In another aspect, 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 comprising a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence in any one of SEQ ID NO: 745-754. In one aspect, the oligonucleotide tag has the nucleotide sequence shown in any one of SEQ ID NO: 745-754.

[0422] In one aspect, the method or kit comprises a set of non-cross-reactive oligonucleotide tags selected from a "parental set" of non-cross-reactive oligonucleotide tags. In one aspect, the set of non-cross-reactive oligonucleotide tags is complementary to a set of non-cross-reactive capturing oligonucleotides. In one aspect, the non-cross-reactive oligonucleotide tags in the set are configured to hybridize with their corresponding complementary sequences in a corresponding set of capturing oligonucleotides. In one aspect, the oligonucleotide tags in the set hybridize with less than 0.05% of the non-complementary sequences in the corresponding set of capturing oligonucleotides relative to the complementary sequences.

[0423] Two or more oligonucleotides may be selected from the parent set to form a “subset” of non-cross-reactive oligonucleotide tags, wherein each oligonucleotide in the subset is a member of the initial parent set. The subset cannot contain oligonucleotide tags from more than one parent set. In one aspect, 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 parent set of non-cross-reactive sequences.

[0424] In one aspect, a first set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 1 (SEQ ID NO: 1-64) is generated. In one aspect, the first set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in Table 13 (SEQ ID NO: 745-808).

[0425] In one aspect, a second set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 2 (SEQ ID NO: 65-122) is generated. In one aspect, the second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in Table 14 (SEQ ID NO: 809-866).

[0426] In one aspect, a third set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 3 (SEQ ID NO: 123-186) is generated. In one aspect, the third set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in Table 15 (SEQ ID NO: 867-930).

[0427] In one aspect, a fourth set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 4 (SEQ ID NO: 187-250) is generated. In one aspect, the fourth set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in Table 16 (SEQ ID NO: 931-994).

[0428] In one aspect, a fifth set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 5 (SEQ ID NO: 251-308) is generated. In another aspect, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in 17 (SEQ ID NO: 995-1052).

[0429] In one aspect, a sixth set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 6 (SEQ ID NO: 309-372) is generated. In another aspect, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in Table 18 (SEQ ID NO: 1053-1116).

[0430] In one aspect, a seventh set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 7 (SEQ ID NO: 373-436) is generated. In another aspect, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in Table 19 (SEQ ID NO: 1117-1180).

[0431] In one aspect, an eighth set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 8 (SEQ ID NO: 437-494) is generated. In another aspect, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in Table 20 (SEQ ID NO: 1181-1238).

[0432] In one aspect, a ninth set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 9 (SEQ ID NO: 495-558) is generated. In another aspect, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in Table 21 (SEQ ID NO: 1239-1302).

[0433] In one aspect, a tenth set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 10 (SEQ ID NO: 559-622) is generated. In another aspect, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in Table 22 (SEQ ID NO: 1303-1366).

[0434] In one aspect, an eleventh set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 11 (SEQ ID NO: 623-680) is generated. In another aspect, a second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in Table 23 (SEQ ID NO: 1367-1424).

[0435] In one aspect, a twelfth set of non-cross-reactive oligonucleotide tags complementary to one or more capture sequences shown in Table 12 (SEQ ID NO: 681-744) is generated. In another aspect, the second set of non-cross-reactive oligonucleotide tags comprises two or more oligonucleotide tags from the parent set shown in Table 24 (SEQ ID NO: 1425-1488).

[0436] In one aspect, the set of non-cross-reactive oligonucleotide tags comprises one or more tags having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of the capturing oligonucleotide listed below: Table 1 (SEQ ID NO: 1-64), Table 2 (SEQ ID NO: 65-122), Table 3 (SEQ ID NO: 123-186), Table 4 (SEQ ID NO: 187-250), Table 5 (SEQ ID NO: 251-308), Table 6 (SEQ ID NO: 309-372), Table 7 (SEQ ID NO: 373-436), Table 8 (SEQ ID NO: 437-494), Table 9 (SEQ ID NO: 495-558), Table 10 (SEQ ID NO: 559-622), Table 11 (SEQ ID NO: 623-680), or Table 12 (SEQ ID NO: 1-64). 681-744). In one aspect, the collection of non-cross-reactive oligonucleotide tags comprises one or more tags having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences shown below: Table 13 (SEQ ID NO: 745-808), Table 14 (SEQ ID NO: 809-866), Table 15 (SEQ ID NO: 867-930), Table 16 (SEQ ID NO: 931-994), Table 17 (SEQ ID NO: 995-1052), Table 18 (SEQ ID NO: 1053-1116), Table 19 (SEQ ID NO: 1117-1180), Table 20 (SEQ ID NO: 1181-1238), Table 21 (SEQ ID NO: 1239-1302), Table 22 (SEQ ID NO: 1303-1366), Table 23 ...3 (SEQ ID NO: 1239-1302), Table 24 (SEQ ID NO: 1239-1302), Table 25 (SEQ ID NO: 1239-1302), Table 26 (SEQ ID NO: 1239-1302), Table 27 (SEQ ID NO: 1239-1302), Table 28 (SEQ ID NO: 1239-1302), Table 29 (SEQ ID NO: 1239-1302), Table 22 (SEQ ID NO: 1239-1302), Table 23 (SEQ ID NO: 1239-1302), Table 23 (SEQ ID NO: 1239-13 (SEQ ID NO: 1367-1424) or Table 24 (SEQ ID NO: 1425-1488).

[0437] In another aspect, the set of non-cross-reactive oligonucleotide tags comprises one or more tags having a nucleotide sequence comprising at least 20, 21, 22, 23, or 24 consecutive nucleotides that are complementary to the sequence of the capture oligonucleotide in Tables 1 (SEQ ID NO: 1-64), 2 (SEQ ID NO: 65-122), 3 (SEQ ID NO: 123-186), 4 (SEQ ID NO: 187-250), 5 (SEQ ID NO: 251-308), 6 (SEQ ID NO: 309-372), 7 (SEQ ID NO: 373-436), 8 (SEQ ID NO: 437-494), 9 (SEQ ID NO: 495-558), 10 (SEQ ID NO: 559-622), 11 (SEQ ID NO: 623-680), or 12 (SEQ ID NO: 681-744). In another respect, the collection of non-cross-reactive oligonucleotide tags comprises one or more tags having a nucleotide sequence comprising at least 20, 21, 22, 23, or 24 consecutive nucleotides of the sequence shown below: Table 13 (SEQ ID NO: 745-808), Table 14 (SEQ ID NO: 809-866), Table 15 (SEQ ID NO: 867-930), Table 16 (SEQ ID NO: 931-994), Table 17 (SEQ ID NO: 995-1052), Table 18 (SEQ ID NO: 1053-1116), Table 19 (SEQ ID NO: 1117-1180), Table 20 (SEQ ID NO: 1181-1238), Table 21 (SEQ ID NO: 1239-1302), Table 22 (SEQ ID NO: 1303-1366), Table 23 (SEQ ID NO: 745-808), Table 24 (SEQ ID NO: 809-866), Table 25 (SEQ ID NO: 867-930), Table 16 (SEQ ID NO: 931-994), Table 17 (SEQ ID NO: 995-1052), Table 18 (SEQ ID NO: 1053-1116), Table 19 (SEQ ID NO: 1117-1180), Table 20 (SEQ ID NO: 1181-1238), Table 21 (SEQ ID NO: 1239-1302), Table 22 (SEQ ID NO: 1303-1366), Table 23 ...3 (SEQ ID NO: (SEQ ID NO: 1367-1424) or Table 24 (SEQ ID NO: 1425-1488).

[0438] In another aspect, the set of non-cross-reactive oligonucleotide tags comprises one or more tags having a sequence of at least 20, 21, 22, 23, or 24 consecutive nucleotides that are at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of the capture oligonucleotides listed below: Table 1 (SEQ ID NO: 1-64), Table 2 (SEQ ID NO: 65-122), Table 3 (SEQ ID NO: 123-186), Table 4 (SEQ ID NO: 187-250), Table 5 (SEQ ID NO: 251-308), Table 6 (SEQ ID NO: 309-372), Table 7 (SEQ ID NO: 373-436), Table 8 (SEQ ID NO: 437-494), Table 9 (SEQ ID NO: 495-558), Table 10 (SEQ ID NO: 559-622), Table 1 ... (NO:623-680) or Table 12 (SEQ ID NO: 681-744). In another respect, the set of non-cross-reactive oligonucleotide tags comprises one or more oligonucleotides having a sequence comprising at least 20, 21, 22, 23, or 24 consecutive nucleotides of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown below: Table 13 (SEQ ID NO: 745-808), Table 14 (SEQ ID NO: 809-866), Table 15 (SEQ ID NO: 867-930), Table 16 (SEQ ID NO: 931-994), Table 17 (SEQ ID NO: 995-1052), Table 18 (SEQ ID NO: 1053-1116), Table 19 (SEQ ID NO: 1117-1180), Table 20 (SEQ ID NO: 1181-1238), Table 21 (SEQ ID NO: 1239-1302), Table 22 (SEQ ID NO: 1053-1116), Table 22 (SEQ ID NO: 1053-1116), Table 20 (SEQ ID NO: 1181-1238), Table 21 (SEQ ID NO: 1239-1302), Table 22 (SEQ ID NO: 1053-1116), Table 22 (SEQ ID NO: 1053-1116), Table 23 ... Table 23 (SEQ ID NO: 1303-1366), Table 23 (SEQ ID NO: 1367-1424), or Table 24 (SEQ ID NO: 1425-1488).

[0439] In one aspect, the non-cross-reactive oligonucleotide tags in the set are selected from: oligonucleotide tags having a sequence containing at least 20, 21, 22, 23, or 24 consecutive nucleotides selected from the sequence of SEQ ID No: 745-808; oligonucleotide tags having a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence selected from SEQ ID No: 745-808; oligonucleotide tags having a sequence containing at least 20, 21, 22, 23, or 24 consecutive nucleotides that are at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence selected from SEQ ID No: 745-808; oligonucleotide tags having a sequence selected from SEQ ID No: 745-808; and combinations thereof.

[0440] In one aspect, the non-cross-reactive oligonucleotide tags in the set are selected from: oligonucleotide tags having a sequence containing at least 20, 21, 22, 23, or 24 consecutive nucleotides selected from the sequence of SEQ ID No: 745-754; oligonucleotide tags having a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence selected from SEQ ID No: 745-754; oligonucleotide tags having a sequence containing at least 20, 21, 22, 23, or 24 consecutive nucleotides that are at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence selected from SEQ ID No: 745-754; oligonucleotide tags having a sequence selected from SEQ ID No: 745-754; and combinations thereof.

[0441] [J. Detection of labeled oligonucleotide products]

[0442] In one aspect, methods and kits are provided for labeling and detecting one or more target analytes in a sample. In one aspect, the presence of one or more target analytes in a sample is determined by generating a reaction product containing an oligonucleotide tag. In one aspect, the reaction product contains a tag. Various methods can be used to generate the reaction product. In one aspect, the reaction product is generated by the methods described herein, including (but not limited to) sandwich assays, oligonucleotide ligation assays (OLA), primer extension assays (PEA), direct hybridization assays, polymerase chain reaction (PCR)-based assays or other targeted amplification assays, and nuclease protection assays.

[0443] 【1. Sandwich Analysis】

[0444] In one aspect, a method and kit are provided for detecting, identifying, or quantifying one or more target analytes in a sample using sandwich analysis. In one aspect, the method or kit comprises one or more sets of probes containing a targeting probe and a detection probe. In one aspect, the targeting probe comprises a single-stranded oligonucleotide tag complementary to at least a portion of a capture oligonucleotide immobilized on a carrier surface and a first binding conjugate. In one aspect, the first binding conjugate comprises a first nucleic acid sequence. In one aspect, the first nucleic acid sequence of the first binding conjugate is complementary to a first region of a target nucleotide sequence in the sample. In one aspect, the first nucleic acid sequence of the first binding conjugate comprises a therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide comprises an RNA oligonucleotide sequence. In one aspect, the therapeutic oligonucleotide is selected from miRNA, therapeutic RNA, mRNA, RNA virus, antisense oligonucleotide (ASO), or combinations thereof. In one aspect, the first nucleic acid sequence of the first binding conjugate is specifically bound by an anti-drug antibody (ADA) in the sample. In another aspect, the first binding conjugate comprises an antibody that specifically binds to a target analyte in the sample. In one aspect, the detection probe comprises a label and a second binding conjugate.

[0445] In one aspect, the second binding conjugate comprises a second nucleic acid sequence. In one aspect, the second nucleic acid sequence of the second binding conjugate is complementary to a second region of the target nucleotide sequence. In one aspect, the second nucleic acid sequence of the second binding conjugate comprises a therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide comprises an RNA oligonucleotide sequence. In one aspect, the therapeutic oligonucleotide is selected from miRNA, therapeutic RNA, mRNA, RNA virus, antisense oligonucleotide (ASO), or combinations thereof. In one aspect, the second nucleic acid sequence of the second binding conjugate is specifically bound by an antidrug antibody (ADA) in the sample. In one aspect, the first ASO of the first binding conjugate and the second ASO of the second binding conjugate are specifically bound by the same antidrug antibody.

[0446] In one aspect, the nucleotide sequence of the first ASO of the first binding conjugate and the nucleotide sequence of the second ASO of the second binding conjugate are at least about 95%, 96%, 97%, 98%, 99%, or 100% identical. In one aspect, the second binding conjugate comprises an antibody that specifically binds to a target analyte in the sample. In one aspect, the targeting probe and the detection probe can simultaneously bind to the same target analyte in the sample to form a reaction product. In one aspect, the reaction product is a sandwich complex.

[0447] In one aspect, the method or kit comprises a collection of multiple probes that can be used in a multi-task array to detect, identify, or quantify multiple target analytes in parallel. In one aspect, each probe collection comprises: a targeting probe having a first binding complex that specifically binds to a first target analyte different from that of a targeting probe in another collection; and an oligonucleotide tag having a sequence complementary to a capture oligonucleotide sequence different from that of a targeting probe in another collection. In one aspect, each probe collection comprises a detection probe containing a second binding complex that specifically binds to a first target analyte and a labeled detection probe. In one aspect, the method or kit comprises multiple collections of oligonucleotide probes. In one aspect, each probe collection comprises: a targeting probe, wherein the first binding complex comprises a nucleic acid sequence complementary to a first target nucleotide sequence; and an oligonucleotide tag having a sequence complementary to a capture oligonucleotide sequence, wherein the target nucleotide sequence of the targeting probe in one collection differs from the target nucleotide sequence in the targeting probe in another collection. In one aspect, the sequence of the oligonucleotide tag of the targeting probe in one collection is complementary to a capture oligonucleotide sequence different from that of the oligonucleotide tag of the targeting probe in another collection. In one aspect, the method or kit comprises a detection probe having a second target nucleotide sequence that is complementary to a second target nucleotide sequence in one or more target nucleotides.

[0448] In one aspect, the method includes the step of providing an array comprising one or more carbon-based electrodes having one or more surfaces and one or more non-cross-reactive capturing oligonucleotides as described herein, wherein the one or more non-cross-reactive capturing oligonucleotides are immobilized in one or more binding domains on one or more surfaces of the one or more carbon-based electrodes. In one aspect, the method includes the step of binding one or more target analytes to an oligonucleotide tag and label, said oligonucleotide tag being complementary to at least a portion of the capturing oligonucleotide immobilized on a carrier surface, and subsequently contacting the array with a composition comprising one or more tagged and labeled target analytes or reaction products. As used herein, “associating / associated” means covalently or non-covalently bound to the target analyte. In one aspect, one or more target analytes are bound to an oligonucleotide tag and label in a sandwich complex. In one aspect, the target analyte is used to generate a reaction product comprising an oligonucleotide tag and label. In one aspect, the method includes the following steps: incubating the sandwich complex or reaction product with a carrier surface under conditions in which an oligonucleotide tag of the sandwich complex or reaction product hybridizes with its corresponding complementary capture oligonucleotide, and identifying, detecting or quantifying the target analyte based on the presence or absence of a tag in an array location.

[0449] 2. Oligonucleotide conjugation assay (OLA)

[0450] In one aspect, an array is contacted with a composition comprising a plurality of target analytes, wherein each target analyte binds to an oligonucleotide tag complementary to a different capture oligonucleotide, and the target analyte can be identified, detected, or quantified based on the binding of the oligonucleotide tag at an array location. In another aspect, an array is contacted with a composition comprising a plurality of tagged and labeled reaction products, wherein each target analyte is used to generate a reaction product comprising an oligonucleotide tag complementary to a different capture oligonucleotide, and the target analyte in a sample can be identified, detected, or quantified based on the binding of the reaction product at an array location.

[0451] In one aspect, the tagged and labeled reaction product is prepared by oligonucleotide conjugation assay (OLA) and can be captured and detected to identify, detect, or quantify one or more target nucleotide sequences. In one aspect, conjugation assay is used to detect, identify, or quantify single nucleotide polymorphisms (SNPs) in one or more target nucleotide sequences. In one aspect, conjugation assay is performed after amplification of one or more target nucleotide sequences in a sample. In another aspect, conjugation assay is performed on samples in which one or more target nucleotide sequences have not yet been amplified. In one aspect, the reaction product from the conjugation assay is amplified prior to capture and detection. In another aspect, the reaction product from the conjugation assay is not amplified prior to capture and detection. Known methods can be used to amplify the reaction product from the conjugation assay.

[0452] Methods for performing oligonucleotide conjugation reactions are known and generally include the following steps: contacting a sample containing or potentially containing one or more nucleotide sequences of interest with a single-stranded oligonucleotide probe pair, said single-stranded oligonucleotide probe being complementary to the target nucleotide sequence and allowing hybridization with the target nucleotide sequence; conjugating the probe that hybridizes to adjacent regions of the target nucleotide sequence to form a reaction product. In one aspect, these steps may be repeated to obtain multiple copies of the reaction product. In one aspect, the nucleotide sequence in the conjugation reaction mixture is denatured prior to an annealing step. The target nucleotide sequence can be detected, identified, or quantified based on the presence or quantity of the reaction product in the sample.

[0453] DNA conjugation depends on three events: (1) the oligonucleotide probe must hybridize to a complementary sequence within the target nucleotide sequence; (2) the oligonucleotide probes must be adjacent to each other in the 5'-to-3'- orientation without intercalating nucleotides; and (3) the oligonucleotide probe must have perfect base pair complementarity with the target nucleotide sequence at its conjugation site. Single nucleotide mismatches between the primer and the target can inhibit conjugation.

[0454] In one aspect, probes are generated by identifying nucleic acid sequences containing approximately 40 base pairs (approximately 80 base pairs in total) flanking the SNP site in the target nucleotide sequence, and by constructing upstream and downstream complementary sequences of the SNP with a span of approximately 18 to 28 nucleotides. In another aspect, two targeting probes differ at the SNP location. Typically, only one detection probe is needed to detect wild-type and variant alleles.

[0455] In another aspect, the target nucleotide sequence is a small nucleic acid, for example, having a length of 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 and at most about 20 base pairs, or a length of at most about 25 base pairs, at most about 30 base pairs, at most about 40 base pairs, or at most about 50 base pairs. In one aspect, a probe for detecting such small nucleic acid targets comprises a length of 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 and at most about 20 base pairs, a length of at most about 25 base pairs, a length of at most about 30 base pairs, a length of at most about 40 base pairs, or a length of at most about 50 base pairs, and conjugates the probe and the small nucleic acid target after hybridization to another, as described herein.

[0456] The length of the oligonucleotide probe sequence can be varied based on the binding temperature requirements of the OLA reaction (e.g., between approximately 62°C and approximately 64°C). Bases can be added to or removed from the targeting or detection probe until the probe length is suitable for the given reaction temperature.

[0457] After determining the sequences and lengths of the targeting and detection probes, oligonucleotide tags can be added to the targeting probe. In one aspect, an oligonucleotide tag is added to the 5' end of the upstream targeting probe. In one aspect, each oligonucleotide tag is complementary to a different capturing oligonucleotide immobilized on a carrier surface. In one aspect, the detection probe contains a label. In one aspect, the detection probe contains a 5' phosphate group and a 3' label. In one aspect, the detection probe contains a 5' phosphate group and a 3' biotin label.

[0458] In one aspect, the method involves using more than one pair of probes. In one aspect, a pair of probes is provided for each target sequence in a sample. In one aspect, three probes are prepared for detecting SNP pairs: two targeting probes that differ at single nucleotide polymorphisms and one detection probe. In one aspect, the two targeting probes contain a 5' oligonucleotide tag and a 3' nucleic acid complementary to the wild-type or variant single nucleotide polymorphism in the target nucleic acid of interest, and the detection probe contains a 3' tag. In one aspect, the 3' tag is a primary binding reagent that binds to a detectable secondary binding reagent. In one aspect, the 3' tag contains biotin, and the secondary binding reagent contains MSD SULFO-TAG streptavidin. In one aspect, a pair of probes is prepared for each allele at the polymorphic site; for example, two probes may be prepared, one for the wild-type allele and one for the mutant allele. In one aspect, a conjugation reaction is performed for each target nucleotide sequence. In another aspect, a multi-task conjugation reaction is performed for more than one target nucleotide sequence. In one aspect, a multi-task conjugation reaction is performed targeting between approximately 1 and approximately 100, or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, or 100 target nucleotide sequences. In one aspect, a multi-task conjugation reaction is performed to detect, identify, or quantify up to 10 target nucleotide sequences in each well. In one aspect, multiple allele pairs are detected, identified, or quantified. In one aspect, up to five allele pairs (i.e., wild-type and mutant SNP pairs) in each well are detected, identified, or quantified. In one aspect, detection, identification, or quantification includes determining whether the sample is homozygous, heterozygous, or absent for variant alleles.

[0459] In one aspect, a template-dependent conjugating enzyme, such as a DNA conjugating enzyme (e.g., *E. coli* DNA conjugating enzyme, T4 DNA conjugating enzyme, *Taq* aquatic bacteria conjugating enzyme, *Thermophilus* DNA conjugating enzyme, or *Porcine conjugating enzyme*), is used to conjugate the probe. In one aspect, the conjugating enzyme is a thermostable conjugating enzyme. In another aspect, the probe is conjugated by chemical conjugation. In one aspect, hybridization and conjugation are performed using a combination of steps, such as using multiple thermal cycles and a thermostable conjugating enzyme. In one aspect, the reaction mixture contains at least about 100 U / mL, 500 U / mL, or 1000 U / mL, and at most about 1500 U / mL or 2000 U / mL of conjugating enzyme.

[0460] In one aspect, conjugation analysis is performed by combining a sample with one or more pairs of probes and conjugating enzymes in a conjugation buffer. In another aspect, the sample, probes, and conjugating enzymes are combined with the conjugation buffer to form a conjugation reaction mixture having a volume of at least about 10 µL, 15 µL, or 20 µL and at most about 20 µL, 25 µL, or 50 µL.

[0461] In one aspect, each probe pair comprises a targeting probe and a detection probe. In one aspect, 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 a capture oligonucleotide immobilized on a carrier surface. In one aspect, the detection probe comprises a tag and a nucleotide sequence complementary to a second region of the target nucleotide sequence, the second region being adjacent to a first region complementary to a first nucleic acid sequence of the same type as the targeting probe sequence. In one aspect, the 5' end of the targeting probe is phosphorylated and is adjacent to the 3'-hydroxyl group of the detection probe when the probe pair is annealed to the target nucleotide sequence, such that the ends of the two probes can be joined by forming a phosphodiester bond. In one aspect, the 5' end of the detection probe is phosphorylated and is adjacent to the 3'-hydroxyl group of the targeting probe when the probe pair is annealed to the target nucleotide sequence, such that the ends of the two probes can be joined by forming a phosphodiester bond.

[0462] In one aspect, the targeting probe comprises between about 5 and about 100, between about 10 and about 50, between about 20 and about 30, or at least about 5, 6, 7, 8, 9, 10, 15, 20, or 25 and at most about 30, 35, 40, 45, 50, 75, or 100 nucleotides. In another aspect, the reaction mixture comprises at least about 1 nM, 2 nM, 3 nM, 4 nM, or 5 nM and at most about 5 nM, 10 nM, 25 nM, or 50 nM of targeting probe.

[0463] In one aspect, the entire length of the targeting probe is complementary to the target nucleotide sequence. In another aspect, a portion of the targeting probe is complementary to the target nucleotide sequence. In one aspect, the targeting probe is complementary to the target nucleotide sequence downstream of the polymorphic site. In one aspect, the targeting probe is an allele-specific probe containing a nucleic acid sequence complementary to a region containing a target nucleotide sequence with a single nucleotide variant. In one aspect, the targeting probe is an allele-specific probe containing a nucleic acid sequence complementary to a region containing a target nucleotide sequence with a single nucleotide polymorphism. In one aspect, the 3'-terminal nucleic acid of the targeting probe is complementary to the polymorphic nucleic acid of the target nucleotide sequence. In another aspect, the 3'-terminal nucleic acid of the targeting probe is complementary to the 3' nucleotide of the polymorphic nucleic acid of the target nucleotide sequence.

[0464] In one aspect, the targeting probe includes a tag that specifically binds to the capture molecule. In one aspect, the tag includes a single-stranded oligonucleotide sequence complementary to at least a portion of the nucleotide sequence of the single-stranded capture oligonucleotide. In one aspect, the tag is attached to the 5' end of the targeting probe. In another aspect, the tag is attached to the 3' end of the targeting probe. In one aspect, the tag is not complementary to and does not hybridize with the target nucleotide sequence.

[0465] In one aspect, the tag comprises a nucleotide sequence shorter than the complementary capture oligonucleotide sequence of the following: at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and at most about 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or between about 1 and about 20, or between about 10 and about 15, or between about 12 and about 13 nucleotides. In another aspect, the tag has a nucleotide sequence of the following length: at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 and at most about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or between about 15 and about 40, or between about 20 and about 30 nucleotides.

[0466] In one aspect, the tag comprises a nucleotide sequence complementary to at least a portion of the nucleotide sequence of the capturing oligonucleotide shown in SEQ ID Nos: 1-10. In another aspect, the tag comprises a nucleotide sequence complementary to about 20 to about 25, or about 24, consecutive nucleotides of the sequence of the capturing oligonucleotide shown in SEQ ID Nos: 1-10. In another aspect, a single-stranded oligonucleotide tag is prepared based on the sequence of the capturing oligonucleotide using a known method.

[0467] In one aspect, each pair of oligonucleotide probes comprises a detection probe having between about 5 and about 100, between about 10 and about 50, between about 20 and about 30, or at least about 5, 6, 7, 8, 9, 10, 15, 20 or 25 and at most about 30, 35, 40, 45, 50, 75 or 100 nucleotides.

[0468] In one aspect, the targeting and detection probes have approximately 60 o The melting temperature is between C and approximately 65°C or between approximately 62°C and approximately 64°C. In one aspect, the targeting and detection probes have similar melting temperatures (i.e., within approximately 1°C, 2°C, 3°C, 4°C, or 5°C).

[0469] In one aspect, the conjugation reaction mixture contains a 1:1 ratio of targeting and detection probes of the target nucleotide sequence. In another aspect, an excess of detection probe is included; for example, the conjugation reaction mixture may contain at least about 5×, 10×, or 20× more detection probes than the targeting probe. In one aspect, the reaction mixture contains at least about 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, or 200 nM of detection probes.

[0470] In one aspect, the entire length of the detection probe is complementary to the target nucleotide sequence. In another aspect, a portion of the detection probe is complementary to the target nucleotide sequence. In one aspect, the detection probe is complementary to the target nucleotide sequence upstream of the polymorphic site. In one aspect, the detection probe contains a nucleic acid sequence complementary to a region of the target nucleotide sequence containing a single nucleotide variant. In one aspect, the detection probe contains a nucleic acid sequence complementary to a region of the target nucleotide sequence containing a single nucleotide polymorphism. In one aspect, the 5' terminal nucleic acid of the detection probe is complementary to the polymorphic nucleic acid of the target nucleotide sequence. In another aspect, the 5' terminal nucleic acid of the detection probe hybridizes with the 5' nucleic acid of the polymorphic nucleic acid of the target nucleotide sequence.

[0471] In one aspect, the detection probe includes a label. In one aspect, the label is attached to the 3' end of the detection probe. In one aspect, the label is attached to the 3' end of the detection probe, and the 5' end has a nucleic acid sequence complementary to the sequence of the target nucleotide immediately adjacent to the 3' end of the target nucleotide hybridizing with the 3' end of the target probe. In one aspect, the label is attached to the 5' end of the detection probe, and the 3' end has a nucleic acid sequence complementary to the sequence of the target nucleotide immediately adjacent to the 5' end of the target probe.

[0472] In one aspect, the targeting probe hybridizes with the target nucleotide sequence such that its 3' end is directly located at a polymorphic nucleotide in the target nucleotide sequence, and the detection probe hybridizes with a target nucleotide sequence adjacent to the same polymorphic site, thus providing a 5' end for the conjugation reaction. If the targeting probe is complementary to the polymorphic nucleotide in the target nucleotide sequence, then the first oligonucleotide will hybridize with the target nucleotide sequence at the polymorphic site and conjugation will occur. If the targeting probe is not complementary to the polymorphic nucleotide in the nucleotide sequence, then the first oligonucleotide will not hybridize with the target nucleotide sequence at the polymorphic site and conjugation will not occur.

[0473] In another aspect, the targeting probe hybridizes with the target nucleotide sequence, such that the 5'-base of the targeting probe is directly located on the polymorphic nucleotide of the target nucleotide sequence, and the detection probe hybridizes with the target nucleotide sequence adjacent to the same polymorphic site, thereby providing the 3' end for the conjugation reaction.

[0474] In another aspect, the detection probe hybridizes with the target nucleotide sequence, such that the 5'-base of the detection probe is directly located at the polymorphic nucleotide of the target nucleotide sequence, and the targeting probe hybridizes with the target nucleotide sequence adjacent to the same polymorphic site, thereby providing the 3' end for the conjugation reaction.

[0475] In another aspect, the detection probe hybridizes with the target nucleotide sequence, such that the 3'-base of the detection probe is directly located at the polymorphic nucleotide of the target nucleotide sequence, and the targeting probe hybridizes with the target nucleotide sequence adjacent to the same polymorphic site, thereby providing the 5' end for the conjugation reaction.

[0476] In one aspect, the method comprises: (i) contacting a sample containing one or more target nucleotides with a pair of oligonucleotide probes and a DNA conjugating enzyme to form a conjugation reaction mixture; (ii) hybridizing the pair of probes with a target nucleotide sequence, wherein the pair comprises a capture or detection probe having a terminal 3' or 5' base of a polymorphic nucleotide directly located in the target nucleotide sequence; (iii) conjugating the target and detection probes together to form a labeled and tagged reaction product; (iv) contacting a carrier surface on which one or more capture oligonucleotides are immobilized with the labeled and tagged reaction product; (v) hybridizing the tag with the capture oligonucleotides; and (vi) detecting the presence of the labeled and tagged reaction product.

[0477] In one aspect, the probe used in the conjugation assay contains an excess (i.e., at the nM level) of the target nucleotide sequence, and therefore in some cases, the nonspecific binding of oligonucleotides on the plate to the target can be detected as a positive signal. While not wishing to be bound by theory, it is believed that nonspecific hybridization can result from the hybridization of the probe to the target nucleotide sequence, with the remainder hybridizing in the absence of conjugation, yielding a signal that is not attributed to the conjugation reaction product but is a nonspecific signal (referred to as bridging background).

[0478] In one aspect, the method includes providing one or more capping probes in a conjugation reaction mixture. In one aspect, including one or more capping probes in the conjugation reaction mixture reduces nonspecific bridging background. As used herein, the term "capping probe" refers to a single-stranded nucleotide sequence complementary to a target nucleotide sequence and spanning the probe conjugation site but not containing a tag or label, or a single-stranded nucleotide sequence complementary to a probe designed to hybridize with the target nucleotide sequence. In one aspect, the capping probe is largely collinear with the probe sequence. In one aspect, the capping probe comprises at least about 20, 25, 30, 35, 40, 45, or 50 and at most about 50, 75, 100, 150, or 200 nucleotides, or between about 20 and about 200 or between about 50 and about 100, complementary to the target nucleotide sequence or a probe targeting the target nucleotide sequence. In one aspect, the conjugation reaction mixture includes a pair of capping probes, wherein the first capping probe has a sequence consistent with the conjugation probe but without a complementary oligonucleotide tag; and the second capping probe has a sequence consistent with the detection probe but without a biotin label. In one aspect, up to 2, 3, 4, or 5 additional nucleotides complementary to the target nucleotide sequence adjacent to the same probe sequence may be added to the 5' and 3' ends of the capping probe.

[0479] While not wishing to be bound by theory, it is believed that the presence of capping probes can reduce the formation of complexes in which the target nucleotide sequence serves as a bridge to probes annealed to the target sequence but not conjugated, allowing the complex to generate spurious signals. In one aspect, the conjugation reaction mixture contains a pair of capping probes. In another aspect, the conjugation reaction mixture contains an excess of one or more capping probes relative to the corresponding OLA probe. In one aspect, the conjugation reaction mixture contains an excess of at least about 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, or 100× moles of capping probes relative to the corresponding OLA probe.

[0480] An example of oligonucleotide conjugation assay is schematically illustrated in Figure 1. Briefly, a target nucleotide sequence 1 containing a polymorphic site 2 is contacted with a pair of oligonucleotide probes, said pair comprising a targeting probe 3 having an oligonucleotide tag 4 and a nucleotide complementary to the polymorphic site, and a detection probe 5 having a tag 6. Oligonucleotide probes 3 and 5 are then hybridized to the target nucleotide sequence. Figure 1A Oligonucleotide probes 3 and 5, which hybridize to the perfectly complementary sequence at the polymorphic site, are used to form reaction product 11, tagged with 4 and labeled with 6. Figure 1BThe reaction mixture containing the conjugation product 11, tagged with 4 and labeled with 6, is introduced onto the surface of a vector having one or more capturing oligonucleotides 7 immobilized in one or more binding domains 9. A signal 10 is detected when the conjugation product 11, tagged with 4 and labeled with 6, is immobilized on the vector surface via hybridization between complementary nucleotide sequences contained in the tagged oligonucleotide 4 and the capturing oligonucleotide 7. Figure 1C ).

[0481] In one aspect, a multi-task conjugation enzyme detection reaction is provided. In one aspect, a sample is contacted with one or more allele-specific probes and one or more shared probes. In one aspect, the one or more allele-specific probes comprise an upstream probe containing a 5' oligonucleotide tag and a 3' sequence corresponding to the polymorphism of interest, said tag having a sequence complementary to the capture oligonucleotide sequence. In one aspect, the one or more shared probes are 5' phosphorylated and 3' biotin-labeled downstream probes. In one aspect, the multi-task conjugation probes are contacted with a sample containing one or more target analytes to hybridize, and adjacent probes are conjugated with a DNA conjugation enzyme to form a conjugation product. In one aspect, one or more immobilized capture oligonucleotides are contacted with the conjugation product, and the oligonucleotide tag hybridizes with its corresponding capture oligonucleotide. Labeled streptavidin, such as SULFO-TAG-labeled streptavidin, can be used, for example, to detect the immobilized conjugation product.

[0482] In one aspect, oligonucleotide conjugation analysis (OLA) is used to detect, identify, and / or quantify a target nucleotide sequence contained in a sample, said sample may contain degradation products of the target nucleotide sequence, also referred to as oligonucleotide metabolites. In one aspect, the sample containing the target nucleotide sequence further contains one or more oligonucleotide metabolites. In one aspect, OLA is used to measure the amount of the target nucleotide sequence relative to the oligonucleotide metabolites in the sample. In one aspect, OLA is used to determine pharmacokinetic parameters of the target nucleotide sequence. In one aspect, the measured pharmacokinetic parameters are clearance, volume distribution, plasma concentration, half-life, peak time, peak concentration, available rate, or combinations thereof. The measurement and interpretation of pharmacokinetic parameters are described herein. In one aspect, the target nucleotide sequence is a therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one aspect, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. Therapeutic oligonucleotides, ASOs, and their metabolism and pharmacology are described herein.

[0483] In one aspect, the oligonucleotide metabolite is shorter than the target nucleotide sequence by 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. In one aspect, 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 aspect, the target nucleotide sequence is a therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one aspect, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite.

[0484] Exemplary embodiments in Figure 15 As shown in [the image]. Figure 15In this process, a sample containing a target nucleotide sequence is contacted with a template oligonucleotide. The template oligonucleotide includes a first sequence complementary to the target nucleotide sequence and a second sequence adjacent to the first sequence and complementary to a conjugate of the target nucleotide sequence. In one aspect, the target nucleotide sequence hybridizes with the first sequence of the template oligonucleotide, and the conjugate of the target nucleotide sequence hybridizes with the second sequence of the template oligonucleotide. In one aspect, the target nucleotide sequence and the conjugate hybridize along the entire length of the template oligonucleotide. In one aspect, the target nucleotide sequence and the conjugate hybridize with the template oligonucleotide to form a double-stranded complex. The target nucleotide sequence and the conjugate are conjugated together using the method described herein to form a target nucleotide sequence conjugation product. Subsequently, the target nucleotide sequence conjugation product is contacted with a pair of single-stranded oligonucleotide probes complementary to the target nucleotide sequence conjugation product, and hybridized with the target nucleotide sequence conjugation product. In one aspect, a probe capable of hybridizing with an adjacent region of the target nucleotide sequence conjugation product is added to the target nucleotide sequence conjugation product. In one aspect, two adjacent probes are conjugated to form a reaction product, each of the two adjacent probes hybridizing with an adjacent region of the target nucleotide sequence conjugation product. In one aspect, the probe includes a targeting probe and a detection probe as described herein. In one aspect, the targeting probe and the detection probe hybridize over the entire length of the target nucleotide sequence conjugation product. In one aspect, the targeting probe includes an oligonucleotide tag. The targeting probe and the oligonucleotide tag are further described herein. In one aspect, the oligonucleotide tag is complementary to at least a portion of a capture oligonucleotide immobilized on a carrier surface. In one aspect, the detection probe includes a label. The detection probe and the label are further described herein. In one aspect, the label includes biotin, and the detection reagent is linked to an antibiotic streptavidin. In another aspect, the label includes a hapten, and the detection reagent is linked to a hapten-binding conjugate, 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 aspect, the surface is contacted with the detection reagent to bind to the label. In one aspect, the detection reagent is an electrochemiluminescent reagent. In one aspect, the detection reagent includes an MSD SULFO-TAG. In one aspect, electrochemiluminescence is measured as described herein to detect, identify, and / or quantify the target nucleotide sequence. In one aspect, the amount of a target nucleotide sequence in a sample is measured to determine the pharmacokinetic parameters of the target nucleotide sequence. 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 therapeutic oligonucleotide is detected without amplification of the therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide in a sample is detected without a nucleic acid extraction step.

[0485] In one aspect, the sample containing the target nucleotide sequence also contains one or more oligonucleotide metabolites. In one aspect, the oligonucleotide metabolites interfere with the detection, identification, and / or quantification of the target nucleotide sequence. Therefore, it may be necessary to remove the oligonucleotide metabolites from the sample. Therefore, in one aspect, a nuclease specific for single-stranded oligonucleotides (i.e., a "single-strand-specific nuclease") is added to the sample, and the target nucleotide sequence conjugation product is hybridized to the template oligonucleotide before the probe is added, such as... Figure 15 As outlined in [the document]. Single-stranded specific nucleases specifically remove single-stranded oligonucleotide metabolites, while being largely unreactive to the conjugation products of the target nucleotide sequence and the template oligonucleotide. In one aspect, single-stranded specific nucleases additionally remove excess unhybridized template oligonucleotides. Non-limiting examples of single-stranded specific nucleases include nuclease S1 (e.g., isolated from Aspergillus oryzae). Aspergillus oryzae ), nuclease P1 (e.g., isolated from Penicillium citrinum ( Penicillium citrinum ), nuclease MB (e.g., isolated from mung bean) Vigna radiata )) and isolated from Alternating Monoclonal bacteria ( Alteromonas espejiana), thick neurosporum ( Neurospora crassa ) and corn smut ( Ustilagomaydis Nucleases. Single-stranded specific nucleases may also include, for example, RNases, such as RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V, PNP enzyme, RNase PH, RNase R, RNase D, RNase T, RNase ONE, oligonucleotide nucleases, ribonuclease I, and ribonuclease II. Additional nucleases that may be suitable for the methods of the present invention include certain DNAases. Additional nucleases, including single-stranded specific nucleases, are provided, for example, in Yang, *Biophysical Quarterly Review* (… Q Rev Biophys ))》 44(1):1-93 (2011) and Desai et al., FEMS Microbiology Review ( FEMS Microbiol Rev In 26:457-491 (2003), 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. In one aspect, the target nucleotide sequence contains RNA. In one aspect, the target nucleotide sequence contains miRNA, therapeutic RNA, mRNA, RNA virus, or a combination thereof.

[0486] 3. Primer Extension Analysis (PEA)

[0487] In another aspect, primer extension analysis (PEA) is used to detect, identify, or quantify one or more target nucleotide sequences in a sample. In one aspect, the target nucleotide sequence contains one or more single nucleotide variants (SNVs). In another aspect, the nucleotide sequence contains one or more single nucleotide polymorphisms (SNPs). In one aspect, primer extension is performed after amplifying the target nucleotide sequence in the sample. In another aspect, primer extension is performed on samples that have not yet been amplified.

[0488] Methods for performing primer extension analysis 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 aspect, the entire length of the probe is complementary to the target nucleotide sequence. In another aspect, a portion of the probe is complementary to the target nucleotide sequence. In one aspect, the probe comprises a nucleic acid sequence complementary to a nucleic acid sequence immediately following the 3' end of the target nucleotide sequence of the polymorphism, such that the probe hybridizes to the target nucleotide sequence downstream of the polymorphic nucleotide. In one aspect, the probe comprises between about 5 and about 100, between about 10 and about 50, between about 20 and about 30, or at least about 5, 6, 7, 8, 9, 10, 15, 20, or 25 and at most about 30, 35, 40, 45, 50, 75, or 100 nucleotides.

[0489] In one aspect, the probe is a targeting probe comprising a tag that specifically binds to the capture molecule. In one aspect, the tag comprises a single-stranded oligonucleotide sequence complementary to the nucleotide sequence of the single-stranded capture oligonucleotide. In one aspect, the tag is attached to the 5' end of the targeting probe. In one aspect, the tag is attached to the 5' end of the targeting probe, and the 3' terminal nucleic acid of the targeting probe is complementary to the nucleic acid downstream of the polymorphic site immediately adjacent to the target nucleotide sequence. In one aspect, the single-stranded oligonucleotide tag is prepared by an end-user using a known method based on the sequence of the capture oligonucleotide.

[0490] In one aspect, the tag comprises a nucleotide sequence shorter than the capturing oligonucleotide sequence: at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and at most about 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or between about 1 and about 20, or between about 10 and about 15 nucleotides. In one aspect, the tag has a nucleotide sequence of the following length: at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 and at most about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or between about 15 and about 40, or between about 20 and about 30 nucleotides. In one aspect, the tag comprises a nucleotide sequence complementary to at least a portion of the nucleotide sequence of the capturing oligonucleotide shown in SEQ ID NO: 1-64. In one aspect, the label comprises a nucleotide sequence that is complementary to at least a portion of the nucleotide sequence of the capture oligonucleotide shown in SEQ ID NO: 1-10.

[0491] In one aspect, one or more tag oligonucleotides contain a sequence complementary to the full sequence of their corresponding capture oligonucleotide. In another aspect, one or more tag oligonucleotides contain a sequence complementary only to a portion of the sequence of their corresponding capture oligonucleotide. For example, and not as a limitation, the capture oligonucleotide may contain a linker as described herein, which may consist of or include an oligonucleotide sequence that is not complementary to the tag oligonucleotide sequence and is located close to the surface of the linker (e.g., starting with a terminal nucleotide modified with a thiol group). The length of the complementary region between the tag and the capture oligonucleotide may also vary. In some aspects of the invention, the length of the complementary region between the oligonucleotides is at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides.

[0492] In one aspect, the method includes contacting a sample containing one or more target nucleotide sequences with a target probe, and hybridizing the target probe with a target oligonucleotide in the presence of a primer extension reaction mixture comprising a polymerase and one or more 2'3'-dideoxynucleotide triphosphates (ddNTPs), including, for example, ddA, ddT, ddC, and ddG. In one aspect, ddNTPs complementary to the polymorphic site are labeled. In one aspect, ddNTPs not complementary to the polymorphic site are not labeled. In one aspect, ddNTPs complementary to wild-type polymorphic nucleotides are labeled. In another aspect, ddNTPs complementary to mutant polymorphic nucleotides are labeled. In one aspect, a single ddNTP is extended from the 3' end of the target probe. In one aspect, when a labeled ddNTP is complementary to a polymorphic nucleotide, a primer extends a labeled ddNTP to form a tagged and labeled reaction product. When a labeled ddNTP is not complementary to a polymorphic nucleotide, then the primer extends an unlabeled ddNTP, which is not detected.

[0493] Suitable polymerases include (but are not limited to) DNA polymerases, RNA polymerases, DNA-dependent RNA polymerases (reverse transcriptases), and their active subunits, including, for example, the Klenow fragment of a DNA polymerase. In one aspect, the polymerase is a DNA polymerase. In another aspect, the polymerase is a thermostable polymerase, such as Taq polymerase.

[0494] An embodiment of primer extension analysis is schematically illustrated in Figure 2. Briefly, a target nucleotide sequence 21 containing a polymorphic site 22 is contacted with a targeting probe 23 having an oligonucleotide tag 25 in the presence of a primer extension reaction mixture containing DNA polymerase and 2'3'-dideoxynucleotide triphosphates (ddNTPs), namely ddA, ddT, ddC, and ddG, wherein the ddNTP 25 complementary to the polymorphic site is labeled 26. A single ddNTP is extended from the 3' end of the targeting probe. Figure 2A As shown, when a labeled ddNTP is complementary to a polymorphic nucleotide, the primer extends a labeled ddNTP to form a tagged and labeled reaction product. Figure 2B As shown, when the polymorphic nucleotide is not complementary to the labeled ddNTP, the primer extends to an unlabeled ddNTP, thereby producing an unlabeled reaction product that will not be detected.

[0495] 4. Direct hybridization

[0496] In one aspect, a method or kit is provided for detecting, identifying, or quantifying one or more target analytes in a sample using direct hybridization. In another aspect, the method or kit comprises one or more capturing oligonucleotides (referred herein to as "target-specific capturing oligonucleotides") comprising one or more nucleic acid sequences complementary to a sequence of one or more target nucleic acids in the sample. In yet another aspect, the method or kit comprises multiple target-specific capturing oligonucleotides that can be used in a multi-task array to detect, identify, or quantify multiple target analytes in parallel.

[0497] In one aspect, the method includes the step of providing a carrier surface on which one or more target-specific capture molecules are immobilized. In one aspect, the carrier surface has a flat surface. In one aspect, the carrier surface is a plate with multiple pores, i.e., a "porous plate". The porous plate may contain any number of pores of any size or shape arranged in any pattern or configuration. In another aspect, the carrier surface has a curved surface. In one aspect, the carrier surface contains analytical modules, such as analytical plates, glass slides, tubes, beads, or chips. In one aspect, the carrier surface is provided by one or more particles or "beads". In one aspect, the carrier surface contains color-coded microspheres. See, for example, Yang et al. (2001), "BADGE, BeadsArray for the Detection of Gene Expression, a High-Throughput Diagnostic Bioassay". Genome Research (… Genome Res )》. 11(11):1888-1898. In one aspect, the carrier surface contains one or more beads on which one or more target-specific capture oligonucleotides are immobilized.

[0498] In one aspect, one or more target-specific capture molecules are immobilized in binding domains in an array. In another aspect, the carrier surface comprises one or more carbon-based electrodes having one or more surfaces and one or more target-specific capture oligonucleotides immobilized in one or more binding domains on one or more surfaces of the one or more carbon-based electrodes.

[0499] In one aspect, a sample containing or suspected of containing one or more target analytes is contacted with one or more oligonucleotide probes and labeled primers, wherein the labeled primers hybridize with the target analytes, the one or more oligonucleotide probes comprising one or more sequences complementary to sequences on one or more target nucleic acids, and the primers comprising sequences complementary to one or more target analytes. Subsequently, known techniques, such as PCR amplification, are used to amplify the target analytes to form labeled reaction products.

[0500] In one aspect, under the condition that one or more labeled reaction products can capture oligonucleotide sequences complementary to their corresponding sequences, the surface of a carrier on which one or more target-specific capture oligonucleotide sequences are immobilized is contacted with the labeled reaction products, and the target analyte is identified, detected, or quantified based on the presence or absence of a label in the array position.

[0501] In one respect, direct hybridization is used to detect, identify, or quantify the presence of a virus in a sample. In another respect, direct hybridization can be used for human papillomavirus (HPV) genotyping. Human papillomavirus (HPV) infection is a leading cause of cervical cancer. More than 200 HPV genotypes have been identified, and approximately 40 cause genital infections. HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 are considered carcinogenic. Munoz et al. (2003), "Epidemiologic classification of human papillomavirus types associated with cervical cancer," *The New England Journal of Medicine*. N. Engl. J. Med. )》 3(48):518.

[0502] In one aspect, direct hybridization is used to detect, identify, or quantify the presence of bacteria in a sample. In another aspect, direct hybridization is used to detect, identify, or quantify *Chlamydia trachomatis* in a sample. Chlamydia trachomatis / C. trachomatis In one aspect, direct hybridization is used for detection, identification, or quantification. Trachoma Clothing bacteria One or more of the three main serotypes (serotype AC).

[0503] In one aspect, direct hybridization is used to detect and identify Salmonella enterica in samples. Salmonella entericaThe existence or quantification of serotypes of Salmonella enterica has been studied. More than 2600 different serotypes have been identified, which can be divided into typhoidal and non-typhoidal serotypes. Gal-mor et al. (2014) "Same species, different diseases: how and why typhoidal and non-typhoidal Salmonella enterica serotypes differ." Salmonella enterica sevovars differ ) . Frontier Microbiology ( Front. Microbiol )》.5(391) Numeric object identifier: 10.3389 / fmicb.2014.00391.

[0504] In one aspect, direct hybridization is used to detect, identify, and / or quantify a target nucleotide sequence, such as a therapeutic oligonucleotide, in a sample that may contain an oligonucleotide metabolite. In one aspect, the sample containing the target nucleotide sequence further comprises one or more oligonucleotide metabolites. In one aspect, direct hybridization is used to measure the amount of the target nucleotide sequence relative to the oligonucleotide metabolite in the sample. In one aspect, direct hybridization is used to determine pharmacokinetic parameters of the target nucleotide sequence. In one aspect, the measured pharmacokinetic parameters are clearance, volume distribution, plasma concentration, half-life, peak time, peak concentration, available rate, or combinations thereof. The measurement and interpretation of pharmacokinetic parameters are described herein. In one aspect, the target nucleotide sequence is a therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). Therapeutic oligonucleotides, ASOs, and their metabolism and pharmacology are described herein.

[0505] In one aspect, the oligonucleotide metabolite is shorter than the target nucleotide sequence by 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. In one aspect, 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 aspect, the target nucleotide sequence is a therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one aspect, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite.

[0506] Exemplary embodiments in Figure 16 As shown in [the image]. Figure 16In one aspect, under conditions where the target nucleotide sequence hybridizes with its complementary sequence, a sample containing the target nucleotide sequence is contacted with a complementary sequence of the target nucleotide sequence. In one aspect, the target nucleotide sequence and its complementary sequence hybridize over their entire length. In one aspect, the target nucleotide sequence analyte hybridizes with its complementary sequence 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 ASO) are described herein. In one aspect, the method includes the removal of oligonucleotide metabolites. In one aspect, a single-stranded specific nuclease is added to the sample while the target nucleotide sequence hybridizes with its complementary sequence. In one aspect, the single-stranded specific nuclease specifically removes the single-stranded oligonucleotide metabolite, while being substantially unreactive to the hybridized target nucleotide sequence and its complementary sequence. In one aspect, the single-stranded specific nuclease additionally removes excess unhybridized complementary sequences of the target nucleotide sequence. Examples of suitable nucleases, including single-stranded 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 another aspect, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite.

[0507] In one aspect, after removing oligonucleotide metabolites and / or unhybridized target nucleotide sequence complementary sequences by a single-stranded specific nuclease, a probe capable of hybridizing with adjacent regions of the target nucleotide sequence is added. In one aspect, two adjacent probes are conjugated to form a reaction product, each of the two adjacent probes hybridizing with an adjacent region of the target nucleotide sequence. In one aspect, the probe comprises a targeting probe and a detection probe as described herein. In one aspect, the targeting probe and the detection probe hybridize over the entire length of the target nucleotide sequence. In one aspect, the targeting probe comprises an oligonucleotide tag. Targeting probes and oligonucleotide tags are further described herein. In one aspect, the oligonucleotide tag is complementary to at least a portion of a capture oligonucleotide immobilized on a carrier surface. In one aspect, the detection probe comprises a label. Detection probes and labels are further described herein. In one aspect, the detection probe is capable of binding to a detection reagent. In one aspect, the detection probe comprises a biotin label. In one aspect, the label comprises biotin, and the detection reagent is linked to an antibiotic streptavidin. In another aspect, the label comprises a hapten, and the detection reagent is linked to a hapten-binding conjugate, such as an antibody. This document further describes the label, the detection reagent, and the binding mode between the label and the detection reagent. In one aspect, a surface is contacted with the detection reagent to bind to the label. In one aspect, the detection reagent is an electrochemiluminescent reagent. In one aspect, the detection reagent includes MSD SULFO-TAG. In one aspect, electrochemiluminescence is measured as described herein to detect, identify, and / or quantify a target nucleotide sequence. In one aspect, the amount of the target nucleotide sequence in a sample is measured to determine pharmacokinetic parameters of the target nucleotide sequence. 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. In one aspect, the target nucleotide sequence comprises RNA. In one aspect, the target nucleotide sequence comprises miRNA, therapeutic RNA, mRNA, RNA virus, or a combination thereof.

[0508] 5. Polymerase Chain Reaction (PCR)

[0509] In one aspect, 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 aspect, PCR is used to amplify target nucleic acids. In one aspect, a method or kit is provided comprising one or more sets of PCR primers, wherein each set of primers includes an upstream primer and a downstream primer. In one aspect, one or more upstream and downstream PCR primers are used to amplify a target nucleotide sequence in a sample.

[0510] In one aspect, one or more target nucleotide analytes in a sample are amplified using one or more modified upstream or downstream primers. In one aspect, one or more target nucleotide analytes are amplified using one or more upstream primers containing an oligonucleotide tag sequence configured to hybridize with a capture oligonucleotide having a complementary sequence. In one aspect, one or more target nucleotide analytes are amplified using one or more labeled downstream primers. In one aspect, one or more target nucleotide analytes are amplified using one or more downstream primers containing an oligonucleotide tag sequence configured to hybridize with a capture oligonucleotide having a complementary sequence. In one aspect, one or more target nucleotide analytes are amplified using one or more labeled upstream primers.

[0511] In one aspect, 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 with a capture oligonucleotide immobilized on a vector surface. In another aspect, a target nucleotide sequence is amplified using one or more modified PCR primers to form a PCR reaction product comprising a tag. In yet another aspect, 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 with a capture oligonucleotide immobilized on a vector surface and a tag. The method for tagging the PCR reaction product is known and comprises, for example, a labeled deoxynucleotide triphosphate (dNTP) or a labeled modified upstream or downstream primer.

[0512] In one aspect, one or more capture oligonucleotides are immobilized in binding domains in an array on a vector surface. In another aspect, PCR reaction products are captured on the vector surface by hybridization of the oligonucleotide tag with its corresponding capture oligonucleotide.

[0513] In one aspect, conjugation-mediated amplification (LM PCR) is used to detect, identify, or quantify one or more target analytes. In another aspect, multi-task conjugation-mediated amplification combined with the methods described herein is used to detect, identify, or quantify one or more target analytes. In one aspect, one or more target nucleotide analytes are reverse transcribed using upstream and downstream probes. In one aspect, the upstream probe contains a nucleotide sequence complementary to a universal primer site (e.g., T7), an oligonucleotide tag sequence, and a gene-specific sequence; and the downstream probe contains a gene-specific fragment adjacent to the gene-specific fragment of the upstream probe and a universal primer site (e.g., T3). In one aspect, the downstream probe is 5' phosphorylated. In one aspect, the probe is annealed to its target, the free probe is removed, and the annealed probe is conjugated using a conjugation enzyme to obtain an amplification template. In one aspect, PCR is performed using T3 and a 5' biotin-labeled T7 primer. In one aspect, under conditions in which the oligonucleotide tag hybridizes with its corresponding capture oligonucleotide, a capture oligonucleotide immobilized on the surface of a vector is contacted with a biotin-labeled amplicon. In one aspect, captured tagged amplicones are incubated with a tagged streptavidin, such as a SULFO-TAG-tagged streptavidin, to enable the detection, identification, or quantification of the captured tagged amplicones. See, for example, Peck et al. (2006), “A method for high-throughput gene expression signature analysis.” *Genome Biology* Genome Biol. )》 7(7):R61.

[0514] In one aspect, the target analyte is cDNA. In another aspect, the target analyte is mRNA. In one aspect, cDNA is synthesized from mRNA with a poly-A tail using oligo-dT primers. In another aspect, cDNA can be generated from mRNA using randomly initiated cDNA synthesis.

[0515] 6. Nuclease Protection Assay (NPA)

[0516] In one aspect, 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 aspect, the 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 aspect, the target analyte comprises a single-stranded nucleic acid, such as single-stranded RNA. In one aspect, the target analyte comprises microRNA (miRNA). In one aspect, under conditions in which the target analyte hybridizes with a probe, 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 to form a tagged reaction product. In one aspect, 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 aspect, the hybrid probe comprises a biotin label.

[0517] In one aspect, under conditions where one or more oligonucleotide tag sequences hybridize with their corresponding capture oligonucleotide sequences immobilized on a carrier surface, the carrier surface on which one or more capture oligonucleotides are immobilized is contacted with a mixture containing the tagged reaction product. After hybridizing the oligonucleotide tag with its corresponding capture oligonucleotide on the carrier surface, the carrier surface is washed and, under conditions where an RNase can digest single-stranded RNA molecules, and excess probes bound to unhybridized target RNA spots are removed, as well as any mismatch sites between the cleavage probe and the target RNA, it is contacted with an RNase specific to single-stranded RNA (e.g., RNase A or RNase I).

[0518] In one aspect, miRNA analysis includes a step-down probe hybridization step, wherein a DNA / RNA chimeric probe hybridizes with the target miRNA during a decrease in annealing temperature increments.

[0519] In one aspect, 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 (also referred to as oligonucleotide metabolites) of the target nucleotide sequence. In one aspect, the sample containing the target nucleotide sequence further comprises one or more oligonucleotide metabolites. In one aspect, a direct-surface-coated NPA is used to measure the amount of the target nucleotide sequence relative to the oligonucleotide metabolite in the sample. In one aspect, a direct-surface-coated NPA is used to determine pharmacokinetic parameters of a therapeutic oligonucleotide. In one aspect, the measured pharmacokinetic parameters are clearance, volume distribution, plasma concentration, half-life, peak time, peak concentration, available rate, or combinations thereof. The measurement and interpretation of pharmacokinetic parameters are described herein. In one aspect, the target nucleotide sequence is a therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one aspect, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. Therapeutic oligonucleotides, antisense oligonucleotides, and their metabolism and pharmacology are described herein.

[0520] In one aspect, the oligonucleotide metabolite is shorter than the target nucleotide sequence by 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. In one aspect, 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 aspect, the target nucleotide sequence is a therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one aspect, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. In one aspect, the therapeutic oligonucleotide is detected without amplification of the therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide in a sample is detected without a nucleic acid extraction step.

[0521] Exemplary embodiments in Figure 17 As shown in [the image]. Figure 17In this document, the complementary sequence of the target nucleotide sequence is used as a capture oligonucleotide. The complementary sequence includes a label at one end and a surface linker at the other end. Methods for immobilizing the capture oligonucleotide to a surface are described herein and include, for example, electrostatic interactions, complementary binding complexes, complementary reactive functional groups, linkers (e.g., cross-linking agents containing reactive functional groups), etc. In one aspect, the surface is coated with the complementary sequence of the target nucleotide sequence via the surface linker. In one aspect, the surface linker includes a thiol group. In one aspect, the surface linker includes biotin.

[0522] In one aspect, a surface coated with the target nucleotide sequence complementary sequence is brought into contact with a sample containing the target nucleotide sequence, under conditions in which the target nucleotide sequence complementary sequence hybridizes with the target nucleotide sequence. In one aspect, the target nucleotide sequence and the target nucleotide sequence complementary sequence hybridize over their entire length. In one aspect, the target nucleotide sequence hybridizes with the target nucleotide sequence complementary sequence to form a double-stranded complex. In one aspect, the sample containing the target nucleotide sequence further contains one or more oligonucleotide metabolites. Metabolites of the target nucleotide sequence (e.g., therapeutic oligonucleotides, such as ASO) are described herein. In one aspect, the method includes the removal of oligonucleotide metabolites. In one aspect, a single-stranded specific nuclease is added to the sample while the target nucleotide sequence hybridizes with the target nucleotide sequence complementary sequence. In one aspect, the single-stranded specific nuclease specifically removes single-stranded oligonucleotide metabolites and is substantially unreactive to the hybridized target nucleotide sequence-target nucleotide sequence complementary sequence. Examples of suitable nucleases, including single-stranded specific nucleases, are provided herein.

[0523] In one aspect, after the oligonucleotide metabolite is removed by a single-stranded specific nuclease, the surface is contacted with a detection reagent capable of binding to a label on a complementary sequence to the target nucleotide sequence. In one aspect, the label includes biotin, and the detection reagent is linked to an antibiotic streptavidin. In another aspect, the label includes a hapten, and the detection reagent is linked to a hapten-binding conjugate, 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 aspect, the detection reagent is an electrochemiluminescent reagent. In one aspect, the detection reagent includes MSD SULFO-TAG. In one aspect, electrochemiluminescence is measured as described herein to detect, identify, and / or quantify the target nucleotide sequence. In one aspect, the amount of the target nucleotide sequence in a sample is measured to determine the pharmacokinetic parameters of the target nucleotide sequence. 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. In one aspect, the target nucleotide sequence comprises RNA. In one aspect, the target nucleotide sequence comprises miRNA, therapeutic RNA, mRNA, RNA virus, or a combination thereof.

[0524] In another aspect, the target nucleotide sequence is a small nucleic acid, for example, having a length of 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 and at most about 20 base pairs, or a length of at most about 25 base pairs, at most about 30 base pairs, at most about 40 base pairs, or at most about 50 base pairs. In one aspect, a probe for detecting such small nucleic acid targets comprises a length of 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 and at most about 20 base pairs, a length of at most about 25 base pairs, a length of at most about 30 base pairs, a length of at most about 40 base pairs, or a length of at most about 50 base pairs, and conjugates the probe and the small nucleic acid target after hybridization to another, as described herein.

[0525] 【7. Hybridization / Conservation Analysis】

[0526] In one aspect, hybridization / protection analysis is used to detect, identify, and / or quantify a target nucleotide sequence, such as a therapeutic oligonucleotide, in a sample that may contain an oligonucleotide metabolite. In one aspect, the sample containing the target nucleotide sequence further comprises one or more oligonucleotide metabolites. In one aspect, hybridization / protection analysis is used to measure the amount of the target nucleotide sequence relative to the oligonucleotide metabolite in the sample. In one aspect, hybridization / protection analysis is used to determine the pharmacokinetic parameters of a therapeutic oligonucleotide. In one aspect, the measured pharmacokinetic parameters are clearance, volume distribution, plasma concentration, half-life, peak time, peak concentration, available rate, or combinations thereof. The measurement and interpretation of pharmacokinetic parameters are described herein. In one aspect, the target nucleotide sequence is a therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). Therapeutic oligonucleotides, ASOs, and their metabolism and pharmacology are described herein.

[0527] In one aspect, the oligonucleotide metabolite is shorter than the target nucleotide sequence by 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. In one aspect, 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 aspect, the target nucleotide sequence is a therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). In one aspect, the oligonucleotide metabolite is a therapeutic oligonucleotide metabolite. In one aspect, the therapeutic oligonucleotide is detected without amplification of the therapeutic oligonucleotide. In one aspect, the therapeutic oligonucleotide in a sample is detected without a nucleic acid extraction step.

[0528] Exemplary embodiments in Figure 18 As shown in [the image]. Figure 18In this invention, a sample containing a target nucleotide sequence is contacted with a target nucleotide sequence complementary sequence probe comprising: (i) a target nucleotide 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 complementary sequence probe is complementary to at least a portion of a capture oligonucleotide immobilized on a carrier 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 the capture oligonucleotide immobilized on a carrier surface. The oligonucleotide tag and the capture oligonucleotide are further described herein. In one aspect, the label of the target nucleotide sequence complementary sequence probe is capable of binding 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 conjugate, such as an antibody. The label, the detection reagent, and the binding mode between the label and the detection reagent are further described herein.

[0529] In one aspect, the target nucleotide sequence hybridizes with a target nucleotide sequence complementary sequence probe. In another aspect, the target nucleotide sequence and the target nucleotide sequence complementary sequence probe hybridize along the entire length of the target nucleotide sequence and the target nucleotide sequence complementary sequence. In one aspect, the oligonucleotide tag is double-stranded, and the target nucleotide sequence and the target nucleotide sequence complementary sequence hybridize to form a double-stranded complex. In one aspect, the sample containing the target nucleotide sequence further contains one or more oligonucleotide metabolites. Metabolites of the target nucleotide sequence are described herein, such as therapeutic oligonucleotides, such as ASO. In one aspect, the method includes the removal of oligonucleotide metabolites. In one aspect, a single-stranded specific nuclease is added to the sample while the target nucleotide sequence hybridizes with the target nucleotide sequence complementary sequence. In one aspect, the single-stranded specific nuclease specifically removes the single-stranded oligonucleotide metabolite and is substantially unreactive to the hybridized target nucleotide sequence-target nucleotide sequence complementary sequence. In one aspect, the single-stranded specific nuclease additionally removes excess unhybridized target nucleotide sequence complementary sequence probes. Examples of suitable nucleases, including single-stranded specific nucleases, are provided herein.

[0530] In one aspect, after removing oligonucleotide metabolites and / or unhybridized target nucleotide sequence complementary probes, the hybridized target nucleotide sequence-target nucleotide sequence complementary probe is immobilized onto a carrier surface by binding an oligonucleotide tag on the target nucleotide sequence complementary probe to a capturing oligonucleotide on the surface. In one aspect, before immobilizing the hybridized target nucleotide sequence-target nucleotide sequence complementary probe, oligonucleotide metabolites and / or unhybridized target nucleotide sequence complementary probes are removed to provide improved sensitivity compared to simultaneous removal / immobilization or removal after immobilization. In one aspect, a detection reagent is added to the surface, and the detection reagent binds to a tag on the target nucleotide sequence complementary probe. In one aspect, the detection reagent is an electrochemiluminescent reagent. In one aspect, the detection reagent includes MSD SULFO-TAG. In one aspect, electrochemiluminescence is measured as described herein to detect, identify, and / or quantify the target nucleotide sequence. In one aspect, the amount of the target nucleotide sequence in a sample is measured to determine the pharmacokinetic parameters of the target nucleotide sequence. 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. In one aspect, the target nucleotide sequence comprises RNA. In one aspect, the target nucleotide sequence comprises miRNA, therapeutic RNA, mRNA, RNA virus, or a combination thereof.

[0531]

K. Sample

[0532] The methods or kits described herein are suitable for detecting one or more target analytes in samples containing or suspected of containing one or more target analytes. In one aspect, the target analyte comprises a target nucleotide sequence. In another aspect, the target analyte comprises a target protein. In one aspect, the sample contains or is suspected of containing one or more prokaryotic or eukaryotic DNA or RNA sequences of interest. In one aspect, the sample is a biological sample obtained from: an organism, such as a human or other mammal, including (but not limited to) non-human primates, dogs, cats, cattle, sheep, poultry, horses; or other organisms, such as plants, bacteria, fungi, protozoa, or viruses. In one aspect, the biological sample comprises: solid material, such as tissue, cells, cell extracts, or biopsies; or biological fluids, such as urine, blood, saliva, amniotic fluid, secretions from infected or inflamed areas, oral wash containing buccal cells, cerebrospinal fluid, or synovial fluid. In one aspect, the sample is isolated from an individual. In another aspect, the sample is derived from a group of individuals. In one aspect, the sample comprises one or more individual samples or pooled samples.

[0533] In one aspect, 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 aspect, 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 yet another aspect, the sample contains or is suspected of containing one or more target nucleotide sequences, said one or more target nucleotide sequences being amplicones, such as PCR products, plasmids, granules, DNA libraries, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), synthetic oligonucleotides, restriction enzyme fragments, DNA / RNA hybrids, peptide nucleic acids (PNA), or DNA / RNA mosaic nucleic acids. For double-stranded nucleic acids, the target nucleotide sequence may be present in either strand. In one aspect, the sample does not contain ethylenediaminetetraacetic acid (EDTA).

[0534] In one aspect, the sample contains one or more target nucleotide sequences, such as therapeutic oligonucleotides, and the sample may also contain oligonucleotide metabolites. As used herein, "therapeutic oligonucleotide" refers to an oligonucleotide capable of interacting with a biomolecule to provide a therapeutic effect. In one aspect, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO). An ASO is a single-stranded oligonucleotide, typically ranging in length from about 5, 10, 15, 20, or 25 nucleotides to about 30, 35, 40, 45, or 50 nucleotides. ASOs can influence RNA processing and / or regulate protein expression. An ASO is a single-stranded oligonucleotide that binds to single-stranded RNA to inactivate it. In one aspect, an ASO binds to the messenger RNA (mRNA) of a gene, thereby inactivating the gene. In one aspect, the gene is a disease gene. Therefore, an ASO can inactivate the mRNA of a disease gene to prevent or improve the production of a specific disease-causing protein. In one aspect, an ASO includes DNA, RNA, or a combination thereof.

[0535] Oligonucleotides in a sample, such as therapeutic oligonucleotides (e.g., ASO), can degrade or shorten over time due to various factors, including the presence of nucleases, temperature, pH, and salt concentration. In some respects, the degradation of therapeutic oligonucleotides in a sample indicates a pharmacokinetic response to the therapeutic oligonucleotide. Degraded or shortened therapeutic oligonucleotides, also referred to herein as therapeutic oligonucleotide metabolites, may lose their therapeutic efficacy. In one respect, the sample contains a therapeutic oligonucleotide and one or more therapeutic oligonucleotide metabolites. In one respect, the therapeutic oligonucleotide metabolite is shorter than the therapeutic oligonucleotide by one or more nucleotides, two or more nucleotides, three or more nucleotides, four or more nucleotides, five or more nucleotides, six or more nucleotides, seven or more nucleotides, eight or more nucleotides, nine or more nucleotides, ten or more nucleotides, fifteen or more nucleotides, or twenty or more nucleotides. In one aspect, the therapeutic oligonucleotide metabolites are 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 oligonucleotides.

[0536] In one aspect, the method provided herein is used to measure the amount of a therapeutic oligonucleotide relative to its metabolites in a sample. In one aspect, pharmacokinetic parameters of a therapeutic oligonucleotide are determined by measuring the degradation rate and / or amount of the therapeutic oligonucleotide in a biological environment (e.g., a patient). Therefore, in one aspect, the method provided herein is used to determine the pharmacokinetic parameters of a therapeutic oligonucleotide. In one aspect, the pharmacokinetic parameters measured are clearance, volumetric distribution, plasma concentration, half-life, peak time, peak concentration, available rate, or combinations thereof. The measurement and interpretation of pharmacokinetic parameters are further described herein.

[0537] In one aspect, the sample contains one or more anti-drug antibodies (ADAs). In one aspect, ADA binds to a therapeutic peptide, including (but not limited to) a therapeutic protein or a therapeutic antibody. In one aspect, ADA binds to a therapeutic oligonucleotide, including (but not limited to) an antisense oligonucleotide (ASO), short interfering RNA, microRNA, and a CRISPR / Cas synthesis guide strand. In one aspect, ADA is capable of binding to a biopharmaceutical product. In one aspect, ADA is capable of inhibiting the functional activity of a therapeutic product.

[0538] In one aspect, the sample contains one or more unamplified target nucleotide sequences. In another aspect, 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).

[0539] In one aspect, the sample contains or is suspected of containing one or more target proteins. In one aspect, the target protein contains a DNA-binding protein, such as a protein having a DNA-binding domain capable of binding to single- or double-stranded DNA. Examples of DNA-binding proteins include (but are not limited to) transcription factors, polymerases, nucleases, and histones. In one aspect, the DNA-binding protein binds to a specific DNA sequence (e.g., a transcription factor).

[0540] In one aspect, one or more target analytes are purified from a biological sample. Methods for purifying the target analytes from the sample are known. Methods for purifying nucleotide sequences from a biological sample are known and include, 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 a biological sample are known and include, for example, chromatography, such as size exclusion chromatography, high performance liquid chromatography (HPLC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, affinity chromatography; and electrophoresis.

[0541] In one aspect, the sample contains 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 at most 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, such as genomic DNA purified from whole-genome amplified DNA from a cell line. In one aspect, the sample comprises at least about 0.1 µL, 0.5 µL, 1 µL, 2 µL, 3 µL, 4 µL, 5 µL, and at most about 6 µL, 7 µL, 8 µL, 9 µL, 10 µL, 15 µL, 20 µL, or 25 µL, or between about 1 µL and about 25 µL, or between about 0.1 µL and about 5 µL, containing one or more target analytes, such as samples containing one or more amplification products, such as PCR amplicon samples generated from cell line DNA. In one aspect, the sample has an analyte concentration of at least about 1 ng / µL, 5 ng / µL, or 10 ng / µL, and at most about 25 ng / µL, 50 ng / µL, or 100 ng / µL.

[0542] In one aspect, the sample contains at least one copy of the target analyte. In another aspect, the sample contains fewer than 10 copies. 7 10 6 10 5 10 4 10 3 10 2 Or 10 1 The target nucleic acid. In one aspect, these copies are present in samples between about 0.001 mL and about 1 mL, or in samples smaller than about 1 mL, 0.1 mL, 0.01 mL or 0.001 mL.

[0543] [L. Sample Amplification]

[0544] While the methods or kits described herein can be used with samples in which one or more target nucleotide sequences have not yet been amplified, amplification steps may be required to increase the number of target nucleotides in the sample. For example, amplification of the target nucleotide sequence may be necessary when the target nucleotide sequence contains one or more rare mutations, such as one or more rare or low-allelic fraction mutations associated with cancer.

[0545] In one aspect, the target nucleotide sequence is amplified by polymerase chain reaction (PCR). Methods for PCR amplification are known. See, for example, Saiki et al., "Using Heat-Stable..." DNA polymerase performs DNA synthesis.Primer-directed enzymatic amplification ( Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase (See *Science*, 239:487-491). In short, PCR amplification involves contacting two oligonucleotide primers with a specific nucleotide sequence on each side of the target nucleotide sequence. Repeated cycles of heat denaturation, primer annealing to their complementary sequences, and extension of the annealed primers with DNA polymerase yield approximately 2... n The exponential accumulation, of which n The number of cycles.

[0546] In another aspect, rolling circle amplification (RCA) is used to amplify the target nucleotide sequence. RCA is an isothermal nucleic acid (e.g., DNA or RNA) amplification technique in which a polymerase continuously adds single nucleotides to primers that anneal to a circular template, thereby producing a long single-stranded DNA or RNA sequence containing multiple, for example, dozens to hundreds, tandem repeats complementary to the circular template.

[0547] In another aspect, whole-genome amplification (WGA) is used to amplify genomic DNA samples. Methods for whole-genome amplification are known and include, for example, multiple displacement amplification (MDA), degenerate oligonucleotide PCR (DOP-PCR), and primer extension preamplification (PEP). While DOP-PCR and PEP are based on standard PCR techniques, MDA uses isothermal reaction settings.

[0548] In some aspects, amplification involves initiating DNA or RNA synthesis using one or more oligonucleotide primers employed by a polymerase. Primers may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), DNA containing phosphate-thioester linkages, or combinations thereof, and may contain nucleotide analogs or modified nucleotides. Generally, primers are single-stranded oligonucleotides of length between about 10 and about 100 nucleotides, or between about 15 and about 30 nucleotides, or at least about 10, 15, or 20 and at most about 25, 30, 35, 40, 45, or 50 nucleotides. In some aspects, the oligonucleotide primers are specific primers that are complementary to certain regions of the target nucleotide sequence, such that the region of the template for amplification is defined by the primer. Methods for preparing oligonucleotide primers are known. In one aspect, commercially available amplification primers may be used. In one aspect, the primers are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0549] In one aspect, the sample contains PCR products. In one aspect, the length of the PCR products is between approximately 25 bp and approximately 500 bp, or between approximately 50 bp and approximately 300 bp, or between approximately 75 bp and approximately 200 bp. In one aspect, the PCR primers have a melting temperature similar to that of other primers used in multi-task PCR analysis (i.e., within approximately 5°C or 1°C).

[0550] [M. Detection]

[0551] In one aspect, a target analyte in an array is detected, identified, or quantified. In another aspect, reaction products in the array, including, for example, PCR reaction products, OLA reaction products, PEA reaction products, sandwich complexes, or NPA reaction products as described herein, can be detected, identified, or quantified. In one aspect, the array is a multi-task array, and the target analyte is detected, identified, or quantified by detecting a tag linked to an immobilized target molecule on the array. In one aspect, a carrier surface is contacted with a hybridization mixture containing tagged reaction products, and the reaction products are immobilized on the carrier surface by hybridization of a single-stranded oligonucleotide tag with its corresponding complementary capturing oligonucleotide. In one aspect, the reaction products are amplified before contacting the solid carrier with the reaction products. In one aspect, the amplified reaction products are at least about 10×, 20×, 30×, 40×, or 50×. In one aspect, the hybridization mixture contains a hybridization buffer. In one aspect, the presence or amount of the reaction products can be detected, identified, or quantified based on a tag linked to the reaction products. In one aspect, the carrier surface is washed with a washing buffer after the reaction products are immobilized on the carrier surface.

[0552] In one aspect, the presence, identification, or quantification of one or more target nucleotide sequences is based on the detection of a reaction product immobilized on a carrier surface. In one aspect, the presence of the immobilized reaction product is detected by monitoring light emitted from a label on the reaction product, including (but not limited to) fluorescence, time-varying fluorescence, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), luminescence, chemiluminescence, bioluminescence, phosphorescence, light scattering, or electrode-induced fluorescence. In another aspect, the label comprises an enzyme or other chemically reactive species having chemical activity that induces measurable signals, such as light scattering, absorbance, fluorescence, etc. Examples of enzyme labels include (but are not limited to) horseradish peroxidase or alkaline phosphatase. In one aspect, the label is a detectable hapten, including (but not limited to) biotin, luciferin, or digoxigenin. In one aspect, the reaction product contains a biotin label.

[0553] In one aspect, a reaction product is immobilized on one or more binding domains located on a support surface. In another aspect, one or more binding domains are positioned on one or more electrodes, and detection, identification, or quantification includes applying a voltage waveform to the one or more electrodes to stimulate a label on the captured reaction product to generate an electrochemical or luminescent signal. In one aspect, detection, identification, or quantification includes measuring the electrochemiluminescent signal and correlating the signal with the presence or amount of a target nucleotide sequence in a sample. In one aspect, the intensity of the emitted light is proportional to the amount of the target in the sample, such that the emitted light can provide a quantification of the amount of the target nucleotide in the sample.

[0554] In one aspect, after the reaction product is immobilized on a support surface, the support surface is brought into contact with the detection mixture. In one aspect, the detection mixture comprises an electrochemiluminescent label. Examples of electrochemiluminescent labels include: i) organometallic compounds, wherein the metal is derived from, for example, a noble metal of Group VIII, comprising Ru- and Os-containing organometallic compounds, such as the terpyridylruthenium (RuBpy) moiety; and ii) lumens and related compounds. In one aspect, the detection mixture further comprises one or more electrochemiluminescent co-reactants and one or more additional components, such as pH buffers, detergents, preservatives, defoamers, salts, metal ions, or metal chelators. The term "electrochemiluminescent co-reactant" refers to species that participate in electrochemiluminescent labeling and comprise (but are not limited to) tertiary amines, such as tripropylamine (TPA), oxalate ions, ascorbic acid and persulfate (for RuBpy), and hydrogen peroxide (for lumens). The method for measuring electrochemiluminescence is known, and the instrument used to perform the measurement is commercially available. For example, multi-task measurements of analytes using electrochemiluminescence are used in Meso Scale Diagnostics, LLC, MULTI-ARRAY®, and SECTOR® Imager lines or products (see, for example, U.S. Patent Nos. 7,842,246 and 6,977,722, the disclosures of which are incorporated herein by reference in their entirety).

[0555] In one aspect, biotin is covalently linked to the reaction product, and the detection mixture contains a tag bound to antibiotic streptavidin, said tag being bound to the immobilized reaction product via an avidin protein moiety. In one aspect, the antibiotic streptavidin-bound tag is an electrochemiluminescent (ECL) tag. In one aspect, the electrochemiluminescent tag is an n-hydroxysuccinimide ester, such as Sulfo-TAG NHS-ester (Meso Scale Diagnostics).

[0556] In one aspect, a 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 aspect, the presence of the SNP of interest is detected by determining the ratio between wild-type and variant alleles in a sample. In one aspect, the ratio is determined by determining the ratio of detectable markers of wild-type and variant alleles present in a sample. In one aspect, the ratio of electrochemiluminescent markers of wild-type and variant alleles is determined. The following formula can be used to determine the ratio of wild-type or variant alleles present in a...

Claims

1. A collection of two or more non-cross-reactive capturing oligonucleotides, wherein the collection of capturing oligonucleotides is a subset of a parental collection of capturing oligonucleotides, and one or more capturing oligonucleotides in the collection 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 having one or more nucleotide sequences from the parental collection, wherein the parental collection of capturing oligonucleotides is selected from the following: (a) Capture set 1, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 1-64; (b) Capture set 2, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 65-122; (c) Capture set 3, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 123-186; (d) Capture set 4, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 187-250; (e) Capture set 5, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 251-308; (f) Capture set 6, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 309-372; (g) Capture set 7, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 373-436; (h) Capture set 8, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 437-494; (i) Capture set 9, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 495-558; (j) Capture set 10, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 559-662; (k) Capture set 11, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 623-680; and (l) Capture set 12, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 681-744.

2. A collection of two or more non-cross-reactive capturing oligonucleotides, wherein the collection of capturing oligonucleotides is a subset of a parental collection of capturing oligonucleotides, and one or more capturing oligonucleotides in the collection comprise a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more nucleotide sequences from the parental collection, wherein the parental collection of capturing oligonucleotides is selected from the following: (a) Capture set 1, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 1-64; (b) Capture set 2, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 65-122; (c) Capture set 3, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 123-186; (d) Capture set 4, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 187-250; (e) Capture set 5, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 251-308; (f) Capture set 6, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 309-372; (g) Capture set 7, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 373-436; (h) Capture set 8, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 437-494; (i) Capture set 9, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 495-558; (j) Capture set 10, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 559-662; (k) Capture set 11, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 623-680; and (l) Capture set 12, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 681-744.

3. A set of two or more non-cross-reactive capturing oligonucleotides, wherein the set of capturing oligonucleotides is a subset of a parent set of capturing oligonucleotides, wherein the parent set of capturing oligonucleotides is selected from the following: (a) Capture set 1, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 1-64; (b) Capture set 2, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 65-122; (c) Capture set 3, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 123-186; (d) Capture set 4, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 187-250; (e) Capture set 5, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 251-308; (f) Capture set 6, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 309-372; (g) Capture set 7, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 373-436; (h) Capture set 8, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 437-494; (i) Capture set 9, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 495-558; (j) Capture set 10, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 559-662; (k) Capture set 11, comprising capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 623-680; and (l) Capture set 12, which includes capture oligonucleotides having nucleotide sequences selected from SEQ ID No: 681-744.

4. A collection of two or more non-cross-reactive capturing oligonucleotides according to any one of claims 1 to 3, wherein one or more capturing oligonucleotides in the collection are selected from: (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 selected from the sequence of SEQ ID No: 1-64; (b) Capture oligonucleotides, including sequences having at least 95%, 96%, 97%, 98%, 99% or 100% identity with sequences selected from SEQ ID No: 1-64; (c) 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 having at least 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID No: 1-64; (d) Capture oligonucleotides, including sequences selected from SEQ ID No: 1-64; and (e) Capture oligonucleotides, which are selected from any one of (a)-(d).

5. A collection of two or more non-cross-reactive capturing oligonucleotides according to any one of claims 1 to 3, wherein one or more capturing oligonucleotides in the collection are selected from: (a) Capture oligonucleotides 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 selected from the sequences of SEQ ID No: 1-10; (b) Capture oligonucleotides, including sequences having at least 95%, 96%, 97%, 98%, 99% or 100% identity with sequences selected from SEQ ID No: 1-10; (c) 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 having at least 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID No: 1-10; (d) Capture oligonucleotides, including sequences selected from SEQ ID No: 1-10; and (e) Capture oligonucleotides, which are selected from any one of (a)-(d).

6. A collection of two or more non-cross-reactive capturing oligonucleotides according to any one of claims 1 to 5, wherein one or more capturing oligonucleotides in the collection comprise reactive functional groups.

7. The collection of two or more non-cross-reactive capturing oligonucleotides according to claim 6, wherein the reactive functional group is linked to the capturing oligonucleotide via a linker.

8. The collection of two or more non-cross-reactive trapping oligonucleotides according to claim 6 or 7, wherein the reactive functional group comprises a thiol group.

9. A collection of two or more non-cross-reactive capture oligonucleotides according to any one of claims 6 to 8, wherein the oligonucleotides are immobilized on the surface by the reactive functional groups.

10. The collection of two or more non-cross-reactive capture oligonucleotides according to claim 9, wherein the surface includes an electrode surface.

Images