Detection of target molecules in a dried biological matrix

Abstract

A method is provided for detecting target molecules in a dried biological matrix in a proteomics-based assay. Such a method has broad utility in proteomic applications in research and development, diagnostics, and therapeutics by providing a method for preparing a dried biological matrix for the detection of target molecules in a proteomic assay, and a method for detecting one or more target molecules from a dried biological matrix in a proteomic assay.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 525,341 filed on 6 July 2023 and U.S. Provisional Patent Application No. 63 / 530,113 filed on 1 August 2023, each of which is incorporated herein by reference in whole for all purposes.

[0002] This disclosure generally relates to the field of proteomic assays and methods for detecting target molecules in dried biological matrices. Such methods have broad utility in proteomics applications for research and development, diagnosis, and therapy. Specifically, methods for preparing dried biological matrices for the detection of target molecules in proteomic assays and methods for detecting target molecules from dried biological matrices in proteomic assays are provided. [Background technology]

[0003] Assays targeting the detection and quantification of physiologically important molecules in biological and other sample types are essential tools in scientific research and healthcare. For example, multiplex array assays use surface-bound probes to detect target molecules in a sample. These surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies, aphibodies, aptamers, or other molecules (collectively, biopolymers) that can bind to the target molecule in the sample. These binding interactions form the basis of many methods and devices used in various fields, such as genomics, transcriptomics, and proteomics.

[0004] Assays using solution-based samples typically require maintaining the sample at -80°C prior to the assay, including during transport and storage. In addition, frozen solution-based samples containing cells, such as whole blood, may experience cell lysis during freeze-thaw sample processing, resulting in samples with insufficient cell homogenization. Improper storage of solution-based samples and incomplete cell homogenization within the solution can lead to pre-analysis variations in target molecules within the solution-based samples.

[0005] This disclosure describes a method for eliminating the need to transport and store samples at -80°C before assay. This disclosure describes a method for eliminating or reducing incomplete homogenization of cells in solution. This disclosure describes a method for detecting target molecules in samples extracted from a dried biological matrix. [Overview of the project]

[0006] The aforementioned and other purposes, features, and advantages of this disclosure will become even clearer from the following detailed description, which will proceed with reference to the attached drawings.

[0007] A particular non-exclusive exemplary embodiment is as follows:

[0008] In some embodiments, a method for preparing a biological sample for a multiplex assay is disclosed. This method includes depositing a biological sample containing multiple target molecules onto a collection device, and drying the biological sample on the collection device for a certain period of time to stabilize the dry sample before any temperature fluctuations, wherein the biological sample is dried at or below room temperature for at least 4 hours so that the target molecules in the dried biological sample are detectable by the multiplex assay.

[0009] In some embodiments, the biological sample is dried at or near room temperature, 4°C–8°C, or -20°C. In some embodiments, the dried biological sample is stored at or below room temperature before detection by multiplex assay. In some embodiments, the biological sample is stored at approximately 4°C–8°C, or -20°C.

[0010] In some embodiments, the biological sample is dried for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, 1 day, at least 2 days, or at least 3 days.

[0011] In some embodiments, the biological sample is selected from plasma, serum, urine, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, sputum, tears, mucus, nasal lavage fluid, nasal aspirate, semen, saliva, peritoneal lavage fluid, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph, nipple aspirate, bronchial aspirate, bronchial swab, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid. In some embodiments, the biological sample is selected from plasma, serum, urine, and whole blood. In some embodiments, the multiple target molecules are selected from proteins, peptides, carbohydrates, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, bacteria, metabolites, cofactors, inhibitors, drugs, pigments, nutrients, growth factors, cells, and tissues.

[0012] In some embodiments, the dried biological sample is homogenized by the drying process. In some embodiments, multiple target molecules are extractable from the collection device after the biological sample has dried and are detectable in a multiplex assay.

[0013] In some embodiments, a method for detecting multiple target molecules is disclosed. The method involves extracting target molecules from a dry biological sample from a collection device; diluting the extracted target molecules into a first diluent and a second diluent; if the target molecules are present in the first diluent, contacting the first diluent with a first capture reagent to form a first capture reagent affinity complex with the target molecules; if the target molecules are present in the second diluent, contacting the second diluent with a second capture reagent to form a second capture reagent affinity complex with the target molecules; and incubating the first and second diluted samples separately to enable the formation of the capture reagent affinity complexes; where the first capture Each of the reagent affinity complex and the second capture reagent affinity complex is immobilized on a separate first solid support, and the method includes enabling the formation of a capture reagent affinity complex, releasing the first capture reagent affinity complex to capture on the second solid support, releasing the first capture reagent affinity complex and then releasing the second capture reagent affinity complex to capture on the second solid support, and detecting the presence or level of the first or second capture reagent affinity complex, or detecting the presence or amount of the first or second capture reagent affinity complex.

[0014] In some embodiments, the target molecule is extracted from the collection device in the formulation for at least 5 minutes. In some embodiments, the target molecule is extracted from the collection device in the formulation for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, at least 150 minutes, at least 160 minutes, at least 170 minutes, at least 180 minutes, at least 190 minutes, or at least 200 minutes.

[0015] In some embodiments, the formulation comprises a buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide.

[0016] In some embodiments, one or more salts are independently selected from sodium salts, potassium salts, and magnesium salts.

[0017] In some embodiments, one or more salts include a sodium salt, a potassium salt, and a magnesium salt. In some embodiments, the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2. In some embodiments, the concentration of NaCl in the formulation is about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 75 to 125 mM, or about 100 mM. In some embodiments, the concentration of KCl is about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM. In some embodiments, the concentration of MgCl2 is about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 8 mM.

[0018] In some embodiments, the buffer is selected from HEPES, IVIES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, coramine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS. In some embodiments, the buffer in the formulation is concentrated at a level of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 60 mM, or about 50 mM.

[0019] In some embodiments, the chelating agent is selected from EGTA, EDTA, DTPA, BAPTA, DMPS, and ALA. In some embodiments, the chelating agent in the formulation is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.25 mM.

[0020] In some embodiments, the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80). In some embodiments, the nonionic surfactant is, by volume to volume, about 0.1% to about 5% of the formulation, or about 0.2% to about 4% of the formulation, or about 0.3% to about 3% of the formulation, or about 0.4% to about 2% of the formulation, or about 0.5% of the formulation, or about 1.5% of the formulation, or about 1.2% of the formulation.

[0021] In some embodiments, the pH of the formulation is about pH 5 to about pH 9, or about pH 6 to about pH 8, or about pH 7 to about pH 7.9, or about pH 7.5.

[0022] In some embodiments, the formulation comprises 50 mM HEPES, 100 mM NaCl, 5 mM KCl, 8 mM MgCl2, 1.25 mM EGTA, and 1.2% Tween-20.

[0023] In some embodiments, the formulation has a pH of about 7.5.

[0024] In some embodiments, the protease inhibitor is a reversible protease inhibitor. In some embodiments, the protease inhibitor inhibits a protease selected from trypsin, plasmin, and thrombin. In some embodiments, the protease inhibitor is a serine protease inhibitor. In some embodiments, the protease inhibitor is benzamidine. In some embodiments, the protease inhibitor in the formulation is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.2 mM.

[0025] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is 20 to 100 nucleotides long, or 25 to 80 nucleotides long, or 25 to 70 nucleotides long, or 25 to 50 nucleotides long, or about 30 nucleotides long. In some embodiments, the oligonucleotide contains one or more modified nucleotides. In some embodiments, the oligonucleotide contains one or more C-5 modified pyrimidines. In some embodiments, the oligonucleotide comprises the sequence [(ACXX)7-AC], where X is BndU. In some embodiments, the oligonucleotide in the formulation has a concentration of 5 μM to 100 μM, or 10 μM to 80 μM, or 20 μM to 60 μM, or 30 μM to 50 μM, or about 75 μM, or about 37 μM.

[0026] In some embodiments, the biological sample is dried at or below room temperature for at least 4 hours. In some embodiments, the biological sample is dried at or near room temperature, 4°C to 8°C, or -20°C. In some embodiments, the dried biological sample is stored at or below room temperature before detection. In some embodiments, the biological sample is stored at approximately 4°C to 8°C, or -20°C before detection.

[0027] In some embodiments, the biological sample is dried for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, at least 1 day, at least 2 days, or at least 3 days.

[0028] In some embodiments, the biological sample is selected from plasma, serum, urine, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, sputum, tears, mucus, nasal lavage fluid, nasal aspirate, semen, saliva, peritoneal lavage fluid, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph, nipple aspirate, bronchial aspirate, bronchial swab, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid. In some embodiments, the biological sample is selected from plasma, serum, urine, and whole blood. In some embodiments, the multiple target molecules are selected from proteins, peptides, carbohydrates, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, bacteria, metabolites, cofactors, inhibitors, drugs, pigments, nutrients, growth factors, cells, and tissues.

[0029] In some embodiments, the dried biological sample is homogenized by a drying process.

[0030] In some embodiments, the first and second capture reagent-target molecule affinity complexes are non-covalent complexes.

[0031] In some embodiments, the first diluent is a 0.001% to 0.1% diluent of the elution target molecule sample, and the second diluent is a 0.1% to 10% diluent of the elution target molecule sample.

[0032] In some embodiments, the first diluent is a diluent of the test sample at a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second diluent is at a concentration of 0.01% to 1% ( It is a diluent of the test sample (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.1% to 0.8%, or 0.2% to 0.75%, or about 0.5%. In some embodiments, the first diluent is a diluent of the test sample at a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%; the second diluent is at a concentration of 5% to 39% (or 5%). Dilution of the test sample (which is %, 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%, or 39%), or 15% to 30%, or 15% to 25%, or about 20%.In some embodiments, the first diluent is a diluent of the test sample at a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.1% to 0.8%, or 0.2% to 0.75%, or about 0.5% The first diluent is a 5% to 39% (or 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%, or 39%) diluent of the test sample, or 15% to 30%, or 15% to 25%, or about 20%. In some embodiments, the first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.1% to 0.8%. , or 0.2% to 0.75%, or about 0.5%; the second diluent is a diluent of the test sample of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.In some embodiments, the first diluent is a diluent of the test sample at a concentration of 5% to 39% (or 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%, or 39%), or 15% to 30%, or 15% to 25%, or about 20% The first diluent is a 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%) diluent of the test sample, or 0.1% to 0.8%, or 0.2% to 0.75%, or approximately 0.5%. In some embodiments, the first diluent is a diluent of the test sample at a concentration of 5% to 39% (or 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%, or 39%), or 15% to 3 The first diluent is 0%, or 15% to 25%, or about 20%, and the second diluent is a diluent of the test sample at a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

[0033] In some embodiments, the first diluent is a diluent of the test sample at a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second diluent is at a concentration of 0.01% to 1% (or It is a diluent of the test sample (which is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%, or 0.02% to 0.1%, or approximately 0.05%. In some embodiments, the first diluent is a diluent of the test sample at a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second The diluent is a diluent of the test sample at a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or approximately 2.5%.In some embodiments, the first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%, or 0.0 The first diluent is 2% to 0.1%, or about 0.05%, and the second diluent is a diluent of the test sample at 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%. In some embodiments, the first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%. The first diluent is 0.02% to 0.1%, or about 0.05%, and the second diluent is a diluent of the test sample at a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.In some embodiments, the first diluent is a diluent of the test sample in a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%. The first diluent is 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%) of the test sample, or 0.002% to 0.008%, or 0.003% to 0.007%, or approximately 0.005%. In some embodiments, the first diluent is a diluent of the test sample at a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%, and the second diluent is 0.01 A diluent of the test sample in a concentration of % to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%, or 0.02% to 0.1%, or approximately 0.05%.

[0034] In some embodiments, the method further comprises contacting a third diluted sample with a third capture reagent, wherein if a target molecule is present in the third diluted sample, the interaction of the third capture reagent with its target molecule forms a third capture reagent affinity complex, and the third diluted sample is incubated separately from the first and second diluted samples to allow the formation of a third aptamer with its target molecule. In some embodiments, the method further comprises releasing a second capture reagent affinity complex, followed by releasing a third capture reagent affinity complex and capturing it on a second solid support. In some embodiments, the method further comprises detecting the presence of the third capture reagent in the third capture reagent affinity complex, or determining its level, or detecting the presence or amount of the third capture reagent affinity complex.

[0035] In some embodiments, the third diluent is a different diluent from the first and / or second diluents of the same eluting target molecule sample. In some embodiments, the third diluent is a 0.001% to 0.1% diluent of the eluting target molecule sample. In some embodiments, the third diluent is a 0.001% to 40% diluent of the eluting target molecule sample.

[0036] In some embodiments, the third diluent is 5% to 39% (or 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%, or 39%), 15% to 30%, 15% to 25%, about 20%; 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0. These are dilutions of test samples at concentrations of 0.8%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), 0.1% to 0.8%, 0.2% to 0.75%, approximately 0.5%; and 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, 0.003% to 0.007%, approximately 0.005%.In some embodiments, the third diluent is a diluent of the test sample at a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%; or the third diluent is 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%). The third diluent is 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or approximately 0.005%), or 0.002% to 0.008%, or 0.003% to 0.007%, or approximately 0.005%; or the third diluent is 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or approximately 0.005%), or 0.002% to 0.008%, or 0.003% to 0.007%, or approximately 0.005%.

[0037] In some embodiments, the first and second capture reagents are independently selected from aptamers or antibodies. In some embodiments, the third capture reagent is selected from aptamers or antibodies. In some embodiments, each of the first, second, and third capture reagents is an aptamer. In some embodiments, each aptamer independently comprises at least one 5-modified pyrimidine. In some embodiments, the at least one 5-modified pyrimidine comprises a linker at the 5-position of the pyrimidine and a moiety bound to the linker.

[0038] In some embodiments, the linker is selected from amide linkers, carbonyl linkers, propynyl linkers, alkyne linkers, ester linkers, urea linkers, carbamate linkers, guanidine linkers, amidine linkers, sulfoxide linkers, and sulfone linkers.

[0039] In some embodiments, the portion is a hydrophobic portion. In some embodiments, the portion is selected from naphthyl, benzyl, fluorobenzyl, tyrosyl, indole, morpholino, isobutyl, 3,4-methylenedioxybenzyl, benzothiophenyl, benzofuranyl, phenylbenzyl, 4-phenoxybenzyl, diphenylpropyl, and benzhydryl portions. In some embodiments, the pyrimidine of the 5-position modified pyrimidine is uridine, cytidine, or thymidine.

[0040] In some embodiments, the presence of dissociated first and second capture reagents, or the determination of their levels, is performed by PCR, mass spectrometry, nucleic acid sequencing, next-generation sequencing (NGS), or hybridization.

[0041] In some embodiments, a method for preparing a liquid sample is provided. The method includes drying the sample at a constant temperature of -20°C to room temperature for at least 4 hours to produce a dry sample, and reconstituting the dry sample with a formulation comprising a buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide.

[0042] In some embodiments, one or more salts are independently selected from sodium salts, potassium salts, and magnesium salts.

[0043] In some embodiments, one or more salts include a sodium salt, a potassium salt, and a magnesium salt. In some embodiments, the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2. In some embodiments, the concentration of NaCl in the formulation is about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 75 to 125 mM, or about 100 mM. In some embodiments, the concentration of KCl is about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM. In some embodiments, the concentration of MgCl2 is about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 8 mM.

[0044] In some embodiments, the buffer is selected from HEPES, IVIES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, coramine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS. In some embodiments, the buffer in the formulation is concentrated at a level of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 60 mM, or about 50 mM.

[0045] In some embodiments, the chelating agent is selected from EGTA, EDTA, DTPA, BAPTA, DMPS, and ALA. In some embodiments, the concentration of the chelating agent in the formulation is about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.25 mM.

[0046] In some embodiments, the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80). In some embodiments, the nonionic surfactant is present in volume-to-volume amounts of about 0.1% to about 5% of the formulation, or about 0.2% to about 4% of the formulation, or about 0.3% to about 3% of the formulation, or about 0.4% to about 2% of the formulation, or about 0.5% of the formulation, or about 1.5% of the formulation, or about 1.2% of the formulation.

[0047] In some embodiments, the pH of the formulation is approximately pH 5 to approximately pH 9, or approximately pH 6 to approximately pH 8, or approximately pH 7 to approximately pH 7.9, or approximately pH 7.5.

[0048] In some embodiments, the formulation comprises 50 mM HEPES, 100 mM NaCl, 5 mM KCl, 8 mM MgCl2, 1.25 mM EGTA, and 1.2% Tween-20.

[0049] In some embodiments, the formulation has a pH of about 7.5.

[0050] In some embodiments, the protease inhibitor is a reversible protease inhibitor. In some embodiments, the protease inhibitor inhibits a protease selected from trypsin, plasmin, and thrombin. In some embodiments, the protease inhibitor is a serine protease inhibitor. In some embodiments, the protease inhibitor is benzamidine. In some embodiments, the protease inhibitor in the formulation is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.2 mM.

[0051] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is 20 to 100 nucleotides long, or 25 to 80 nucleotides long, or 25 to 70 nucleotides long, or 25 to 50 nucleotides long, or about 30 nucleotides long. In some embodiments, the oligonucleotide contains one or more modified nucleotides. In some embodiments, the oligonucleotide contains one or more C-5 modified pyrimidines. In some embodiments, the oligonucleotide comprises the sequence [(ACXX)7-AC], where X is BndU. In some embodiments, the oligonucleotide in the formulation has a concentration of 5 μM to 100 μM, or 10 μM to 80 μM, or 20 μM to 60 μM, or 30 μM to 50 μM, or about 75 μM, or about 37 μM.

[0052] In some embodiments, the sample is dried at a constant temperature of approximately 4°C to approximately 8°C. In some embodiments, the sample is dried for approximately 4 hours to approximately 48 hours.

[0053] In some embodiments, the sample is diluted in a first diluent and a second diluent, the first diluent being a diluent of the test sample at a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second diluent being , a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.1% to 0.8%, or 0.2% to 0.75%, or approximately 0.5%. In some embodiments, the sample is diluted in a first diluent and a second diluent, the first diluent being a 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%) diluent of the test sample, or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%; the second diluent being 5 Dilution of a test sample of %~39% (or 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%, or 39%), or 15%~30%, or 15%~25%, or about 20%.In some embodiments, the sample is diluted in a first diluent and a second diluent, the first diluent being a 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%) diluent of the test sample, or 0.1% to 0.8%, or 0.2% to 0.7%. The first diluent is 5%, or about 0.5%; the second diluent is a 5%–39% (or 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%, or 39%) diluent of the test sample, or 15%–30%, or 15%–25%, or about 20%. In some embodiments, the sample is diluted in a first diluent and a second diluent, the first diluent being a diluent of the test sample at a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or The first diluent is 0.1% to 0.8%, or 0.2% to 0.75%, or about 0.5%; the second diluent is a diluent of the test sample in a 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%) concentration, or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.In some embodiments, the sample is diluted in a first diluent and a second diluent, the first diluent being a 5% to 39% (or 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%, or 39%) diluent of the test sample, or 15% to 30%, or 15% The first diluent is ~25%, or about 20%, and the second diluent is a 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%) diluent of the test sample, or 0.1% to 0.8%, or 0.2% to 0.75%, or about 0.5%. In some embodiments, the sample is diluted in a first diluent and a second diluent, the first diluent being a diluent of the test sample at concentrations of 5% to 39% (or 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%, or 39%). The first diluent is 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%) of the test sample, or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.In some embodiments, the sample is diluted in a third diluent, the third diluent being 5% to 39% (or 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%, or 39%), 15% to 30%, 15% to 25%, approximately 20%, 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%). This is a diluent for a test sample selected from %, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), 0.1%~0.8%, 0.2%~0.75%, approximately 0.5%, and 0.001%~0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002%~0.008%, 0.003%~0.007%, or approximately 0.005%.

[0054] In some embodiments, the first diluent is a diluent of the test sample at a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second diluent is at a concentration of 0.01% to 1% (or It is a diluent of the test sample (which is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%, or 0.02% to 0.1%, or approximately 0.05%. In some embodiments, the first diluent is a diluent of the test sample at a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second The diluent is a diluent of the test sample at a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or approximately 2.5%.In some embodiments, the first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%, or 0.0 The first diluent is 2% to 0.1%, or about 0.05%, and the second diluent is a diluent of the test sample at 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%. In some embodiments, the first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%. The first diluent is 0.02% to 0.1%, or about 0.05%, and the second diluent is a diluent of the test sample at a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.In some embodiments, the first diluent is a diluent of the test sample in a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%. The first diluent is 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%) of the test sample, or 0.002% to 0.008%, or 0.003% to 0.007%, or approximately 0.005%. In some embodiments, the first diluent is a diluent of the test sample at a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%, and the second diluent is 0.01 A diluent of the test sample in a concentration of % to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%, or 0.02% to 0.1%, or approximately 0.05%.

[0055] In some embodiments, compositions are provided. The compositions comprise a dry sample and a formulation comprising a buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide, wherein the dry sample was obtained from a liquid sample that was dried at a constant temperature of about -20°C to room temperature for at least 4 hours.

[0056] In some embodiments, a method is provided for detecting an analyte in a sample. The method includes drying the sample at a constant temperature of about -20°C to room temperature for at least 4 hours to produce a dried sample, reconstituting the dried sample with a formulation comprising a buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide, and detecting the analyte from the reconstituted sample.

[0057] In some embodiments, detection is performed using a protein-binding reagent or a mass spectrometer.

[0058] In some embodiments, the protein-binding reagent is selected from aptamers or antibodies.

[0059] In some embodiments, detection is performed using a multiplex assay. In some embodiments, the multiplex assay detects at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 of the analytes.

[0060] The aforementioned and other purposes, features, and advantages of this disclosure will become even clearer from the following detailed description, which will proceed with reference to the attached drawings. [Brief explanation of the drawing]

[0061] [Figure 1] The linear range for dilution group 1 is shown. The x-axis represents the concentration (%) of the DBS extract on a log10 scale (from 20% to 0), and the y-axis counts the number of analytes based on the linear signal response at that dilution. [Figure 2]A and B represent the linear ranges for dilution group 2(A) and dilution group 3(B), respectively. The y-axis represents the number of analytes within the linear range. The x-axis represents the concentration (%) of the DBS extract on a log10 scale. [Figure 3A] For dilution group 1, the analytes with signals above the background are shown. [Figure 3B] For dilution group 2, the analytes with signals above the background are shown. [Figure 3C] For dilution group 3, the analytes with signals above the background are shown. [Figure 4A] The collective coefficient of variation for all analytes tested is shown. [Figure 4B] The collective coefficient of variation for all analytes tested is shown. [Figure 5A] This shows a concurring plot of median (n=3) relative fluorescence units (RFU) between a standard serum sample and the same sample after staining and extraction using water as a volume-expanding agent. [Figure 5B] This shows a matched plot of median (n=3) relative fluorescence units (RFU) between a standard serum sample and the same sample after staining and extraction using PBS as a volume-expanding agent. [Figure 5C] This shows a concurring plot of median (n=3) relative fluorescence units (RFU) when comparing PBS with water. [Figure 6] A and B show the analyte recovery rates over different extraction times in two different sample collections. [Figure 7A] This shows the mean absolute error of signals from multiplex assays performed on dried plasma at 4°C and under temperature stress, compared to liquid plasma. [Figure 7B] This shows the mean absolute error of signals from multiplex assays performed on dried plasma at room temperature and under temperature stress, compared to liquid plasma. [Figure 7C] This shows the mean absolute error of signals from multiplex assays performed on dried plasma at -20°C and under temperature stress, compared to liquid plasma. [Figure 8A]We compare the concordance rate between frozen plasma and dried blood spots. [Figure 8B] Compare the concordance rate between frozen serum and dried blood spots. [Figure 8C] We will compare the concordance rate between frozen serum and frozen plasma. [Figure 8D] We compare the concordance rates between two pooled DBS samples. [Figure 9] This shows the correlation between DBS and plasma / serum protein measurements. [Figure 10] A and B show significant correlations from paired observations mapped to coincident plots. A shows correlations from paired observations, and B shows correlations using a 5% FDR. [Figure 11] Figures 10A and 10B show significant overlap in analytes between the two methods. [Figure 12A] This demonstrates analytes that show a significant correlation between DBS and plasma. [Figure 12B] This demonstrates analytes that show a significant correlation between DBS and plasma. [Figure 12C] This demonstrates analytes that show a significant correlation between DBS and plasma. [Figure 12D] This demonstrates analytes that show a significant correlation between DBS and plasma. [Figure 13A] A significant correlation between DBS and plasma is shown when mapped onto a coincident serum-versus-plasma plot. [Figure 13B] A significant correlation between DBS and plasma is shown when mapped onto a coincident serum-versus-plasma plot. [Figure 13C] A significant correlation between DBS and plasma is shown when mapped onto a coincident serum-versus-plasma plot. [Figure 13D] A significant correlation between DBS and plasma is shown when mapped onto a coincident serum-versus-plasma plot. [Figure 14] This shows the classification of analytes based on their biological function. [Figure 15] The CDF plot shows 2,941,335 random correlations between plasma and serum. [Figure 16] This shows the probability distribution of false positives based on the Pearson cutoff. [Figure 17] An example of finding the weighted average of false positives is shown. [Figure 18] The plot shows significant correlation versus spurious correlation. [Figure 19] The plot of the mean FDR at a given Pearson cutoff is shown. [Figure 20] This shows a plot of the number of significant correlations as a function of FDR. [Figure 21] The following are some specific exemplary 5-position modified uridines and cytidines that may be incorporated into aptamers. [Figure 22] The following shows certain exemplary modifications that may be present at the 5-position of uridine. The chemical structures of the C-5 modifications include an exemplary amide bond linking the modification to the 5-position of uridine. The 5-position moieties shown contain two phenyl groups covalently bonded to each other. Examples of the 5-position moieties shown include phenylbenzyl moieties (e.g., BPE, PBnd, DBM), 4-phenoxybenzyl moieties (e.g., POP), diphenylpropyl moieties (e.g., DPP), and benzhydryl moieties (e.g., BH). [Figure 23] The following shows certain exemplary modifications that may be present at the 5-position of cytidine. The chemical structures of the C-5 modifications include an exemplary amide bond linking the modification to the 5-position of cytidine. The 5-position moieties shown contain two phenyl groups covalently bonded to each other. Examples of the 5-position moieties shown include phenylbenzyl moieties (e.g., BPE, PBnd, DBM), 4-phenoxybenzyl moieties (e.g., POP), diphenylpropyl moieties (e.g., DPP), and benzhydryl moieties (e.g., BH). [Figure 24]Exemplary modifications that may be present at the 5-position of uridine are shown. The chemical structures of the C-5 modifications include an exemplary amide bond linking the modification to the 5-position of uridine. Examples of 5-position moieties shown include benzyl moieties (e.g., Bn, PE, and PP), naphthyl moieties (e.g., Nap, 2Nap, NE), butyl moieties (e.g., iBu), fluorobenzyl moieties (e.g., FBn), tyrosyl moieties (e.g., Tyr), 3,4-methylenedioxybenzyl (e.g., MBn), morpholino moieties (e.g., MOE), benzofuranyl moieties (e.g., BF), indole moieties (e.g., Trp), and hydroxypropyl moieties (e.g., Thr). [Figure 25] The following shows certain exemplary modifications that may be present at the 5-position of cytidine. The chemical structures of the C-5 modifications include exemplary amide bonds linking the modification to the 5-position of cytidine. Examples of 5-position moieties shown include benzyl moieties (e.g., Bn, PE, and PP), naphthyl moieties (e.g., Nap, 2Nap, NE, and 2NE), and tyrosyl moieties (e.g., Tyr). [Modes for carrying out the invention]

[0062] Unless otherwise specified, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may also be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9), Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0063] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. The singular terms “a,” “an,” and “the” may be used interchangeably with the plural forms, “at least one” and “one or more,” unless otherwise clearly indicated by the context. “A or B” means A, or B, or A and B. It should be further understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for illustrative purposes only.

[0064] Furthermore, it should be understood that the ranges provided herein are abbreviated representations of all values ​​within that range. For example, the range 1 to 50 should be understood to include any number, combination of numbers, or subrange (and fractions thereof, unless otherwise explicitly stated by the context) in the group consisting of 1, 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, or 50. Any concentration range, percentage range, ratio range, or integer range should be understood to include any integer within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated. Similarly, any numerical ranges listed herein relating to any physical characteristics, such as polymer subunits, size, or thickness, should be understood to include any integer within the listed range, unless otherwise indicated. Where used herein, “approximately” or “essentially from” means ±20% of the indicated range, value, or structure, unless otherwise indicated.

[0065] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “contains,” and “containing,” and any variations thereof, are intended to encompass non-exclusive inclusions, thereby including, any process, method, product-by-process, or composition of substance that comprises, includes, or contains an element or set of elements, which may include other elements not expressly enumerated.

[0066] Similar or equivalent methods and materials may be used in the implementation or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including the definition of terms, shall prevail. In addition, the materials, methods, and examples are illustrative and not intended to limit the scope of this disclosure.

[0067] As used herein, the term “nucleotide” refers to ribonucleotides or deoxyribonucleotides, or modified forms thereof, and their analogues. Nucleotides include species such as purines (e.g., adenine, hypoxanthine, guanine, and their derivatives and analogues), as well as pyrimidines (e.g., cytosine, uracil, thymine, and their derivatives and analogues). As used herein, the term “cytidine” is generally used to refer to ribonucleotides, deoxyribonucleotides, or modified ribonucleotides containing a cytosine base, unless otherwise specifically indicated. The term “cytidine” includes 2'-modified cytidines, such as 2'-fluoro, 2'-methoxy, etc. Similarly, the term “modified cytidine” or a specific modified cytidine also refers to ribonucleotides, deoxyribonucleotides, or modified ribonucleotides (2'-fluoro, 2'-methoxy, etc.) containing a modified cytosine base, unless otherwise specifically indicated. The term "uridine" is generally used to refer to ribonucleotides, deoxyribonucleotides, or modified ribonucleotides containing a uracil base, unless otherwise specified. The term "uridine" includes 2'-modified uridines, such as 2'-fluoro and 2'-methoxy. Similarly, the term "modified uridine" or a specific modified uridine also refers to ribonucleotides, deoxyribonucleotides, or modified ribonucleotides (such as 2'-fluoro and 2'-methoxy) containing a modified uracil base, unless otherwise specified.

[0068] As used herein, the terms “C-5 modified carboxamide cytidine,” “cytidine-5-carboxamide,” “5-position modified cytidine,” or “C-5 modified cytidine” refer to cytidine with a carboxyamide (-C(O)NH-) modification at the C-5 position, for example, those portions (R) illustrated herein. X1This refers to cytidines having modifications including, but not limited to, 5-(N-benzylcarboxamide)-2'-deoxycytidine (referred to as "BndC" and shown in Figure 25), 5-(N-2-phenylethylcarboxamide)-2'-deoxycytidine (referred to as "PEdC" and shown in Figure 25), 5-(N-3-phenylpropylcarboxamide)-2'-deoxycytidine (referred to as "PPdC" and shown in Figure 25), 5-(N-1-naphthylmethylcarboxamide)-2'-deoxycytidine (referred to as "NapdC" and shown in Figure 22), 5 Examples include, but are not limited to, C5-modified cytidines (N-2-naphthylmethylcarboxamide)-2'-deoxycytidine (referred to as "2NapdC" and shown in Figure 25), 5-(N-1-naphthyl-2-ethylcarboxamide)-2'-deoxycytidine (referred to as "NEdC" and shown in Figure 25), 5-(N-2-naphthyl-2-ethylcarboxamide)-2'-deoxycytidine (referred to as "2NEdC" and shown in Figure 25), and 5-(N-tyrosylcarboxamide)-2'-deoxycytidine (referred to as "TyrdC" and shown in Figure 25). In some embodiments, C5-modified cytidines can be incorporated into oligonucleotides by polymerase (e.g., KOD DNA polymerase), for example, in their triphosphate form.

[0069] The chemical modifications of C-5 modified cytidine described herein may also be combined, either alone or in any combination, with 2' sugar modifications, modifications with extracyclic amines, and substitutions of 4-thiocytidine.

[0070] As used herein, the terms “C-5 modified carboxamide cytosine,” “cytosine-5-carboxamide,” “5-position modified cytosine,” or “C-5 modified cytosine” refer to cytosine with a carboxyamide (-C(O)NH-) modification at the C-5 position, for example, those portions (R) illustrated herein. X1This refers to cytosine bases having modifications including, but not limited to, C-5 modified carboxamide cytosines. Examples of C-5 modified carboxamide cytosines include, but are not limited to, the modified cytidine shown in Figure 25.

[0071] As used herein, the terms “C-5 modified uridine” or “5-position modified uridine” refer to uridine (typically deoxyuridine) having a carboxyamide (-C(O)NH-) modification at the C-5 position, as shown, for example, in Figure 21. In some embodiments, C5-modified uridines can be incorporated into oligonucleotides by polymerase (e.g., KOD DNA polymerase), for example, in their triphosphate form. Non-limiting and exemplary 5-position modified uridines include: 5-(N-benzylcarboxyamide)-2'-deoxyuridine (BndU), 5-(N-benzylcarboxamide)-2'-O-methyluridine, 5-(N-benzylcarboxamide)-2'-fluorouridine, 5-(N-phenethylcarboxamide)-2'-deoxyuridine (PEdU), 5-(N-thiophenylmethylcarboxamide)-2'-deoxyuridine (ThdU), 5-(N-isobutylcarboxyamide)-2'-deoxyuridine (iBudU), 5-(N-tyrosylcarboxamide)-2'-deoxyuridine (TyrdU), 5-(N-3,4-methylenedioxybenzylcarboxamide)-2'-deoxyuridine (MBndU), 5-(N-4-fluorobenzylcarboxyamide)-2'-deoxyuridine (FBndU), 5-(N-3-phenylpropylcarboxamide)-2'-deoxyuridine (PPdU), 5-(N-imidizolylethylcarboxamide)-2'-deoxyuridine (ImdU), 5-(N-isobutylcarboxamide)-2'-O-methyluridine, 5-(N-isobutylcarboxyamide)-2'-fluorouridine, 5-(N-tryptaminocarboxamide)-2'-deoxyuridine (TrpdU), 5-(NR-threoninylcarboxamide)-2'-deoxyuridine (ThrdU), 5-(N-tryptaminocarboxamide)-2'-O-methyluridine, 5-(N-tryptaminocarboxamide)-2'-fluorouridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamide)-2'-deoxyuridine chloride, 5-(N-naphthylmethylcarboxamide)-2'-deoxyuridine (NapdU), 5-(N-naphthylmethylcarboxamide)-2'-O-methyluridine, 5-(N-naphthylmethylcarboxamide)-2'-fluorouridine, 5-(N-[1-(2,3-dihydroxypropyl)]carboxamide)-2'-deoxyuridine), 5-(N-2-naphthylmethylcarboxamide)-2'-deoxyuridine (2NapdU), 5-(N-2-naphthylmethylcarboxamide)-2'-O-methyluridine, 5-(N-2-naphthylmethylcarboxamide)-2'-fluorouridine, 5-(N-1-naphthylethylcarboxamide)-2'-deoxyuridine (NEdU), 5-(N-1-naphthylethylcarboxamide)-2'-O-methyluridine, 5-(N-1-naphthylethylcarboxamide)-2'-fluorouridine, 5-(N-2-naphthylethylcarboxamide)-2'-deoxyuridine (2NEdU), 5-(N-2-naphthylethylcarboxamide)-2'-O-methyluridine, 5-(N-2-naphthylethylcarboxamide)-2'-fluorouridine, 5-(N-3-benzofuranylethylcarboxamide)-2'-deoxyuridine (BFdU), 5-(N-3-benzofuranylethylcarboxamide)-2'-O-methyluridine, 5-(N-3-benzofuranylethylcarboxamide)-2'-fluorouridine, 5-(N-3-benzothiophenylethylcarboxamide)-2'-deoxyuridine (BTdU), 5-(N-3-benzothiophenylethylcarboxamide)-2'-O-methyluridine, and 5-(N-3-benzothiophenylethylcarboxamide)-2'-fluorouridine. An additional C-5 modification can be found in WO / 2022 / 221241.

[0072] As used herein, the terms “modified,” “modified,” and any variant thereof, when used in relation to oligonucleotides, mean that at least one of the four constituent nucleotide bases (i.e., A, G, T / U, and C) of the oligonucleotide is an analogue or ester of a naturally occurring nucleotide. In some embodiments, the modified nucleotide confers nuclease resistance to the oligonucleotide. Additional modifications include skeletal modifications, methylation, and combinations of rare base pairings, such as isobases isocytidine and isoguanidine. Other modifications include 3' and 5' modifications such as capping. Other modifications include substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as modifications by uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and modifications by charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), modifications by intercalators (e.g., acridine, psoralens, etc.), modifications containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), modifications containing alkylating agents, and modifications by modified bonds (e.g., alpha-anomeric nucleic acids, etc.). Furthermore, any of the hydroxyl groups normally present on the sugar of a nucleotide may be replaced with a phosphonate group or a phosphate group, protected with a standard protecting group, or activated to prepare additional binding to additional nucleotides or to a solid support. The 5' and 3' terminal OH groups may be phosphorylated or replaced with amines, organic capping groups of about 1 to about 20 carbon atoms, polyethylene glycol (PEG) polymers in the range of about 10 to about 80 kDa in one embodiment, PEG polymers in the range of about 20 to about 60 kDa in another embodiment, or other hydrophilic or hydrophobic biopolymers or synthetic polymers.

[0073] As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide” are interchangeable terms used to refer to polymers of nucleotides, including DNA, RNA, DNA / RNA hybrids, and modified forms of these types of nucleic acids, oligonucleotides, and polynucleotides, which involve the binding of various entities or parts to nucleotide units at arbitrary positions. The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” include triple-helical molecules in addition to double-stranded or single-stranded molecules. Nucleic acid, oligonucleotide, and polynucleotide are broader terms than aptamer, and therefore the terms nucleic acid, oligonucleotide, and polynucleotide include polymers of nucleotides that are aptamers, but are not limited to aptamers.

[0074] Polynucleotides also contain analogous forms of ribose or deoxyribose sugars generally known in the art, such as 2'-O-methyl, 2'-O-allyl, 2'-O-ethyl, 2'-O-propyl, 2'-O-CH2CH2OCH3, 2'-fluoro, 2'-NH2 or 2'-azide, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars, such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs and debasic nucleoside analogs, such as methylriboside. As described herein, one or more phosphodiester bonds may be replaced with alternative linking groups. These alternative linking groups may include phosphate groups P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR X 2 ("Amidate"), P(O)R X , P(O)OR X ', replaced by CO or CH2 ("formacetal"), in the formula, each R X or R XEmbodiments include those in which ' is independently H, or optionally contains an ether (-O-) bond and is a substituted or unsubstituted alkyl (C1-C20), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl alkyl (C1-C20), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl alkyl group. Not all bonds in the polynucleotide need to be identical. Substitutions of sugars, purines, and pyrimidines in similar forms may be advantageous in designing the final product, as may alternative skeletal structures such as a polyamide skeleton.

[0075] Polynucleotides may also contain analogues of carbocyclic sugars, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogues, and debasic nucleoside analogues such as methyl riboside.

[0076] If present, modifications to the nucleotide structure may be introduced before or after polymer assembly. The nucleotide sequence may be interposed by non-nucleotide components. After polymerization, polynucleotides may be further modified, for example, by complexing with labeling components.

[0077] As used herein, the term “at least one nucleotide” means, when referring to the modification of a nucleic acid, one, some, or all of the nucleotides in the nucleic acid, indicating that the presence of any or all of A, C, T, G, or U in the nucleic acid may or may not be modified.

[0078] The term "antibody" refers to full-length antibodies of any species that possess the ability to bind to an antigen, as well as fragments and derivatives of such antibodies (including Fab fragments, F(ab')2 fragments, single-chain antibodies, Fv fragments, and single-chain Fv fragments). The term "antibody" also includes synthetically derived antibodies, such as phage display-derived antibodies and fragments, aphibodies, and nanobodies.

[0079] As used herein, “nucleic acid ligand,” “aptamer,” “SOMAmer,” “modified aptamer,” and “clone” are interchangeable and refer to non-naturally occurring nucleic acids that have a desired effect on a target molecule. Desired effects include, but are not limited to, binding to a target, catalytically altering a target, reacting with a target in a way that modifies or alters the target or its functional activity, covalent binding to a target (as in suicide inhibitors), and promoting a reaction between a target and another molecule. In one embodiment, the effect is specific binding affinity to a target molecule, such a target molecule being a three-dimensional chemical structure other than a polynucleotide that binds to the aptamer by a mechanism independent of Watson / Crick base pairing or triple helix formation, and the aptamer is not a nucleic acid with a known physiological function to which the target molecule is bound. An aptamer for a given target comprises a nucleic acid identified from a candidate mixture of nucleic acids, and the aptamer is a ligand for the target by a method comprising: (a) contacting the candidate mixture with the target so that nucleic acids having a higher affinity for the target compared to other nucleic acids in the candidate mixture can be separated from the remainder of the candidate mixture; (b) separating the high-affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the high-affinity nucleic acids to produce a ligand-enriched mixture of nucleic acids, thereby identifying the aptamer of the target molecule. While affinity interactions are recognized as a matter of degree, in this context, the “specific binding affinity” of an aptamer for its target means that the aptamer binds to that target with a generally much higher degree of affinity than the aptamer would bind to other non-target components in the mixture or sample. An “aptamer,” “SOMAmer,” or “nucleic acid ligand” is one type or copy set of one type of nucleic acid molecule having a specific nucleotide sequence. An aptamer may contain any preferred number of nucleotides. An "aptamer" refers to two or more sets of such molecules. Different aptamers may have the same or different numbers of nucleotides. Aptamers may be DNA or RNA, and may be single-stranded, double-stranded, or may contain double-stranded or triple-stranded regions.In some embodiments, the aptamers are prepared using SELEX processes as described herein or known in the art.

[0080] As used herein, “SOMAmer” or slow off-rate modified aptamer refers to an aptamer having improved off-rate properties. SOMAmer may be produced using the improved SELEX method described in U.S. Patent No. 7,947,447, entitled “Method for Generating Aptamers with Improved Off-Rates”.

[0081] As used herein, aptamers comprising two different 5-position modified pyrimidines or C-5-position modified pyrimidines may also be referred to as “double-modified aptamers,” “aptamers having two modified bases,” “aptamers having two base modifications” or “double-modified” aptamers, or “aptamers having two modified bases,” all of which may be used interchangeably. The same terminology may also be used for libraries of aptamers or aptamer libraries. Thus, in some embodiments, an aptamer comprises two different 5-position modified pyrimidines, and the two different 5-position modified pyrimidines may be NapdC and NapdU, NapdC and PPdU, NapdC and MOEdU, NapdC and TyrdU, NapdC and ThrdU, PPdC and PPdU, PPdC and NapdU, PPdC and MOEdU, PPdC and TyrdU, PPdC and ThrdU, NapdC and 2NapdU, NapdC and T Selected from rpdU, 2NapdC and NapdU, as well as 2NapdC and 2NapdU, 2NapdC and PPdU, 2NapdC and TrpdU, 2NapdC and TyrdU, PPdC and 2NapdU, PPdC and TrpdU, PPdC and TyrdU, TyrdC and TyrdU, TrydC and 2NapdU, TyrdC and PPdU, TyrdC and TrpdU, TyrdC and TyrdU, and TyrdC and TyrdU. In some embodiments, the aptamer comprises at least one modified uridine and / or thymidine and at least one modified cytidine, wherein the at least one modified uridine and / or thymidine is modified at position 5 by a moiety selected from naphthyl moiety, benzyl moiety, fluorobenzyl moiety, tyrosyl moiety, indole moiety, morpholino moiety, isobutyl moiety, 3,4-methylenedioxybenzyl moiety, benzothiophenyl moiety, and benzofuranyl moiety, and the at least one modified cytidine is modified at position 5 by a moiety selected from naphthyl moiety, tyrosyl moiety, and benzyl moiety.In certain embodiments, the portion is covalently bonded to the 5-position of the base via a linker containing a group selected from amide linkers, carbonyl linkers, propynyl linkers, alkyne linkers, ester linkers, urea linkers, carbamate linkers, guanidine linkers, amidine linkers, sulfoxide linkers, and sulfone linkers. See Figure 21 for further examples of exemplary linkers that may be used to covalently bond the portion to the 5-position of the pyrimidine.

[0082] As used herein, “hydrophobic group” and “hydrophobic moiety” are interchangeable herein and refer to any uncharged group or moiety, the majority of which are hydrogen and carbon atoms, the group or moiety having a small dipole, and / or being repelled by water. These groups or moieties may include aromatic hydrocarbons or planar aromatic hydrocarbons. Methods for determining whether a molecule (or group or moiety) is hydrophobic are well known in the art and include calculation methods in addition to empirically obtained methods. An exemplary method is described in Zhu Chongqin et al. (2016) Characterizing hydrophobicity of amino acid side chains in a protein environment via measuring contact angle of a water nanodroplet on planar peptide network. Proc. Natl. Acad. Sci., 113(46) pgs. 12946-12951. As disclosed herein, exemplary hydrophobic moieties include, but are not limited to, groups I, II, III, IV, V, VII, VIII, IX, XI, XII, XIII, XV, and XVI in Figure 21. Further exemplary hydrophobic moieties include the hydrophobic moieties in Figure 25 (e.g., Bn, Nap, PE, PP, iBu, 2Nap, Try, NE, MBn, BF, BT, Trp).

[0083] As used herein, aptamers containing a single type of 5-position modified pyrimidine or C-5 modified pyrimidine may be referred to as “single-modified aptamers,” “aptamers having a single modified base,” “aptamers having a single base modification,” or “aptamers that are single-base modified,” and all of these may be used interchangeably. Libraries of aptamers or aptamer libraries may also use the same terminology. As used herein, “protein” is used synonymously with “peptide,” “polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein, peptide, or peptide fragment is substantially free of cellular material or other contaminating proteins from cells, tissues, or cell-free sources from which the amino acid sequence is obtained, or, if chemically synthesized, substantially free of chemical precursors or other chemicals.

[0084] In certain embodiments, the aptamer comprises a first 5-position modified pyrimidine and a second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine contains a tyrosyl moiety at position 5 of the first 5-position modified pyrimidine, and the second 5-position modified pyrimidine contains a naphthyl or benzyl moiety at position 5 of the second 5-position modified pyrimidine. In related embodiments, the first 5-position modified pyrimidine is uracil. In related embodiments, the second 5-position modified pyrimidine is cytosine. In related embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the uracil in the aptamer is modified at position 5. In related embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosine in the aptamer is modified at position 5.

[0085] Those skilled in the art of nucleic acid hybridization will recognize that factors commonly used to give or control the stringency of hybridization include formamide concentration (or other chemical denaturing reagents), salt concentration (i.e., ionic strength), hybridization temperature, surfactant concentration, pH, and the presence or absence of chaotrope. The optimal stringency of a probe / target sequence combination is often found by the well-known technique of fixing some of the aforementioned stringency factors and then determining the effect of varying a single stringency factor. Except for the fact that PNA hybridization is entirely independent of ionic strength, the same stringency factors can be modulated to control the stringency of PNA hybridization to nucleic acids. The optimal stringency of an assay may be experimentally determined by testing each stringency factor until the desired degree of discrimination is achieved.

[0086] As used herein, the terms “hybridization,” “hybridizing,” “binding,” and similar terms may be used interchangeably in the context of nucleotide sequences. The ability of two nucleotide sequences to hybridize with one another is based on the degree of complementarity between the two sequences and the proportion of similarly matched complementary nucleotide pairs. The more nucleotides in a given sequence that are complementary to another sequence, the more stringent the conditions for hybridization can be, and the more specific the binding of the two sequences becomes. High stringency can be achieved by increasing the temperature, increasing the ratio of cosolvents, decreasing the salt concentration, etc. While hybridization of complementary Watson / Crick base pairs of probe and target materials on a microarray is generally preferred, non-Watson / Crick base pairings can also occur during hybridization.

[0087] Conventional hybridization solutions and hybridization processes are described in J. Sambrook, Molecular Cloning: A Laboratory Manual (above) and are incorporated herein by reference. Hybridization conditions typically include (1) a high ionic strength solution, (2) a controlled temperature, and (3) the presence of carrier DNA, a surfactant, and a divalent cation chelating agent, all of which are known in the art.

[0088] As used herein, “biopolymer” is a polymer of one or more repeating units. Biopolymers typically include polysaccharides (such as carbohydrates), as well as peptides (this term is used to include polypeptides and proteins, whether or not they are bound to polysaccharides), and polynucleotides, as well as their analogues, such as compounds composed of or containing amino acid analogues or non-amino acid groups, or nucleotide analogues or non-nucleotide groups. Thus, this term includes polynucleotides in which the conventional backbone is not naturally occurring or has been replaced by a synthetic backbone, and nucleic acids (or synthetic or naturally occurring analogues) in which one or more of the conventional bases have been replaced by groups (natural or synthetic) that can participate in Watson-Crick hydrogen bond interactions. Polynucleotides may include single-stranded or multi-stranded configurations, with one or more strands being fully aligned or not aligned with other strands. In particular, “biopolymer” includes deoxyribonucleic acid or DNA (including cDNA), ribonucleic acid or RNA, and oligonucleotides, regardless of their source.

[0089] As used herein, “array” includes any one-dimensional, two-dimensional, or three-dimensional arrangement of an addressable region having one or more specific chemical moieties associated with that region (e.g., biopolymers such as peptide nucleic acid molecules, peptides, or polynucleotide sequences), where one or more chemical moieties are immobilized on the surface of the region. “Immobilized” means that one or more moieties stably associate with the substrate surface within the region so that they do not separate from the region under the conditions under which the array is used, for example, under hybridization and washing and stripping conditions. As is known in the art, one or more moieties may be covalently or non-covalently bonded to the surface of the region. For example, each region may extend three-dimensionally if the substrate is porous, but may have no substantial three-dimensional measurement (thickness) if the substrate is non-porous. An array may contain more than 10, more than 100, more than 1,000, more than 10,000, or more than 100,000 features in a region less than 20 cm or less than 10 cm. For example, a feature may have a width (i.e., diameter in the case of a circular spot) in the range of about 10 μm to about 1.0 cm. In other embodiments, each feature may have a width in the range of about 1.0 μm to about 1.0 mm, for example, about 5.0 μm to about 500 μm, and about 10 μm to about 200 μm. Non-circular features may have a regional range equivalent to the range of circular features having the aforementioned width (diameter) range. A given feature consists of a chemical moiety, e.g., a peptide nucleic acid molecule, a peptide, or a nucleic acid, that binds to (e.g., hybridizes with) a target molecule (e.g., a target nucleic acid or aptamer) so that the given feature corresponds to a specific target.

[0090] In the case of an array, the “target” refers to a portion of the mobile phase (usually a fluid) that is detected by probes (“target probes”) bound to the substrate in various regions. However, either the “target” or the “target probe” may be detected by the other. In some embodiments, the target is an oligonucleotide or aptamer. In some embodiments, the probe is a peptide nucleic acid molecule, a peptide, a protein, an oligonucleotide, or an aptamer.

[0091] The terms “biological sample,” “sample,” and “test sample” are interchangeable herein and refer to any material, biological fluid, tissue, or cell obtained from or otherwise derived from an individual, environment, animal, or food sample. This includes blood (including whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, and serum), sputum, tears, mucus, nasal lavage fluid, nasal aspirate, exhaled breath, urine, semen, saliva, peritoneal lavage fluid, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph, papillary aspirate, bronchial aspirate (e.g., bronchoalveolar lavage fluid), bronchial swabs, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid. This also includes all of the experimentally separated fractions mentioned above. For example, a blood sample may be fractionated into serum, plasma, or fractions containing specific types of blood cells, such as red blood cells or white blood cells (leukocytes). In some embodiments, the sample may be a combination of samples from an organism, such as a combination of tissue and liquid samples. The term “biological sample” also includes materials containing homogenized solid material from, for example, fecal samples, tissue samples, or tissue biopsies. The term “biological sample” also includes materials derived from tissue culture or cell culture. Any preferred method for obtaining a biological sample may be used, exemplary methods including, for example, venotomy, swabbing (e.g., cheek swabs), and fine-needle aspiration cytology procedures. Exemplary tissues that can be fine-needled include lymph nodes, lungs, lung lavage fluid, BAL (bronchial alveolar lavage fluid), thyroid gland, breast, pancreas, and liver. Samples may also be collected by, for example, microdissection (e.g., laser capture microdissection (LCM) or laser microdissection (LMD)), bladder lavage, smear (e.g., PAP smear), or tubal lavage. A “biological sample” obtained from or derived from an organism includes any such sample that has been processed in any preferred manner after being obtained from the organism.

[0092] The collection devices used herein may be any suitable absorbent device capable of absorbing the sample to be dried. Non-limiting examples include Whatman®, Guthrie Card, MITRA® microsampling device, Capitainer® microsampling device, TAPII microsampling device, Tasso device, dry fluid card, and similar devices.

[0093] As used herein, “target protein level,” “analyte level,” and “level” or “target protein value,” “analyte value,” and “value” refer to measurements made using any analytical method for detecting an analyte (e.g., target protein) in a biological sample, indicating the presence, absence, absolute amount or concentration, relative amount or concentration, titer, level, expression level, ratio of measured levels, etc., of the analyte in the biological sample, or its corresponding value. The exact nature of “level” or “value” depends on the specific design and components of the particular analytical method used to detect the analyte.

[0094] As used herein, “capturer” or “capture reagent” refers to a molecule capable of specifically binding to an analyte such as a biomarker, protein, and / or peptide. “Target protein capture reagent” refers to a molecule capable of specifically binding to a target protein. Non-limiting exemplary capture reagents include aptamers, antibodies, adonectin, ankyrin, other antibody mimetic and other protein scaffolds, autoantibodies, chimeras, small molecules, nucleic acids, lectins, ligand-binding receptors, imprinted polymers, avimers, peptide mimetic, hormone receptors, cytokine receptors, synthetic receptors, and modifications and fragments of any of the aforementioned capture reagents. In some embodiments, the capture reagent is selected from aptamers and antibodies.

[0095] The “control level” or “control value” of a target molecule refers to the level of the target molecule in the same sample type from individuals without disease or condition, from individuals not suspected of having or at risk of having disease or condition, or from individuals with a non-progressive form of disease or condition. Furthermore, the “control level” or “control value” may refer to a baseline based on the mean or what is considered to be within normal or healthy parameters. The “control level” or “control value” may also refer to a baseline level used for comparison with previously measured and subsequently measured or detected levels of the target. For example, the level of the target may be detected at time A and then at time B, where time B is after time A. In a more specific example, time A can be thought of as zero hours (0) or zero days (0), and time B can be a few minutes (e.g., 10, 20, 30, 40, 50, 60 minutes after time A), a few hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after time A), or a few days (e.g., 1, 2, 3, 4, This could be 5, 6, or 7 days later, several weeks later (e.g., 1, 2, 3, or 4 weeks after time point A), several months later (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after time point A), and even several years later (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 years after time point A). The “control level” of the target molecule does not need to be determined each time the method is performed, and may be a predetermined level used as a reference value or threshold to determine whether the level in a particular sample is higher or lower than the normal level.

[0096] The "corresponding correlation" or "coincident correlation coefficient" measures the agreement between two continuous variables X and Y (e.g., prediction, estimate or decision, and observed value). The "corresponding correlation" is calculated using a pair of 45 coefficients. oThis evaluates the degree to which the results fall on a line and includes measurements of accuracy and precision (or "Lin's coincident correlation coefficient"). Further information can be found in Lin, Biometrics, Vol. 45, No. 1 (March, 1989), 255-268, which is incorporated herein by reference. Other methods for determining correlation that may be used herein include, but are not limited to, Pearson correlation coefficient, paired t-test, least squares analysis of slope (=1) and intercept (=0), coefficient of variation, and intraclass correlation coefficient. In certain embodiments, paired correlation is determined by a method selected from Lin's coincidence, Pearson correlation coefficient, paired t-test, least squares analysis of slope (=1) and intercept (=0), coefficient of variation, and intraclass correlation coefficient.

[0097] As used herein, “detecting” or “determining” includes the use of both instruments used to observe and record a signal corresponding to an analyte level, and the substances / substances(s) necessary to generate that signal. In various embodiments, biomarker levels are detected using any preferred method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection methods, nuclear magnetic resonance, quantum dots, and the like.

[0098] The terms "dilution," "dilution series," and their variations encompass, but are not limited to, several different types of dilution, including serial dilution, serial dilution, and combinations thereof. As an example of serial dilution, if the dilution ratio is 1000 (1:1000 dilution), the user can first perform a 1:10 dilution (dilution ratio 10), followed by a 1:100 dilution (dilution ratio 100) using 1 part solute and 99 parts diluent from the 1:10 dilution, thereby obtaining a solute dilution of dilution ratio 1000 or 1:1000. Serial dilution includes a series of serial dilutions, each having the same dilution ratio, where subsequent dilutions are prepared using the diluent material from the previous step. As an example of serial dilution, to prepare a 5-point 1:2 serial dilution, one part of the solute is mixed with one part of the diluent to prepare the first dilution of the series (the first point of the 5-point dilution). Then, one part of the solute from the first dilution is used to prepare the second dilution of the successive series (the second point of the 5-point dilution). This procedure is repeated until the fifth serial dilution is reached.

[0099] The "dilution factor" refers to the ratio of the solute to the diluent. For example, a dilution factor of 2 means a 1:2 dilution, resulting in 1 part solute and 1 part diluent for a total of 2 parts; a dilution factor of 10 means a 1:10 dilution, resulting in 1 part solute and 9 parts diluent for a total of 10 parts.

[0100] "Target," "target molecule," and "analyte" are used interchangeably herein to refer to any target molecule that may be present in a sample. This term includes any slight alteration of a particular molecule, for example, in the case of a protein, slight alteration of the amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, for example, conjugation with a labeling component that does not substantially alter the identity of the molecule. "Target molecule," "target," or "analyte" refers to one type or one set of copies of a single molecule or multimolecular structure. "Target molecule," "target," and "analyte" refer to two or more types of molecules or multimolecular structures. Exemplary target molecules include proteins, polypeptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, aphibodies, antibody mimetic compounds, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, pigments, nutrients, growth factors, cells, tissues, and fragments or parts of any of the above. In some embodiments, the target molecule is a protein, in which case the target molecule may be referred to as a "target protein."

[0101] As used herein, “test sample” means a material, solution, or mixture containing or derived from a biological sample. In some embodiments, the test sample is produced from a biological sample. In some embodiments, the test sample is produced from a biological sample or a solution containing a biological sample by performing a buffer exchange on the biological sample or a solution containing a biological sample. As used herein, “adjusted test sample” is a test sample that has been adjusted, such as by changing the total protein concentration.

[0102] The terms “oligonucleotide bound to the surface of a solid support,” “probe bound to a solid support,” or “target bound to a solid support” refer to peptide nucleic acid molecules, oligonucleotides, aptamers, e.g., PNA (peptide nucleic acid), LNA (locked nucleic acid), or UNA (unlocked nucleic acid) molecules immobilized on the surface of a solid substrate, where the substrate may have various configurations, e.g., sheets, beads, particles, slides, wafers, webs, fibers, tubes, capillaries, microfluidic channels or reservoirs, or other structures. In certain embodiments, the collection of oligonucleotides or target elements used herein is present, for example, in the form of an array, on the surface of the same planar support. The terms “probe” and “target” are relative terms and should be understood that a molecule considered a probe in one assay may function as a target in another assay. Immobilization of oligonucleotides onto a substrate or surface can be achieved by well-known techniques commonly available in the literature. For example, see ACPease, et al., Proc. Nat. Acad. Sci, USA, 91:5022-5026 (1994), Z. Guo, et al., Nucleic Acids Res, 22, 5456-65 (1994), and M. Schena, et al., Science, 270, 467-70 (1995), each incorporated herein by reference.

[0103] The aforementioned chemistry of polynucleotide synthesis is described in detail, for example, Caruthers, Science 230:281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53:323-356; Hunkapillar et al., Nature 310:105-110, 1984; and “Synthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivatives”, CRC Press, Boca Raton, Fla., pages 100 et seq.; U.S. Patents No. 4,458,066, No. 4,500,707, No. 5,153,319, No. 5,869,643, EP0294196, and elsewhere. While the phosphoramidite and phosphite triester approaches are the most widely used, other approaches include the phosphodiester approach, the phosphotryester approach, and the H-phosphonate approach. The substrate is typically functionalized to bind to the first deposited monomer. Preferred techniques for functionalizing substrates containing such binding sites are described, for example, in Southern, EM, Maskos, U., and Elder, JK, Genomics, 13, 1007-1017, 1992. For array fabrication, different monomers and activators may be deposited at different addresses on the substrate during any given cycle, so that different features of the completed array have different desired biopolymer sequences. For in situ fabrication of polynucleotide arrays, one or more further intermediate steps, such as conventional oxidation, capping, and washing steps, may be required in each cycle (these steps may also be performed in a flooding procedure).

[0104] Multiplex assay Multiplex aptamer assays in solution-based targeted interaction and separation steps are described, for example, in U.S. Patents No. 7,855,054 and 7,964,356 and PCT application PCT / US2013 / 044792. In one embodiment, a multiplex assay is described in Example 1 of this specification.

[0105] In a multiplex assay format where multiple target proteins are measured by multiple capture reagents, the ability of a particular capture reagent to measure a particular target protein may be limited by the natural variation in the abundance of different target proteins (for example, high abundances of a target protein may saturate the assay, interfering with or reducing the ability of an assay to measure low abundances of a target protein). To address this variation in biological samples, aptamer reagents may be divided into at least two distinct groups (DIL1 capture reagents and DIL2 capture reagents), preferably three distinct groups (A3-DIL1 capture reagents, A2-DIL2 capture reagents, and A1-DIL3 capture reagents) based on the abundance of their respective protein targets in the biological sample. Each of the capture reagent groups A1, A2, and A3 has a different set of aptamers, each of which has a specific affinity for the target protein. Biological samples are diluted into two (Diluent 1 or DIL1 and Diluent 2 or DIL2), preferably three different dilution groups (Diluent 1 or DIL1, Diluent 2 or DIL2 and Diluent 3 or DIL3), based on their relative concentrations detected by the protein target capture reagents, to create individual test samples. Thus, the biological samples are diluted into high, medium, and low, or high, medium, and low abundance target protein dilution groups, with the least abundant protein target being measured in the minimum dilution group and the most abundant protein target being measured in the maximum dilution group. The capture reagents for each dilution group of the capture reagents are incubated together (for example, A3 set of aptamers is incubated with the test sample in Diluent 1 or DIL1, A2 set of aptamers is incubated with the test sample in Diluent 2 or DIL2, and A1 set of aptamers is incubated with the test sample in Diluent 3 or DIL3). The total number of aptamers A1, A2, and A3 may be 4,000, 4,500, or 5,000 or more. In some embodiments, dilution group 1 may be 2.5%, dilution group 2 may be 0.05%, and dilution group 3 may be 0.005%. Other dilutions from the three dilution groups may also be used.

[0106] This disclosure describes a method for preparing a dry biological matrix for the detection of target molecules in a multiplex assay, and a method for detecting target molecules from a dry biological matrix in a multiplex assay.

[0107] The biological matrix may be prepared for use in multiplex assays as follows: The biological matrix can be collected from subjects following standard procedures such as blood collection, fluid collection, or biopsy collection. Once collected, the biological matrix can be spotted or adsorbed onto a collection device and dried.

[0108] drying temperature In some embodiments, the drying temperature may be around -20°C to room temperature (about 20 to 24°C). In some embodiments, the drying temperature may be about -20°C, about -15°C, about -10°C, about -5°C, about 0°C, about 2°C, about 4°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 20°C, about 22°C, or about 24°C.

[0109] Drying time In some embodiments, the drying time may range from about 1 hour to about 10 days. In some embodiments, the trial time may range from about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours, about 52 hours, about 56 hours, about 60 hours, about 64 hours, about 68 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In some embodiments, the drying time may range from at least 1 hour to at least 10 days. In some embodiments, the trial time may be at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, at least 24 hours, at least 26 hours, at least 28 hours, at least 30 hours, at least 32 hours, at least 34 hours, at least 36 hours, at least 40 hours, at least 44 hours, at least 48 hours, at least 52 hours, at least 56 hours, at least 60 hours, at least 64 hours, at least 68 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days. In some embodiments, the drying time may be at a constant temperature until the sample is dry, prior to temperature fluctuations. In some embodiments, temperature fluctuations may occur due to transport and handling. In some embodiments, the dried sample is suitable for multiplex assays if the drying temperature is constant until the sample is dry.

[0110] Extraction time In some embodiments, the target molecule may be extracted from the collection device in the formulation for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, at least 150 minutes, at least 160 minutes, at least 170 minutes, at least 180 minutes, at least 190 minutes, or at least 200 minutes.

[0111] Extracted preparations In some embodiments, the extraction formulation may include a buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide. In some embodiments, the extraction formulation may include 20% PBS in addition to the buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide. In some embodiments, one or more salts are independently selected from sodium salts, potassium salts, and magnesium salts.

[0112] In some embodiments, one or more salts include a sodium salt, a potassium salt, and a magnesium salt. In some embodiments, the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2. In some embodiments, the concentration of NaCl in the formulation is about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 75 to 125 mM, or about 100 mM. In some embodiments, the concentration of KCl is about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM. In some embodiments, the concentration of MgCl2 is about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 8 mM.

[0113] In some embodiments, the buffer is selected from HEPES, IVIES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, coramine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS. In some embodiments, the buffer in the formulation is concentrated at a level of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 60 mM, or about 50 mM.

[0114] In some embodiments, the chelating agent is selected from EGTA, EDTA, DTPA, BAPTA, DMPS, and ALA. In some embodiments, the concentration of the chelating agent in the formulation is about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.25 mM.

[0115] In some embodiments, the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80). In some embodiments, the nonionic surfactant is present in volume-to-volume amounts of about 0.1% to about 5% of the formulation, or about 0.2% to about 4% of the formulation, or about 0.3% to about 3% of the formulation, or about 0.4% to about 2% of the formulation, or about 0.5% of the formulation, or about 1.5% of the formulation, or about 1.2% of the formulation.

[0116] In some embodiments, the pH of the formulation is approximately pH 5 to approximately pH 9, or approximately pH 6 to approximately pH 8, or approximately pH 7 to approximately pH 7.9, or approximately pH 7.5.

[0117] In some embodiments, the formulation comprises 50 mM HEPES, 100 mM NaCl, 5 mM KCl, 8 mM MgCl2, 1.25 mM EGTA, and 1.2% Tween-20.

[0118] In some embodiments, the formulation has a pH of about 7.5.

[0119] In some embodiments, the protease inhibitor is a reversible protease inhibitor. In some embodiments, the protease inhibitor inhibits a protease selected from trypsin, plasmin, and thrombin. In some embodiments, the protease inhibitor is a serine protease inhibitor. In some embodiments, the protease inhibitor is benzamidine. In some embodiments, the protease inhibitor in the formulation is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.2 mM.

[0120] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is 20 to 100 nucleotides long, or 25 to 80 nucleotides long, or 25 to 70 nucleotides long, or 25 to 50 nucleotides long, or about 30 nucleotides long. In some embodiments, the oligonucleotide contains one or more modified nucleotides. In some embodiments, the oligonucleotide contains one or more C-5 modified pyrimidines. In some embodiments, the oligonucleotide comprises the sequence [(ACXX)7-AC], where X is BndU. In some embodiments, the oligonucleotide in the formulation has a concentration of 5 μM to 100 μM, or 10 μM to 80 μM, or 20 μM to 60 μM, or 30 μM to 50 μM, or about 75 μM, or about 37 μM.

[0121] The volume of sample to be dried may be determined by the device being used. As a non-limiting example, the mitra VAMS microsampling device is packed with 30 μL, so the initial sample volume is greater than 30 μL. In some embodiments, the packed sample volume may be less than 30 μL, for example, 20 μL, 10 μL, or 5 μL. Depending on the sample collection device, sample volumes greater than 30 μL may also be used.

[0122] As used herein, “Catch-1” refers to the splitting of an aptamer-target affinity complex or an aptamer-target covalent complex. The objective of Catch-1 is to remove substantially all components in the test sample that do not associate with the aptamer. Removing most of such components can improve target tagging efficiency by removing non-target molecules from the target tagging step, which is typically used for Catch-2 capture, and may lead to a reduction in assay background. In one embodiment, the tag is bound to the aptamer by attaching the tag to the aptamer either before the assay, during assay preparation, or during the assay. In one embodiment, the tag is a freeable tag. In one embodiment, the freeable tag includes a cleavable linker and the tag. As described above, the tagged aptamer may be captured on a solid support, which contains a capture element suitable for the tag. The solid support may then be washed as described herein before equilibration with the test sample to remove all unwanted material (Catch-0).

[0123] As used herein, “Catch-2” refers to the splitting of an aptamer-target affinity or aptamer-target covalent complex based on the capture of the target molecule. The purpose of the Catch-2 step is to remove free or non-complexed aptamers from the test sample before detection and any quantification. Removal of free aptamers from the sample enables detection of the aptamer-target affinity or aptamer-target covalent complex by any suitable nucleic acid detection technique. When using Q-PCR for detection and any quantification, removal of free aptamers is necessary for accurate detection and quantification of the target molecule.

[0124] In one embodiment, the target molecule is a protein or peptide, and the free aptamer is separated from the aptamer-target affinity (or covalent) complex (and the remainder of the test sample) using a complex containing a reagent that can be incorporated into the protein (and peptide) and the protein (or peptide), such as an aptamer-target affinity (or covalent) complex. The tagged protein (or peptide) and the aptamer-target affinity (or covalent) complex may be immobilized on a solid support to enable the separation of the protein (or peptide) and the aptamer-target affinity (or covalent) complex from the free aptamer. Such tagging may include, for example, a biotin moiety that can be incorporated into the protein or peptide.

[0125] In one embodiment, the Catch-2 tag is bound to a protein (or peptide) by chemically conjugating the tag to the target either before the assay, during assay preparation, or during the assay. In one embodiment, the Catch-2 tag is a releaseable tag. In one embodiment, the releaseable tag includes a cleavable linker and tag. However, it is generally not necessary to release the protein (or peptide) from the Catch-2 solid support. As described above, the tagged target may be captured on a second solid support, the solid support containing a capture element suitable for the target tag. The solid support is then washed with a variety of buffer solutions, including buffer solutions containing organic solvents and buffer solutions containing salts and / or surfactants containing salts and / or surfactants.

[0126] After washing the second solid support, the aptamer-target affinity complex is then subjected to a dissociation step in which the complex is broken down to produce free aptamers, while the target molecule generally remains bound to the solid support by binding interactions between the capture element and the target capture tag. The aptamer can be released from the aptamer-target affinity complex by any method that breaks down the structure of either the aptamer or the target. This may be achieved by washing the support-bound aptamer-target affinity complex in a high-salt buffer that dissociates the non-covalently bound aptamer-target complex. The eluted free aptamers are collected and detected. In another embodiment, high or low pH is used to break down the aptamer-target affinity complex. In another embodiment, high temperature is used to dissociate the aptamer-target affinity complex. In another embodiment, any combination of the above methods may be used. In another embodiment, proteolytic digestion of the protein portion of the aptamer-target affinity complex is used to release the aptamer component.

[0127] In the case of aptamer-target covalent complexes, the release of the aptamer for subsequent quantification is achieved using a cleavable linker in the aptamer construct. In another embodiment, a cleavable linker in the target tag results in the release of the aptamer-target covalent complex.

[0128] For example, a proteomics affinity assay (multiplex assay) may be performed as follows:

[0129] A 7.5% streptavidin-agarose slurry of 133 in Catch-0:1x SB17, Tw (40mM HEPES, 102mM NaCl, 1mM EDTA, 5mM MgCl2, 5mM KCl, 0.05% Tween-20) was added to the wells of a filter plate (0.45 μm Millipore HV plate (Durapore catalog number MAHVN4550)). A suitable 1.1x aptamer mixture (all aptamers containing a Cy3 fluorophore and a photocleavable biotin moiety at the 5' end) was thawed and then vortexed. The 1.1x aptamer mixture was then boiled for 10 minutes, vortexed for 30 seconds, and cooled to 20°C in a water bath for 20 minutes. Next, the liquid in the filter plate containing the streptavidin agarose slurry was removed by centrifugation (1000 × g for 1 minute). 100 μL of the aptamer mixture was added to the wells of the filter plate (using robotic control). The mixture was incubated at 25°C for 20 minutes on a light-shielded shaker set to 850 rpm.

[0130] Catch-0 Wash: After incubation for 20 minutes, the solution was removed by vacuum filtration. 190 μL of 1x CAPS aptamer pre-wash buffer (50 mM CAPS, 1 mM EDTA, 0.05% Tw-20, pH 11.0) was added, and the mixture was incubated for 1 minute with shaking. The CAPS wash solution was then removed by vacuum filtration. The CAPS wash was then repeated once. 190 μL of 1x SX17-Tween was added, and the mixture was incubated for 1 minute with shaking. The 1x SB17-Tween was then removed by vacuum filtration. Another 190 μL of 1x SX17-Tw was added, and the mixture was incubated for 1 minute with shaking. The 1x SB17-Tw was then removed by centrifugation (1000 × g for 1 minute). After removing 1x SB17 and Tw, 150 μL of Catch-0 preservation buffer (150 mM NaCl, 40 mM HEPES, 1 mM EDTA, 0.02% sodium azide, 0.05% Tween-20) was added, the filter plate was carefully sealed only around the outer edge of the plate, and stored in a dark place at 4°C until use.

[0131] Sample preparation: 75 microliters of 40% sample diluent were placed in a 40% sample plate (the final 40% sample contained 20 μM Z-block, 1 mM benzamidine, 1 mM EGTA, 40 mM HEPES, 5 mM MgCl2, 5 mM KCl, and 1% Tween-20). 195 microliters of 1x SB17-Tw were placed in a 1% sample plate. 90 microliters of 1x SB17-Tw were placed in a 1:10 dilution plate. 133 microliters of 1x SB17-Tw were placed in a 0.005% sample plate. The samples were thawed on a rack thawing station in a 25°C incubator for 10 minutes, then vortexed at 1000 × g for 1 minute. The caps were removed from the tubes. The samples were mixed (50 μL, 5 times), and 50 μL of the 100% sample was transferred to a 40% sample plate containing the sample diluent. The 40% sample was then mixed on the sample plate by pipetting (110 μL, 10 times). Next, 5 μL of the 40% sample was transferred to a 1% sample plate containing 1x SB17-Tw. This sample was mixed again by pipetting (120 μL, 10 times). After mixing, 10 μL of the 1% sample was transferred to a 1:10 dilution plate containing 1x SB17-Tw, and this was mixed by pipetting (75 μL, 10 times). 7 microliters of the 0.1% sample from the 1:10 dilution plate was transferred to a 0.005% sample plate containing 1x SB17-Tw, and this was mixed by pipetting (110 μL, 10 times).

[0132] Plate preparation before incubation: The Catch-0 storage solution was removed from the filter plate by vacuum filtration. Then, 190 microliters of 1x SB17-Tw were added and subsequently removed from the filter plate by vacuum filtration. Then, another 190 μL of 1x SB17-Tw was added to the filter plate.

[0133] Incubation: 1x SB17-Tw buffer was removed from the filter plate by centrifugation (1000 x g for 1 minute). 100 microliters of appropriate sample diluent were added to the filter plate (three filter plates, i.e., one for each of the 40%, 20%, 1%, or 0.005% sample diluents). The filter plate was carefully sealed only around the periphery of the plate to avoid pressurizing the wells. Pressure would cause leakage during incubation. The plate was then incubated at 28°C for 3.5 hours on a light-shielded thermoshaker set to 850 rpm.

[0134] Processing of filter plates: After incubation, the filter plates were placed on a vacuum manifold and the sample was removed by vacuum filtration. 190 microliters of biotin washing solution (100 μM biotin in 1x SB17-Tw) was added, and the liquid was removed by vacuum filtration. Next, the sample was washed five times with 190 μL of 1x SB17-Tw (vacuum filtration). 100 microliters of 1 mM NHS-biotin (freshly prepared) in 1x SB17-Tw was added, the filter plates were adsorbed onto an absorption pad, and the mixture was incubated for 5 minutes with shaking. The liquid was removed by vacuum filtration. 125 microliters of 20 mM glycine in 1x SB17-Tw was added, and the liquid was removed by vacuum filtration. Again, 125 μL of 20 mM glycine in 1x SB17-Tw was added, and the liquid was removed by vacuum filtration.

[0135] Next, the samples were washed six times with 190 μL of 1x SB17-Tw, and the liquid was removed by vacuum filtration. Then, 85 microliters of photocutting buffer (2 μM Z-block in 1x SB17-Tw) was added to each of the filter plates.

[0136] Light sectioning: The filter plate was adsorbed onto an absorbent pad and irradiated with a BlackRay UV lamp for 6 minutes while shaking (800 rpm, 25°C). The plate was rotated 180 degrees and irradiated for another 6 minutes under the BlackRay light source. The 40% filter plate was placed on an empty 96-well plate. The 1% filter plate was placed on top of the 40% filter plate, and the 0.005% filter plate was placed on top of the 1% filter plate. The plate assembly was rotated at 1000 × g for 1 minute. The 96-well plate containing the eluted sample was placed on the robotic deck. 60 percent glycerol in 1x SB17-Tw was placed on the robotic deck from a 37°C incubator.

[0137] Catch-2: During assay setup, 50 μL of 10 mg / mL MyOne SA beads (500 μg) were added to a 96-well plate in an ABgene Omni tube for Catch-2 and placed in a Cytomat. The 96-well bead plate for Catch-2 was suspended for 90 seconds and then placed on a magnetic block for 60 seconds, after which the supernatant was removed. Simultaneously or sequentially, the Catch-1 eluates from each dilution group were transferred to the Catch-2 bead plate and incubated on a Peltier thermoshaker (1350 rpm, 5 minutes, 25°C). The plate was then placed on a 25°C magnet for 2 minutes, and the supernatant was removed. Next, 75 μL of 1x SB17-Tw was added, and the sample was incubated on a Peltier shaker at 1350 rpm, 37°C for 1 minute. Next, 75 μL of 60% glycerol (heated to 37°C) in 1x SB17-Tw was added, and the sample was incubated again on a Peltier shaker at 1350 rpm and 37°C for 1 minute. The plate was transferred to a magnet heated to 37°C and incubated for 2 minutes, after which the supernatant was removed. This 37°C 1x SB17-Tw and glycerol washing cycle was repeated two more times. Next, the sample was washed on a Peltier shaker (1350 rpm, 1 minute, 25°C) with 150 μL of 1x SB17-Tw to remove any remaining glycerol, and then placed on a magnetic block at 25°C for 1 minute. The supernatant was removed, 150 μL of 1x SB17-Tw replaced with 0.5 M NaCl was added, incubated at 1350 rpm for 1 minute (25°C), and then placed on a magnetic block at 25°C for 1 minute. The supernatant was removed, and 75 μL of perchlorate elution buffer (1.8 M NaClC-4, 40 mM PIPES, 1 mM EDTA, 0.05% Triton X-100, 1x hybridization control, pH=6.8) was added, followed by incubation on a Peltier shaker for 10 minutes (25°C, 1350 rpm). The plate was then transferred to a magnetic separator and incubated for 90 seconds to collect the supernatant.

[0138] Hybridization: 20 microliters of eluted sample were added to an empty 96-well plate using robotic control. 5 microliters of 10x Agilent blocking buffer containing a second set of hybridization control were added to the eluted sample using robotic control. Then, 25 μL of 2x Agilent HiRPM hybridization buffer was manually added to the wells. 40 microliters of the hybridization mixture was placed on an Agilent gasket slide. An Agilent 8×15k array was added to the gasket slide, and the sandwich was clamped. The sandwich was then incubated at 55°C for 19 hours while rotating (20 rpm).

[0139] Washing after hybridization: Slide processing after hybridization was performed on a Little Dipper Processor (SciGene, catalog number 1080-40-1). Approximately 750 mL of Wash Buffer 1 (Oligo aCGH / ChlP-on-chip Wash Buffer 1, Agilent Technologies) was placed in one glass staining dish. Approximately 750 mL of Wash Buffer 1 (Oligo aCGH / ChlP-on-chip Wash Buffer 1, Agilent Technologies) was placed in tank 1 of the Little Dipper Processor. Approximately 750 mL of Wash Buffer 2 (Oligo aCGH / ChlP-on-chip Wash Buffer 1, Agilent Technologies), heated to 37°C, was placed in tank 2 of the Little Dipper Processor. The magnetic stirring speed of both tanks was set to 5. The temperature controller for tank 1 was not activated, but the temperature controller for tank 2 was set to 37°C. Up to 12 slide / gasket assemblies were sequentially disassembled in a first staining dish containing wash buffer 1, and the slides, still immersed in wash buffer 1, were placed in a slide rack. Once all slide / gasket assemblies were disassembled, the slide rack was quickly transferred to tank 1 of the Little Dipper Processor to initiate the automated washing protocol. The Little Dipper Processor incubated the slides in tank 1 at a speed of 250 for 300 seconds, then transferred them to tank 2 at 37°C containing Agilent Wash 2 (Oligo aCGH / ChlP-on-chip Wash Buffer 2, Agilent Technologies), where they were incubated at a speed of 100 for 300 seconds. The Little Dipper Processor then transferred the slide rack to its built-in centrifuge, where the slides were rotated at a speed of 690 for 300 seconds.

[0140] Microarray imaging: Microarray slides were imaged in the Cy3 channel using a microarray scanner (Agilent G2565CA Microarray Scanner System, Agilent Technologies) at 5 μm resolution and 100% PMT settings, with the XRD option enabled at 0.05. The resulting TIFF images were processed using Agilent feature extraction software version 10.7.3.1 with the GE1_107_Sep09 protocol.

[0141] As used herein, “free-movable” or “cleavable” elements, parts, or linkers refer to molecular structures that can be broken to produce two distinct components. A free-movable (or cleavable) element may contain a single molecule whose chemical bond can be cleaved (referred to herein as an “in-line cleavable linker”), or the element may contain two or more molecules whose non-covalent interactions can be cleaved or broken (referred to herein as a “hybridization linker”).

[0142] In some embodiments, it is necessary to spatially isolate certain functional groups from others to prevent interference with individual functional groups. For example, the presence of a label closest to a photocleavable group may absorb certain wavelengths of light, hindering the efficiency of photocleavage. Therefore, it is desirable to isolate such groups using non-interfering regions that provide sufficient spatial isolation to restore the full activity of photocleavage. In some embodiments, a "spacing linker" is introduced into the aptamer having both labeling and photocleavage functions.

[0143] "Solid support" refers to any substrate having a surface to which molecules may be directly or indirectly bound, either covalently or non-covalently. The solid support may include any substrate material capable of providing physical support to a capture element or probe bound to its surface. The material is generally able to withstand conditions associated with the binding of the capture element or probe to the surface and any subsequent procedures, handling, or processing it may face during the performance of the assay. The material may be naturally occurring, synthetic, or a modified version of a naturally occurring material. Suitable solid support materials include silicon, silicon wafer chips, graphite, mirrors, laminates, films, ceramics, plastics (polymers, e.g., poly(vinyl chloride), cycloolefin copolymers, agarose gels or beads, polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), polytetrafluoroethylene (PTFE or Teflon®), nylon, poly(vinyl butyrate), etc.), germanium, gallium arsenide, gold, silver, Langmuir Blodgett films, flow-through chips, etc., which may be used alone or in combination with other materials. Additional rigid materials, such as glass, may also be considered, including silica and, for example, glass available as bioglass. Other materials that may be used include porous materials such as controlled pore glass beads, crosslinked beads Sepharose® or agarose resin, or crosslinked bisacrylamide and azalactone copolymers. Other types of beads include nanoparticles, polymer beads, solid core beads, paramagnetic beads, or microbeads. Any other materials known in the art may also be considered, for example, having one or more functional groups incorporated on the surface of the material, such as amino, carboxyl, thiol, or hydroxyl functional groups.

[0144] The materials used for solid supports can take on a variety of configurations, ranging from simple to complex. Solid supports can have one of a number of shapes, including strips, plates, disks, rods, particles, beads, tubes, and wells (microtiters). Solid supports may be porous or nonporous, magnetic, paramagnetic, or nonmagnetic, polydisperse or monodisperse, hydrophilic or hydrophobic. Solid supports may also be in the form of a gel or slurry of densely packed (as in a column matrix) or loosely packed particles.

[0145] In one embodiment, a solid support to which a capture element is bound is used to capture a tagged aptamer-target affinity complex or aptamer-target covalent complex from a test mixture. In one particular example, if the tag is a biotin moiety, the solid support is streptavidin-coated beads or resins, e.g., Dynabeads M-280 streptavidin, Dynabeads MyOne streptavidin, Dynabeads M-270 streptavidin (Invitrogen), streptavidin agarose resin (Pierce), streptavidin Ultralink resin, MagnaBind streptavidin beads (ThermoFisher Scientific), BioMag streptavidin, ProMag streptavidin, silica streptavidin (Bangs Laboratories), streptavidin Sepharose High Performance (GE Healthcare),

[0146] This could be streptavidin polystyrene microspheres (Microspheres-Nanospheres), streptavidin-coated polystyrene particles (Spherotech), or any other streptavidin-coated beads or resins commonly used by those skilled in the art to capture biotin-tagged molecules.

[0147] As described above, one object of the present invention is to convert protein signals into aptamer signals. As a result, the amount of aptamers collected / detected may indicate and be directly proportional to the amount of bound target molecules and the amount of target molecules in the sample. Numerous detection schemes can be used without eluting the aptamer-target affinity complex or aptamer-target covalent complex from the second solid support after catch-2 splitting. In addition to the embodiments of the detection methods described below, other detection methods are known to those skilled in the art.

[0148] Many detection methods require explicit labeling incorporated into the aptamer before detection. In these embodiments, the label, such as a fluorescent or chemiluminescent dye, may be incorporated into the aptamer either during or after synthesis using standard techniques for nucleic acid synthesis. Radiolabeling may be incorporated either during or after synthesis using standard enzymatic reactions with appropriate reagents. Labeling may also be performed after catch-2 splitting and elution by using suitable enzymatic techniques. For example, using primers with the label described above, the label will be incorporated into the amplified product of the eluted aptamer by PCR. When gel techniques are used for quantification, different sized mass labels may also be incorporated using PCR. These mass labels may also incorporate different fluorescent or chemiluminescent dyes for further multiplexing capability. Labeling may also be indirectly added to the aptamer by using a specific tag incorporated into the aptamer either during or after synthesis, and then adding a probe that associates with the tag and holds the label. In addition to those described above, labeling may include, for example, enzymes used in standard assays for colorimetric analysis readings. These enzymes act in combination with enzyme substrates and include, for example, enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (AP). Labels may also include substances or compounds that are electrochemical functional groups for electrochemical detection.

[0149] For example, the aptamer may be labeled with a radioactive isotope such as 32P as described above before contacting the test sample. Using any one of the four basic assays and their variations as described above, aptamer detection may be easily achieved by quantifying the radioactivity on the second solid support at the end of the assay. The radioactivity value will be directly proportional to the amount of the target in the original test sample. Similarly, labeling the aptamer with a fluorescent dye as described above before contacting the test sample allows for direct and simple fluorescence reading on the second solid support. Likewise, chemiluminescent labeling or quantum dots may be used for direct reading from the second solid support without requiring aptamer elution.

[0150] Additional detection schemes may be used in addition to those described above, by eluting aptamers from a second solid support or releasing photo-aptamer-target covalent complexes. For example, the released aptamers, photo-aptamers, or photo-aptamer-target covalent complexes may be detected by electrophoresis on a PAGE gel and optionally quantified with nucleic acid dyes such as SYBR Gold. Alternatively, the released aptamers, photo-aptamers, or photo-aptamer covalent complexes may be detected and quantified using capillary gel electrophoresis (CGE) with fluorescent labels incorporated into the aptamers as described above. Another detection scheme uses quantitative PCR, for example, SYBR Green, to detect and quantify the eluted aptamers. Alternatively, the Invader® DNA assay may be used to detect and quantify the eluted aptamers. Another alternative detection scheme uses next-generation sequencing.

[0151] In another embodiment, the amount or concentration of the aptamer-target affinity complex (or aptamer-target covalent complex) is determined using a “molecular beacon” during the replication process (see, e.g., Tyagi et ah, Nat. Biotech. J_6:49 53, 1998, U.S. Patent No. 5,925,517). A molecular beacon is a specific nucleic acid probe that folds into a hairpin loop, containing a fluorophore at one end of the hairpin structure and a quencher at the other end, such that little or no signal is generated by the fluorophore when the hairpin is formed. The loop sequence is specific to the target polynucleotide sequence, and upon hybridization with the aptamer sequence, the hairpin's folded structure unfolds, thereby generating a fluorescent signal.

[0152] For the multiplexed detection of a small number of aptamers still bound to the second solid support, fluorescent dyes with different excitation / emission spectra may be used to detect and quantify two, three, five, or up to ten individual aptamers.

[0153] Similarly, quantum dots of different sizes can be used for multiplexed readings. Quantum dots can be introduced after the free aptamers have been separated from a second solid support. By using aptamer-specific hybridization sequences coupled to specific quantum dots, multiplexed readings of 2, 3, 5, and up to 10 aptamers can be performed. Labeling of different aptamers with different radioactive isotopes that can be detected individually, such as 32P, 3H, 13C, and 35S, can also be used for limited multiplexed readings.

[0154] For the multiplexed detection of aptamers released from the second solid support of Catch-2, a single fluorescent dye incorporated into each aptamer as described above can be used in conjunction with a quantification method that enables identification of the aptamer sequence along with quantification at the aptamer level. Such methods include, but are not limited to, DNA chip hybridization, microbead hybridization, next-generation sequencing, and CGE analysis.

[0155] In one embodiment, a standard DNA hybridization array or chip is used to hybridize each aptamer or photoaptamer to a specific probe or a set of specific probes immobilized on a slide or chip, such as an Agilent array, Illumina BeadChip array, NimbleGen array, or a custom-printed array. Each specific probe is complementary to a sequence on the aptamer. The complementary sequence may be a specific hybridization tag incorporated into the aptamer, a portion of the aptamer sequence, or the entire aptamer sequence. The aptamers released from the Catch-2 solid support are added to a suitable hybridization buffer and processed using a standard hybridization method. For example, the aptamer solution is incubated with the DNA hybridization array at approximately 60°C for 12 hours to ensure stringency of hybridization. The array is washed and then scanned with a fluorescence slide scanner to generate an image of the aptamer hybridization intensity for each feature of the array. Image segmentation and quantification are achieved using image processing software such as ArrayVision. In one embodiment, a multiplex aptamer assay may be performed using up to 25 aptamers, up to 50 aptamers, up to 100 aptamers, up to 200 aptamers, up to 500 aptamers, up to 1000 aptamers, and up to 10,000 aptamers.

[0156] In one embodiment, addressable microbeads having a specific DNA probe complementary to the aptamer described above are used for hybridization. The microbeads may be addressable using a specific fluorescent dye, such as in Luminex bead technology, or they may be labeled with a barcode, such as in Illumina VeraCode technology, or they may use a laser-powered transponder. In one embodiment, the aptamers released from the solid support of Catch-2 are added to a suitable hybridization buffer and processed using a standard microbead hybridization method. For example, the aptamer solution is incubated with a set of microbeads at approximately 60°C for 2 hours to ensure stringency for hybridization. The solution is then processed on a Luminex instrument that counts individual bead types and quantifies the aptamer fluorescence signal. In another embodiment, VeraCode beads are contacted with the aptamer solution and hybridized at approximately 60°C for 2 hours, then deposited on a grid-filled surface and scanned using a slide scanner for identification and fluorescence quantification. In another embodiment, transponder microbeads are incubated with the aptamer sample at approximately 60°C, and then quantified using a device appropriate for the transponder microbeads. In one embodiment, a multiplex aptamer assay may be performed using up to 25 aptamers, up to 50 aptamers, up to 100 aptamers, up to 200 aptamers, and up to 500 aptamers, and detection may be performed by hybridization to microbeads.

[0157] Samples containing eluted aptamers can be processed to incorporate a unique mass tag along with the fluorescent labeling described above. The mass-labeled aptamers are then injected into a CGE instrument, which is essentially a DNA sequencer, to identify the aptamers by their unique mass and quantify them using fluorescence from the dye incorporated during the labeling reaction. One exemplary example of this technique has been developed by Althea Technologies.

[0158] In many of the methods described above, the aptamer solution may be amplified and, in some cases, tagged before quantification. Standard PCR amplification may be used with the aptamer solution eluted from the Catch-2 solid support. Such amplification may be used before DNA array hybridization, microbead hybridization, and CGE reading.

[0159] In another embodiment, the aptamer-target affinity complex (or aptamer-target covalent complex) is detected and / or quantified using Q-PCR. As used herein, "Q-PCR" refers to a PCR reaction performed in such a manner and under such controlled conditions that the results of the assay are quantitative, i.e., the assay can quantify the amount or concentration of aptamers present in the test sample.

[0160] In one embodiment, the amount or concentration of the aptamer-target affinity complex (or aptamer-target covalent complex) in the test sample is determined using TaqMan® PCR. This technique generally relies on the 5'-3' exonuclease activity of an oligonucleotide replicase to generate a signal from the target sequence. TaqMan probes are selected based on the sequence of the aptamer to be quantified and generally contain a 5' terminal fluorophore, e.g., 6-carboxyfluorescein, and a 3' terminal quencher, e.g., 6-carboxytetramethylfluorescein, which generate a signal when the aptamer sequence is amplified using polymerase chain reaction (PCR). When the polymerase copies the aptamer sequence, the exonuclease activity releases the fluorophore from the probe annealed downstream of the PCR primer, thereby generating a signal. The signal increases as the replica product is produced. The amount of PCR product depends on both the number of replication cycles performed and the initial concentration of the aptamer.

[0161] In another embodiment, the amount or concentration of the aptamer-target affinity complex (or aptamer-target covalent complex) is determined using an intercalating fluorescent dye during the replication process. For example, an intercalating dye such as SYBR® green generates a greater number of fluorescent signals in the presence of double-stranded DNA compared to the fluorescent signal generated in the presence of single-stranded DNA. If a double-stranded DNA product is formed during PCR, the signal generated by the dye increases. The magnitude of the generated signal depends on both the number of PCR cycles and the initial concentration of the aptamer.

[0162] In another embodiment, the aptamer-target affinity complex (or aptamer-target covalent complex) is detected and / or quantified using mass spectrometry. A specific mass tag may be introduced using the enzymatic techniques described above. For mass spectrometry readings, no detection label is required at all; rather, the mass itself is used for identification, and both quantification is performed based on the position and area under the mass peak generated during analysis by mass spectrometry, using techniques commonly used by those skilled in the art. An example of using mass spectrometry is the MassARRAY® system developed by Sequenom.

[0163] A computer program may be used to perform one or more steps of any of the methods disclosed herein. Another aspect of this disclosure is a computer program product including a computer-readable storage medium on which the computer program is stored, which, when loaded into a computer, performs or assists in performing any of the methods disclosed herein.

[0164] One aspect of this disclosure is the product of any of the methods disclosed herein, i.e., assay results, which may be evaluated at the test site or, if desired, transported to another location for evaluation and communication with relevant parties in a remote location. As used herein, “remote location” means a location physically different from the location where the results are obtained. Thus, the results may be sent to a different room, a different building, a different area of ​​a city, a different city, etc. The data may be transmitted by any preferred means, e.g., facsimile, mail, next-day delivery, email, FTP, voicemail, etc.

[0165] "Communicating" information means the transmission of data representing that information as electrical signals over a suitable communication channel (e.g., a private or public network). "Transferring" an item means any means of moving the item from one place to another, either by physical transport or by other means (if possible), and, at least in the case of data, by physical transport of the medium holding the data or the medium on which the data is transmitted.

[0166] Modified nucleotides In certain embodiments, the disclosure provides oligonucleotides such as aptamers, which comprise two different types of base-modified nucleotides. In some embodiments, the oligonucleotide comprises two different types of 5-position modified pyrimidines. In some embodiments, the oligonucleotide comprises at least one C5-modified cytidine and at least one C5-modified uridine. In some embodiments, the oligonucleotide comprises two different C5-modified cytidines. In some embodiments, the oligonucleotide comprises two different C5-modified uridines. Non-limiting exemplary C5-modified uridines and cytidines are shown, for example, in Figure 21. Certain non-limiting exemplary C5-modified uridines are shown in Figures 22 and 24, and certain non-limiting exemplary C5-modified cytidines are shown in Figures 23 and 25.

[0167] Preparation of oligonucleotides Automated synthesis of oligodeoxynucleosides is routinely performed in many laboratories (see, for example, Matteucci, MD and Caruthers, MH, (1990) J. Am. Chem. Soc., 103:3185-3191, the entire contents of which are incorporated herein by reference). The synthesis of oligoribonucleosides is also well known (see, for example, Scaringe, SA, et al., (1990) Nucleic Acids Res. 18:5433-5441, the entire contents of which are incorporated herein by reference). As described herein, phosphoramidites are useful for the chemical synthesis of modified nucleosides into oligonucleotides, and triphosphates are useful for the enzymatic synthesis of modified nucleosides into oligonucleotides. For example, see Vaught, J. Det al. (2004) J. Am. Chem. Soc., 126: 11231-11237, Vaught, J. V. et al. (2010) J. Am. Chem. Soc. 132, 4141-4151, Gait, M. J. “Oligonucleotide Synthesis a practical approach” (1984) IRL Press (Oxford, UK), Herdewijn, P. “Oligonucleotide Synthesis” (2005) Humana Press, Totowa, NJ (each of these in their entirety is incorporated herein by reference).

[0168] "Target" or "target molecule" or "target" as used herein means any compound on which nucleic acids can act in a desired or intended manner. Target molecules may be, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, pigments, nutrients, growth factors, cells, tissues, or any part or fragment of any of the foregoing. Substantially, virtually any chemical or biological effector may be a suitable target. Molecules of any size can function as targets. Targets may also be modified in certain ways to increase the likelihood or intensity of interaction between the target and nucleic acids. Targets may also include any slight changes to a particular compound or molecule, for example, in the case of proteins, slight changes in the amino acid sequence, disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or any other operation or modification, such as complexing with a labeling component that does not substantially alter the identity of the molecule. A “target molecule” or “target” is one type or set of copies of a molecule or multimolecular structure that can bind to an aptamer. A “target molecule” or “target” refers to two or more sets of such molecules. Embodiments of the SELEX process where the target is a peptide are described in U.S. Patent No. 6,376,190, entitled “Modified SELEX Processes Without Purified Protein.” In some embodiments, the target is a protein.

[0169] As used herein, “competitive molecule” and “competitor” are used interchangeably and refer to any molecule that can form a nonspecific complex with a non-target molecule. In this context, the non-target molecule includes a free aptamer, and a competitor may be used, for example, to inhibit the nonspecific binding (recombination) of an aptamer to another non-target molecule. A “competitive molecule” or “competitor” is one type or one copy set of a molecule. A “competitive molecule” or “competitor” refers to two or more sets of such molecules. Examples of competitive molecules include, but are not limited to, oligonucleotides and polyanions (e.g., heparin, herring sperm DNA, salmon sperm DNA, tRNA, dextran sulfate, polydextran, debasic phosphodiester polymers, dNTPs, and pyrophosphates). In various embodiments, a combination of one or more competitors may be used.

[0170] As used herein, “non-specific complex” refers to a non-covalent association between two or more molecules other than an aptamer and its target molecule. Non-specific complexes represent interactions between classes of molecules. Non-specific complexes include complexes formed between an aptamer and a non-target molecule, a competitor and a non-target molecule, a competitor and a target molecule, and a target molecule and a non-target molecule.

[0171] In another embodiment, a polyanion competitor (e.g., dextran sulfate or another polyanion material) is used in a slow off-rate enrichment process to facilitate the identification of aptamers that are persistent in the presence of polyanions. In this context, a “polyanionally persistent aptamer” is an aptamer that can form an aptamer / target complex that is less likely to dissociate in a solution also containing a polyanionally persistent material than an aptamer / target complex containing a non-polyanionally persistent aptamer. Thus, a polyanionally persistent aptamer may be used in the implementation of an analytical method for detecting the presence, amount, or concentration of a target in a sample, the detection method comprising the use of a polyanion material (e.g., dextran sulfate) to which the aptamer is persistent.

[0172] Thus, in one embodiment, a method for producing a polyanion-resistant aptamer is provided. In this embodiment, after contacting a candidate mixture of nucleic acids with a target, the target and the nucleic acids in the candidate mixture are brought to equilibrium. A polyanion competitor is introduced and incubated in solution for a time sufficient for most of the high off-rate aptamers in the candidate mixture to dissociate reliably from the target molecule. Also, the aptamers in the candidate mixture that may dissociate in the presence of the polyanion competitor will be released from the target molecule. The mixture is split to isolate the high-affinity slow off-rate aptamers that remain associated with the target molecule, and all non-complexed material is removed from the solution. The aptamers can then be released from and isolated from the target molecule. Amplification of the isolated aptamers can also be performed, and additional rounds of selection can be applied to enhance the overall performance of the selected aptamers. This process may be used with a minimal incubation time if selection of slow off-rate aptamers is not required for a particular application.

[0173] Salt It may be convenient or desirable to prepare, purify, and / or handle the corresponding salts of the compound, such as pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts are described in Berge et al. (1977) “Pharmaceutically Acceptable Salts” J. Pharm. Sci. 66:1-19.

[0174] For example, if the compound is anionic or has a functional group that can be anionic (e.g., -COOH can be -COO - ), the salt may be formed with a suitable cation. Examples of suitable inorganic cations include alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ , but are not limited thereto. Examples of suitable organic cations include ammonium ions (i.e., NH4 +) and substituted ammonium ions (e.g., NH3R X+ NH2R X 2 + NHR X 3 + , NR X 4 + Examples of suitable substituted ammonium ions include, but are not limited to, ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those obtained from amino acids, such as lysine and arginine. A common example of a quaternary ammonium ion is N(CH3)4 + That is the case.

[0175] If a compound is cationic or has a functional group that can be cationic (for example, -NH2 is -NH3 + The salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those obtained from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrite, phosphoric acid, and phosphorous acid.

[0176] Suitable organic anions include, but are not limited to, those obtained from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphor sulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucoic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, and valeric acid. Suitable polymer organic anions include, but are not limited to, those obtained from the following polymer acids: tannic acid, carboxymethylcellulose.

[0177] Unless otherwise specified, references to specific compounds include their salt forms.

[0178] Other Embodiments In some embodiments, the method comprises: a) contacting a first test sample with a first set of aptamers to form a first mixture, wherein the first test sample is a Z% dilution of a biological sample, where Z is a 0.1% to 10% dilution of the biological sample, and at least A3 different aptamers are present in the first set of aptamers; b) contacting a second test sample with a second set of aptamers to form a second mixture, wherein the second test sample is a Y% dilution of a biological sample, where Y is less than Z, where Y is 0.001% to 0.1%, and at least A2 different aptamers are present in the second set of aptamers; c) contacting a third test sample with a third set of aptamers to form a third mixture, wherein the third test sample is an X% dilution of a biological sample, where X is 0.001% to 0.1%, and the aptamers A method is disclosed comprising: d) contacting the first, second, and third sets of aptamers, wherein at least one different aptamer A1 is present in the third set; e) incubating the first, second, and third mixtures to enable the formation of aptamer-protein complexes and removing the majority of aptamers that did not form aptamer-protein complexes; f) collecting aptamers from the aptamer-protein complexes by dissociating the aptamers; and f) detecting or quantifying the collected aptamers, wherein the majority of aptamers in the first set of aptamers, the second set of aptamers, and the third set of aptamers each have affinity for a different target protein in the test sample and are capable of forming aptamer-protein complexes with that target protein, A3 is greater than A2, A2 is greater than A1, and the sum of A1, A2, and A3 is at least 4,000.

[0179] In one embodiment, the sum of A1, A2, and A3 is at least 4,500 or 5,000.

[0180] In one embodiment, A3 is 50% to 90% (or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) of the sum of A1, A2, and A3, or 60% to 85% of the sum of A1, A2, and A3, or approximately 80% or 81% of the sum of A1, A2, and A3.

[0181] In one embodiment, A2 is 10% to 49% (or 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 49%) of the sum of A1, A2, and A3, or 12% to 35% of the sum of A1, A2, and A3, or 15% to 30% of the sum of A1, A2, and A3, or approximately 15% or 16% of the sum of A1, A2, and A3.

[0182] In one embodiment, A1 is 1% to 9% (or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%) of the sum of A1, A2, and A3, or 2% to 7% of the sum of A1, A2, and A3, or 3% to 6% of the sum of A1, A2, and A3, or approximately 3% or 4% of the sum of A1, A2, and A3.

[0183] In one embodiment, A3 is at least 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4200, 4270, 4500, 5000 (or 900-16,500 or 2000-15,000 or 3,000-12,000 or 4,000-10,000).

[0184] In one embodiment, A2 is at least 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820, 900 (or 500-3500, 700-2500, or 800-2000).

[0185] In one embodiment, A1 is at least 100, 110, 120, 130, 140, 150, 160, 170, 173 (or 100-700 or 100-650).

[0186] In one embodiment, at least different aptamers of A3 are distinguished from one another by at least one nucleotide difference and / or at least one nucleotide modification.

[0187] In one embodiment, at least different aptamers of A2 are distinguished from one another by at least one nucleotide difference and / or at least one nucleotide modification.

[0188] In one embodiment, at least different aptamers of A1 are distinguished from one another by at least one nucleotide difference and / or at least one nucleotide modification.

[0189] In one embodiment, at least different aptamers of A3, at least different aptamers of A2, and at least different aptamers of A1 are distinguished from each other by at least one nucleotide difference and / or at least one nucleotide modification.

[0190] In some embodiments, the system comprises: a) a first container having a first mixture containing a first test sample together with a first set of aptamers, wherein the first test sample is a Z% dilution of the test sample and at least A3 different aptamers are present in the first set of aptamers; b) a second container having a second mixture containing a second test sample together with a second set of aptamers, wherein the second test sample is a Y% dilution of the test sample, where Y is less than or equal to Z and at least A2 different aptamers are present in the second set of aptamers; c) a third container having a third mixture containing a third test sample together with a third set of aptamers; A third container is disclosed, wherein the third test sample is an X% dilution of the test sample, where X is less than or equal to Y, and at least A1 different aptamers are present in the third set of aptamers; the majority of the aptamers in the first set of aptamers, the second set of aptamers and the third set of aptamers have affinity for the protein in the test sample and are capable of forming aptamer-protein complexes, where A3 is greater than A2 and A2 is greater than A1; the sum of A1, A2 and A3 is at least 4,000; the system is used to detect the protein in the test sample, and the first, second and third test samples are different dilutions of the same test sample.

[0191] In one embodiment, the number of first capture reagents is approximately 100, 110, 120, 130, 140, 150, 160, 170 or 173; or 100 to 700; or 100 to 650 capture reagents.

[0192] In one embodiment, the number of second capture reagents is approximately 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820 or 900; or 500 to 3500; or approximately 700 to 2500; or 800 to 2000; or approximately 828 capture reagents.

[0193] In one embodiment, the multiple third capture reagents are approximately 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4200, 4270, 4500 or 5000; or approximately 900-16,500; or approximately 2000-15,000; or approximately 3,000-12,000; or approximately 4,000-10,000; or approximately 4271 capture reagents. [Examples]

[0194] The following embodiments are provided to better illustrate some embodiments of the present disclosure. However, they should not be construed as limiting the broad scope of the present disclosure. Those skilled in the art will readily adopt the principles underlying the present discovery and design a variety of mixtures without departing from the spirit of the present disclosure.

[0195] Example 1. Multiplex aptamer assay analysis of the sample Multiplex aptamer assays were used to analyze test and control samples and investigate the detection of target molecules from dried biological matrices. The multiplex analysis used in this experiment contained aptamers to detect approximately 5,000 proteins eluted from dried biological matrix samples with a low detection limit (median 1 pM), a dynamic range of approximately 7 log, and a median coefficient of variation of approximately 5%. Multiplex aptamer assays are generally described, for example, in Gold et al. (2010) Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery. PLoS ONE 5(12):e15004; and U.S. Patent Application Publications 2012 / 0101002 and 2012 / 0077695; and PCT Publication WO2019 / 246289.

[0196] Example 2: Optimization of multiplex assay diluents using dried blood spot (DBS) extracts This embodiment provides a description of optimizing the diluent of dried blood spot extract in a multiplex assay to maximize the number of analytes within the linear range while still maintaining the maximum median signal-to-background signal ratio in a multiplex assay.

[0197] In a multiplex assay format where multiple target proteins are measured by multiple capture reagents, the ability of a particular capture reagent to measure a particular target protein may be limited by the natural variation in the abundance of different target proteins (for example, high abundances of a target protein may saturate the assay, interfering with or reducing the ability of an assay to measure a low abundance of a target protein). To address this variation in the biological sample, the aptamer reagents are divided into at least two distinct groups, preferably three distinct groups, based on the abundance of each of their respective protein targets in the biological sample. The biological sample is diluted into at least two, preferably three distinct dilution groups, and separate test samples are created based on their relative concentrations detected by the protein target capture reagents. Thus, the biological sample may be diluted into high and low abundance target protein dilution groups, or into high, medium, and low abundance target protein dilution groups, with the least abundant protein target being measured in the lowest dilution group and the most abundant protein target being measured in the highest dilution group. In this example, the aptamers were grouped into three distinct mixtures, Dil1, Dil2, and Dil3.

[0198] Methods: Venous blood was collected from three healthy volunteers into two 4 mL BD Vacutainer tubes containing K2EDTA. One tube from each donor was centrifuged at 2,200 × g for 15 minutes to produce approximately 2 mL of platelet-poor plasma. The second tube was not centrifuged and was used for collecting and drying whole venous blood.

[0199] Whole blood (100 μL) was spotted onto Parafilm paper, and the tip of the Mitra VAMS® microsampling device was immersed in the whole blood sample until the sample collection device was completely saturated, followed by a 2-second residence time. This was repeated four times, once for each tip contained in the sample collection device's cramb package. The same procedure was repeated for plasma. The Mitra package device was closed and placed in a sealed container with eight DRIERITE desiccants at room temperature for 3 days.

[0200] Dry tips from each crumb were placed in 3 mL tubes (four per tube) containing 600 μL of elution buffer (80% plasma diluent and 20% PBS). The plasma diluent consisted of 50 mM Hepes, pH 7.5, 100 mM NaCl, 8 mM MgCl2, 5 mM KCl, 1.25 mM EGTA, 1.2 mM benzamidine, 37.5 μM Z-block, and 1.2% Tween-20. The samples were eluted at room temperature for 1 hour with shaking at 1000 rpm. After 1 hour, the buffer was gently mixed by pipetting up and down, and then diluted. Extracts (450 μL) from each donor were pooled into the Western Bound master stock and the plasma master stock.

[0201] To determine the appropriate dilution for Dilution Group 1, serial dilution (1:2) with plasma diluent was performed by transferring 400 μL of whole blood extract to the first 1 mL well of a 96-well plate, and then transferring 200 μL to a subsequent well containing 200 μL of 80% plasma diluent and 20% PBS. This was repeated 14 times. These served as the diluents for Dilution Group 1. Dilutions for Dilution Group 2 (40-fold dilution from Diluent Group 1) and Dilution Group 3 (100-fold dilution from Diluent Group 2) were then performed for the multiplex assay. [Table 1]

[0202] To determine the appropriate dilutions for dilution groups 2 and 3, serial dilutions were prepared in 1 mL volumes of 96-well plates. Serial dilution with assay buffer (1:2) was performed by starting with 800 μL of 20% extract in plasma diluent and PBS, transferring 400 μL of the extract, and mixing it with 400 μL of assay buffer. The multiplex assay was performed by directly pipetting 100 μL of each dilution into each catch plate.

[0203] The diluted solution was divided into two submissions for the multiplex assay. [Table 2] [Table 3]

[0204] result: The optimal concentration for performing the assay is within the linear range. This avoids problems with saturation, background noise measurements, or unknown factors that could interfere with the assay results. Therefore, it is important to choose a matrix dilution that places as many analytes as possible within the linear range.

[0205] The linearity algorithm examines each analyte across all 15 dilution steps to determine whether the signal doubles (plus or minus 25%) when the plasma concentration doubles. An analyte must contain at least three dilutions in which the signal is linear before being counted by the algorithm. For each dilution, the number of signals within the linear range is tallied to generate Figures 1, 2A, and 2B. The optimal concentration (dilution in which most analytes undergo a linear signal response) was determined by diluting dried blood spot (DBS) extracts with plasma diluent and PBS (80 / 20). Based on the plot shown in Figure 1, the optimal concentration range for dilution group 1 is between 0.3% and 5%. The optimal concentrations for dilution groups 2 and 3 (dilutions in which most analytes undergo a linear signal response) were determined by employing DBS extracted with plasma diluent and PBS (80 / 20) and diluting them with assay buffer. Based on the plots shown in Figures 2A and 2B, the optimal concentration range for dilution group 2 is between 0.005% and 0.038%, and the optimal concentration range for dilution group 3 is between 0.005% and 0.038%.

[0206] The maximum acceptable dilution of a sample in a multiplex assay is indicated by the loss of a significant amount of analyte signal, at which point the analyte signal falls below the level of background noise. Background in a multiplex assay can be defined as a multiple of the standard deviation of repeated signal measurements when analyzing only the buffer solution.

[0207] Every analyte in a multiplex assay exhibits a background signal in buffer, and all assays are performed using at least three buffer samples to determine the standard deviation of this background signal. For any given dilution of DBS or plasma, a signal is considered "above background" if it is 3.3 standard deviations above the mean background signal. In statistical terms, an analyte with a signal intensity of this magnitude is only 0.1% likely to be due to noise.

[0208] As shown in Figure 3A, in dilution group 1, where the DBS extract concentration is 20% or 10%, there is little signal loss due to noise. In dilution group 1, no significant number of analytes are lost due to background in the DBS until the extract is diluted to less than 1.25%. As shown in Figure 3B, in dilution group 2, there is little signal loss due to noise until the extract concentration falls below the DBS extract concentration of 0.3%. In dilution group 2, no significant number of analytes are lost due to background in the extract until the extract is diluted to less than 0.038%. As shown in Figure 3C, in dilution group 3, there is virtually no signal loss due to noise until the extract concentration falls below the whole blood extract concentration of 0.018%. In dilution group 3, no significant number of analytes are lost due to background in the whole blood extract until the extract is diluted to less than 0.005%.

[0209] Example 3: Accuracy of dried blood spots Materials: Whatman903 (trademark), Guthrie card. Plasma diluent. PBS. BD Vacutainer tubing (K2EDTA).

[0210] Methods: Blood was collected from a small group of six donors using K2EDTA vacuum tubes, and the blood was divided into five assay replicas to obtain in-plate assay CVs. Plasma was also collected from these donors, and three replicas were analyzed.

[0211] Blood collection / preparation / handling: Blood samples were collected from six donors and placed into one of three K2EDTA, BD vacutaine tubes (whole blood, plasma, and spare). The plasma tubes from each donor were centrifuged at 2,200xg for 15 minutes, and approximately 2 mL of plasma (PPP) was extracted and stored in two 1.5 mL Eppendorf tubes, which were immediately frozen on dry ice.

[0212] Filling and drying gas cards: Spots were made on three Guthrie cards (Whatman903®, LOT7211021) for each donor. Using a micropipette, 40 μL of whole blood was spotted in the center of the dotted circle on three different Guthrie cards (five spots per card, three cards per donor). The cards were dried in a laminar flow hood for one hour (folding the cards to ensure that the back of the spotted paper did not come into contact with any surface), and then placed in six different sealed bags (three cards per bag), each bag containing two desiccants. The cards were dried at room temperature for three days.

[0213] Elution: After drying for three days, one Guthrie card was removed from each bag. The remaining cards were stored at -20°C as a backup. The dried blood spots were carefully removed from the cards using scissors, ensuring 100% removal. Five whole blood circles were placed in five 2 mL screw-cap tubes. 1.6 mL of extraction buffer (80% plasma dil and 20% PBS) was added to each tube, and they were shaken at 1000 rpm at room temperature for 1 hour. This was done for each donor, for a total of 30 tubes.

[0214] The samples from dilution group 1 (200 μL from each extract) were transferred to 30 matrix tubes and frozen at -80°C. An additional 600 μL was transferred to 30 different matrix tubes and used for dilution groups 2 and 3.

[0215] One plasma sample from each donor was thawed, and 50 μL from each donor was transferred to three matrix tubes (sample replication). An additional 200 μL of extraction buffer was added to dilute to 20%. Three plasma QCs, eight blanks (five consecutive numbers unrecorded), and five calibrators were similarly diluted to 20% with extraction buffer. One diluted sample was thawed for the multiplex assay. All extracts and plasma samples were stored on ice.

[0216] The linearity of the multiplex assay signal increases with the optimization dilution of whole blood to a lower concentration than the optimal dilution of plasma or serum. Dried whole blood samples were prepared from all six donors at dilutions of 2.5%, 0.05%, and 0.005%. Liquid plasma was prepared from all six donors at dilutions of 20%, 0.5%, and 0.005%. The samples were refrozen and subjected to analysis by multiplex assay. [Table 4]

[0217] Results: The overall average precision of all tested analytes is shown in Figures 4A and 4B. The population coefficient of variation (CV) of the analytes showed a bimodal distribution, with the majority of analytes (>70%) having a CV of less than 5%. The second population of analytes had a broader CV distribution and a higher degree of inaccuracy. As shown in Figure 4A, more than 95% of the analytes showed an inaccuracy of less than 15% between replicas.

[0218] The inaccuracies in whole blood analysis can be divided into two groups: high-precision and moderate-precision. The high-precision group represents the majority of the tested analytes, densely distributed with a CV of around 3% and a distribution of 2.5% to 7.5%. Moderate-precision analytes account for approximately 25% of the library, ranging from 7.5% to 17.5%, centered around 11%. The bulk of these analytes has a CV of less than 15%, which is promising for medical applications.

[0219] Both CV populations can be seen in the CDF plot shown in Figure 4B. The CDF plot is an integral histogram (area under the curve) and conveys the same information.

[0220] Example 4: Robustness of whole blood protein signals determined by protein spikes Methods: A large protein library was spiked into DBS extracts to determine whether an increase in protein concentration could be measured between analytes in this matrix.

[0221] Blood collection / preparation / handling: Venous blood was collected from donor 1301 into two 6 mL BD Vacutainer tubes containing K2EDTA, stored at room temperature, and processed within 2 hours. One tube was centrifuged at 2,200 × g for 15 minutes to create a platelet-poor plasma layer.

[0222] Filling Mitra devices: 100 μL each of whole blood and plasma were spotted onto Parafilm paper. The tip of the Mitra VAMS collection device was placed in the whole blood and plasma until the collection device was completely saturated, and then left in for an additional 2 seconds after saturation. This was repeated 16 times for the whole blood (4 crams) and 4 times for the plasma (1 cram). These devices were placed in a sealed container with 2 DRIERITE desiccants per cram at room temperature for 3 days.

[0223] Protein doping of WB extract Three days later, whole blood-treated Mitra tips were immersed in plasma extraction buffer (20% PBS, 80% plasma diluent, 300 μL per tip) for 1 hour while shaking. A 10-fold (10%) extract was dispensed into aliquots in double strips, and the protein spike library (Appendix II) was spiked. Plasma-filled tips were prepared in the same manner. Samples were prepared in 150 μL aliquots by replacing the spike library with replacement rates of 10%, 5%, 1%, and 0%.

[0224] The samples were frozen and subjected to a multiplex assay.

[0225] Results: Two non-spiked dried whole blood samples show a high level of agreement between dried whole blood extracts collected by the Mitra VAMs system when derived from the same donor. When the non-spiked whole blood samples are compared to those spiked from a protein library with approximately 5 nM or 5% substitution rates, the added proteins "lift" the line of identity. This trend is similarly observed in spiked plasma samples.

[0226] Titration curves of diplicated whole blood and plasma extracts were measured while increasing the protein substitution percentage. Subtracting the background signal reveals a sigmoid-type dose-response pattern. Interestingly, not all elements of the spike library appear to undergo signal increments. This may be due to the fact that these proteins denature more readily in whole blood or undergo a higher level of inaccuracy. Additionally, there may be sensitivity issues with a subset of proteins in the spike library, with certain members not being detected.

[0227] The median signal increase for spike library targets was measured and compared between whole blood and plasma. A median signal increase of 232 was obtained relative to baseline. The signal increase was nearly identical between whole blood and plasma.

[0228] The signal increase for each of the 232 spike analytes between 0 nM and 5 nM in plasma was divided by the equivalent increase in whole blood. If plasma and whole blood respond similarly to protein spikes, the signal distribution should be around 1. Both copies of whole blood demonstrate that, for each analyte, the two matrices respond similarly to proteins spiked in the extract.

[0229] Example 5: Extraction Optimization Study The dried sample needs to be reconstituted to achieve buffer conditions nearly identical to those of a typical plasma or serum sample subjected to the assay. In a typical multiplex assay scheme, one portion of the biological sample is added to four portions of serum diluent (50 mM Hepes (pH 7.5), 100 mM NaCl, 8 mM MgCl2, 5 mM KCl, 1.25 mM EGTA, 1.2 mM benzamidine, 75 μM Z-block, and 1.2% Tween-20) or plasma diluent (50 mM Hepes, pH 7.5, 100 mM NaCl, 8 mM MgCl2, 5 mM KCl, 1.25 mM EGTA, 1.2 mM benzamidine, 37.5 μM Z-block, and 1.2% Tween-20), and 100 μL is added to a well containing one bead of the mixture. Therefore, for a 30 μL dry sample, extraction was performed in 150 μL of buffer containing 4 parts diluent and 1 part volume expander. The volume expanders tested were water and phosphate-buffered saline (PBS). An experiment was conducted to compare these two types of volume expanders. Here, 12 serum samples (QC2) were thawed, Mitra VAMS samples were collected, and dried at 4°C for 2.5 days. Half of the tips were extracted with water at room temperature (RT) for 1 hour, and the other half were extracted with PBS at room temperature for 1 hour. Extracts from the two sets of tips were combined to obtain three sets of samples for each condition. Multiplex assays from the two extraction methods were compared with each other and with a standard QC2 serum sample (undried) that underwent an additional freeze-thaw cycle to equalize the number of freeze-thaw cycles for all samples.

[0230] The matching plots of relative fluorescence units (RFU) between the two extraction conditions show almost no difference (Figure 5). Similarly, the cumulative distribution plots of median RFU for the standard method and the two extraction conditions from dry spots are nearly superimposed, and there is a slight advantage in fitting symmetry when using the PBS method (not shown). Furthermore, the cumulative distribution plot of the coefficient of variation (%CV) also reveals a slight advantage when using the PBS method. Both methods for extracting dry spots showed an increase in %CV compared to the standard method. PBS and water are suitable volume-expanding agents for the extraction of dry samples.

[0231] Example 6: Determination of protein extraction time Methods: Ten QC1 serum samples and ten QC2 serum samples (v4.0) were thawed, Mitra samples were collected, and dried at 4°C for 2.5 days. Samples were extracted using PBS as a volume expander and serum diluent as described above. A double Mitra tip was removed from each QC sample, placed in a microtiter well, and extracted at room temperature for 10, 30, 60, 120, or 180 minutes with rotation. The extracts from the double tips (at each time point) were mixed and frozen together with standard QC1 and QC2 serum samples (undried) that had undergone additional freeze-thaw cycles to equalize the number of freeze-thaw cycles, before being tested in a multiplex assay. As a blank control, Mitra VAMS samples of the extraction buffer were collected, dried in the same manner, and tested in a multiplex assay with a standard multiplex assay blank.

[0232] Results: The recovery of the analyte signal was found to be almost independent of the extraction time over the range of 10 - 180 minutes. Figure 6 shows the median (interquartile range) of the ratio of the analyte signal from the dried spot (Mitra) to the analyte signal obtained from the same samples analyzed by the multiplex assay method (not dried). The median ratio changed little over the range of extraction times and was slightly greater than 1 in all cases. The cumulative distribution plots of the RFU signals obtained for dried serum samples extracted at room temperature for the indicated lengths of time showed that the analyte signals obtained from the five extraction times were almost superimposable on each other and almost superimposable on the analyte signal obtained from a standard serum sample (not dried). The analyte signals obtained from the QC1 blank samples that had undergone the drying and extraction process were superimposable on the signals obtained using a standard multiplex blank sample. Extraction times between about 10 minutes and 200 minutes are acceptable for the multiplex assay. In some embodiments, an extraction time of 60 minutes is used.

[0233] Example 7: Demonstration of Accuracy and Consistency for Dried Serum Methods: To better understand the reproducibility of dried plasma spots, experiments were conducted using fresh serum obtained from 6 normal volunteers, where all Mitra VAMS samples were dried under the same conditions. Blood was collected and allowed to clot at room temperature for 1 hour. The clotted samples were centrifuged at 2,200 × g for 15 minutes, and aliquots of the serum were transferred to Eppendorf tubes. From each serum sample, duplicate Mitra VAMS chips (30 μL) were collected and placed in a sealed container containing DRIERITE desiccant and dried at room temperature in the dark for 4 days. In addition, aliquots of fresh serum were frozen until assayed for comparison with the dried spots. Extraction was performed for 1 hour using water as the volume expander, the extracts from the duplicate Mitra VAMS chips were combined, and frozen at -80 °C until assayed. The extracts and controls were assayed by multiplex assay.

[0234] Results: Agreement plots between the standard method and the dried method for each of the six individual serum samples show that all analytes still have a signal after drying and reconstitution. The agreement is not perfect, but most importantly, whether the ratio of the signals of each analyte is constant between samples, e.g., whether a certain analyte always returns a signal that is half or twice the signal obtained by the standard method. These ratios are constant, i.e., there is an acceptable CV for all ratios, so the results of the dried spots can be mathematically transformed, i.e., "lifted", to match the expected results of the non-dried samples.

[0235] Determine the %CV of the ratio for each analyte and plot it as a cumulative distribution function. The median %CV of the ratio is 6.99, and the 10th percentile and 90th percentile are 3.59 and 18.8, respectively, demonstrating that most analyte ratios remain reasonably constant between samples when dried and processed under the same conditions.

[0236] Example 8: Stability of dried plasma to temperature changes based on drying time and temperature Methods:

[0237] Blood collection / preparation / handling: Venous blood was collected from healthy volunteers into two 6-mL BD Vacutainer tubes containing K2EDTA, stored at room temperature, and processed within 2 hours. One tube was centrifuged at 2,200 × g for 15 minutes to remove platelet-poor plasma. Plasma (400 μL) was frozen at -80 °C for use as a control.

[0238] Filling the Mitra device: 80 μL of whole blood or plasma was spotted onto parafilm paper, and the tip of the Mitra VAMS was placed into the whole blood and plasma until the sample collection device was completely saturated (about 2 seconds).

[0239] Drying conditions: Plasma samples were placed in sealed bags (two drierite containers per Mitra crumb) and dried under three different temperature conditions: -20°C, 4°C, and 25°C. After drying the Mitra VAMS devices for 1–3 days, they were removed and extracted with 20% PBS and 80% plasma diluent, or placed at stress temperatures.

[0240] Stress temperature: After drying for 1-3 days, the samples were removed from the drying conditions and allowed to reach room temperature. They were then placed under stress conditions of -20°C, 37°C, 50°C, or 60°C for either 1 or 2 days. After either 1 or 2 days, the samples were removed and the plasma was extracted with 20% PBS and 80% plasma diluent.

[0241] Extraction: Mitra tips were individually placed in Eppendorf tubes containing 300 μL of extraction buffer (80% plasma diluent and 20% PBS), and extraction was performed for 1 hour with shaking. The contents were transferred to matrix tubes and frozen at -80°C. [Table 5]

[0242] Results: Plasma-filled Mitra VAMS devices were dried at different temperatures, stressed at different temperatures as shown above, and compared via a match plot with liquid plasma stored at -80°C. Best results are achieved when plasma is dried at a cold temperature, which may simply be due to the higher degree of temperature stability found in refrigerators and freezers. While the agreement between all dried samples is high, dried plasma extracts best match liquid plasma when samples are dried at 4°C or -20°C rather than room temperature, although the agreement at room temperature is also acceptable for multiplex assays.

[0243] The agreement of cardiovascular disease analytes (CVD2) between samples dried at 4°C, -20°C, and room temperature was measured. The CVD2 test has 28 analytes, and a subset of these proteins appears to best match liquid plasma when dried at -20°C. In multiplex assays, agreement with liquid plasma is acceptable even with samples dried at 4°C and room temperature.

[0244] Plasma was dried at 4°C, then subjected to temperature stress by exposure to -20°C, 37°C, 50°C, or 60°C, and subjected to a multiplex assay compared to liquid plasma. Figure 7A shows that drying at 4°C for at least one day appears to protect the sample from freezing. Drying the plasma at 4°C for at least two days appears to protect it from freezing and exposure to 37°C for at least one day. Figure 7B shows the results for plasma dried at room temperature and then subjected to temperature stress. Drying at room temperature results in a lower agreement with liquid plasma compared to drying at 4°C. As shown in Figure 7C, the advantage of drying at -20°C lies between drying at 4°C and drying at room temperature. Drying the sample at 4°C, -20°C, and room temperature all provide samples suitable for multiplex assays.

[0245] Example 9: Determination of analytes from DBS samples measuring changes in plasma proteins research methodology A cohort of 16 healthy donors was sampled using three Tasso M20 smart sampling devices, one EDTA (plasma) BD vacuum, and one serum BD vacuum tube. After processing, plasma and serum samples were transferred to cryotubes and frozen at -80°C. The drying tabs of the Tasso M20 devices were immediately removed and placed in sealed plastic containers with desiccant. One device from each donor was placed in a refrigerated room at 4-8°C for 2 hours, then packaged for transport, while the remaining two were left at room temperature. The packages were immediately transported overnight to SomaLogic in Boulder. Two days after blood collection, the refrigerated Tasso devices and the room-temperature device were opened and extracted with 80% serum diluent and 20% PBS for 1 hour. The extracts were frozen at a 2.5% concentration at -80°C. The third Tasso device was kept at room temperature for 7 days after blood collection, and then extracted in the same manner. The SOMAScan® assay was performed on all samples on the same plate to minimize inter-assay variability. Serum and plasma samples were diluted according to 20%, 0.5%, and 0.005% dilution protocols. Dried blood spots were diluted using 2.5%, 0.05%, and 0.005% dilution protocols.

[0246] result Distribution of correlations between matrices Figures 8A–8D compare the concordance rates between frozen plasma versus dried blood spots (8A), frozen serum versus dried blood spots (8B), frozen serum versus frozen plasma (8C), and two pooled DBS samples (8D). In the comparison between (8A) and (8B), cellular proteins in DBS tend to outperform the signaling potential of proteins in plasma or serum, which are often present in smaller amounts. This contrasts with two DBS replicas (8D) or even the matrix of frozen serum versus frozen plasma (8C) prepared from pooled samples.

[0247] Based solely on the data in Figures 8A–8D, one might be tempted to conclude that DBS is completely inconsistent with both the plasma and serum matrices. Adding whole blood cellular components, which contain proteins in higher abundances than in plasma, obscures most plasma protein measurements in DBS. However, some proteins are so highly concentrated in plasma and / or depleted intracellularly that the additional protein abundance does not obscure the signals from the whole blood liquid component. Figure 9 shows that while most DBS signals are inconsistent with plasma, some of them exhibit matrix-to-matrix correlations. The cumulative distribution function of all DBS Pearson correlation coefficients for frozen plasma is shown on the left, along with the cumulative distribution function for serum on the right. For comparison, plasma vs. serum correlations are included (direct and randomized). While the masking effect of whole blood cellular components is very clear in Figure 9, some analytes appear to correlate strongly with both plasma and serum.

[0248] Number of significant correlations between serum / plasma and DBS Statistically significant correlations between v4.1 analytes in plasma, serum, and DBS were determined using two methods. In this example, analytes are defined as individual measurements in the SOMAScan® assay: thus, some blood proteins are measured by multiple “analyte signals”.

[0249] One method (correlation significance by paired observations) involved using a significance threshold for p-values ​​generated by combinations of similar observations. As Figure 9 shows, plasma and serum are not independent of each other but correlate with each other. Observations in one matrix are useful for observations in the other matrix. In addition, similar studies, referred to herein as “DBS accuracy studies” (see below), showed correlations between DBS prepared from anticoagulant treatment, IV-collected blood, and frozen plasma. The p-values ​​from the correlations found from each of these observations were combined and compared to a significance value of 0.1 (multiple comparisons and adjustments for observations as described below). The results can be seen in Tables 1, 2, and 3.

[0250] The second method used to determine which of the correlations shown in FIG. 9 are significant employs the false discovery rate. In simulations with randomization of donors across the entire matrix, nearly 3 million spurious correlations were generated for each DBS condition / matrix match. From these, the average number of spurious correlations for the menu of 7335 human analytes can be calculated for a given threshold of the Pearson correlation coefficient. Next, the number of significant correlations is calculated by subtracting the average number of spurious correlations due to chance from the number of correlations having coefficients exceeding the threshold. The detailed method can be found below, and the results are shown in Table 4.

[0251] Number of significant correlations using corresponding observations The number of significant correlations between plasma / serum and DBS occurring under various drying conditions is presented in the following table using corresponding observations without, corresponding observations across all plasma and serum, and corresponding observations across all plasma and serum combined with results from the DBS accuracy study. The detailed method can be found in "Correlations by Corresponding Observations" below. The "average" condition found in each of these tables is the result of treating all three conditions between DBS and plasma and serum as technical replicates and then taking the average of those three correlations.

Table 6

[0252] In contrast to a single correlation between DBS and serum or plasma, a significant increase in the number of significant correlations can be seen when corresponding observations are used. Note that the Holm-Bonferroni correction used in this method is a conservative filter for spurious correlations when comparing across a large number of analytes. Combining the observed values in serum and plasma more than doubles the number of significant correlations. Adding the DBS accuracy study to the analysis increased the number of significant analytes by an average of 25 analytes. This small benefit is understandable considering the small cohort involved in that study.

Table 7

[0253] The number of significant correlations (above) between serum and plasma and all three DBS conditions was used to classify the dilution groups. [Table 8]

[0254] Dilution group 1 contains 6027 human analytes, dilution group 2 contains 1106 human analytes, and dilution group 3 contains 190 human analytes. When viewed as a percentage of the total number of analytes in each group, the abundance of significant analytes in the SOLMAscan® assay increases with increasing dilution factor. This may be due to the presence of many proteins secreted from plasma and found in high abundance in dilution groups 2 and 3, which can be more easily read over interference from cellular analytes in whole blood.

[0255] In Figure 10A, signals from DBS retained for 7 days before extraction are plotted against signals from the corresponding frozen plasma. Analytes colored gray are those generated from corresponding observations in plasma, serum, and DBS precision studies (500 total) and considered to correlate with plasma. Plasma analytes are concentrated in the lower half of the graph, which is consistent with previous analyses.

[0256] Number of significant correlations using FDR filter The following results are determined by selecting an arbitrary false detection rate (FDR) value and counting the number of analytes that satisfy the Pearson coefficient threshold required to achieve that value. [Table 9]

[0257] Table 4 compares all DBS conditions for plasma and serum, showing the number of matrix correlations based on the tolerance for false positives within the population. The total population of analytes can be calculated by adding the significance correlations (SigCorr's) to the false positives (FalsePos). Dividing the number of false positives by this number gives the FDR. The minimum Pearson coefficient that a correlation can have and is considered significant is obtained by the "minimum Pearson" value. Conditions and tolerances that result in more than 500 analytes are shown in bold.

[0258] In Figure 10B, signals from DBS retained for 7 days prior to extraction are plotted against signals from the corresponding frozen plasma. The gray-colored analytes were generated using an FDR-based algorithm (described below for correlation significance filtered by false-find rate) and produced 557 significant correlations. The plasma analytes were clustered similarly by both methods (Figures 10A and 10B), demonstrating a high level of agreement between the two.

[0259] Characterization of analytes Comparing the groups of analytes labeled as significant by each method reveals considerable overlap (Figure 11). This comparison provides confidence that either method, or a combination of methods, can be used to produce a larger list of significant correlations.

[0260] In Figure 11, the left-hand region shows a significant correlation between frozen plasma and DBS (7-day drying time), selected by choosing a Pearson threshold that achieves a 5% FDR. The right-hand region shows the number of the same correlations using three-way correspondence observations between DBS and plasma and serum, as well as observations from DBS precision studies. The overlap of these two results gives us confidence that these analytes are indeed measuring plasma proteins.

[0261] Once a list of significant correlations between DBS and plasma was generated, we investigated the characteristics of plasma / serum analytes that were likely to be measurable by DBS. Consistent plots between plasma and DBS (Figures 12A–12D), excluding all signals that did not correlate significantly, showed direct relationships between the matrices of these selected analytes. Analytes with lower signal intensity appeared to have a weaker linear relationship with DBS. While we do not wish to be bound by any particular theory, this may be due to background from DBS having a more pronounced effect on plasma signals of lower intensity. Mapping significant analytes to a coincident plot between serum and plasma (Figures 13A–13D) revealed that analytes with greater agreement between these two matrices were also more likely to correlate between plasma / serum and DBS.

[0262] In Figures 12A–12D, each coincident plot compares the relationship between plasma signals and DBS signals in analytes characterized by a significant correlation between DBS (7-day dried) and frozen plasma. As the acceptable FDR increases, as follows: (12A) 5%, (12B) 10%, (12C) 15%, and (12D) 20%, the number of analytes also increases.

[0263] As shown in Figures 13A–13D, the gray analytes have Pearson coefficients that exceed the threshold where the FDR is (13A) 5%, (13B) 10%, (13C) 15%, and (13D) 20% (7-day dried DBS vs. frozen plasma). These are mapped to the serum vs. plasma signal agreement plot. Figures 13A–13D show that analytes with higher agreement between plasma and serum are more likely to agree between plasma and DBS.

[0264] Another way to categorize a list of significant correlations is by classifying the function of the analytes measuring plasma proteins. Princeton University has created a list of gene ontology terms for the human proteome. One way to use the Princeton University dataset is to compare the abundance of selected plasma analytes associated with each category to the abundance of the analytes in the human proteome. Figure 14 shows that the proteins used in plasma measurements are involved in immunology, cell adhesion, cell motility, defense responses, inflammatory responses, cell junctions, circulatory system, wound healing, and the extracellular matrix. The percentages of plasma analytes (left) categorized into various categories of biological function are compared to the abundance of proteins sorted into those categories in the entire human proteome (right). Each of these categories is selected to show enrichment (more than 2x) in plasma proteins identified by paired observation and FDR filtering.

[0265] method Correlation based on paired observations: Several observational findings can be used to address the question of how many plasma / serum analytes have a significant correlation with whole blood. In this study, each patient was sampled through two additional matrices (frozen plasma and serum) in addition to DBS and compared with each DBS sample. Plasma and serum are not independent matrices, and more than 2500 analytes showed a significant correlation between the two matrices in this cohort (α=0.1, Bonferroni-Holm corrected). Therefore, the probability that the correlation between plasma and DBS is significant can be corrected by the measured significance (p-value) of the same correlation between serum and DBS.

[0266] In another study (referred to as the DB precision study) involving a small cohort of six donors, blood was collected intravenously into EDTA vacuum tubes and divided into five assay replicas. Plasma was also collected from these donors, and three replicas were analyzed to determine which plasma analytes were measurable in whole blood. Plasma analytes correlating with DBS in the DBS precision study can support similar observations in this study. A detailed description of the methods used in this study is provided in the protocol section below. Analyte signals were normalized to the median.

[0267] To ignore the null hypothesis that individual correlations are due to chance, a method was developed to evaluate consistent observations across multiple matrices and experiments. Positive correlations were determined using a cutoff of the Pearson coefficient, which was determined by calculating the correlations between matrices after randomization across the entire library (to prevent further matching of donors). This was performed 401 times, yielding a total of 2.94 million random correlations. This number was considered sufficient because the iterative calculations demonstrated high accuracy.

[0268] Figure 15 shows a cumulative distribution function (CDF) plot of the distribution of Pearson coefficients generated by comparing random plasma / serum values ​​with 7-day dried DBS. The dotted line represents the cutoff (0.45) where 95% of random Pearson coefficients fall. The solid line represents the cutoff (0.56) where 98% of random Pearson coefficients fall. In DBS accuracy studies, the likelihood of accidental donor alignment is much higher when randomizing DBS and plasma from 6 individuals than when randomizing from 16 individuals; therefore, the 95% and 98% thresholds are represented by much larger Pearson coefficients (0.75 and 0.86, respectively). Considering these limiting values, the 95% threshold was used.

[0269] For a correlation to be considered valid, the Pearson coefficient must be greater than the 98% cutoff for plasma-serum correlations and the 95% cutoff (0.75) for DBS-precision studies. Since there was no functional difference between the results obtained from the 98% or 95% cutoffs in 16 donor studies, a stricter threshold was used. The threshold value for the Pearson coefficient was determined individually for all matrices and conditions. A p-value of 1 was manually assigned to correlations where the correlation coefficient was below the threshold. The p-values ​​obtained from the paired plasma-serum correlations were multiplied to obtain the probability that both results were due to chance. This is illustrated in Table 5. [Table 10]

[0270] Two comparative versions of the algorithm are shown in Table 5 as an example. In this representation of the paired observation algorithm, the threshold for significant correlation is shown at the bottom of the table. Pearson coefficients below the threshold are highlighted in italics, while Pearson coefficients above the cutoff are highlighted in bold. The p-values ​​of Pearson coefficients below the acceptable threshold are given a value of 1, so they do not contribute to the overall probability of spurious correlation when multiplied with the corresponding values ​​in the other matrices. The obtained p-values ​​are then corrected using the Bonferroni-Holm α. 2 Compare this to (α=0.1). In this algorithm, α is a power of the number of correlations being combined (2 in this case). Thus, to classify plasma analytes, a significant correlation in only one matrix is ​​sufficient. Alternatively, multiple weak correlations can be combined to identify a plasma analyte. By reducing alpha by a power of the number of correlations, we prevent p-values ​​from simply combining until a p-value becomes significant by chance. Results indicated by * are examples of analytes that did not meet the acceptance criteria when using only plasma or serum correlations. Values ​​indicated by # remained unchanged.

[0271] Correlation significance filtered by false detection rate: The null hypothesis can also be rejected by requiring that every significant set of correlations in the dataset contains the maximum false detection rate for spurious correlations. Randomized correlations can be used as described above to select a Pearson coefficient threshold to adjust for the false detection rate of spurious correlations. A specific example of random correlations between plasma and 7-day dried DBS shows that 99.9% of Pearson coefficients are less than 0.87. If that cutoff is used in this example, the false positive rate, or the probability that individual correlations are due to chance, is expected to be 1 in 1000. If analyte correlations are sorted from the maximum to the minimum Pearson coefficient, the mean number of spurious correlations in the group with the minimum coefficient value can be determined. Dividing by the number of analytes in this group gives the false detection rate (FDR).

[0272] The following method determines the number of important analytes that can be classified as having a significant correlation between matrices at various confidence levels. The probability distribution of false positives or spurious correlations is given by Equation 1.

number

[0273] When comparing plasma samples with DBS samples dried for 7 days, probability curves showing different probability density functions for the possible number of analytes exhibiting spurious correlations were generated using Pearson cutoffs of 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, and 0.99, and are shown in Figure 16. A higher acceptable Pearson cutoff indicates a lower number of false positives. These curves can be used to determine the weighted average of the expected number of false positives given a specific threshold.

[0274] The weighted average of false correlations was calculated by multiplying the number of false positives by their probability density functions (shown in Figure 17) and finding the area under the resulting curve (Equation 2).

number

[0275] This integral can only be estimated using integer values ​​of FP by employing the trapezoidal rule to find the area under the curve. In practice, the precise scale of the integral can be determined by bounding the integral to a region where the curve is considerably above the x-axis.

[0276] The weighted average of spurious correlations for a given threshold of the Pearson coefficient is given by the white circles and derived from the area under the curve obtained by multiplying the number of spurious correlations by the probability density function (Equation 2 and Figure 18). The number of significant correlations (black circles) is the sum of all correlations between the matrix (where the Pearson coefficient exceeds the cutoff) and the DBS sample (in this case, 7-day dried DBS) minus the number of spurious correlations. The number of significant correlations appears to increase linearly.

number

[0277] Figure 19 shows the false detection rate of spurious correlations (calculated by Equation 3), which clearly illustrates the dangers of arbitrarily setting a high cutoff for the Pearson coefficient when attempting to eliminate spurious correlations. Any Pearson threshold that moves the FDR beyond its minimum value reduces the number of analytes and increases the rate of spurious correlations.

[0278] As shown in Figure 20, each FDR (x-axis) can be mapped to the number of significant analytes (total phase function - pseudophase function). The curve shown in Figure 20 was created from the correlation between plasma and DBS dried for 7 days. The values ​​in Table 4 were calculated in this manner.

[0279] DBS accuracy study protocol Whole blood samples were dried and extracted using a Whatman903® Guthrie card and closely aligned with a device that could be used in situ. After drying the whole blood spots, they were cut from the card and extracted with plasma diluent and PBS. A 2.5% extract was used to allow for a volume of extraction buffer exceeding 1 mL. This minimized the percentage of volume distortion caused by cutting Guthrie cards of varying sizes. After 1 hour, the extract was transferred to a matrix tube and frozen at -80°C. On the day of the assay, the samples were thawed and diluted to concentrations of 2.5%, 0.05%, and 0.005%. By the end of the extraction, the Guthrie cuts appeared to be completely free of whole blood. Plasma samples were immediately frozen at -80°C and diluted according to standard procedures.

Claims

1. A method for preparing biological samples for a multiplex assay, Depositing the biological sample containing multiple target molecules onto a collection device; This includes: drying the biological sample on the collection device for a certain period of time in order to stabilize the dried sample before any temperature fluctuations; The method comprising drying the biological sample at or below room temperature for at least 4 hours so that the target molecule in the dried biological sample can be detected by the multiplex assay.

2. The method according to claim 1, wherein the biological sample is dried at room temperature, 4°C to 8°C, or -20°C.

3. The method according to claim 1 or 2, wherein the dried biological sample is stored at or below room temperature before being detected by the multiplex assay.

4. The method according to any one of claims 1 to 3, wherein the biological sample is stored at approximately 4°C to 8°C or -20°C.

5. The method according to any one of claims 1 to 4, wherein the biological sample is dried for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, 1 day, at least 2 days, or at least 3 days.

6. The method according to any one of claims 1 to 5, wherein the biological sample is selected from plasma, serum, urine, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, sputum, tears, mucus, nasal lavage fluid, nasal aspirate, semen, saliva, peritoneal lavage fluid, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, papillary aspirate, bronchial aspirate, bronchial swab, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid.

7. The method according to any one of claims 1 to 5, wherein the biological sample is selected from plasma, serum, urine, and whole blood.

8. The method according to any one of claims 1 to 7, wherein the plurality of target molecules are selected from proteins, peptides, carbohydrates, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, bacteria, metabolites, cofactors, inhibitors, drugs, pigments, nutrients, growth factors, cells, and tissues.

9. The method according to any one of claims 1 to 8, wherein the dried biological sample is homogenized by the drying process.

10. The method according to any one of claims 1 to 9, wherein the plurality of target molecules can be extracted from the collection device after the biological sample has dried and can be detected in the multiplex assay.

11. A method for detecting multiple target molecules, the following: Extracting target molecules from a dried biological sample using a collection device; Diluting the extracted target molecule in a first diluent and a second diluent; The first diluent is brought into contact with the first capture reagent to form a first capture reagent affinity complex with the target molecule, in the presence of the target molecule in the first diluent; The second diluent is brought into contact with the second capture reagent to form a second capture reagent affinity complex with the target molecule, in the case that the target molecule is present in the second diluent; The first diluted sample and the second diluted sample are incubated separately to enable the formation of a capture reagent affinity complex; the formation of the capture reagent affinity complex is such that each of the first capture reagent affinity complex and the second capture reagent affinity complex is immobilized on a separate first solid support; The first capture reagent affinity complex is released and captured on the second solid support; After releasing the first capture reagent affinity complex, the second capture reagent affinity complex is released and captured on the second solid support; The method comprising detecting the presence or level of the first capture reagent and the second capture reagent of the first capture reagent affinity complex or the second capture reagent affinity complex, or detecting the presence or amount of the first capture reagent affinity complex or the second capture reagent affinity complex.

12. The method according to claim 11, wherein the target molecule is extracted from the collection device in the formulation for at least 5 minutes.

13. The method according to claim 11 or 12, wherein the target molecule is extracted from the collection device in the formulation for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, at least 150 minutes, at least 160 minutes, at least 170 minutes, at least 180 minutes, at least 190 minutes, or at least 200 minutes.

14. The method according to claim 12 or 13, wherein the formulation comprises a buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide.

15. The method according to claim 14, wherein one or more of the salts are independently selected from a sodium salt, a potassium salt, and a magnesium salt.

16. The method according to claim 14, wherein the one or more salts include a sodium salt, a potassium salt, and a magnesium salt.

17. The sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl ２ The method according to claim 15 or 16.

18. The method according to claim 17, wherein the concentration of NaCl in the formulation is approximately 10 mM to approximately 500 mM, or approximately 50 mM to approximately 250 mM, or approximately 100 mM to approximately 200 mM, or approximately 75 to 125 mM, or approximately 100 mM.

19. The method according to claim 17 or claim 18, wherein the concentration of KCl in the formulation is approximately 0.5 mM to approximately 30 mM, or approximately 1 mM to approximately 20 mM, or approximately 2 mM to approximately 15 mM, or approximately 4 mM to approximately 10 mM, or approximately 5 mM.

20. The MgCl in the above-mentioned formulation ２ The method according to any one of claims 17 to 19, wherein the concentration is approximately 0.5 mM to approximately 30 mM, or approximately 1 mM to approximately 20 mM, or approximately 2 mM to approximately 15 mM, or approximately 4 mM to approximately 10 mM, or approximately 8 mM.

21. The method according to any one of claims 14 to 20, wherein the buffer is selected from HEPES, IVIES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, coramine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

22. The method according to any one of claims 14 to 21, wherein the buffering agent in the formulation has a concentration of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 60 mM, or about 50 mM.

23. The method according to any one of claims 14 to 22, wherein the chelating agent is selected from EGTA, EDTA, DTPA, BAPTA, DMPS, and ALA.

24. The method according to any one of claims 14 to 23, wherein the chelating agent in the formulation is concentrated at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.25 mM.

25. The method according to any one of claims 14 to 24, wherein the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80).

26. The method according to any one of claims 14 to 25, wherein the nonionic surfactant is in volume-to-volume proportions of the formulation, approximately 0.1% to approximately 5%, or approximately 0.2% to approximately 4%, or approximately 0.3% to approximately 3%, or approximately 0.4% to approximately 2%, or approximately 0.5%, or approximately 1.5%, or approximately 1.2%.

27. The method according to any one of claims 14 to 26, wherein the pH of the formulation is approximately pH 5 to approximately pH 9, or approximately pH 6 to approximately pH 8, or approximately pH 7 to approximately pH 7.9, or approximately pH 7.

5.

28. The aforementioned formulation contains 50 mM HEPES, 100 mM NaCl, 5 mM KCl, and 8 mM MgCl. ２ The method according to any one of claims 14 to 28, comprising 1.25 mM EGTA and 1.2% Tween-20.

29. The method according to claim 28, wherein the formulation has a pH of about 7.

5.

30. The method according to any one of claims 14 to 29, wherein the protease inhibitor is a reversible protease inhibitor.

31. The method according to any one of claims 14 to 30, wherein the protease inhibitor inhibits a protease selected from trypsin, plasmin, and thrombin.

32. The method according to any one of claims 14 to 31, wherein the protease inhibitor is a serine protease inhibitor.

33. The method according to any one of claims 14 to 32, wherein the protease inhibitor is benzamidine.

34. The method according to any one of claims 14 to 33, wherein the protease inhibitor in the formulation is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.2 mM.

35. The method according to any one of claims 14 to 34, wherein the oligonucleotide is a single-stranded oligonucleotide.

36. The method according to any one of claims 14 to 35, wherein the oligonucleotide is 20 to 100 nucleotides long, or 25 to 80 nucleotides long, or 25 to 70 nucleotides long, or 25 to 50 nucleotides long, or about 30 nucleotides long.

37. The method according to any one of claims 14 to 36, wherein the oligonucleotide comprises one or more modified nucleotides.

38. The method according to any one of claims 14 to 37, wherein the oligonucleotide comprises one or more C-5 modified pyrimidines.

39. The aforementioned oligonucleotide has the sequence [(A-C-X-X) ７ The method according to any one of claims 14 to 38, comprising [-A-C], wherein X is BndU.

40. The method according to any one of claims 14 to 39, wherein the oligonucleotide in the formulation has a concentration of 5 mM to 100 mM, or 10 mM to 80 mM, or 20 mM to 60 mM, or 30 mM to 50 mM, or about 75 mM, or about 37 mM.

41. The method according to any one of claims 11 to 40, wherein the biological sample is dried at or below room temperature for at least 4 hours.

42. The biological sample is dried at room temperature, 4°C to 8°C, or -20°C. The method according to any one of claims 11 to 41.

43. The method according to any one of claims 11 to 42, wherein the dried biological sample is stored at or below room temperature before detection.

44. The method according to any one of claims 11 to 43, wherein the biological sample is stored at approximately 4°C to 8°C or -20°C before detection.

45. The method according to any one of claims 11 to 44, wherein the biological sample is dried for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, at least 1 day, at least 2 days, or at least 3 days.

46. The method according to any one of claims 11 to 45, wherein the biological sample is selected from plasma, serum, urine, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, sputum, tears, mucus, nasal lavage fluid, nasal aspirate, semen, saliva, peritoneal lavage fluid, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, bronchial swab, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid.

47. The method according to any one of claims 11 to 45, wherein the biological sample is selected from plasma, serum, urine, and whole blood.

48. The method according to any one of claims 11 to 47, wherein the plurality of target molecules are selected from proteins, peptides, carbohydrates, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, bacteria, metabolites, cofactors, inhibitors, drugs, pigments, nutrients, growth factors, cells, and tissues.

49. The method according to any one of claims 11 to 48, wherein the dried biological sample is homogenized by the drying process.

50. The method according to any one of claims 11 to 49, wherein the first capture reagent-target molecule affinity complex and the second capture reagent-target molecule affinity complex are non-covalent complexes.

51. The method according to any one of claims 11 to 50, wherein the first diluent is a 0.001% to 0.1% diluent of the elution target molecule sample, and the second diluent is a 0.1% to 10% diluent of the elution target molecule sample.

52. The first diluent is a diluent of the test sample in a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second diluent is in a concentration of 0.01% to 1% (or 0.01%, 0.02%, The method according to any one of claims 11 to 50, wherein the dilution of the test sample is 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%, or is 0.1% to 0.8%, or 0.2% to 0.75%, or about 0.5%.

53. The first diluent is a diluent of the test sample in a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%; the second diluent is a diluent in a concentration of 5% to 39% (or 5%, 6%, 7%, 8%, 9%, or 1%). The method according to any one of claims 11 to 50, wherein the dilution of the test sample is 0%, 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%, or 39%, or is 15% to 30%, or 15% to 25%, or about 20%.

54. The first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.1% to 0.8%, or 0.2% to 0.75%, or about 0.5%; the second diluent is The method according to any one of claims 11 to 50, wherein the dilution of the test sample is 5% to 39% (or 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%, or 39%), or 15% to 30%, or 15% to 25%, or about 20%.

55. The first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.1% to 0.8%, or 0.2% to 0.75%. The method according to any one of claims 11 to 50, wherein the second diluent is a diluent of the test sample in an amount of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

56. The first diluent is a diluent of the test sample in a concentration of 5% to 39% (or 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%, or 39%), or 15% to 30%, or 15% to 25%, or about 20%, and the second diluent is 0. The method according to any one of claims 11 to 50, wherein the dilution of the test sample is 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or is 0.1% to 0.8%, or 0.2% to 0.75%, or about 0.5%.

57. The first diluent described above is a diluent of the test sample in a concentration of 5% to 39% (or 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%, or 39%), or 15% to 30%, or 15% to 25%. The method according to any one of claims 11 to 50, wherein the second diluent is a diluent of the test sample in an amount of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

58. The first diluent is a diluent of the test sample in a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second diluent is in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.0 The method according to any one of claims 11 to 50, wherein the dilution of the test sample (which is 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%) is 0.01% to 0.5%, or 0.02% to 0.1%, or about 0.05%.

59. The first diluent is a diluent of the test sample in a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second diluent is 0.5% to 5% ( The method according to any one of claims 11 to 50, wherein the dilution of the test sample is (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or is 0.5% to 4%, or 1% to 3%, or about 2.5%.

60. The first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%, or 0.02% to 0.1%, or about 0. The method according to any one of claims 11 to 50, wherein the second diluent is a diluent of the test sample in an amount of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%.

61. The first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%, or 0.02% to 0.1%. The method according to any one of claims 11 to 50, wherein the second diluent is a diluent of the test sample in an amount of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

62. The first diluent is a diluent of the test sample in a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%, and the The method according to any one of claims 11 to 50, wherein the diluent 2 is a diluent of the test sample in an amount of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

63. The first diluent is a diluent of the test sample in a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%, and the second diluent is in a concentration of 0.01% to 1% (or 0.01%, The method according to any one of claims 11 to 50, wherein the dilution of the test sample is 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%; or 0.01% to 0.5%; or 0.02% to 0.1%; or about 0.05%.

64. The method according to any one of claims 11 to 63, further comprising contacting a third diluted sample with a third capture reagent, wherein the interaction of the third capture reagent with its target molecule forms a third capture reagent affinity complex when the target molecule is present in the third diluted sample, and the third diluted sample is incubated separately from the first diluted sample and the second diluted sample to enable the formation of a third aptamer affinity complex with its target molecule.

65. The method according to claim 64, further comprising releasing the second capture reagent affinity complex, then releasing the third capture reagent affinity complex and capturing it on the second solid support.

66. The method according to claim 65, further comprising detecting the presence of the third capture reagent in the third capture reagent affinity complex, determining its level, or detecting the presence or amount of the third capture reagent affinity complex.

67. The method according to claim 66, wherein the third diluent is a different diluent from the first and / or second diluents of the same elution target molecule sample.

68. The method according to claim 66, wherein the third diluent is a 0.001% to 0.1% diluent of the elution target molecule sample.

69. The method according to claim 66, wherein the third diluent is a 0.001% to 40% diluent of the elution target molecule sample.

70. The third diluent described above is 5% to 39% (or 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%, or 39%), 15% to 30%, 15% to 25%, about 20%; 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0. The method according to claim 66, wherein the dilution of the test sample is 1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%, 0.1% to 0.8%, 0.2% to 0.75%, about 0.5%; and 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, 0.003% to 0.007%, about 0.005%.

71. The third diluent is a diluent of the test sample in a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%; or the third diluent is 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.2%) The method according to claim 66, wherein the third diluent is 5%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%, or 0.01% to 0.5%, or 0.02% to 0.1%, or about 0.05%; or the method according to claim 66, wherein the third diluent is 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

72. The method according to any one of claims 11 to 71, wherein the first capture reagent and the second capture reagent are independently selected from an aptamer or an antibody.

73. The method according to any one of claims 64 to 72, wherein the third capture reagent is selected from an aptamer or an antibody.

74. The method according to claim 73, wherein each of the first capture reagent, the second capture reagent, and the third capture reagent is an aptamer.

75. The method according to claim 74, wherein each aptamer independently comprises at least one 5-position modified pyrimidine.

76. The method according to claim 75, wherein the at least one 5-position modified pyrimidine comprises a linker at the 5-position of the pyrimidine and a portion bonded to the linker.

77. The method according to claim 76, wherein the linker is selected from amide linkers, carbonyl linkers, propynyl linkers, alkyne linkers, ester linkers, urea linkers, carbamate linkers, guanidine linkers, amidine linkers, sulfoxide linkers, and sulfone linkers.

78. The method according to claim 76 or 77, wherein the aforementioned portion is a hydrophobic portion.

79. The method according to claim 78, wherein the portion is selected from naphthyl, benzyl, fluorobenzyl, tyrosyl, indole, morpholino, isobutyl, 3,4-methylenedioxybenzyl, benzothiophenyl, benzofuranyl, phenylbenzyl, 4-phenoxybenzyl, diphenylpropyl, and benzhydryl.

80. The method according to any one of claims 75 to 79, wherein the pyrimidine in the 5-position modified pyrimidine is uridine, cytidine, or thymidine.

81. The method according to any one of claims 11 to 80, wherein the detection of the presence of the dissociated first capture reagent and the dissociated second capture reagent, or the determination of the level thereof, is performed by PCR, mass spectrometry, nucleic acid sequencing, next-generation sequencing (NGS), or hybridization.

82. A method for preparing a liquid sample, The aforementioned sample is dried at a constant temperature of -20°C to room temperature for at least 4 hours to produce a dried sample; The method comprising reconstituting the dried sample with a formulation comprising a buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide.

83. The method according to claim 82, wherein one or more of the salts are independently selected from a sodium salt, a potassium salt, and a magnesium salt.

84. The method according to claim 82 or claim 83, wherein the one or more salts include a sodium salt, a potassium salt, and a magnesium salt.

85. The sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl ２ The method according to claim 83 or 84.

86. The method according to claim 85, wherein the concentration of NaCl in the formulation is approximately 10 mM to approximately 500 mM, or approximately 50 mM to approximately 250 mM, or approximately 100 mM to approximately 200 mM, or approximately 75 to 125 mM, or approximately 100 mM.

87. The method according to claim 85 or claim 86, wherein the concentration of KCl in the formulation is about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM.

88. The MgCl in the above-mentioned formulation ２ The method according to any one of claims 85 to 87, wherein the concentration is approximately 0.5 mM to approximately 30 mM, or approximately 1 mM to approximately 20 mM, or approximately 2 mM to approximately 15 mM, or approximately 4 mM to approximately 10 mM, or approximately 8 mM.

89. The method according to any one of claims 82 to 88, wherein the buffer is selected from HEPES, IVIES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, coramine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

90. The method according to any one of claims 82 to 89, wherein the buffering agent in the formulation has a concentration of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 60 mM, or about 50 mM.

91. The method according to any one of claims 82 to 90, wherein the chelating agent is selected from EGTA, EDTA, DTPA, BAPTA, DMPS, and ALA.

92. The method according to any one of claims 82 to 91, wherein the chelating agent in the formulation is concentrated at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.25 mM.

93. The method according to any one of claims 82 to 92, wherein the nonionic surfactant is selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-80).

94. The method according to claim 93, wherein the nonionic surfactant is in volume-to-volume proportions of the formulation, approximately 0.1% to approximately 5%, or approximately 0.2% to approximately 4%, or approximately 0.3% to approximately 3%, or approximately 0.4% to approximately 2%, or approximately 0.5%, or approximately 1.5%, or approximately 1.2%.

95. The method according to any one of claims 82 to 94, wherein the pH of the formulation is approximately pH 5 to approximately pH 9, or approximately pH 6 to approximately pH 8, or approximately pH 7 to approximately pH 7.9, or approximately pH 7.

5.

96. The aforementioned formulation contains 50 mM HEPES, 100 mM NaCl, 5 mM KCl, and 8 mM MgCl. ２ The method according to any one of claims 82 to 95, comprising 1.25 mM EGTA and 1.2% Tween-20.

97. The method according to claim 96, wherein the formulation has a pH of approximately 7.

5.

98. The method according to any one of claims 82 to 97, wherein the protease inhibitor is a reversible protease inhibitor.

99. The method according to any one of claims 82 to 98, wherein the protease inhibitor inhibits a protease selected from trypsin, plasmin, and thrombin.

100. The method according to any one of claims 82 to 99, wherein the protease inhibitor is a serine protease inhibitor.

101. The method according to any one of claims 82 to 100, wherein the protease inhibitor is benzamidine.

102. The method according to any one of claims 82 to 101, wherein the protease inhibitor in the formulation is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.2 mM.

103. The method according to any one of claims 82 to 102, wherein the oligonucleotide is a single-stranded oligonucleotide.

104. The method according to any one of claims 82 to 103, wherein the oligonucleotide is 20 to 100 nucleotides long, or 25 to 80 nucleotides long, or 25 to 70 nucleotides long, or 25 to 50 nucleotides long, or about 30 nucleotides long.

105. The method according to any one of claims 82 to 104, wherein the oligonucleotide comprises one or more modified nucleotides.

106. The method according to any one of claims 82 to 105, wherein the oligonucleotide comprises one or more C-5 modified pyrimidines.

107. The aforementioned oligonucleotide has the sequence [(A-C-X-X) ７ The method according to any one of claims 82 to 106, comprising -A-C], wherein X is BndU.

108. The method according to any one of claims 82 to 107, wherein the oligonucleotide in the formulation has a concentration of 5 mM to 100 mM, 10 mM to 80 mM, 20 mM to 60 mM, 30 mM to 50 mM, or about 37 mM.

109. The method according to any one of claims 82 to 108, wherein the sample is dried at a constant temperature of about 4°C to about 8°C.

110. The method according to any one of claims 82 to 109, wherein the sample is dried for about 4 hours to about 48 hours.

111. The sample is diluted in a first diluent and a second diluent, wherein the first diluent is a diluent of the test sample in a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second diluent is 0.01% to 1% (or The method according to any one of claims 82 to 110, wherein the dilution of the test sample is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% or a dilution of the test sample, or a dilution of 0.1% to 0.8%, or a dilution of 0.2% to 0.75%, or a dilution of about 0.5%.

112. The sample is diluted into a first diluent and a second diluent, the first diluent being a 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%) diluent of the test sample, or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%; the second diluent being 5% to 39% (or 5%, 6%) The method according to any one of claims 82 to 110, wherein the dilution of the test sample is (15% to 30%, or 15% to 25%, or about 20%), and is 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%, or 39%.

113. The sample is diluted in a first diluent and a second diluent, the first diluent being a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.1% to 0.8%, or 0.2% to 0.75%, or about 0.5%. The method according to any one of claims 82 to 110, wherein the second diluent is a diluent of the test sample in an amount of 5% to 39% (or 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%, or 39%), or 15% to 30%, or 15% to 25%, or about 20%.

114. The sample is diluted in a first diluent and a second diluent, wherein the first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.1% to 0.8%, or 0 The method according to any one of claims 82 to 110, wherein the amount is 2% to 0.75%, or about 0.5%; the second diluent is a diluent of the test sample in an amount of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

115. The sample is diluted into a first diluent and a second diluent, wherein the first diluent is a diluent of the test sample in an amount of 5% to 39% (or 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%, or 39%), or 15% to 30%, or 15% to 25%, or about 20%, The method according to any one of claims 82 to 110, wherein the second diluent is a diluent of the test sample in an amount of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.1% to 0.8%, or 0.2% to 0.75%, or about 0.5%.

116. The sample is diluted in a first diluent and a second diluent, wherein the first diluent is a diluent of the test sample in an amount of 5% to 39% (or 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%, or 39%), or 15% to 30%. The method according to any one of claims 82 to 110, wherein the second diluent is a diluent of the test sample in an amount of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

117. The sample is diluted in a third diluent, the third diluent being 5% to 39% (or 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%, or 39%), 15%–30%, 15%–25%, approximately 20%, 0.01%–1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%) The method according to any one of claims 82 to 110, wherein the diluent of the test sample is selected from %, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), 0.1% to 0.8%, 0.2% to 0.75%, about 0.5%, and 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005%.

118. The first diluent is a diluent of the test sample in a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second diluent is in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0. The method according to any one of claims 82 to 110, wherein the dilution of the test sample is 0.3%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%, or is 0.01% to 0.5%, or 0.02% to 0.1%, or about 0.05%.

119. The first diluent is a diluent of the test sample in a concentration of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%, and the second diluent is 0.5% to 5% ( The method according to any one of claims 82 to 110, wherein the dilution of the test sample is (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or is 0.5% to 4%, or 1% to 3%, or about 2.5%.

120. The first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%, or 0.02% to 0.1%, or about 0.0 The method according to any one of claims 82 to 110, wherein the second diluent is a diluent of the test sample in an amount of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%.

121. The first diluent is a diluent of the test sample in a concentration of 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 0.01% to 0.5%, or 0.02% to 0.1%. The method according to any one of claims 82 to 110, wherein the second diluent is a diluent of the test sample in an amount of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

122. The first diluent is a diluent of the test sample in a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%, and the second The method according to any one of claims 82 to 110, wherein the dilution is a dilution of the test sample in an amount of 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

123. The first diluent is a diluent of the test sample in a concentration of 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%), or 0.5% to 4%, or 1% to 3%, or about 2.5%, and the second diluent is in a concentration of 0.01% to 1% (or 0.01%, The method according to any one of claims 82 to 110, wherein the dilution of the test sample is 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%; or 0.01% to 0.5%; or 0.02% to 0.1%; or about 0.05%.

124. The solution further comprises a third diluent of the test sample, wherein the third diluent is selected from 0.5% to 5% (or 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.7%, or 5%). The third diluent is a diluent of the test sample (which is %), or 0.5% to 4%, or 1% to 3%, or about 2.5%; or the third diluent is 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0. The method according to any one of claims 111 to 123, wherein the third diluent is 2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%, or 0.01% to 0.5%, or 0.02% to 0.1%, or about 0.05%; or the method according to any one of claims 111 to 123, wherein the third diluent is 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

125. A composition, The formulation comprises a dried sample, a buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide. The aforementioned dried sample is obtained from a liquid sample that has been dried at a constant temperature of approximately -20°C to room temperature for at least 4 hours, and is the composition.

126. A method for detecting analytes in a sample, The aforementioned sample is dried at a constant temperature of approximately -20°C to room temperature for at least 4 hours to produce a dried sample; Reconstitute the dried sample with a formulation comprising a buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide; The method comprising detecting the analyte from the reconstituted sample.

127. The method according to claim 126, wherein the detection is performed using a protein-binding reagent or a mass spectrometer.

128. The method according to claim 126 or claim 127, wherein the protein-binding reagent is selected from an aptamer or an antibody.

129. The method according to any one of claims 126 to 128, wherein the detection is performed by a multiplex assay.

130. The multiplex assay is performed at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 The method according to claim 129, which detects analytes in quantities of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000.

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