Detection of target molecules in dry biological matrices

Abstract

A method of detecting a target molecule in a dry biological matrix in a proteomic-based assay is provided. Such methods are useful in research and development by providing methods for preparing a dried biological matrix for detecting target molecules in proteomic assays and methods for detecting one or more target molecules from a dried biological matrix in proteomic assays, the invention has wide utility in proteomic applications of diagnostics and therapeutics.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 525,341, filed July 6, 2023, and U.S. Provisional Patent Application No. 63 / 530,113, filed August 1, 2023, each of which is incorporated by reference herein in its entirety for any purpose. TECHNICAL FIELD

[0003] The present disclosure relates generally to the field of proteomics assays, and methods for detecting target molecules in dried biological matrices. Such methods have broad utility in proteomics applications for research and development, diagnostics, and therapeutics. In particular, methods of preparing dried biological matrices for detecting target molecules in proteomics assays, and methods of detecting target molecules from dried biological matrices in proteomics assays are provided. BACKGROUND

[0004] A number of assays for the detection and quantification of physiologically important molecules in biological samples and other sample types are important tools in scientific research and health care fields. For example, multiplex array assays employ surface-bound probes to detect target molecules in a sample. The surface-bound probes can be oligonucleotides, peptides, polypeptides, proteins, antibodies, affimers, aptamers, or other molecules (collectively referred to as biopolymers) that are capable of binding to target molecules from a sample. These binding interactions are the basis of many methods and devices used in various different fields, such as genomics, transcriptomics, and proteomics.

[0005] Assays using solution-based samples often require the solution-based samples to be maintained at -80°C prior to the assay, including transport and storage. In addition, solution-based samples containing cells, such as whole blood, can have cell lysis upon freeze-thaw sample processing, and the sample can have insufficient cell homogenization. Improper storage of solution-based samples and incomplete homogenization of cells within the solution can introduce pre-analytical variation on target molecules in the solution-based sample.

[0006] The present disclosure describes methods that eliminate the need for -80°C transport and storage of samples prior to the assay. The present disclosure describes methods that eliminate or reduce incomplete homogenization of cells within the solution. The present disclosure describes methods for detecting target molecules within a sample extracted from a dried biological matrix. SUMMARY

[0007] The foregoing and other objects, features and advantages of the present application will become more readily apparent from the following detailed description, which proceeds with reference to the accompanying drawings.

[0008] Some non-limiting exemplary embodiments are as follows:

[0009] In some embodiments, a method of preparing a biological sample for multiplexed assay is disclosed. The method comprises depositing a biological sample comprising a plurality of target molecules onto a collection device, drying the biological sample on the collection device for a period of time to stabilize the dried sample prior to any temperature fluctuations, wherein the biological sample is dried at about room temperature or below room temperature for at least 4 hours, such that the target molecules in the dried biological sample are detectable in a multiplexed assay.

[0010] In some embodiments, the biological sample is dried at about room temperature, 4-8°C, or -20°C. In some embodiments, the dried biological sample is stored at about room temperature or below room temperature prior to detection in a multiplexed assay. In some embodiments, the dried biological sample is stored at about 4-8°C or -20°C.

[0011] 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, one day, at least two days, or at least three days.

[0012] In some embodiments, the biological sample is selected from the group consisting of plasma, serum, urine, whole blood, white blood cells, peripheral blood mononuclear cells, buffy coat, sputum, tears, mucus, nasal lavage, nasal aspirate, semen, saliva, peritoneal lavage, ascites, cyst fluid, meningeal fluid, amniotic fluid, glandular fluid, lymphatic fluid, nipple aspirate, bronchial aspirate, bronchial brushings, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid. In some embodiments, the biological sample is selected from the group consisting of plasma, serum, urine, and whole blood. In some embodiments, the plurality of target molecules is selected from the group consisting of proteins, peptides, carbohydrates, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, bacteria, metabolites, co-factors, inhibitors, drugs, dyes, nutrients, growth factors, cells, and tissues.

[0013] In some embodiments, the dried biological sample is homogenized by the drying process. In some embodiments, the plurality of target molecules is extractable from the collection device after the biological sample is dried and is detectable in a multiplexed assay.

[0014] In some embodiments, methods of detecting a plurality of target molecules are disclosed. The methods comprise extracting target molecules in a dried biological sample from a collection device; diluting the extracted target molecules into a first dilution and a second dilution, contacting the first dilution with a first capture reagent to form a first capture reagent affinity complex with its target molecule if the target molecule is present in the first dilution, contacting the second dilution with a second capture reagent to form a second capture reagent affinity complex with its target molecule if the target molecule is present in the second dilution, separately incubating the first dilution sample and the second dilution sample to allow capture reagent affinity complex formation; wherein each of the first and second capture reagent affinity complexes are immobilized on separate first solid supports, releasing and capturing the first capture reagent affinity complex on a second solid support, releasing and capturing the second capture reagent affinity complex on the second solid support after releasing the first capture reagent affinity complex; and detecting the presence or determining the level of the first or second capture reagent of the first or second capture reagent affinity complex, or the presence or amount of the first or second capture reagent affinity complex.

[0015] In some embodiments, the target molecules are extracted from the collection device in the formulation for at least 5 minutes. In some embodiments, the target molecules are 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.

[0016] In some embodiments, the formulation comprises a buffer, one or more salts, a chelating agent, a protease inhibitor, a non-ionic surfactant, and an oligonucleotide.

[0017] In some embodiments, the one or more salts are each independently selected from the group consisting of sodium salts, potassium salts, and magnesium salts.

[0018] In some embodiments, one or more salts include sodium, potassium, and magnesium salts. 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 mM to 125 mM, or about 100 mM. In some embodiments, 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. In some embodiments, the concentration of MgCl2 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 8 mM.

[0019] In some embodiments, the buffer is selected from HEPES, IVIES, Bistris methane, ADA, ACES, Bistris propane, PIPES, MOPSO, choline chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, glycamide, glycylglycine, HEPBS, Bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS. In some embodiments, the concentration of the buffer in the formulation is 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.

[0020] In some embodiments, the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA. In some embodiments, the concentration of the chelating agent in the formulation is from about 0.1 mM to about 10 mM, or from about 0.5 mM to about 5 mM, or about 1.25 mM.

[0021] In some embodiments, the nonionic surfactant is selected from polyoxyethylene (20) sorbitol monolaurate (Tween-20), polyoxyethylene (40) sorbitol monolaurate (Tween-40), and polyoxyethylene (80) sorbitol monolaurate (Tween-80). In some embodiments, the nonionic surfactant is present at 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% or about 1.5% of the formulation, or about 1.2% of the formulation, on a volume-to-volume basis.

[0022] In some embodiments, the pH of the formulation is from about pH 5 to about pH 9, or from about pH 6 to about pH 8, or from about pH 7 to about pH 7.9 or about pH 7.5.

[0023] 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.

[0024] In some implementations, the formulation has a pH of approximately 7.5.

[0025] 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 an inhibitor of serine proteases. In some embodiments, the protease inhibitor is benzomidine. In some embodiments, the concentration of the protease inhibitor in the formulation is about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.2 mM.

[0026] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is 20 to 100 nucleotides in length, or 25 to 80 nucleotides in length, or 25 to 70 nucleotides in length, or 25 to 50 nucleotides in length, or about 30 nucleotides in length. In some embodiments, the oligonucleotide comprises one or more modified nucleotides. In some embodiments, the oligonucleotide comprises 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.

[0027] In some embodiments, the biological sample has been dried at approximately room temperature or below for at least 4 hours. In some embodiments, the biological sample has been dried at approximately room temperature, 4°C–8°C, or -20°C. In some embodiments, the dried biological sample has been stored at approximately room temperature or below prior to detection. In some embodiments, the dried biological sample has been stored at approximately 4°C–8°C or -20°C prior to detection.

[0028] In some implementations, the biological sample has been 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, one day, at least two days, or at least three days.

[0029] In some embodiments, the biological samples are selected from plasma, serum, urine, whole blood, leukocytes, peripheral blood mononuclear cells, erythrocyte sedimentation rate (ESR), sputum, tears, mucus, nasal wash, nasal aspirate, semen, saliva, peritoneal lavage fluid, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph, papillary aspirate, bronchial aspirate, bronchial brushing fluid, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid. In some embodiments, the biological samples are selected from plasma, serum, urine, and whole blood. In some embodiments, a variety of target molecules are selected from proteins, peptides, carbohydrates, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, bacteria, metabolites, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, and tissues.

[0030] In some implementations, the dried biological samples have been homogenized through a drying process.

[0031] In some implementations, the first capture reagent-target molecule affinity complex and the second capture reagent-target molecule affinity complex are non-covalent complexes.

[0032] In some embodiments, the first diluent is a diluent of 0.001% to 0.1% of the eluted target molecule sample, and the second diluent is a diluent of 0.1% to 10% of the eluted target molecule sample.

[0033] In some embodiments, 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%), 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005% of the test sample, and the second diluent is 0.01% to 1% 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% dilution. In some embodiments, 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%), 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005% of the test sample; and the second diluent is 5% to 39% of the test sample. (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% dilution.In some embodiments, the first 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%, 0.25%, 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%, or about 0.5% of the test sample; and the second diluent is 5% to 39% of the test sample. (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% dilution. In some embodiments, the first 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%, 0.25%, 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%, or about 0.5% of the test sample; and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 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%, or about 20% of the test sample, and the second diluent is 0.01% to 1% 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% dilution. In some embodiments, the first 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%, or about 20% of the test sample, and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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%.

[0034] In some embodiments, 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%), 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005% of the test sample, and the second diluent is 0.01% to 1% 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 a dilution of 0.01% to 0.5%, or 0.02% to 0.1% or about 0.05%. In some embodiments, 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%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005% of the test sample, and the second diluent is 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% of the test sample. In some embodiments, the first 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%, 0.25%, 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% of the test sample, and the second diluent is 0.5% to 5% of the test sample. (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% dilution.In some embodiments, the first 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%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), 0.01% to 0.5%, 0.02% to 0.1%, or about 0.05% of the test sample, and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 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% of the test sample, and the second 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% of the test sample. In some implementations, the first diluent is 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% of the test sample, and the second diluent is 0.01% to 1% 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 a dilution of 0.01% to 0.5%, or 0.02% to 0.1% or about 0.05%.

[0035] In some embodiments, the method further includes contacting a third diluent sample with a third capture reagent, wherein if a target molecule is present in the third diluent sample, a third capture reagent affinity complex is formed through the interaction of the third capture reagent with its target molecule; wherein the third diluent sample is incubated separately from the first and second diluent samples to allow the formation of a capture reagent affinity complex between the third aptamer and its target molecule. In some embodiments, the method further includes releasing and capturing the third capture reagent affinity complex on a second solid support after releasing the second capture reagent affinity complex. In some embodiments, the method further includes detecting the presence of the third capture reagent in the third capture reagent affinity complex or determining the level of the third capture reagent, or the presence or amount of the third capture reagent affinity complex.

[0036] In some embodiments, the third diluent is a different diluent from the first and / or second diluents of the same eluted target molecule sample. In some embodiments, the third diluent is 0.001% to 0.1% of the eluted target molecule sample. In some embodiments, the third diluent is 0.001% to 40% of the eluted target molecule sample.

[0037] In some implementations, 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%, or about 20% of the test sample; or 0.01% to 1% (or Diluted solutions of 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%), 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%. In some implementations, the third diluent is 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% of the test sample; 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%, 0.25%, 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%; or the third dilution 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%.

[0038] 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-position modified pyrimidine. In some embodiments, at least one 5-position modified pyrimidine comprises a 5-position linker of the pyrimidine and a portion connected to said linker.

[0039] In some embodiments, the connector is selected from amide connectors, carbonyl connectors, propynyl connectors, alkyne connectors, ester connectors, urea connectors, urethane connectors, guanidine connectors, amidine connectors, sulfoxide connectors, and sulfone connectors.

[0040] In some embodiments, the portion is a hydrophobic portion. In some embodiments, the portion is selected from naphthyl, benzyl, fluorobenzyl, tyrosinyl, indole, morpholino, isobutyl, 3,4-methylenedioxybenzyl, benzo[a]phenylthio, benzo[a]furanyl, phenylbenzyl, 4-phenoxybenzyl, diphenylpropyl, and diphenylmethyl portions. In some embodiments, the pyrimidine of the 5-position modified pyrimidine is uridine, cytidine, or thymidine.

[0041] In some implementations, the presence of the dissociated first and second capture reagents or the level of the dissociated first and second capture reagents are detected by PCR, mass spectrometry, nucleic acid sequencing, next-generation sequencing (NGS), or hybridization.

[0042] In some embodiments, a method for preparing a liquid sample is provided. The method includes drying the sample at a constant temperature from -20°C to room temperature for at least four (4) hours to produce a dried sample, and reconstituted 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.

[0043] In some implementations, one or more salts are independently selected from sodium, potassium, and magnesium salts.

[0044] In some embodiments, one or more salts include sodium, potassium, and magnesium salts. 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 mM to 125 mM, or about 100 mM. In some embodiments, 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. In some embodiments, the concentration of MgCl2 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 8 mM.

[0045] In some embodiments, the buffer is selected from HEPES, IVIES, Bistris methane, ADA, ACES, Bistris propane, PIPES, MOPSO, choline chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, glycamide, glycylglycine, HEPBS, Bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS. In some embodiments, the concentration of the buffer in the formulation is 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.

[0046] In some embodiments, the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA. In some embodiments, the concentration of the chelating agent in the formulation is from about 0.1 mM to about 10 mM, or from about 0.5 mM to about 5 mM, or about 1.25 mM.

[0047] In some embodiments, the nonionic surfactant is selected from polyoxyethylene (20) sorbitol monolaurate (Tween-20), polyoxyethylene (40) sorbitol monolaurate (Tween-40), and polyoxyethylene (80) sorbitol monolaurate (Tween-80). In some embodiments, the nonionic surfactant is present at 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% or about 1.5% of the formulation, or about 1.2% of the formulation, on a volume-to-volume basis.

[0048] In some embodiments, the pH of the formulation is from about pH 5 to about pH 9, or from about pH 6 to about pH 8, or from about pH 7 to about pH 7.9 or about pH 7.5.

[0049] 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.

[0050] In some implementations, the formulation has a pH of approximately 7.5.

[0051] 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 an inhibitor of serine proteases. In some embodiments, the protease inhibitor is benzomidine. In some embodiments, the concentration of the protease inhibitor in the formulation is about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.2 mM.

[0052] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is 20 to 100 nucleotides in length, or 25 to 80 nucleotides in length, or 25 to 70 nucleotides in length, or 25 to 50 nucleotides in length, or about 30 nucleotides in length. In some embodiments, the oligonucleotide comprises one or more modified nucleotides. In some embodiments, the oligonucleotide comprises 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.

[0053] In some embodiments, the sample is dried at a constant temperature of about 4°C to about 8°C. In some embodiments, the sample is dried for about 4 hours to about 48 hours.

[0054] In some embodiments, the sample is diluted into a first diluent and a second diluent, wherein 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%), 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005% of the test sample, and the second diluent is 0.01% to 1% 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% dilution. In some embodiments, the sample is diluted into a first diluent and a second diluent, wherein 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%), 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005% of the test sample; and the second diluent is 5% to 39% of the test sample. (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% dilution.In some embodiments, the sample is diluted into a first diluent and a second diluent, wherein the first 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%, 0.25%, 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%, or 0.2% to 0.75%, or about 0.5% of the test sample; and the second diluent is 5% to 39% of the test sample. (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% dilution. In some embodiments, the sample is diluted into a first diluent and a second diluent, wherein the first 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%, 0.25%, 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% of the test sample; and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 sample is diluted into a first diluent and a second diluent, wherein the first 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%, or about 20% of the test sample, and the second diluent is 0.01% to 1% 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% dilution. In some embodiments, the sample is diluted into a first diluent and a second diluent, wherein the first 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%, or about 20% of the test sample, and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 sample is diluted to a third diluent, wherein the third diluent is a fraction of the test sample selected from 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%, or 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%), 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% dilutions.

[0055] In some embodiments, 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%), 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005% of the test sample, and the second diluent is 0.01% to 1% 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 a dilution of 0.01% to 0.5%, or 0.02% to 0.1% or about 0.05%. In some embodiments, 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%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005% of the test sample, and the second diluent is 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% of the test sample. In some embodiments, the first 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%, 0.25%, 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% of the test sample, and the second diluent is 0.5% to 5% of the test sample. (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% dilution.In some embodiments, the first 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%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), 0.01% to 0.5%, 0.02% to 0.1%, or about 0.05% of the test sample, and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 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% of the test sample, and the second 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% of the test sample. In some implementations, the first diluent is 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% of the test sample, and the second diluent is 0.01% to 1% 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 a dilution of 0.01% to 0.5%, or 0.02% to 0.1% or about 0.05%.

[0056] In some embodiments, a composition is provided. The composition comprises a dried sample and a formulation, the formulation comprising a buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide, wherein the dried sample is derived from a liquid sample dried at a constant temperature from about -20°C to room temperature for at least four (4) hours.

[0057] In some embodiments, methods for detecting analytes in a sample are provided. The methods include drying the sample at a constant temperature from about -20°C to room temperature for at least four (4) hours to produce a dried sample, reconstituted 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.

[0058] In some implementations, the detection is performed using protein binding reagents or mass spectrometry.

[0059] In some implementations, the protein binding agent is selected from aptamers or antibodies.

[0060] In some implementations, the detection is performed in multiple assays. In some implementations, multiplex assays detect 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 analytes.

[0061] The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description, which is taken into account in the accompanying drawings. Attached Figure Description

[0062] Figure 1 The linear range of dilution group 1 is shown. The x-axis represents the concentration (%) of the DBS extract on a log 10 scale (from 20% to 0%), and the y-axis counts the number of analytes that have a linear signal response at that dilution.

[0063] Figure 2A and Figure 2B Diluent group 2 is shown. Figure 2A ) and diluent group 3 ( Figure 2BThe linear range of the analyte is shown on the y-axis. The y-axis represents the amount of analyte within its linear range. The x-axis represents the concentration (%) of the DBS extract according to the log 10 scale.

[0064] Figures 3A-3C The analytes with signals higher than the background are shown in dilution groups 1, 2 and 3, respectively.

[0065] Figure 4A and Figure 4B The overall coefficient of variation for all tested analytes is shown.

[0066] Figures 5A-5C The graph shows the median (n=3) relative fluorescence units (RFU) between the same samples after staining and extraction, using water (5A) or PBS (5B) as a volume swelling agent and PBS versus water (5C).

[0067] Figure 6A and Figure 6B The analyte recoveries for two different samples collected at different extraction times are shown.

[0068] Figures 7A-7C The mean absolute error of the signal from multiple measurements of dried plasma at 4°C (7A), room temperature (7B), -20°C (7C), and under temperature stress is shown compared to that of liquid plasma.

[0069] Figures 8A-8D Compare the consistency between frozen plasma and dried blood spots (8A), frozen serum and dried blood spots (8B), frozen serum and frozen plasma (8C), and two pooled DBS samples (8D).

[0070] Figure 9 The correlation between DBS and protein measurements in plasma / serum is shown.

[0071] Figure 10A and Figure 10B The results show significant correlations between pairs of observations plotted to the consistency map. Figure 10A The correlations from paired observation data are shown, and Figure 10B The correlation is shown using 5% FDR.

[0072] Figure 11 Show Figure 10A and Figure 10B The two methods shown have significant overlap in the analytes.

[0073] Figures 12A-12D The consistency of analytes with a significant correlation between DBS and plasma is shown.

[0074] Figures 13A-13DThe diagram shows a significant correlation between DBS and plasma plotted on a serum-to-plasma concordance map.

[0075] Figure 14 The analytes are classified according to their biological function.

[0076] Figure 15 The CDF plot shows 2,941,335 random correlations between plasma and serum.

[0077] Figure 16 The probability distribution of false positives based on the Pearson cutoff value is shown.

[0078] Figure 17 An example is shown for calculating the weighted average of false positives.

[0079] Figure 18 A graph showing the comparison between significant correlation and spurious correlation.

[0080] Figure 19 A plot showing the average FDR for a given Pearson cutoff value.

[0081] Figure 20 A graph showing the number of significant correlations as a function of FDR.

[0082] Figure 21 Some exemplary 5-position modified uridine and cytidine that can be incorporated into aptamers are shown.

[0083] Figure 22 Some exemplary modifications that may be present at the 5-position of uridine are shown. The chemical structure of the C-5 modification includes an exemplary amide bond linking the modification to the 5-position of uridine. The 5-position moiety shown includes two phenyl groups covalently linked to each other. The 5-position moiety shown includes a phenyl benzyl moiety (e.g., BPE, PBnd, DBM), a 4-phenoxybenzyl moiety (e.g., POP), a diphenylpropyl moiety (e.g., DPP), and a diphenylmethyl moiety (e.g., BH).

[0084] Figure 23 Some exemplary modifications that may be present at the 5-position of cytidine are shown. The chemical structure of the C-5 modification includes an exemplary amide bond linking the modification to the 5-position of cytidine. The 5-position moiety shown includes two phenyl groups covalently linked to each other. The 5-position moiety shown includes a phenyl benzyl moiety (e.g., BPE, PBnd, DBM), a 4-phenoxybenzyl moiety (e.g., POP), a diphenylpropyl moiety (e.g., DPP), and a diphenylmethyl moiety (e.g., BH).

[0085] Figure 24Some exemplary modifications that may be present at the 5-position of uridine are shown. The chemical structure of the C-5 modification includes an exemplary amide bond linking the modification to the 5-position of uridine. The 5-position moiety shown includes a benzyl moiety (e.g., Bn, PE, and PP), a naphthyl moiety (e.g., Nap, 2Nap, NE), a butyl moiety (e.g., iBu), a fluorobenzyl moiety (e.g., FBn), a tyrosinyl moiety (e.g., Tyr), a 3,4-methylenedioxybenzyl moiety (e.g., MBn), a morpholino moiety (e.g., MOE), a benzofuranyl moiety (e.g., BF), an indole moiety (e.g., Trp), and a hydroxypropyl moiety (e.g., Thr).

[0086] Figure 25 Some exemplary modifications that may be present at the 5-position of cytidine are shown. The chemical structure of the C-5 modification includes an exemplary amide bond that links the modification to the 5-position of cytidine. The 5-position moiety shown includes a benzyl moiety (e.g., Bn, PE, and PP), a naphthyl moiety (e.g., Nap, 2Nap, NE, and 2NE), and a tyrosine acyl moiety (e.g., Tyr). Detailed Implementation

[0087] Unless otherwise stated, technical terms are used in accordance with common usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0088] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless the context clearly indicates otherwise, the singular forms “a / an” and “described” include the plural forms and are used interchangeably with “at least one” and “one or more”. “Comprising A or B” means comprising 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 values ​​and are provided for descriptive purposes.

[0089] Furthermore, the ranges provided herein should be understood as abbreviations of all values ​​within the range. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or subrange that comes from the following groups: 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 (and their fractions, unless the context clearly specifies otherwise). Unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the enumerated range, and, where appropriate, its fraction (such as one-tenth and one-hundredth of an integer). Furthermore, unless otherwise stated, any numerical ranges relating to any physical characteristic (such as polymer subunits, size, or thickness) described herein should be understood to include any integers within the range. As used herein, unless otherwise stated, “about” or “consistently of” means ±20% of the indicated range, value, or structure.

[0090] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof are intended to cover non-exclusive inclusion, such that a process, method, process defining a product or substance composition that includes, comprises, or contains an element or a list of elements may include other elements not expressly listed.

[0091] Although similar or equivalent methods and materials can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including the explanation of terminology) shall prevail. Furthermore, the materials, methods, and examples described are illustrative only and not intended to be limiting.

[0092] As used herein, the term "nucleotide" refers to a ribonucleotide or deoxyribonucleotide, or a modified form thereof, and analogues thereof. Substances included in nucleotides include purines (e.g., adenine, hypoxanthine, guanine, and their derivatives and analogues) and pyrimidines (e.g., cytosine, uracil, thymine, and their derivatives and analogues). As used herein, unless otherwise expressly stated, the term "cytidine" generally refers to a ribonucleotide, deoxyribonucleotide, or modified ribonucleotide containing a cytosine base. The term "cytidine" includes 2'-modified cytidines, such as 2'-fluoro, 2'-methoxy, etc. Similarly, unless otherwise expressly stated, the term "modified cytidine" or a specific modified cytidine also refers to a ribonucleotide, deoxyribonucleotide, or modified ribonucleotide (such as 2'-fluoro, 2'-methoxy, etc.) containing a modified cytosine base. Unless otherwise explicitly stated, the term "uridine" generally refers to a ribonucleotide, deoxyribonucleotide, or modified ribonucleotide containing a uracil base. The term "uridine" includes 2'-modified uridines, such as 2'-fluoro, 2'-methoxy, etc. Similarly, unless otherwise explicitly stated, the term "modified uridine" or a specific modified uridine also refers to a ribonucleotide, deoxyribonucleotide, or modified ribonucleotide (such as 2'-fluoro, 2'-methoxy, etc.) containing a modified uracil base.

[0093] As used herein, the terms “C-5 modified formamide cytidine” or “cytidine-5-formamide” or “5-position modified cytidine” or “C-5 modified cytidine” refer to cytidines having formamide (-C(O)NH-) modification at the C-5 position, including but not limited to those moieties shown herein (R X1 Exemplary C-5 modified formamide cytidines include, but are not limited to, 5-(N-benzylformamide)-2'-deoxycytidine (referred to as "BndC" and...). Figure 25 (shown in the image); 5-(N-2-phenylethylformamide)-2′-deoxycytidine (referred to as "PEdC" and in...) Figure 25 (shown in the image); 5-(N-3-phenylpropylformamide)-2'-deoxycytidine (referred to as "PPdC" and in...) Figure 25 (shown in the image); 5-(N-1-naphthylmethylformamide)-2'-deoxycytidine (referred to as "NapdC" and in...) Figure 22(shown in the image); 5-(N-2-naphthylmethylformamide)-2'-deoxycytidine (referred to as "2NapdC" and in...) Figure 25 (shown in the image); 5-(N-1-naphthyl-2-ethylformamide)-2'-deoxycytidine (referred to as "NEdC" and in...) Figure 25 (shown in the image); 5-(N-2-naphthyl-2-ethylformamide)-2'-deoxycytidine (referred to as "2NEdC" and in...) Figure 25 (shown in the image); and 5-(N-tyrosinylformamide)-2′-deoxycytidine (called TyrdC and in...) Figure 25 (as shown in the figure). In some embodiments, C5-modified cytidine (e.g., in its triphosphate form) can be incorporated into oligonucleotides by a polymerase (e.g., KOD DNA polymerase).

[0094] The chemical modifications of C-5 modified cytidine described in this article can also be combined alone or in any combination with modifications of 2'-sugars, modifications of exocyclic amines, and substitution of 4-thiouridines.

[0095] As used herein, the terms “C-5 modified formamide cytosine” or “cytosine-5-formamide” or “5-position modified cytosine” or “C-5 modified cytosine” refer to cytosine bases having a formamide (-C(O)NH-) modified at the C-5 position, including but not limited to those moieties shown herein (R X1 Exemplary C-5 modified formamide cytosines include, but are not limited to, those shown below. Figure 25 The modified cytidine shown is illustrated.

[0096] As used herein, the terms "C-5 modified uridine" or "5-position modified uridine" refer to uridines (usually deoxyuridines) with formamide (-C(O)NH-) modification at the C-5 position, for example, such as Figure 21 As shown in the diagram. In some embodiments, C5-modified uridine (e.g., in its triphosphate form) can be incorporated into oligonucleotides via a polymerase (e.g., KOD DNA polymerase). Non-limiting exemplary 5-position modified uridines include:

[0097] 5-(N-Benzylformamide)-2'-Deoxyuridine (BndU),

[0098] 5-(N-Benzylformamide)-2'-O-methyluridine,

[0099] 5-(N-Benzylformamide)-2'-Fluorouracil,

[0100] 5-(N-phenylethylformamide)-2'-deoxyuridine (PEdU),

[0101] 5-(N-phenylthiomethylformamide)-2'-deoxyuridine (ThdU),

[0102] 5-(N-isobutylcarboxamide)-2'-deoxyuridine (iBudU),

[0103] 5-(N-tyrosinylformamide)-2'-deoxyuridine (TyrdU),

[0104] 5-(N-3,4-methylenedioxybenzylformamide)-2'-deoxyuridine (MBndU),

[0105] 5-(N-4-fluorobenzylformamide)-2'-deoxyuridine (FBndU),

[0106] 5-(N-3-phenylpropylformamide)-2'-deoxyuridine (PPdU),

[0107] 5-(N-Imidazolylethylformamide)-2'-deoxyuridine (ImdU)

[0108] 5-(N-isobutylcarboxamide)-2'-O-methyluridine,

[0109] 5-(N-isobutylformamide)-2'-fluorouridine,

[0110] 5-(N-chromocarbamate)-2'-deoxyuridine (TrpdU),

[0111] 5-(NR-threonylformamide)-2'-deoxyuridine (ThrdU),

[0112] 5-(N-chromocarbamate)-2'-O-methyluridine,

[0113] 5-(N-chromocarbamoamide)-2'-fluorouridine,

[0114] 5-(N-[1-(3-trimethylammonium)propyl]formamide)-2'-deoxyuridine chloride,

[0115] 5-(N-naphthylmethylformamide)-2'-deoxyuridine (NapdU)

[0116] 5-(N-naphthylmethylformamide)-2'-O-methyluridine,

[0117] 5-(N-naphthylmethylformamide)-2'-fluorouridine,

[0118] 5-(N-[1-(2,3-dihydroxypropyl)]formamide)-2'-deoxyuridine),

[0119] 5-(N-2-naphthylmethylformamide)-2'-deoxyuridine (2NapdU),

[0120] 5-(N-2-naphthylmethylformamide)-2'-O-methyluridine,

[0121] 5-(N-2-naphthylmethylformamide)-2'-fluorouridine,

[0122] 5-(N-1-Naphthylethylformamide)-2'-deoxyuridine (NEdU),

[0123] 5-(N-1-Naphthylethylformamide)-2'-O-methyluridine,

[0124] 5-(N-1-Naphthylethylformamide)-2'-Fluorouracil,

[0125] 5-(N-2-naphthylethylformamide)-2'-deoxyuridine (2NEdU),

[0126] 5-(N-2-naphthylethylformamide)-2'-O-methyluridine,

[0127] 5-(N-2-naphthylethylformamide)-2'-fluorouridine,

[0128] 5-(N-3-benzofuranylethylformamide)-2'-deoxyuridine (BFdU),

[0129] 5-(N-3-benzofuranylethylformamide)-2'-O-methyluridine,

[0130] 5-(N-3-benzofuranylethylformamide)-2'-fluorouridine,

[0131] 5-(N-3-Benzophenylthioethylformamide)-2'-deoxyuridine (BTdU),

[0132] 5-(N-3-benzophenylthioethylformamide)-2'-O-methyluridine, and

[0133] 5-(N-3-benzothioethylformamide)-2'-fluorouridine.

[0134] The other C-5 modification can be found in WO / 2022 / 221241.

[0135] As used herein, when referring to oligonucleotides, the terms “modify,” “modified,” “modification,” and any variations thereof mean that at least one of the four constitutive nucleotide bases (i.e., A, G, T / U, and C) of the oligonucleotide is an analog or ester of a naturally occurring nucleotide. In some embodiments, the modified nucleotide confers oligonucleotide nuclease resistance. Additional modifications may include backbone modifications, methylation, unusual base pairings such as isobasic cytosine and isoguanidine. Modifications may also include 3' and 5' modifications, such as capping. Other modifications may include replacing one or more naturally occurring nucleotides with analogs; internucleotide modifications, such as those with uncharged linkages (e.g., methyl phosphonate, triphosphate, phosphoamidate, cabamate, etc.) and charged linkages (e.g., thiophosphate, dithiophosphate, etc.); those with intercalating agents (e.g., acridine, psoralen, etc.); those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.); those containing alkylating agents; and those with modified linkages (e.g., α-anomeric nucleic acids, etc.). Furthermore, any hydroxyl groups normally present on the sugars of nucleotides may be replaced by phosphonate groups, phosphate groups; protected by standard protecting groups; or activated to prepare additional linkages with other nucleotides or with a solid support. The 5′ and 3′ terminal OH groups may be phosphorylated or replaced by an amine, an organic capping group of about 1 to about 20 carbon atoms, a polyethylene glycol (PEG) polymer in the range of about 10 to about 80 kDa in one embodiment, a PEG polymer in the range of about 20 to about 60 kDa in another embodiment, or other hydrophilic or hydrophobic bio or synthetic polymers.

[0136] As used herein, the terms “nucleic acid,” “oligonucleotide,” and “polynucleotide” are used interchangeably to refer to polymers of nucleotides and include modifications of DNA, RNA, DNA / RNA hybrids, and these kinds of nucleic acids, oligonucleotides, and polynucleotides, including the linkage of various entities or portions with nucleotide units at any position. The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” include double-stranded or single-stranded molecules as well as triple-helical molecules. Nucleic acid, oligonucleotide, and polynucleotide are broader terms than the term aptamer, and therefore the terms nucleic acid, oligonucleotide, and polynucleotide include polymers of nucleotides that are aptamers, but the terms nucleic acid, oligonucleotide, and polynucleotide are not limited to aptamers.

[0137] Polynucleotides may also contain similar forms of ribose or deoxyribose known in the art, including 2'-O-methyl-, 2'-O-allyl, 2'-O-ethyl, 2'-O-propyl, 2'-O-CH2CH2OCH3, 2'-fluorine, 2'-NH2 or 2'-azido, carbocyclic sugar analogs, α-terminal isomers, epimeric sugars (such as arabinose, xylose or lythose), pyranose, furanose, sedoheptulose, acyclic analogs, and baseless nucleoside analogs (such as methyl nucleosides). As described herein, one or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include those in which the phosphate ester is replaced by P(O)S (“thioester”), P(S)S (“dithioester”), (O)NR X 2 ("amidide"), P(O)R X P(O)OR X An implementation scheme involving the replacement of ', CO or CH2 ("methyl acetal"), wherein each R X or R X Independently, it is a substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl group with an ether (-O-) bond. Not all bonds in a polynucleotide need to be identical. Similar substitutions of sugars, purines, and pyrimidines may be advantageous in designing the final product; for example, alternative backbone structures such as polyamide backbones may also be advantageous in designing the final product.

[0138] Polynucleotides can also contain carbon-cyclic sugar analogs, α-terminal isomers, epimeric sugars (such as arabinose, xylose, or lysoose), pyranose, furanose, sedoheptulose, acyclic analogs, and similar forms of non-base nucleoside analogs (such as methyl nucleosides).

[0139] If present, modifications to the nucleotide structure can be imposed before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.

[0140] As used herein, when referring to modifications of nucleic acids, the term "at least one nucleotide" means one, several, or all of the nucleotides in a nucleic acid, thus indicating that any or all of any or all of the A, C, T, G, or U in the nucleic acid may be modified or not modified.

[0141] The term "antibody" refers to full-length antibodies of any species, as well as fragments and derivatives of such antibodies that retain the ability to bind to antigens, 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, affinity molecules, and nanobodies.

[0142] As used herein, the terms “nucleic acid ligand,” “aptamer,” “SOMAmer,” “modified aptamer,” and “clone” are used interchangeably to refer to non-naturally occurring nucleic acids that have the desired effect on a target molecule. The desired effect includes, but is not limited to, binding to a target, catalytically altering a target, reacting with a target in a manner that modifies or alters the target or its functional activity, covalently attaching to a target (as in suicide inhibitors), and facilitating a reaction between a target and another molecule. In one embodiment, the effect is a specific binding affinity for a target molecule that binds to the aptamer’s three-dimensional chemical structure other than a polynucleotide via a mechanism independent of Watson / Crick base pairing or triple helix formation, wherein the aptamer is not a nucleic acid with a known physiological function that is being bound by the target molecule. Aptamers for a given target include nucleic acids identified from a mixture of candidate nucleic acids by a method comprising: (a) contacting the candidate mixture with the target, wherein nucleic acids having a higher affinity for the target relative to other nucleic acids in the candidate mixture can be allocated from the remainder of the candidate mixture; (b) allocating the higher-affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the higher-affinity nucleic acids to produce a ligand-enriched mixture of nucleic acids, thereby identifying aptamers of the target molecule. It should be recognized that affinity interactions are a matter of degree; however, in this context, the “specific binding affinity” of an aptamer to its target means that the aptamer typically binds to its target with a much higher degree of affinity than it binds to other non-target components in the mixture or sample. An “aptamer,” “SOMAmer,” or “nucleic acid ligand” is a set of copies of a type or class of nucleic acid molecule having a specific nucleotide sequence. An aptamer may include any suitable number of nucleotides. An “aptamer” refers to more than one such set of molecules. Different aptamers may have the same or different numbers of nucleotides. Aptamers can be DNA or RNA, and can be single-stranded, double-stranded, or contain double-stranded or triple-stranded regions. In some embodiments, aptamers are prepared using the SELEX method described herein or known in the art.

[0143] As used herein, "SOMAmer" or slow dissociation rate modified aptamer refers to an aptamer with improved dissociation rate characteristics. SOMAmer can be generated using the improved SELEX method described in U.S. Patent 7,947,447 entitled "Method for Generating Aptamers with Improved Off-Rates".

[0144] As used herein, an aptamer containing two different types of 5-position modified pyrimidines or C-5 modified pyrimidines may be referred to as a "double-modified aptamer," an aptamer with "two modified bases," an aptamer with "two base modifications" or "two modified bases," or an aptamer with "double-modified bases," all of which are used interchangeably. The same terminology may also be used for "a library of aptamers" or "aptamer library." Therefore, in some embodiments, the aptamer comprises two different 5-position modified pyrimidines, wherein the two different 5-position modified pyrimidines are selected from 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 TrpdU, 2NapdC and NapdU, and 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 partially modified at the 5-position by a portion selected from a naphthyl moiety, benzyl moiety, fluorobenzyl moiety, tyrosinyl moiety, indole moiety, morpholino moiety, isobutyl moiety, 3,4-methylenedioxybenzyl moiety, benzo[a]phenylthio] moiety, and benzo[a]furanyl moiety, and wherein the at least one modified cytidine is partially modified at the 5-position by a portion selected from a naphthyl moiety, tyrosinyl moiety, and benzyl moiety. In some embodiments, the portion is covalently linked to the 5-position of the base via a connector comprising a group selected from amide connectors, carbonyl connectors, propynyl connectors, alkyne connectors, ester connectors, urea connectors, carbamate connectors, guanidine connectors, amidine connectors, sulfoxide connectors, and sulfone connectors. For other examples of exemplary connectors that can be used to covalently link portions to the 5-position of pyrimidines, see [link to relevant documentation]. Figure 21 .

[0145] As used herein, the terms “hydrophobic group” and “hydrophobic moiety” are used interchangeably and refer to any uncharged group or moiety whose majority atoms are hydrogen and carbon, which has a small dipole and / or which tends to repel water. These groups or moiety may comprise 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 empirically derived methods as well as computational methods. Exemplary methods are 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 planarpeptide network. Proc. Natl. Acad. Sci., 113(46), pp. 12946-12951. Exemplary hydrophobic moiety includes, but is not limited to, those disclosed herein. Figure 21 Groups I, II, III, IV, V, VII, VIII, IX, XI, XII, XIII, XV, and XVI. Other exemplary hydrophobic portions include... Figure 25 Those (e.g., Bn, Nap, PE, PP, iBu, 2Nap, Try, NE, MBn, BF, BT, Trp).

[0146] As used herein, an aptamer containing a single type of 5-position modified pyrimidine or a C-5 modified pyrimidine may be referred to as a “single-modified aptamer,” an aptamer with a “single modified base,” an aptamer with a “single base modification,” or an aptamer with “single bases modified,” all of which are used interchangeably. The same terminology may also be used for a library of aptamers or an aptamer library. 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 which its amino acid sequence is derived, or substantially free of chemical precursors or other chemicals during its chemical synthesis.

[0147] In some embodiments, the aptamer comprises a first 5-position modified pyrimidine and a second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine comprises a tyrosine moiety at the 5-position, and the second 5-position modified pyrimidine comprises a naphthyl moiety or a benzyl moiety at the 5-position. In one related embodiment, the first 5-position modified pyrimidine is uracil. In one related embodiment, the second 5-position modified pyrimidine is cytosine. In one related embodiment, 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 the 5-position. In one relevant embodiment, 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 of the aptamer is modified at the 5-position.

[0148] Those skilled in the art of nucleic acid hybridization will recognize that factors commonly used to apply or control hybridization strictness include formamide concentration (or other chemical denaturing agents), salt concentration (i.e., ionic strength), hybridization temperature, detergent concentration, pH, and the presence of a dissociative agent. Optimal strictness for probe / target sequence combinations is typically discovered using well-known techniques that fix several of the aforementioned strictness factors and then determine the effect of altering individual strictness factors. The same strictness factor can be tuned to control the strictness of PNA hybridization with nucleic acids, except that PNA hybridization is completely independent of ionic strength. Optimal strictness for the assay can be experimentally determined by examining each strictness factor until the desired discrimination is achieved.

[0149] As used herein, in the context of nucleotide sequences, the terms “hybridization,” “hybridizing,” “binding,” and similar terms are used interchangeably. The ability of two nucleotide sequences to hybridize is based on the degree of complementarity between the two sequences, which in turn is based on the fraction of matched complementary nucleotide pairs. The more nucleotides in a given sequence that are complementary to another sequence, the more stringent the hybridization conditions may be, and the more specific the binding of the two sequences will be. Increased stringency can be achieved by increasing the temperature, increasing the proportion of the co-solvent, decreasing the salt concentration, etc. Hybridization of complementary Watson / Crick base pairs between the probe and target material on the microarray is generally preferred, but non-Watson / Crick base pairing can also occur during hybridization.

[0150] Conventional hybridization solutions and methods for hybridization are described in J. Sambrook, Molecular Cloning: A Laboratory Manual, which is incorporated herein by reference. Conditions for hybridization typically include (1) solutions with high ionic strength, (2) controlled temperatures, and (3) the presence of vector DNA and surfactants and chelating agents having divalent cations, all of which are known in the art.

[0151] As used herein, a “biopolymer” is a polymer of one or more types of repeating units. Biopolymers are commonly found in biological systems and particularly include polysaccharides (such as carbohydrates) and peptides (the term is used to include polypeptides and proteins, whether or not linked to polysaccharides) and polynucleotides and their analogues, such as those compounds that consist of or contain 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 has been replaced by a backbone that is not naturally occurring or synthetic, and nucleic acids (or synthetic or naturally occurring analogues) in which one or more conventional bases have been replaced by groups (natural or synthetic) capable of participating in Watson-Crick type hydrogen bonding interactions. Polynucleotides include single-stranded or multi-stranded configurations in which one or more strands may or may not be perfectly aligned with another strand. Specifically, “biopolymers” include deoxyribonucleic acid or DNA (including cDNA), ribonucleic acid or RNA, and oligonucleotides, regardless of origin.

[0152] As used herein, an "array" includes any one-dimensional, two-dimensional, or three-dimensional arrangement of said regions having one or more specific chemical portions (e.g., biopolymers, such as peptide nucleic acid molecules, peptides, or polynucleotide sequences) associated with an addressable region, wherein said one or more chemical portions are anchored to the surface of said region. "Anchored" means that the one or more portions are stably associated with the substrate surface in the region such that they do not separate from said region under the conditions of using the array (e.g., hybridization and washing, and stripping conditions). As is known in the art, said one or more portions may be covalently or non-covalently bound to the surface of said region. For example, in the case of a porous substrate, each region may extend into a third dimension, while in the case of a non-porous substrate, each region may not have any substantial third-dimensional measurement (thickness). Arrays may contain more than ten, more than one hundred, more than one thousand, more than ten thousand features, or even more than one hundred thousand features in regions less than 20 cm or even less than 10 cm. For example, features may have a width (i.e., diameter for a circular spot) ranging from about 10 μm to about 1.0 cm. In other embodiments, each feature may have a width ranging from about 1.0 μm to about 1.0 mm, such as from about 5.0 μm to about 500 μm, and including a range from about 10 μm to about 200 μm. Non-circular features may have an area range equal to the area range of circular features having the aforementioned width (diameter) range. A given feature consists of a chemical portion (e.g., a peptide nucleic acid molecule, peptide, nucleic acid) that binds (e.g., hybridizes) to a target molecule (e.g., a target nucleic acid or aptamer) such that the given feature corresponds to a specific target.

[0153] In the case of an array, the "target" will be referred to as a portion (typically a fluid) of the mobile phase that is detected by probes ("target probes") that bind to the substrate in various regions. However, either the "target" or the "target probe" may be the one 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, peptide, protein, oligonucleotide, or aptamer.

[0154] The terms “biological sample,” “sample,” and “test sample” are used interchangeably herein to refer to any material, biological fluid, tissue, or cell obtained from an individual or otherwise, as well as environmental, animal, or food samples. This includes blood (including whole blood, white blood cells, peripheral blood mononuclear cells, erythrocyte sedimentation rate (ESR), plasma, and serum), sputum, tears, mucus, nasal wash, nasal aspirate, respiration, urine, semen, saliva, peritoneal lavage, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph, papillary aspirate, bronchial aspirate (e.g., bronchoalveolar lavage fluid), bronchial brushing material, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid. This also includes experimentally separated portions of all the foregoing substances. For example, blood samples may be graded into serum, plasma, or fractions containing specific types of blood cells such as red blood cells or white blood cells (leukocytes). In some implementations, the sample may be a combination of samples from an individual, such as a combination of tissue and fluid samples. The term "biological sample" also includes materials containing homogenized solid material, such as materials from fecal samples, tissue samples, or tissue biopsies. The term "biological sample" also includes materials derived from tissue cultures or cell cultures. Any suitable method for obtaining a biological sample may be used; exemplary methods include, for example, venipuncture, swabs (e.g., oral swabs), and fine-needle aspiration biopsy procedures. Exemplary tissues readily suitable for fine-needle aspiration include lymph nodes, lungs, bronchoalveolar lavage fluid, BAL (bronchial alveolar lavage fluid), thyroid gland, breast, pancreas, and liver. Samples may also be collected, for example, by microdissection (e.g., laser capture microdissection (LCM) or laser microdissection (LMD)), bladder washing, smears (e.g., PAP smears), or catheter irrigation. "Biological sample" obtained from or derived from an individual includes any such sample that has been processed in any suitable manner after being obtained from the individual.

[0155] The collection device used herein can be any suitable absorption device capable of absorbing the sample to be dried. Non-limiting examples include filter paper such as Whatman filters. ® Guthrie card, MITRA ® Micro-sampling device, Captainer ® Micro-sampling devices, TAPII micro-sampling devices, Tasso devices, dry fluid cards and similar devices.

[0156] As used herein, “target protein level,” “analyte level,” and “level,” or “target protein value,” “analyte value,” and “value” refer to a measurement performed using any analytical method for detecting an analyte (such as a target protein) in a biological sample and indicate the presence, absence, absolute amount or concentration, relative amount or concentration, titer, level, expression level, ratio of the measured level, etc., of the analyte in the biological sample, the analyte used in the biological sample, or the analyte corresponding to the analyte in the biological sample. The exact nature of a “level” or “value” depends on the specific design and composition of the specific analytical method used to detect the analyte.

[0157] As used herein, "capture agent" 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, adnectin, ankyrins, other antibody mimics and other protein scaffolds, autoantibodies, chimeras, small molecules, nucleic acids, lectins, ligand-binding receptors, imprinted polymers, high-affinity multimers (avimers), peptide mimics, hormone receptors, cytokine receptors, synthetic receptors, and any modifications and fragments of the above capture reagents. In some embodiments, the capture reagent is selected from aptamers and antibodies.

[0158] 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 who do not have the disease or condition, or from individuals who are not suspected of having the disease or condition or are not at risk of having the disease or condition, or from individuals with a non-progressive disease or condition. Furthermore, the “control level” or “control value” can refer to a reference value based on an average or considered within normal or healthy parameters. The “control level” or “control value” can also refer to a reference level obtained at a previous time and used for comparison with the target level measured or detected later. For example, the level of the target may be detected at time point A, and then at time point B, where time point B is after time point A. In a more specific instance, time point A can be considered time zero (0) or day zero (0), and time point B can be minutes (e.g., 10, 20, 30, 40, 50, 60 minutes after time point A), 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 point A), or days (e.g., ... The target molecule can be measured at time points A (1, 2, 3, 4, 5, 6, or 7 days after time point A), weeks (e.g., 1, 2, 3, or 4 weeks after time point A), months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after time point A), or even years (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 measured each time the method of this invention is performed, and can be a previously determined level used as a reference or threshold to determine whether the level in a particular sample is higher or lower than normal.

[0159] "Correspondence correlation" or "consistency correlation coefficient" measures the agreement between two continuous variables X and Y (e.g., predicted, estimated, or measured values ​​and actual values). Correspondence correlation evaluates the degree to which pairs fall on the 45° line and includes measures of accuracy and precision (or "Lin's Condordance"). Further information can be found in Lin, Biometrics, Vol. 45, No. 1 (March 1989), pp. 255-268, which is incorporated herein by reference. Other methods that may be used in this paper to determine correlation include, but are not limited to, Pearson correlation coefficient, paired t-test, least squares analysis with slope (=1) and intercept (=0), coefficient of variation, and within-group correlation coefficient. In some implementations, correspondence correlation is determined by a method selected from: Lin's condordance, Pearson correlation coefficient, paired t-test, least squares analysis with slope (=1) and intercept (=0), coefficient of variation, and within-group correlation coefficient.

[0160] As used herein, “detection” or “determination” includes both the use of instruments for observing and recording a signal corresponding to the analyte level and the materials required to generate that signal. In various embodiments, the level is detected using any suitable method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic wave, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection methods, nuclear magnetic resonance, quantum dots, etc.

[0161] "Dilution," "dilution series," and their variations encompass several different types of dilution, including but not limited to dispersive dilution, sequential dilution, and combinations thereof. Taking stepwise dilution as an example, if the dilution factor is 1000 (1:1000 dilution), the user can first perform a 1:10 dilution (dilution factor 10), and then use 1 part solute and 99 parts diluent from the 1:10 dilution to perform a 1:100 dilution (dilution factor 100), thus producing a dilution factor of 1000 or a 1:1000 dilution of the solute. Sequential dilution involves a series of stepwise dilutions, each with the same dilution factor, where the diluent material from the previous step is used for subsequent dilutions. For example, to perform a 5-point 1:2 serial dilution, you need to use 1 part solute and combine it with 1 part diluent to prepare the first dilution in the series (point 1 of 5), then use 1 part solute from the first dilution and combine it with 1 part diluent to prepare the second dilution in the series (point 2 of 5), and so on, until the fifth serial dilution is reached.

[0162] "Dilution factor" refers to the ratio of the number of parts of solute to the number of parts of diluent. For example, a dilution factor of 2 means a 1:2 dilution, where 1 part of solute and 1 part of diluent make a total of 2 parts; and a dilution factor of 10 means a 1:10 dilution, where 1 part of solute and 9 parts of diluent make a total of 10 parts.

[0163] The terms “target,” “target molecule,” and “analyte” are used interchangeably herein to refer to any target molecule that may be present in a sample. The term includes any minor variations in a particular molecule, such as, in the case of proteins, minor variations in amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component that substantially does not alter the molecular identity. “Target molecule,” “target,” or “analyte” refers to a set of copies of a molecule or multimolecular structure of one type or kind. “Target molecule,” “target,” and “analyte” refer to more than one type or kind of molecule or multimolecular structure. Exemplary target molecules include proteins, peptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, affinities, antibody mimics, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, and any fragment or portion of any of the foregoing substances. In some embodiments, the target molecule is a protein, in which case the target molecule may be referred to as a “target protein.”

[0164] 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 generated from a biological sample. In some embodiments, the test sample is generated from a biological sample or a solution containing a biological sample by buffer exchange. As used herein, "adjusted test sample" is a test sample that has been adjusted, such as by changes in total protein concentration.

[0165] The phrases “oligonucleotide bound to a solid support surface,” “probe bound to a solid support,” or “target bound to a solid support” refer to peptide nucleic acid molecules, oligonucleotides, aptamers (e.g., PNAs), LNAs (locked nucleic acids), or UNAs (unlocked nucleic acids) molecules immobilized on a solid substrate surface, wherein the substrate may have various configurations, such as sheets, beads, particles, glass slides, wafers, meshes, fibers, tubes, capillaries, microfluidic channels, reservoirs, or other structures. In some embodiments, the collection of oligonucleotide or target elements used herein is present on the surface of a planar support, for example, in an array. It should be understood that the terms “probe” and “target” are relative terms, and a molecule considered a probe in one assay may act as a target in another. The immobilization of oligonucleotides on a substrate or surface can be accomplished using well-known techniques, which are generally available in the literature. See, for example, AC Pease, 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 of which is incorporated herein by reference.

[0166] The aforementioned chemical descriptions of polynucleotide synthesis are found, for example, in 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., p. 100, and so on; U.S. Patents 4,458,066, 4,500,707, 5,153,319, 5,869,643, EP 0294196, and others. The phosphorous acid and phosphite methods are most widely used, but other methods include phosphodiester methods, phosphotriester methods, and H-phosphonate methods. The substrate is typically functionalized to bind to the monomers deposited in the first deposition. Suitable techniques for functionalizing the substrate with such connecting portions are described, for example, in Southern, EM, Maskos, U., and Elder, JK, Genomics, 13, 1007–1017, 1992. In the case of array fabrication, different monomers and activators can be deposited at different sites on the substrate during any given cycle, such that different features of the complete array will have different desired biopolymer sequences. One or more intermediate steps may be required in each cycle, such as the conventional oxidation, capping, and washing steps in the case of in-situ fabrication of polynucleotide arrays (again, these steps can be performed during the implantation process).

[0167] Multiple determination

[0168] Multiple assays based on target interactions and separation steps in solutions are described, for example, in U.S. Patent Nos. 7,855,054 and 7,964,356 and PCT application PCT / US2013 / 044792. In one embodiment, multiple assays are described in Example 1 herein.

[0169] In multiplex assays that measure multiple target proteins using various capture reagents, the natural variation in the abundance of different target proteins can limit the ability of certain capture reagents to measure certain target proteins (e.g., high-abundance target proteins can saturate the assay and prevent or reduce its ability to measure low-abundance target proteins). To address this variation in biological samples, aptamer reagents can be grouped into at least two distinct groups (capture reagents for DIL1 and capture reagents for DIL2), preferably three distinct groups (A3 – capture reagents for DIL1; A2 – capture reagents for DIL2; and A1 – capture reagents for DIL3), 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 distinct set of aptamers that have specific affinity for the target protein. Biological samples are diluted into two (dilution 1 or DIL1 and dilution 2 or DIL2), preferably three different dilution groups (dilution 1 or DIL1; dilution 2 or DIL2 and dilution 3 or DIL3) to create individual test samples based on the relative concentrations of protein targets to be detected by their capture reagents. Thus, biological samples are diluted into high and low, or high, medium and low abundance target protein dilution groups, where the lowest abundance protein target is measured in the lowest dilution group and the highest abundance protein target is measured in the highest dilution group. The capture reagents used for their respective dilution groups are incubated together (e.g., A3 aptamers are incubated with the test sample in dilution 1 or DIL1; A2 aptamers are incubated with the test sample in dilution 2 or DIL2; and A1 aptamers are incubated with the test sample in dilution 3 or DIL3). The total number of aptamers A1, A2, and A3 can be 4,000; 4,500; 5,000, or more. In some implementations, diluent group 1 may be 2.5%, diluent group 2 may be 0.05%, and diluent group 3 may be 0.005%. Other diluents in the three diluent groups may also be used.

[0170] This disclosure describes methods for preparing a dried biological matrix for detecting target molecules in multiplex assays, and methods for detecting target molecules from a dried biological matrix in multiplex assays.

[0171] Biomatrixes can be prepared for multiple assays as follows. Biomatrixes can be collected from subjects according to standard procedures, such as blood draws, fluid collections, biopsy collections, etc. Once collected, the biomatrix can be spotted or adsorbed onto a collection device and dried.

[0172] Drying temperature

[0173] In some embodiments, the drying temperature may be about -20°C to about room temperature (about 20°C-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.

[0174] Drying time

[0175] In some embodiments, the drying time can be from about 1 hour to about 10 days. In some embodiments, the drying time can be 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 can be from at least 1 hour to at least 10 days. In some embodiments, the drying time can 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 can be at a constant temperature until the sample is dry before any temperature fluctuations occur. In some embodiments, temperature fluctuations may occur during transport and handling. In some embodiments, when the drying temperature is constant until the sample is dry, the dried sample is suitable for multiple determinations.

[0176] Extraction time

[0177] 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.

[0178] Extracts

[0179] In some embodiments, the extraction formulation may comprise a buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide. In some embodiments, in addition to the buffer, one or more salts, a chelating agent, a protease inhibitor, a nonionic surfactant, and an oligonucleotide, the extraction formulation may also comprise 20% PBS. In some embodiments, one or more salts are each independently selected from sodium, potassium, and magnesium salts.

[0180] In some embodiments, one or more salts include sodium, potassium, and magnesium salts. 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 mM to 125 mM, or about 100 mM. In some embodiments, 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. In some embodiments, the concentration of MgCl2 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 8 mM.

[0181] In some embodiments, the buffer is selected from HEPES, IVIES, Bistris methane, ADA, ACES, Bistris propane, PIPES, MOPSO, choline chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, glycamide, glycylglycine, HEPBS, Bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS. In some embodiments, the concentration of the buffer in the formulation is 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.

[0182] In some embodiments, the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA. In some embodiments, the concentration of the chelating agent in the formulation is from about 0.1 mM to about 10 mM, or from about 0.5 mM to about 5 mM, or about 1.25 mM.

[0183] In some embodiments, the nonionic surfactant is selected from polyoxyethylene (20) sorbitol monolaurate (Tween-20), polyoxyethylene (40) sorbitol monolaurate (Tween-40), and polyoxyethylene (80) sorbitol monolaurate (Tween-80). In some embodiments, the nonionic surfactant is present at 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% or about 1.5% of the formulation, or about 1.2% of the formulation, on a volume-to-volume basis.

[0184] In some embodiments, the pH of the formulation is from about pH 5 to about pH 9, or from about pH 6 to about pH 8, or from about pH 7 to about pH 7.9 or about pH 7.5.

[0185] 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.

[0186] In some implementations, the formulation has a pH of approximately 7.5.

[0187] 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 an inhibitor of serine proteases. In some embodiments, the protease inhibitor is benzomidine. In some embodiments, the concentration of the protease inhibitor in the formulation is about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1.2 mM.

[0188] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is 20 to 100 nucleotides in length, or 25 to 80 nucleotides in length, or 25 to 70 nucleotides in length, or 25 to 50 nucleotides in length, or about 30 nucleotides in length. In some embodiments, the oligonucleotide comprises one or more modified nucleotides. In some embodiments, the oligonucleotide comprises 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.

[0189] The volume of sample to be dried can be determined by the device used. As a non-limiting example, the mitra VAMS micro-sampling device loads 30 μL of sample, thus the initial sample volume is greater than 30 μL. In some embodiments, the loaded 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.

[0190] As used herein, “Catch-1” refers to the partitioning of an aptamer-target affinity complex or an aptamer-target covalent complex. The purpose of Catch-1 is to substantially remove all test sample components that are not associated with the aptamer. Removal of most of these components will generally improve target labeling efficiency by removing non-target molecules from the target labeling step used for Catch-2 capture and may result in reduced assay background. In one embodiment, the tag is attached to the aptamer by affixing the tag to the aptamer before assay, during assay preparation, or during assay. In one embodiment, the tag is a releasable tag. In one embodiment, the releasable tag comprises a cleavable connector and a tag. As described above, the tagged aptamer may be captured on a solid support, wherein the solid support includes a capture element adapted for the tag. The solid support may then be washed as described herein to remove any unwanted material (Catch-0) prior to equilibration with the test sample.

[0191] As used herein, “Catch-2” refers to the partitioning of aptamer-target affinity complexes or aptamer-target covalent complexes based on target molecule capture. The purpose of the Catch-2 step is to remove free or unreintegrated aptamers from the test sample prior to detection and optional quantification. Removing free aptamers from the sample allows for the detection of aptamer-target affinity or aptamer-target covalent complexes using any suitable nucleic acid detection technique. When using Q-PCR for detection and optional quantification, the removal of free aptamers is necessary for accurate detection and quantification of the target molecule.

[0192] In one embodiment, the target molecule is a protein or peptide, and a reagent that can be incorporated into the protein (and peptide) and a complex comprising the protein (or peptide) (e.g., an aptamer-target affinity (or covalent) complex) is used to dispense the free aptamer from the aptamer-target affinity (or covalent) complex (and the remainder of the test sample). The labeled protein (or peptide) and aptamer-target affinity (or covalent) complex can be immobilized on a solid support, thereby enabling the dispensing of the protein (or peptide) and aptamer-target affinity (or covalent) complex from the free aptamer. Such labeling may include, for example, a biotin moiety that can be incorporated into the protein or peptide.

[0193] In one embodiment, a Catch-2 tag is attached to a protein (or peptide) by chemically attaching the tag to a target before, during, or during assay. In one embodiment, the Catch-2 tag is a releasable tag. In one embodiment, the releasable tag comprises a cleavable linker and a tag. However, it is generally not necessary to release the protein (or peptide) from the Catch-2 solid carrier. As described above, the tagged target can be captured on a second solid carrier, wherein the solid carrier includes a capture element suitable for the target tag. The solid carrier is then washed with various buffer solutions, including buffer solutions containing organic solvents and buffer solutions containing salts and / or salt-containing detergents and / or cleaning agents.

[0194] After washing the second solid support, the aptamer-target affinity complex undergoes a dissociation step, wherein the complex is disrupted to produce free aptamers, while the target molecules generally remain bound to the solid support through binding interactions between the capture elements and the target capture tag. The aptamer can be released from the aptamer-target affinity complex by any method that disrupts the structure of the aptamer or target. This can be achieved by washing the carrier-bound aptamer-target affinity complex in a high-salt buffer solution 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 disrupt 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 can be used. In another embodiment, proteolytic digestion of the protein portion of the aptamer-target affinity complex is used to release the aptamer component.

[0195] In the case of aptamer-target covalent complexes, a cleavable linker in the aptamer construct is used to release the aptamer for subsequent quantification. In another embodiment, a cleavable linker in the target will induce the release of the aptamer-target covalent complex.

[0196] For example, proteomic affinity assays (multiplex assays) can be performed as follows:

[0197] Catch-0: Add 133 7.5% streptavidin-agarose syrup from lxSB17,Tw (40 mM HEPES, 102 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 5 mM KCl, 0.05% Tween-20) to multiple wells of a filter plate (0.45 μm Millipore HV plate (Durapore, catalog number MAHVN4550)). Thaw an appropriate mixture of l.lx aptamers (all aptamers containing a Cy3 fluorophore and a photolyzable biotin moiety at the 5' end) and then vortex. Boil the l.lx aptamer mixture for 10 min, vortex for 30 sec, and cool to 20°C in a water bath for 20 min. Remove the liquid containing streptavidin-agarose syrup from the filter plate by centrifugation (1000 x g, 1 min). Add 100 μL of the aptamer mixture to multiple wells of the filter plate (automatically). Incubate the mixture in the dark for 20 minutes at 25°C on a shaker set to 850 rpm.

[0198] Catch-0 Wash: After 20 minutes of incubation, remove the solution via vacuum filtration. Add 190 lx CAPS aptamer pre-wash buffer (50 mM CAPS, 1 mM EDTA, 0.05% Tw-20, pH 11.0) and incubate the mixture for 1 minute with shaking. Then remove the CAPS wash solution via vacuum filtration. Repeat the CAPS wash once. Add 190 μL lx SX17-Tween and incubate the mixture for 1 minute with shaking. Then remove lx SB17-Tween via vacuum filtration. Add another 190 μL lx SX17-Tw and incubate the mixture for 1 minute with shaking. Then remove lx SB17-Tw by centrifugation (1 minute, 1000 x g). After removing lx SB17,Tw, add 150 μL of Catch-0 storage buffer (150 mM NaCl, 40 mM HEPES, 1 mM EDTA, 0.02% sodium azide, 0.05% Tween-20), and carefully seal the filter plate only around the perimeter of the plate, and store it in the dark at 4°C until use.

[0199] Sample preparation: 75 μL of 40% sample dilution was plated out in a 40% sample plate (final 40% sample contained: 20 μM Z-block, 1 mM benzalkonium chloride, 1 mM EGTA, 40 mM HEPES, 5 mM MgCl2, 5 mM KCl, 1% Tween-20). 195 μL of 1x SB17-Tw was plated out in a 1% sample plate. 90 μL of 1x SB17-Tw was plated out in a 1:10 dilution plate. 133 μL of 1x SB17-Tw was plated out in a 0.005% sample plate. The samples were thawed for 10 minutes in a 25°C incubator on a rack thawing station, then vortexed and centrifuged at 1000 x g for 1 minute. The caps on the tubes were removed. Mix the sample (5 times, 50 μL), and transfer 50 μL of 100% sample to a 40% sample plate containing sample diluent. Then mix the 40% sample on the sample plate by up-and-down pipetting (110 μL, 10 times). Then transfer five (5) μL of 40% sample to a 1% sample plate containing 1x SB17-Tw. Mix this sample again by up-and-down pipetting (120 μL, 10 times). After mixing, transfer 10 μL of 1% sample to a 1:10 dilution plate containing 1x SB17-Tw and mix the sample by up-and-down pipetting (75 μL, 10 times). Transfer seven (7) μL of 0.1% sample from the 1:10 dilution plate to a 0.005% sample plate containing 1x SB17-Tw and mix by up-and-down pipetting (110 μL, 10 times).

[0200] Plate preparation prior to incubation: Catch-0 storage solution was removed from the filter plate via vacuum filtration. Then 190 μL of SB17-Tw was added, which was subsequently removed from the filter plate via vacuum filtration. Then 190 μL of SB17-Tw was added to the filter plate.

[0201] Incubation: Remove 1 x SB17-Tw buffer from the filter plate by centrifugation (1 min, 1000 x g). Add 100 μL of the appropriate sample dilution to the filter plate (three filter plates, each for 40% or 20%, 1% or 0.005% of the sample dilution). Carefully seal the filter plate only around the perimeter to avoid pressurizing the wells. Pressure will cause leakage during incubation. Then incubate the plate in the dark for 3.5 h at 28 °C on a thermostatic shaker set to 850 rpm.

[0202] Filter plate preparation: After incubation, the filter plate was placed on a vacuum manifold and the sample was removed by vacuum filtration. 190 μL of biotin wash buffer (lx SB17-Tw containing 100 μM biotin) was added, and the liquid was removed by vacuum filtration. The sample was then washed 5 times with 190 μL lx SB17-Tw (vacuum filtration). 100 μL of freshly prepared lx SB17-Tw containing 1 mM NHS-biotin was added, and the filter plate was blotted dry on an adsorption pad and incubated with the mixture for 5 minutes with shaking. The liquid was removed by vacuum filtration. 125 μL of lx SB17-Tw containing 20 mM glycine was added, and the liquid was removed by vacuum filtration. 125 μL of lx SB17-Tw containing 20 mM glycine was added again, and the liquid was removed by vacuum filtration.

[0203] The sample was then washed six times with 190 μL lx SB17-Tw and the liquid was removed by vacuum filtration. Then, 85 μL of photolysis buffer (containing 2 μM Z-block lx SB17-Tw) was added to each filter plate.

[0204] Photolysis: The filter plate was blotted dry on the adsorption pad and irradiated with a BlackRay UV lamp for 6 minutes under shaking (800 rpm, 25°C). The plate was rotated 180 degrees and irradiated again for 6 minutes under a BlackRay light source. The 40% filter plate was placed on an empty 96-well plate. The 1% filter plate was stacked on top of the 40% filter plate, and the 0.005% filter plate was stacked on top of the 1% filter plate. The plate assembly was centrifuged at 1000 x g for 1 minute. The 96-well plate containing the eluted sample was placed on the automated machine platform. 1 x SB17-Tw containing 60% glycerol was placed on the automated machine platform from the 37°C incubator.

[0205] Catch-2: During assay setup, 50 μL of 10 mg / mL MyOne SA beads (500 μg) was added to an ABgene Omni-tube 96-well plate for Catch-2 and placed under a Cytomat. The Catch-2 96-well bead plate was suspended for 90 seconds, placed on a magnetic block for 60 seconds, and the supernatant was removed. Simultaneously or sequentially, Catch-1 eluent from each dilution group was transferred to the Catch-2 bead plate and incubated on a Peltier thermostatic shaker (1350 rpm, 5 min, 25°C). The plate was transferred to a 25°C magnet and held for 2 min, and the supernatant was removed. Next, 75 μL of lxSB17-Tw was added, and the sample was incubated at 37°C on a Peltier shaker at 1350 rpm for 1 min. Then add lx SB17-Tw containing 75 μL of 60% glycerol (heated to 37°C) and incubate the sample again at 37°C on a Peltier shaker at 1350 rpm for 1 min. Transfer the plate to a magnet heated to 37°C and incubate for 2 min, then remove the supernatant. Repeat this 37°C lx SB17-Tw and glycerol wash cycle twice more. Then wash the sample with 150 μL lx SB17-Tw to remove residual glycerol on a Peltier shaker (1350 rpm, 1 min, 25°C), followed by 1 min on a magnet block at 25°C. Remove the supernatant and add 150 μL lx SB17-Tw replaced with 0.5 M NaCl, and incubate at 1350 rpm for 1 min (25°C), followed by 1 min on a magnet block at 25°C. Remove the supernatant and add 75 μL of perchlorate elution buffer (1.8 M NaClC-4, 40 mM PIPES, 1 mM EDTA, 0.05% Triton X-100, lx hybridization control, pH=6.8), then incubate on a Peltier shaker for 10 min (25°C, 1350 rpm). Afterward, transfer the plate to a magnetic separator and incubate for 90 sec, then recover the supernatant.

[0206] Hybridization: Twenty (20) μL of eluted sample was automatically added to an empty 96-well plate. Five (5) μL of 10x Agilent blocking buffer containing a second hybridization control was automatically added to the eluted sample. Then, 25 μL of 2x Agilent HiRPM hybridization buffer was manually added to each well. Forty (40) μL of hybridization mixture was loaded onto an Agilent spacer slide. An Agilent 8×15k array was added to the spacer slide, and the sandwich structure was clamped. The sandwich structure was then incubated at 55°C with rotation (20 rpm) for 19 hours.

[0207] Post-hybridization washing: Post-hybridization slides were processed 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 a glass staining dish. Approximately 750 mL of Wash Buffer 1 (Oligo aCGH / ChlP-on-chip Wash Buffer 1, Agilent Technologies) was placed in bath 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 bath 2 of the Little Dipper Processor. The magnetic stirring speed of both baths was set to 5. The temperature controller of bath 1 was not turned on, while the temperature controller of bath 2 was set to 37°C. Up to twelve slide / pad assemblies were sequentially disassembled into a first staining dish containing Wash Buffer 1, and then the slides were placed in a slide holder while still immersed in Wash Buffer 1. Once all slide / pad assemblies were disassembled, the slide holder was quickly transferred to Bath 1 of the Little Dipper Processor, and the automated washing protocol was initiated. The Little Dipper Processor incubated the slides in Bath 1 at 250 for 300 seconds, then transferred them to Bath 2 at 37°C containing AgilentWash 2 (Oligo aCGH / ChlP-on-chip Wash Buffer 2, Agilent Technologies), and incubated for 300 seconds at 100. Afterward, the Little Dipper Processor transferred the slide holder to the built-in centrifuge, where the slides were spun at 690 for 300 seconds.

[0208] Microarray imaging: The microarray slides were imaged in the Cy3 channel at a resolution of 5 μm with 100% PMT setting using a microarray scanner (Agilent G2565CA microarray scanner system, Agilent Technologies), and the XRD option was activated at 0.05. The resulting TIFF format images were processed using Agilent Feature Extraction Software version 10.7.3.1 with the GEl_107_Sep09 scheme.

[0209] As used herein, a “releasable” or “cleavable” element, part, or connector refers to a molecular structure that can be broken to produce two separate components. A releasable (or cleavable) element may comprise a single molecule in which chemical bonds can be broken (referred to herein as an “intrachain cleavable connector”), or may comprise two or more molecules in which non-covalent interactions can be broken or disrupted (referred to herein as a “hybrid connector”).

[0210] In some embodiments, it is necessary to spatially separate certain functional groups from others to prevent interference with individual functionalities. For example, the presence of a marker absorbing certain wavelengths of light near a photolytically cleavable group may interfere with photolysis efficiency. Therefore, it is necessary to separate the groups with, for example, non-interfering portions that provide sufficient spatial separation to restore full photolysis activity. In some embodiments, "spacer connectors" have been introduced into the aptamer using markers and photolysis functionality.

[0211] "Solid support" refers to any substance having a surface through which molecules can be directly or indirectly linked by covalent or non-covalent bonds. Solid supports may include any material capable of providing physical support for a capture element or probe attached to the surface. The material is generally able to withstand the conditions encountered during the assay related to the attachment of the capture element or probe to the surface and any subsequent handling, disposal, or processing. The material may be naturally occurring, synthetic, or a modified version of a naturally occurring material. Suitable solid carrier materials may include silicon, silicon wafer chips, graphite, mirrors, laminates, films, ceramics, and plastics (including polymers such as poly(vinyl chloride), cyclic olefin copolymers, agarose gels or beads, polyacrylamide, polyacrylates, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, polyethylene terephthalate, polytetrafluoroethylene (PTFE or Teflon®), nylon, poly(vinyl butyrate)), germanium, gallium arsenide, gold, silver, Langmuir-Blodgett films, current-carrying chips, etc. Other rigid materials may be considered, such as glass including silica, and also including, for example, glass that can be used as bioglass. Other materials that may be used include porous materials, such as, for example, controlled-pore glass beads, bead-crosslinked Sepharose® or agarose resin, or crosslinked copolymers of bisacrylamide and azalactone. Other beads include nanoparticles, polymer beads, solid core beads, paramagnetic beads, or microspheres. Also included are any other materials known in the art capable of having one or more functional groups (such as any one of amino, carboxyl, thiol, or hydroxyl functional groups) incorporated into their surface.

[0212] Materials used for solid supports can take on any of a range of configurations, from simple to complex. Solid supports can have any of many shapes, including strips, plates, discs, rods, particles, beads, tubes, pores (microtitering), etc. Solid supports can be porous or non-porous, magnetic, paramagnetic or non-magnetic, polydisperse or monodisperse, hydrophilic or hydrophobic. Solid supports can also be in the form of tightly packed gels or slurries (e.g., in matrix form) or loosely packaged particles.

[0213] In one embodiment, a solid carrier connected to a capture element is used to capture a labeled aptamer-target affinity complex or aptamer-target covalent complex from a test mixture. In a specific example, when the label is a biotin portion, the solid carrier may be streptavidin-coated beads or resin, such as Dynabeads M-280 streptavidin, Dynabeads MyOne streptavidin, Dynabeads M-270 streptavidin (Invitrogen), streptavidin agarose resin (Pierce), streptavidin hyperlinked resin, MagnaBind streptavidin beads (ThermoFisherScientific), BioMag streptavidin, ProMag streptavidin, silica streptavidin (BangsLaboratories), and high-performance streptavidin sepharose (GE Healthcare).

[0214] Streptavidin-coated polystyrene microspheres (Nanospheres), streptavidin-coated polystyrene particles (Spherotech), or any other streptavidin-coated beads or resin commonly used by those skilled in the art to capture biotin-tagged molecules.

[0215] As described above, one object of the present invention is to convert protein signals into aptamer signals. Therefore, the amount of aptamer collected / detected indicates the amount of target molecules bound and the amount of target molecules in the sample, and may be proportional to the amount of target molecules bound and the amount of target molecules in the sample. Many detection schemes can be employed after Catch-2 partitioning without eluting the aptamer-target affinity or aptamer-target covalent complex from the second solid support. Other detection methods will be known to those skilled in the art in addition to the detection method embodiments described below.

[0216] Many detection methods require the incorporation of specific tags into the aptamer prior to detection. In these implementations, labels, such as fluorescent or chemiluminescent dyes, can be incorporated into the aptamer during or after synthesis using standard nucleic acid synthesis techniques. Radioactive labels can be incorporated during or after synthesis using standard enzyme reactions with appropriate reagents. Labeling can also occur after Catch-2 partitioning and elution using suitable enzymatic techniques. For example, using primers with the aforementioned labels, PCR incorporates the labels into the amplified products of the eluted aptamer. When using gel electrophoresis for quantification, PCR can also be used to incorporate mass markers of different sizes. These mass markers can also be incorporated with different fluorescent or chemiluminescent dyes to obtain additional multiplex assay capabilities. Labeling can be indirectly added to the aptamer by using a specific tag incorporated into the aptamer during or after synthesis and subsequently adding a tag-associated and tag-carrying probe. Labels include those described above as well as enzymes used in standard assays for, for example, colorimetric readings. These enzymes work in combination with their substrates and include enzymes such as, for example, horseradish peroxidase (HRP) and alkaline phosphatase (AP). Labeling may also include materials or compounds with electrochemical functional groups for electrochemical detection.

[0217] For example, the aptamer can be labeled with a radioactive isotope such as 32P, as described above, before contact with the test sample. Using any of the four basic assays and their variations discussed above, aptamer detection can be easily achieved by quantifying the radioactivity of the second solid support at the end of the assay. The radioactivity count will be proportional to the amount of the target in the original test sample. Similarly, labeling the aptamer with a fluorescent dye before contact with the test sample, as described above, allows for a simple direct fluorescence reading of the second solid support. Chemiluminescent labels or quantum dots can be similarly used for direct readings from the second solid support without the need for aptamer elution.

[0218] In addition to the methods described above, other detection methods can be employed by eluting aptamers or releasing photoaptor-target covalent complexes from a second solid carrier. For example, the released aptamers, photoaptamers, or photoaptor-target covalent complexes can be run on a PAGE gel and detected and optionally quantified using a nucleic acid staining agent such as SYBR Gold. Alternatively, the released aptamers, photoaptamers, or photoaptor covalent complexes can be detected and quantified using capillary gel electrophoresis (CGE) with fluorescent labels incorporated into the aptamers as described above. Another detection method employs quantitative PCR, using, for example, SYBR Green to detect and quantify the eluted aptamers. Alternatively, the Invader® DNA assay can be used to detect and quantify the eluted aptamers. Another alternative detection method employs next-generation sequencing.

[0219] In another embodiment, a “molecular beacon” is used during replication to determine the amount or concentration of the aptamer-target affinity complex (or aptamer-target covalent complex) (see, for example, Tyagi et al., Nat. Biotech. J_6:4953, 1998; U.S. Patent No. 5,925,517). The molecular beacon is a specific nucleic acid probe folded into a hairpin loop, containing a fluorophore at one end of the hairpin structure and a quenching group at the other end, such that the fluorophore produces little or no signal when the hairpin is formed. The loop sequence is specific to the target polynucleotide sequence, and upon hybridization with the aptamer sequence, the hairpin unfolds and thus produces a fluorescent signal.

[0220] For multiplex detection of a small number of aptamers still bound to a second solid support, fluorescent dyes with different excitation / emission spectra can be used to detect and quantify two, three, five, or up to ten individual aptamers.

[0221] Similarly, quantum dots of different sizes can be used for multiple readouts. Quantum dots can be introduced after the dispensing of free aptamers from a second solid carrier. Multiple readouts of 2, 3, 5, and up to 10 aptamers can be performed by using aptamer-specific hybridization sequences linked to unique quantum dots. Restricted multiple readouts can also be performed using antibodies labeled with different individually detectable radioisotopes such as 32P, 3H, 113JC, and 3J5JS.

[0222] To enable multiplex detection of aptamers released from the Catch-2 second solid vector, a single fluorescent dye incorporated into each aptamer, as described above, can be used for quantitative methods that allow for the identification of aptamer sequences and the quantification of aptamer levels. These methods include, but are not limited to, DNA microarray hybridization, microbead hybridization, next-generation sequencing, and CGE analysis.

[0223] In one implementation, a standard DNA hybridization array or chip is used to hybridize each aptamer or optical aptamer to a unique or a series of unique probes immobilized on a slide or chip, such as an Agilent array, Illumina BeadChip array, NimbleGen array, or custom-printed array. Each unique probe is complementary to a sequence on the aptamer. The complementary sequence can be a unique 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 vector are added to an appropriate hybridization buffer and processed using standard hybridization methods. For example, the aptamer solution is incubated with the DNA hybridization array at approximately 60°C for 12 hours to ensure hybridization tightness. The array is washed and then scanned in a fluorescence slide scanner to produce images of the aptamer hybridization intensity for each feature of the array. Image segmentation and quantification are performed using image processing software such as ArrayVision. In one implementation, multiple aptamer assays can 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.

[0224] In one embodiment, addressable microbeads with unique DNA probes complementary to the aptamers, as described above, are used for hybridization. The microbeads can be addressed using unique fluorescent dyes (such as Luminex bead technology) or by using barcode labeling (such as in Illumina VeraCode technology) or laser-driven transponders. In one embodiment, the aptamer released from the Catch-2 solid vector is added to a suitable hybridization buffer and processed using standard microbead hybridization methods. For example, the aptamer solution is incubated with a set of microbeads at approximately 60°C for two hours to ensure hybridization tightness. The solution is then processed on a Luminex instrument that can count individual bead types and quantify the aptamer fluorescence signal. In another embodiment, VeraCode beads are contacted with the aptamer solution and hybridized at approximately 60°C for two hours, then deposited on a gridded surface and scanned using a slide scanner for identification and fluorescence quantification. In yet another embodiment, transponder microbeads are incubated with the aptamer sample at approximately 60°C, and then quantified using a device suitable for transponder microbeads. In one implementation, the multiple aptamer assay can be performed by hybridization with microbeads using up to 25, 50, 100, 200, and 500 aptamers.

[0225] Samples containing the eluted aptamers can be processed to incorporate unique quality tags and fluorescent markers as described above. The quality-tagged aptamers are then injected into a CGE instrument (essentially a DNA sequencer), and the aptamers are identified by their unique quality and quantified using fluorescence from the dye incorporated during the labeling reaction. Althea Technologies has developed an exemplary instance of this technology.

[0226] Among the many methods described above, the aptamer solution can be amplified and optionally labeled before quantification. Standard PCR amplification can be used with the aptamer solution eluted from the Catch-2 solid vector. This amplification can be used prior to DNA array hybridization, bead hybridization, and CGE readout.

[0227] In another embodiment, Q-PCR is used to detect and / or quantify aptamer-target affinity complexes (or aptamer-target covalent complexes). As used herein, “Q-PCR” refers to a PCR reaction performed under controlled conditions so that the assay result is a quantitative result (i.e., the assay can quantify the amount or concentration of aptamers present in the test sample).

[0228] In one implementation, TaqMan® PCR is used to determine the amount or concentration of an aptamer-target affinity complex (or aptamer-target covalent complex) in a test sample. This technique generally relies on the 5'–3' exonuclease activity of an oligonucleotide replicase that generates a signal from a target sequence. TaqMan probes are selected based on the sequence of the aptamer to be quantified and typically include a 5' fluorophore (such as 6-carboxyfluorescein) and a 3' quencher group (such as, for example, 6-carboxytetramethylfluorescein) to generate a signal when the aptamer sequence is amplified using polymerase chain reaction (PCR). As the polymerase copies the aptamer sequence, the exonuclease activity releases the fluorophore from the probe, which anneals downstream of the PCR primers, thereby generating a signal. The amount of PCR product depends on the number of replication cycles performed and the initial concentration of the aptamer.

[0229] In another embodiment, an inserted fluorescent dye is used during replication to determine the amount or concentration of the aptamer-target affinity complex (or aptamer-target covalent complex). The inserted dye (such as, for example, SYBR® green) produces a larger fluorescent signal in the presence of double-stranded DNA compared to the fluorescence signal generated in the presence of single-stranded DNA. The signal generated by the dye is increased because double-stranded DNA products are formed during PCR. The intensity of the generated signal depends on the number of PCR cycles and the initial concentration of the aptamer.

[0230] In another implementation, mass spectrometry is used to detect and / or quantify the aptamer-target affinity complex (or aptamer-target covalent complex). The enzymatic techniques described above can be used to introduce unique mass tags. For mass spectrometry readout, no detection label is required; instead, identification is based on the position and area under the mass peak generated during mass spectrometry analysis, using mass itself for identification and quantification using techniques commonly used by those skilled in the art. An example of the use of mass spectrometry is the MassARRAY® system developed by Sequenom.

[0231] A computer program can 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 comprising 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.

[0232] One aspect of this disclosure is the product of any method disclosed herein, namely, the measurement result, which can be evaluated at the testing site or transported to another site for evaluation and, if necessary, transmitted to relevant parties at a remote location. As used herein, "remote location" means a location physically different from the location where the result is obtained. Therefore, the result can be sent to different rooms, different buildings, different parts of a city, different cities, etc. Data can be transmitted by any means, such as, for example, fax, mail, overnight delivery, email, FTP, voicemail, etc.

[0233] "Transmitting" information means the transmission of data representing the information as electronic signals through a suitable transmission channel (e.g., a private or public network). "Forwarding" an item means any means of moving the item from one location to another, whether by physically transporting the item or by other means (where possible), and includes, at least in the case of data, the physical transport of a medium carrying the data or the transmission of the data.

[0234] Modified nucleotides

[0235] In some embodiments, this disclosure provides oligonucleotides, such as aptamers, comprising 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 in, for example... Figure 21 In the middle. Certain non-limiting exemplary C5-modified uridines are shown. Figure 22 and Figure 24In, and certain non-limiting exemplary C5-modified cytidines are shown Figure 23 and Figure 25 middle.

[0236] Preparation of oligonucleotides

[0237] The automated synthesis of oligodeoxynucleotides is a routine practice in many laboratories (see, for example, Matteucci, MD and Caruthers, MH, (1990) J. Am. Chem. Soc., 103 (The contents of 3185-3191 are hereby incorporated in their entirety by reference). The synthesis of oligonucleotides is also well known (see, for example, Scaringe, SA, et al., (1990) Nucleic Acids Res.). 18 (5433-5441, the contents of which are hereby incorporated in their entirety by reference). As described herein, phosphoramides can be used to incorporate modified nucleosides into oligonucleotides via chemical synthesis, and triphosphates can be used to incorporate modified nucleosides into oligonucleotides via enzymatic synthesis. (See, for example, Vaught, JD et al. (2004) J.Am. Chem. Soc., 126 :11231-11237; Vaught, JV, et al. (2010) J. Am. Chem. Soc. 132 , 4141-4151; Gait, MJ “Oligonucleotide Synthesis a practical approach” (1984) IRL Press (Oxford, UK); Herdewijn, P. “Oligonucleotide Synthesis” (2005) (Humana Press, Totowa, NJ) (each is incorporated herein by reference in its entirety).

[0238] In this document, "target" or "target molecule" refers to any compound on which nucleic acids can act in a desired or anticipated manner. Target molecules can be, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, pathogens, toxic substances, substrates, metabolites, transformed analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, any part or fragment of any of the foregoing. Essentially, any chemical or biological effector can be a suitable target. Molecules of any size can serve as targets. Targets can also be modified in some way to enhance the likelihood or strength of the interaction between the target and the nucleic acid. Targets can also include any minor changes to a specific compound or molecule, such as, in the case of proteins, minor changes in the amino acid sequence, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component that substantially does not alter the identity of the molecule. A "target molecule" or "target" is a set of copies of a type or class of molecule or multimolecular structure capable of binding to an aptamer. "Target molecule" or "target" refers to more than one such group of molecules. An embodiment of the SELEX method in which the target is a peptide is described in U.S. Patent No. 6,376,190, entitled "Modified SELEX Processes Without Purified Protein". In some embodiments, the target is a protein.

[0239] As used herein, the terms "competitive molecule" and "competitive" are used interchangeably to refer to any molecule that can form a nonspecific complex with a nontarget molecule. In this document, nontarget molecules include free aptamers, where, for example, a competitor can be used to inhibit the nonspecific binding (rebinding) of the aptamer to another nontarget molecule. A "competitive molecule" or "competitive" is a set of copies of a type or class of molecules. A "competitive molecule" or "competitive" refers to more than one such set of molecules. Competitive molecules include oligonucleotides, polyanions (e.g., heparin, herring sperm DNA, salmon sperm DNA, tRNA, dextran sulfate, dextran, non-basic phosphodiester polymers, dNTPs, and pyrophosphates). In various embodiments, combinations of one or more competitors may be used.

[0240] As used herein, a “nonspecific complex” refers to a non-covalent association between two or more molecules other than an aptamer and their target molecule. Nonspecific complexes represent interactions between molecular classes. Nonspecific 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.

[0241] In another embodiment, a polyanionic competitor (e.g., dextran sulfate or another polyanionic material) is used during a slow dissociation rate enrichment process to facilitate the identification of aptamers that are difficult to contain polyanions. In this context, a "polyanionic refractory aptamer" is an aptamer capable of forming an aptamer / target complex that is less likely to dissociate in a solution also containing a polyanionic refractory material compared to an aptamer / target complex containing a non-polyanionic refractory aptamer. In this way, polyanionic refractory aptamers can be used to perform analytical methods to detect the presence, amount, or concentration of a target in a sample, wherein the detection method includes using a polyanionic material (e.g., dextran sulfate) to which the aptamer is intolerant.

[0242] Therefore, in one embodiment, a method for generating polyanion-intolerant aptamers is provided. In this embodiment, after contacting a candidate nucleic acid mixture with a target, the nucleic acids in the target and the candidate mixture are brought to equilibrium. A polyanion competitor is introduced and incubated in solution for a sufficient period of time to ensure that most fast-dissociation-rate aptamers in the candidate mixture dissociate from the target molecule. Furthermore, aptamers in the candidate mixture that may dissociate in the presence of the polyanion competitor are released from the target molecule. The mixture is dispensed to separate high-affinity, slow-dissociation-rate aptamers that are still associated with the target molecule and to remove any uncomplexed material from the solution. The aptamers can then be released and separated from the target molecule. The separated aptamers can also be amplified, and additional selection rounds can be applied to improve the overall performance of the selected aptamers. If it is not necessary to select slow-dissociation-rate aptamers for a specific application, this method can also be used with the shortest possible incubation time.

[0243] Salt

[0244] Salts that allow for the convenient or desirable preparation, purification, and / or treatment of compounds, such as pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts are found in Berge et al. (1977), “Pharmaceutically Acceptable Salts”, J. Pharm. Sci. 66 This was discussed in :1-19.

[0245] For example, if the compound is an anion or has a functional group that can be an anion (e.g., -COOH can be -COO). − If a suitable inorganic cation is present, then the salt can be formed using a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions, such as Na+. + and K + Alkaline earth metal cations, such as Ca 2+ and Mg 2+ ; and other cations, such as Al+3 Examples of suitable organic cations include, but are not limited to, 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 are derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. A common example of a quaternary ammonium ion is N(CH3)4. + .

[0246] If the compound is a cation or has a functional group that can be a cation (e.g., -NH2 can be -NH3). + If a salt can be formed from a suitable anion, then the salt can be formed from a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0247] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphor sulfonic acid, cinnamic acid, citric acid, edetate, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheponic acid, gluconic acid, glutamic acid, glycolic acid, oxaloacetic acid, hydroxynaphthylcarboxylic acid, hydroxyethyl sulfonic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucoic acid, oleic acid, oxalic acid, palmitic acid, pyruvic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, p-aminobenzenesulfonic acid, tartaric acid, toluenesulfonic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid and carboxymethyl cellulose.

[0248] Unless otherwise specified, references to a particular compound include its salt form.

[0249] Other implementation plans

[0250] In some embodiments, a method is disclosed, the method comprising: 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, wherein Z is a 0.1% to 10% dilution of the biological sample, and the first set of aptamers contains at least A3 different 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 the biological sample, wherein Y is less than Z, wherein Y is 0.001% to 0.1%, and wherein the second set of aptamers contains at least A2 different 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 the biological sample. d) Incubate the first, second, and third mixtures to allow the formation of aptamer-protein complexes and remove most of the aptamers that do not form aptamer-protein complexes; e) Collect the aptamers from the aptamer-protein complexes by dissociating the aptamer-protein complexes; f) Detect or quantify the collected aptamers; wherein most of the aptamers in the first, second, and third groups of aptamers each have an affinity for different target proteins in the test sample and are capable of forming aptamer-protein complexes with their target proteins, and wherein A3 is greater than A2, and A2 is greater than A2; and wherein the sum of A1, A2, and A3 is at least 4,000.

[0251] In one respect, the sum of A1, A2 and A3 is at least 4,500 or 5,000.

[0252] In one respect, A3 is A 1、A2 50% to 90% of the sum of A1, A2 and A3 (or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%); 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.

[0253] In one respect, 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.

[0254] In one respect, 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 about 3% or 4% of the sum of A1, A2 and A3.

[0255] In one aspect, 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 to 16,500, or 2000 to 15,000, or 3,000 to 12,000 or 4,000 to 10,000).

[0256] In one respect, A2 is at least 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820, 900 (or 500 to 3500, or 700 to 2500, or 800 to 2000).

[0257] In one respect, A1 is at least 100, 110, 120, 130, 140, 150, 160, 170, 173 (or 100 to 700 or 100 to 650).

[0258] In one respect, at least A3 different aptamers are distinguished from each other by at least one nucleotide difference and / or at least one nucleotide modification.

[0259] In one respect, at least A2 different aptamers are distinguished from each other by at least one nucleotide difference and / or at least one nucleotide modification.

[0260] In one respect, at least A1 different aptamers are distinguished from each other by at least one nucleotide difference and / or at least one nucleotide modification.

[0261] In one respect, at least A3 different aptamers, at least A2 different aptamers, and at least A1 different aptamers are distinguished from each other by at least one nucleotide difference and / or at least one nucleotide modification.

[0262] In some embodiments, a system is disclosed, the system comprising: a) a first container having a first mixture containing a first test sample having a first set of aptamers, wherein the first test sample is a Z% dilution of a test sample, and the first set of aptamers contains at least A3 different aptamers; b) a second container having a second mixture containing a second test sample having a second set of aptamers, wherein the second test sample is a Y% dilution of the test sample, wherein Y is less than or equal to Z, and the second set of aptamers contains at least A2 different aptamers; c) a third container having a third mixture containing a third set of aptamers. The system comprises a third test sample, wherein the third test sample is an X% dilution of the test sample, wherein X is less than or equal to Y, and at least A1 different aptamers are present in the third group of aptamers; wherein most of the aptamers in the first, second, and third groups of aptamers have an affinity for the protein in the test sample and are capable of forming an aptamer-protein complex, wherein A3 is greater than A2 and A2 is greater than A1; wherein the sum of A1, A2, and A3 is at least 4,000; and wherein 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.

[0263] In one aspect, the number of first capture agents is about 100, 110, 120, 130, 140, 150, 160, 170 or 173; or 100 to 700; or 100 to 650 capture agents.

[0264] In one aspect, the multiple second capture agents are about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820 or 900; or 500 to 3500; or about 700 to 2500; or 800 to 2000; or about 828 capture agents.

[0265] In one aspect, the various third capture agents are about 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 about 900 to 16,500; or about 2000 to 15,000; or about 3,000 to 12,000; or about 4,000 to 10,000; or about 4271 capture agents.

[0266] Example

[0267] The following examples are provided to illustrate some embodiments of this disclosure more fully. However, these examples should not in any way be construed as limiting the broad scope of this disclosure. Those skilled in the art can readily employ the basic principles of this discovery to design various compounds without departing from the spirit of this disclosure.

[0268] Example 1. Multiple aptamer determination and analysis of samples

[0269] Multiplex aptamer assays were used to analyze test and control samples to examine the detection of target molecules in dried biomatrices. The multiplex analysis used in this experiment included aptamers to detect approximately 5,000 proteins eluted from dried biomatric samples with a low limit of detection (median 1 pM), a dynamic range of approximately 7 log, and a median coefficient of variation of approximately 5%. Multiplex aptamer assays are commonly described in Gold et al. (2010) Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery. PLoS ONE 5(12): e15004; US Publications: 2012 / 0101002 and 2012 / 0077695; and PCT Publication WO 2019 / 246289.

[0270] Example 2: Optimization of multiplex assay diluent using dried blood spot (DBS) extract

[0271] This embodiment provides a description of the optimization of a diluent for dried blood spot extract in multiplex assays, which maximizes the amount of analyte within a linear range while maintaining the highest median signal-to-background signal ratio in multiplex assays.

[0272] In multiplex assays that measure multiple target proteins using various capture reagents, natural variations in the abundance of different target proteins can limit the ability of certain capture reagents to measure certain target proteins (e.g., high-abundance target proteins can saturate the assay and prevent or reduce its ability to measure low-abundance target proteins). To address this variation in biological samples, aptamer reagents are grouped into at least two distinct groups, preferably three, based on the abundance of their respective protein targets in the biological sample. The biological sample is diluted into at least two, preferably three distinct groups of dilutions to produce individual test samples based on the relative concentration of the protein targets to be detected by their capture reagents. Thus, the biological sample can be diluted into high- and low-abundance target protein dilution groups, or high, medium, and low-abundance target protein dilution groups, wherein the lowest-abundance protein target is measured in the lowest dilution group, and the highest-abundance protein target is measured in the highest dilution group. In this embodiment, the aptamers are grouped into three distinct mixtures, Dil1, Dil2, and Dil3.

[0273] Methods: Venous blood from three healthy volunteers was collected into 2.4 mL BD vacuum blood collection tubes containing K2EDTA. One tube from each donor was centrifuged at 2,200 xg for 15 minutes, producing approximately 2 mL of anemic platelet plasma. The second tube was not centrifuged but used for the collection and drying of whole venous blood.

[0274] Spot 100 µL of whole blood onto paraffin paper and place the tip of the Mitra VAMS™ microsampling device into the whole blood sample until the sample collection device is fully saturated, pausing for an additional 2 seconds. This process was repeated four times, once for each tip included in the sample collection device clam package. The same procedure was repeated for plasma. The Mitra package was sealed and placed at room temperature in a sealed container containing eight DRIERITE desiccants for three days.

[0275] Place the dried tip from each clam into four 3 mL tubes 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 benzidine, 37.5 µM Z-Block, and 1.2% Tween-20. Elute the sample at room temperature with shaking at 1000 rpm for 1 hour. After 1 hour, gently pipette the buffer up and down to mix, then dilute. Combine the extracts (450 µL) from each donor into the master stock solution for Western blotting and the master stock solution for plasma.

[0276] To determine the appropriate dilution for diluent group 1, serial dilutions (1:2) were performed in plasma diluent by transferring 400 µL of whole blood extract to the first 1 mL well of a 96-well plate, followed by 200 µL to subsequent wells containing 200 µL of 80% plasma diluent and 20% PBS. This process was repeated fourteen times. These served as diluents for diluent group 1. Diluent groups 2 (40-fold dilution of diluent 1) and 3 (100-fold dilution of diluent 2) were also diluted in multiplex assays.

[0277]

[0278] To determine the appropriate dilutions for dilution groups 2 and 3, serial dilutions were prepared in 1 mL volumes of 96-well plates. Serial dilutions (1:2) were performed in assay buffer 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. Multiple assays were performed by directly pipetting 100 µL of each dilution onto each capture plate.

[0279] The diluted solution was divided into two portions for multiple assays.

[0280] Submission I

[0281]

[0282] Submission II

[0283]

[0284] result:

[0285] The optimal concentration for the assay is within the linear range. This avoids problems such as saturation, measurement background noise, or unknown factors that could confound the results. Therefore, it is important to select the matrix dilution so that as many of its analytes as possible are within the linear range.

[0286] A linear algorithm examines each analyte across all 15 dilutions and determines whether doubling the plasma concentration doubles the signal (by 25%). Before the algorithm can count the signals, the analyte must contain at least three dilutions where the signal is linear. At each dilution, the number of signals within the linear range is counted, generating... Figure 1 as well as Figure 2A and Figure 2B The optimal concentration was determined by diluting dried blood spot (DBS) extract in plasma diluent and PBS (80 / 20) (most analytes undergo dilution with a linear signal response). Figure 1As shown in the figure, the optimal concentration range for dilution group 1 is between 0.3% and 5%. The optimal concentrations for dilution groups 2 and 3 were determined by obtaining DBS extracted in plasma diluent and PBS (80 / 20) and diluting it in assay buffer (most analytes undergo dilutions with a linear signal response). Based on... Figure 2A and Figure 2B As shown in the figure, the optimal concentration range for diluent group 2 is between 0.005% and 0.038%, and the optimal concentration range for diluent group 3 is between 0.005% and 0.038%.

[0287] The maximum permissible dilution of a sample is defined as the excessive loss of analyte signal when the analyte signal drops below the background noise level in multiplex assays. Background in multiplex assays can be defined as a multiple of the standard deviation of repeated measurements of the signal when only the buffer solution is analyzed.

[0288] In multiplex assays, each analyte generates a background signal in buffer, and each assay is run with at least three buffer samples to determine the standard deviation of the background signal. For any given dilution of DBS or plasma, a signal is considered "above background" when it is 3.3 standard deviations above the average background signal. Statistically, an analyte with this level of signal intensity has only a 0.1% chance of being due to noise.

[0289] like Figure 3A As shown, at concentrations of 20% or 10% of the DBS extract, there is almost no signal loss due to noise in dilution group 1. Dilution group 1 does not introduce significant analyte loss into the DBS background until the extract is diluted below 1.25%. Figure 3B As shown, in dilution group 2, almost no signal is lost due to noise until the extract concentration drops below 0.3% of the DBS extract concentration. Dilution group 2 does not cause significant analyte loss into the extract background until the extract is diluted below 0.038%. Figure 3C As shown, in dilution group 3, almost no signal is lost due to noise until the extract concentration drops below 0.018% of the whole blood extract concentration. Dilution group 3 does not lose a significant amount of analyte to the whole blood background until the extract is diluted to below 0.005%.

[0290] Example 3: Accuracy of dried blood spots

[0291] Materials: Whatman 903™, Guthrie card, plasma diluent, PBS, BD vacuum blood collection tube (K2EDTA).

[0292] Methods: A small group of six donors were drawn using K2EDTA vacuum blood collection tubes, and their blood was divided into five assay replicates to obtain in-plate assay CV. Plasma was also collected from these donors, and three replicates were analyzed.

[0293] Blood collection / preparation / processing:

[0294] Six donors were drawn into one of three K2EDTA BD vacuum blood collection tubes (whole blood, plasma, and spare). The plasma tube from each donor was centrifuged at 2,200 xg for 15 minutes and approximately 2 mL of plasma (PPP) was collected and stored in two 1.5 mL Eppendorf tubes and immediately frozen on dry ice.

[0295] Load Guthrie cards and dry them:

[0296] For each donor, three Guthrie cards (Whatman 903™, lot number 7211021) were spotted. Using a micropipette, 40 µL of whole blood was spotted into the center of the dashed circle on three different Guthrie cards (five spots per card, three cards per donor). The cards were allowed to dry in a laminar flow hood for 1 hour (folding the cards to ensure the back of the spotted paper did not contact any surface), and then placed in six different sealed bags (three cards per bag), each bag containing two desiccants. The cards were allowed to dry at room temperature for three days.

[0297] Washout:

[0298] After drying for three days, remove one Guthrie card from each bag. Store the rest at -20°C as backup. Use scissors to cut the dried blood spot from its card, ensuring 100% removal of the dried blood spot. Place five rings of whole blood into five 2 mL threaded cap tubes. Add 1.6 mL of extraction buffer (80% plasma dip and 20% PBS) to each tube and vortex at 1000 rpm for 1 hour at room temperature. Perform this procedure for each donor, for a total of thirty tubes.

[0299] Diluent group 1 samples (200 µL each) were transferred into 30 matrix tubes and frozen at -80°C. An additional 600 µL was transferred into 30 different matrix tubes for diluent groups 2 and 3.

[0300] Thaw one plasma sample from each donor and transfer 50 µL to three matrix tubes for each donor (sample replicates). Add an additional 200 µL of extraction buffer to dilute to 20%. In the same manner, dilute three plasma QC samples, eight blank samples (five without sequence numbers), and five calibration samples to 20% in extraction buffer. Thaw the diluted sample for multiplex assays. Store all extracts and plasma samples on ice.

[0301] The linearity of the multiplex assay signal increased with optimized whole blood dilution (concentrations lower than the optimal dilution for plasma or serum). Dry whole blood samples from all six donors were prepared at dilutions of 2.5%, 0.05%, and 0.005%. Liquid plasma from all six donors was prepared at dilutions of 20%, 0.5%, and 0.005%. Samples were refrozen and submitted for analysis via multiplex assay.

[0302]

[0303] Results: The overall mean precision of the analytes for all tests is described as follows: Figure 4A and Figure 4B The overall coefficient of variation (CV) of the analytes shows a bimodal distribution, with most analytes (>70%) having a CV of less than 5%. The second analyte population exhibits greater imprecision and a wider CV distribution. Figure 4A As shown, over 95% of the analytes showed an imprecision of less than 15% between replicates.

[0304] The imprecision of whole blood analytes can be divided into two groups: high precision and intermediate precision. The high precision group represents the majority of analytes tested, closely distributed around 3% CV and ranging from 2.5% to 7.5%. Intermediate precision analytes comprise approximately 25% of the library, ranging from 7.5% to 17.5%, with a central value of approximately 11%. Most of these analytes have CVs less than 15%, which is promising for medical applications.

[0305] exist Figure 4B Two CV groups can be observed in the CDF plot shown. The CDF plot is an integral histogram (area under the curve) and conveys the same information.

[0306] Example 4: Robustness of protein signal determination in whole blood by protein spiking

[0307] Methods: Large protein libraries were spiked into DBS extracts to determine whether an increase in protein concentration in the analyte could be measured in the matrix.

[0308] Blood collection / preparation / processing:

[0309] Venous blood from donor 1301 was collected into 2.6 mL BD vacuum blood collection tubes containing K2EDTA, stored at room temperature, and processed within two hours. One tube was centrifuged at 2,200 xg for 15 minutes to produce anemic platelet plasma.

[0310] Loading Mitra device:

[0311] Spot 100 µL of whole blood and plasma (separately) onto paraffin-coated paper and place the tip of a Mitra VAMS collection device into it until the device is fully saturated, then wait 2 seconds. Repeat this procedure 16 times for whole blood (four clams) and 4 times for plasma (one clam). Store these devices in a sealed container at room temperature for three days, with two DRIERITE desiccants per clam.

[0312] Proteins were incorporated into the WB extract.

[0313] Three days later, Mitra tips treated with whole blood were immersed in plasma extraction buffer (20% PBS, 80% plasma diluent, 300 μL per tip) under shaking for 1 hour. The 10x (10%) extract was aliquoted into two portions and spiked with a protein-spiked library (Appendix II). Plasma-loaded tips were prepared in the same manner. Samples were prepared in 150 µL aliquots, and the spiked library was replaced with 10%, 5%, 1%, and 0% substitutions.

[0314] The sample was frozen and run in a multiplex assay.

[0315] Results: The two unspiked dried whole blood samples demonstrated a high degree of consistency between dried whole blood extracts collected via the Mitra VAMs system when derived from the same donor. When the unspiked whole blood samples were compared to spiked whole blood samples from a protein library with approximately 5 nM or 5% substitution, the added proteins “elevated” from the identity line. This trend was also observed in similarly spiked plasma samples.

[0316] Titration curves were measured for both whole blood and plasma extracts as the percentage of protein substitution increased. After subtracting the background signal, an S-shaped dose-response pattern was observed. Interestingly, not all elements in the spiked library appeared to experience an increase in signal. This could be due to these proteins being more prone to denaturation in whole blood or experiencing higher levels of inaccuracy. Sensitivity issues may also exist for subsets of proteins within the spiked library, leading to a lack of detection for some members.

[0317] The median signal increase of the spiked library targets was measured and compared between whole blood and plasma. A median of 232 signal increases exceeding baseline was obtained. The signal increases were nearly identical between whole blood and plasma.

[0318] The signal increase of each of the 232 spiked analytes between 0 nM and 5 nM in plasma was divided by the equivalent increase in whole blood. If plasma and whole blood react to protein spikes in the same manner, the signal distribution should be around 1. Two replicate whole blood samples showed that, on an analyte basis, both matrices reacted identically to proteins spiked into their extracts.

[0319] Example 5: Extraction Optimization Study

[0320] The dried sample must be reconstituted to achieve buffer conditions substantially the same as those of a typical plasma or serum sample submitted for assay. In a typical multiplex assay protocol, one biological sample is added to four serum diluents (50 mM Hepes (pH 7.5), 100 mM NaCl, 8 mM MgCl2, 5 mM KCl, 1.25 mM EGTA, 1.2 mM benzidine, 75 µM Z-Block, and 1.2% Tween-20) or plasma diluents (50 mM Hepes (pH 7.5), 100 mM NaCl, 8 mM MgCl2, 5 mM KCl, 1.25 mM EGTA, 1.2 mM benzidine, 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 30 µL of dried sample, extraction occurred in 150 µL of buffer solution containing 4 parts diluent and 1 part volume expander. The volume expander tested was water and phosphate-buffered saline (PBS). An experiment was performed to compare the two types of volume expanders. Twelve serum samples (QC2) were thawed and collected using Mitra VAMS 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. The extracts from the duplicate tips were combined to obtain three replicates for each condition. Multiple assays from both extraction methods were compared with each other and with each standard QC2 serum sample (undried) that had undergone additional freeze-thaw cycles to ensure that the number of freeze-thaw cycles was equal for all samples.

[0321] The concordance plots of relative fluorescence units (RFU) between the two extraction conditions showed small differences (Figure 5). Similarly, the cumulative distribution plots of median RFU from the standard method and the two extraction conditions from the dry spots almost overlapped, with a slight benefit in fitting symmetry when using the PBS method (not shown). Furthermore, the cumulative distribution plots of coefficient of variation percentage (CV%) also revealed a slight benefit 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 extracting dried samples.

[0322] Example 6: Determination of protein extraction time

[0323] Methods: Ten QC1 and ten QC2 serum samples (v4.0) were thawed and Mitra samples were collected and dried at 4°C for 2.5 days. Samples were extracted using PBS and serum diluent as volume expansion agents, as described above. A duplicate Mitra tip was removed from each QC sample and placed in a microtiter well, and extracted by rotation at room temperature for 10, 30, 60, 120, or 180 minutes. Extracts from the duplicate tips (at each time point) were combined and frozen until tested in multiplex assays with standard QC1 and QC2 serum samples (undried) that had undergone additional freeze-thaw cycles to ensure equal freeze-thaw cycles for all samples. As a blank control, Mitra VAMS samples in extraction buffer were collected and dried in the same manner and tested in multiplex assays with a standard multiplex assay blank.

[0324] Results: Analytical signal recoveries were largely independent of extraction time, ranging from 10 to 180 minutes. Figure 6 shows the median (interquartile range) ratio of analyte signals from the dry spot (Mitra) to those obtained from the same sample (undried) analyzed by multiplexing. The median ratio did not vary significantly across the extraction time range and was slightly greater than 1 in all cases. The cumulative distribution of RFU signals obtained from dried serum samples extracted at room temperature for specified time lengths showed that analyte signals obtained from the five extraction times almost overlapped with each other and were also overlapping with analyte signals obtained from standard serum samples (undried). Analyte signals obtained from QC1 blank samples that underwent drying and extraction processes were overlapping with signals obtained using standard multiplex blank samples. For multiplexing, extraction times between approximately 10 minutes and 200 minutes are acceptable. In some embodiments, an extraction time of 60 minutes was used.

[0325] Example 7: Proof of accuracy and consistency with dried serum

[0326] Methods: To better understand the reproducibility of dried plasma spots, experiments were conducted using fresh serum obtained from six healthy volunteers, with all Mitra VAMS samples dried under the same conditions. Blood was collected and allowed to coagulate at room temperature for 1 hour. The coagulated samples were centrifuged at 2,200 xg for 15 minutes, and aliquots of serum were transferred to Eppendorf tubes. Duplicate Mitra VAMS tips (30 µL) were collected from each serum sample and placed in sealed containers containing DRIERITE desiccant to dry in the dark at room temperature for 4 days. Additionally, aliquots of fresh serum were frozen until assay to compare with dried spots. Extraction was performed for 1 hour using water as a swelling agent, and extracts from duplicate Mitra VAMS tips were combined and frozen at -80°C until assay. Extracts and controls were determined in multiple assays.

[0327] Results: Concordance plots between the standard method and the drying method for each of the six individual serum samples showed that all analytes retained signal after drying and reconstruction. While the concordance was not perfect, the most important factor was whether the signal ratio of each analyte remained constant in the sample; for example, whether the analyte always returned half or twice the signal obtained by the standard method. Because these ratios were constant, i.e., all ratios had acceptable CVs, the results from the dried spots could be mathematically transformed to “boost” them to match the expected results from the undried samples.

[0328] The CV% of the ratio for each analyte was determined and plotted as a cumulative distribution function. The median CV% of the ratio was 6.99, with the 10th and 90th percentiles being 3.59 and 18.8, respectively, indicating that the ratios of most analytes remained reasonably constant across samples when dried and processed under the same conditions.

[0329] Example 8: Stability of dried blood plasma to temperature changes based on drying time and temperature

[0330] method:

[0331] Blood collection / preparation / processing: Venous blood from healthy volunteers was collected into 2.6 mL BD vacuum blood collection tubes containing K2EDTA, stored at room temperature, and processed within two hours. One tube was centrifuged at 2,200 xg for 15 minutes, and anemic platelet plasma was collected. The plasma (400 µL) was frozen at -80°C as a control.

[0332] Loading the Mitra device: Spot 80 µL of whole blood or plasma onto paraffin paper and place the tip of the Mitra VAMS into it until the sample collection device is fully saturated (approximately 2 seconds).

[0333] Drying conditions: Placing plasma samples in sealed bags (two desiccant containers per Mitra clam) and drying at three different temperatures: -20°C, 4°C, and 25°C. The Mitra VAMS device was dried for one to three days, after which it was removed and extracted with 20% PBS and 80% plasma diluent, or placed under stress temperature.

[0334] Stress temperature: After drying for 1 to 3 days, remove the samples from their drying environment and allow them to reach room temperature. Then place them under their stress conditions of -20°C, 37°C, 50°C, or 60°C for 1 or 2 days. After 1 or 2 days, remove the samples and extract plasma using 20% ​​PBS and 80% plasma diluent.

[0335] Extraction: Place the Mitra tip into an Eppendorf tube containing 300 µL of extraction buffer (80% plasma diluent and 20% PBS) and extract with shaking for 1 hour. Transfer the contents to a matrix tube and freeze at -80°C.

[0336]

[0337] Results: Plasma-loaded Mitra VAMS devices were dried at different temperatures, as shown above, and then compared with liquid plasma that had been stored at -80°C using a consistency graph. Optimal results were achieved when the plasma was dried at low temperatures; however, this may simply be due to the higher temperature stability in refrigerators and freezers. Consistency was high among all dried samples, but the best consistency between the dried plasma extract and liquid plasma was observed when the samples were dried at 4°C or -20°C instead of room temperature, although consistency at room temperature was acceptable for multiplex assays.

[0338] The concordance of cardiovascular disease analytes (CVD2) among samples dried at 4°C, -20°C, and room temperature was measured. There are 28 analytes in the CVD2 assay, and this subset of proteins appeared to show the highest concordance with liquid plasma when dried at -20°C. For multiplex assays, the concordance between samples dried at 4°C and room temperature and liquid plasma was also acceptable.

[0339] The plasma was dried at 4°C and then exposed to -20°C, 37°C, 50°C or 60°C to subject the sample to temperature stress and run in multiple assays compared to liquid plasma. Figure 7A The results showed that drying at 4°C for at least one day appeared to protect the samples from freezing. Drying plasma at 4°C for at least two days and exposure to 37°C for at least one day appeared to protect it from freezing. Figure 7BThe results show the plasma dried at room temperature and subsequently subjected to temperature stress. Drying at room temperature resulted in decreased concordance with liquid plasma compared to drying at 4°C. Figure 7C As shown, the benefits of drying at -20°C fall between those of drying at 4°C and drying at room temperature. Drying samples at 4°C, -20°C, and room temperature all provide suitable samples for multiple determinations.

[0340] Example 9: Identifying analytes from DBS samples by measuring changes in plasma proteins.

[0341] Study Methods

[0342] A cohort of 16 healthy donors was sampled using three Tasso M20 smart sampling devices, one EDTA (plasma) BD vacuum blood collection tube, and one serum BD vacuum blood collection tube. After processing, plasma and serum samples were transferred to cryovials and frozen at -80°C. The dried Tasso M20 devices were immediately removed and placed in sealed plastic containers with desiccant. One device from each donor was placed in a 4°C–8°C freezer for 2 hours before being packaged for transport, while the other two devices were kept at room temperature. The packages were immediately shipped overnight to SomaLogic in Boulder. Two days after extraction, the refrigerated Tasso devices and the devices kept at room temperature were opened and extracted for 1 hour with 80% serum diluent and 20% PBS. The extracts were frozen at -80°C to a concentration of 2.5%. A third Tasso device was kept at room temperature for 7 days after extraction and then extracted in a similar manner. All samples on the same plate were assayed using SOMAscan® to mitigate inter-assay variability. Serum and plasma samples were diluted using 20%, 0.5%, and 0.005% dilution protocols. Dried blood spots were diluted using 2.5%, 0.05%, and 0.005% dilution protocols.

[0343] Results

[0344] Distribution of inter-matrix correlations

[0345] Figures 8A-8D The consistency between frozen plasma and dried blood spots (8A), frozen serum and dried blood spots (8B), frozen serum and frozen plasma (8C), and two pooled DBS samples (8D) was compared. In the comparisons of (8A) and (8B), cellular proteins in DBS tended to have stronger signaling potential than proteins that are typically present in smaller amounts in plasma or serum. This contrasts with two DBS replicates prepared from the pooled sample (8D) or even the frozen serum versus frozen plasma matrix (8C).

[0346] Based on only Figures 8A-8DThe data readily leads to the conclusion that DBS is completely inconsistent with plasma and serum matrices. The addition of cellular components from whole blood (which contain a greater abundance of proteins than plasma) causes the measurement of most plasma proteins in DBS to be masked. However, some proteins are highly concentrated in plasma and / or reduced in cells, ensuring that the abundance of the additional proteins does not mask signals from the liquid components of whole blood. Figure 9 This indicates that while most DBS signals are not correlated with plasma, a small subset exhibit matrix-related correlations. The cumulative distribution functions of the Pearson correlation coefficients for all DBS signals with frozen plasma are shown on the left, and the cumulative distribution functions of the Pearson correlation coefficients with serum are shown on the right. For comparison, correlations between plasma and serum (direct and random) were included. Figure 9 The masking effect of cellular components in whole blood can be observed very clearly, but some analytes appear to be strongly correlated with both plasma and serum.

[0347] Number of significant correlations between serum / plasma and DBS

[0348] Two methods were used to determine statistically significant correlations among v4.1 analytes in plasma, serum, and DBS. In this example, analytes were defined as individual measurements in the SOMAscan® assay. In this way, some blood proteins are measured via multiple “analyte signals.”

[0349] One approach (based on the significance of correlation between paired observations) involves using a significance threshold for p-values ​​generated from combinations of similar observations. For example... Figure 9 As shown, plasma and serum are not independent but correlated. Observational data in one matrix can inform observational data in another matrix. Furthermore, a similar study, referred to herein as the “DBS Precision Study” (described below), investigated the correlation between DBS prepared from anticoagulated IV-extracted blood and frozen plasma. The p-values ​​of the correlations found for each of these observational data were pooled and compared with a significance value of 0.1 (corrected for multiple comparisons and observational data as described below). The results are shown in Tables 1, 2, and 3.

[0350] Used to determine Figure 9 The second method, which shows which correlations are significant, employs a false discovery rate. Simulations involving donor-matrix randomization generated nearly 3 million spurious correlations for each DBS condition / matrix pairing. Thus, for a given Pearson correlation coefficient threshold, the average number of spurious correlations for a menu of 7335 human analytes can be calculated. The number of significant correlations is then calculated by subtracting the average number of spurious correlations due to random chance from the number of correlations with coefficients above the threshold. Detailed methods can be found below, and the results are given in Table 4.

[0351] Number of significant correlations using paired observations data

[0352] Using unpaired observation data, paired observation data between plasma and serum, and paired observation data between serum and plasma, combined with results from the DBS precision study, the number of significant correlations between plasma / serum and DBS occurring under various drying conditions is presented in the table below. Detailed methodology can be found below in "Correlation by Paired Observation Data". The "mean" condition in each of these tables is the result of repeating all three conditions as a technique and then averaging the three correlations between DBS and plasma and serum.

[0353] Table 1. Number of statistically significant correlations in paired observation data

[0354]

[0355] In contrast to the single correlation between DBS and serum or plasma, a significant increase in the number of significant correlations can be observed when using paired observation data. It should be noted that the Holm-Bonferroni correction used in this method is a conservative filter for erroneous correlations when comparing a large number of analytes. By combining observation data from serum and plasma, the number of significant correlations more than doubled. Adding the DBS precision study to the analysis increased the number of significant analytes by an average of 25. This small benefit is understandable given the small cohort involved in this study.

[0356] Table 2. Statistically significant correlations among paired observation data (by dilution group)

[0357]

[0358] The number of significant correlations between serum and plasma with all three DBS conditions (top) was subdivided into dilution groups.

[0359] Table 3. Statistically significant correlations among paired observation data (subdivided by dilution group coverage)

[0360]

[0361] Diluent group 1 contained 6027 human analytes, diluent group 2 contained 1106 human analytes, and diluent group 3 contained 190 human analytes. The abundance of significant analytes increased proportionally to the total number of analytes in each group with increasing dilution factor in the SOMAscan® assay. This is likely due to the presence of many excreted plasma proteins in high abundance in diluent groups 2 and 3, which are more easily read despite interference from cellular analytes in whole blood.

[0362] exist Figure 10A In the figure, signals from DBS stored for 7 days prior to extraction are plotted against signals from the corresponding frozen plasma. Gray analytes (500 in total) generated from paired observation data in plasma, serum, and DBS accuracy studies are considered relevant to plasma. Plasma analytes cluster in the lower half of the figure, consistent with previous analyses.

[0363] The number of significant correlations using the FDR filter

[0364] The following results were determined by selecting an arbitrary false discovery rate (FDR) value and counting the number of analytes that met the required Pearson coefficient threshold to achieve that value.

[0365] Table 4. Number of significant correlations between DBS and serum / plasma using FDR.

[0366]

[0367] Table 4 compares all DBS conditions with plasma and serum, and gives the number of correlations between matrices based on the tolerance for false positives within the population. The total population of analytes that will appear can be calculated by adding the significant correlations (SigCorr's) to the false positives (False Pos). The FDR is obtained by dividing the number of false positives by this number. The minimum Pearson coefficient that a correlation can have and be considered significant is given by the "Min Pearson" value. Conditions and tolerances that produce more than 500 analytes are marked in bold.

[0368] exist Figure 10B In the above, signals from DBS held for 7 days prior to extraction were plotted against signals from the corresponding frozen plasma. Gray analytes were generated using an FDR-based algorithm (described below in the section on correlation significance filtered by false discovery rate) to produce 557 significant correlations. For both methods, plasma analytes clustered in the same manner ( Figure 10A and Figure 10B This indicates a high degree of consistency between the two.

[0369] Characterization of analytes

[0370] Comparisons of analyte groups marked as significant by each method showed considerable overlap. Figure 11 This comparison increases the confidence level that either method or a combination of methods can be used to create a larger list of significant correlations.

[0371] exist Figure 11 In the diagram, the left region represents the significant correlation between frozen plasma and DBS (7-day drying time) selected by choosing a Pearson threshold to achieve a 5% FDR. The right region represents the number of similar correlations derived using three-way pairwise observations between DBS and plasma and serum, as well as observations from a DBS precision study. This overlap between the two results increases the confidence that these analytes actually measure plasma proteins.

[0372] Once a list of significant correlations between DBS and plasma is generated, the characteristics of potentially measurable plasma / serum analytes in DBS are investigated. A concordance plot between plasma and DBS is then created. Figures 12A-12D Filtering out all insignificantly correlated signals indicates a direct relationship between the matrices of these selected analytes. Analytes with lower signal intensities appear to have a weaker linear relationship with DBS. It is undesirable to be bound by any particular theory, as the background from DBS may have a more significant impact on smaller-order plasma signals when significant analytes are mapped onto a serum-plasma concordance plot. Figures 13A-13D It is clear that analytes with greater consistency between the two matrices are also more likely to be correlated with DBS in plasma / serum.

[0373] like Figures 12A-12D As shown, each concordance plot compares the relationship between plasma and DBS signals in the analytes, which have been characterized as having a significant correlation with DBS (7-day dried) and frozen plasma. The number of analytes also increases with the allowed FDR: (12A) 5%, (12B) 10%, (12C) 15%, and (12D) 20%.

[0374] like Figures 13A-13D As shown, the gray analytes have Pearson coefficients greater than the thresholds of (13A) 5%, (13B) 10%, (13C) 15%, and (13D) 20% (DBS versus frozen plasma after 7 days of drying). These are mapped to a concordance plot of serum versus plasma signals. Figures 13A-13D The results showed that the higher the consistency of the analyte between plasma and serum, the more likely the analyte is to be consistent between plasma and DBS.

[0375] Another way to categorize the list of significant correlations is to categorize the functions of analytes that measure plasma proteins. Princeton University has compiled 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 with the abundance of analytes in the human proteome. Figure 14 The results show that the proteins used in plasma measurements are involved in immunology, cell adhesion, cell motility, defense responses, inflammatory responses, cell junctions, the circulatory system, wound healing, and the extracellular matrix. The percentage of plasma analytes belonging to each of the various biological function categories (left) is compared to the abundance of proteins in the entire human proteome that have been classified into those categories (right). Each of these categories was selected because they were representative (greater than 2x) in terms of enrichment among plasma proteins identified through paired observation data and FDR filtering.

[0376] method

[0377] By analyzing the correlation between paired observation data:

[0378] Multiple observational data points are available to address the question of how many plasma / serum analytes are significantly correlated with whole blood. In this study, each patient was sampled using two additional matrices besides DBS (frozen plasma and serum) for comparison with their respective DBS samples. Plasma and serum are not independent matrices, and over 2500 analytes in this cohort showed significant correlations between the two matrices (α = 0.1, Bonferroni-holm corrected). Therefore, the probability that the correlation between plasma and DBS is significant can be corrected for the significance (p-value) of a measure of the same correlation between serum and DBS.

[0379] A separate study (referred to as the DBS Accuracy Study) involved a small cohort of six donors, whose blood was drawn via IV into EDTA vacuum blood collection tubes and aliquoted into five replicates for measurement. Plasma was also collected from the donors, and three replicate samples were analyzed to determine which plasma analytes could be measured in whole blood. Plasma analytes associated with DBS in the DBS Accuracy Study support similar observational data in this study. A detailed description of the methods used in this study is provided below in the Protocol section. Median normalization was performed on the analyte signals.

[0380] To rule out the null hypothesis that individual correlations are due to random chance, a method was developed that rewards consistent observational data across the matrix and the experiment. Positive correlations were determined using a Pearson coefficient cutoff value, which was determined by calculating the correlations between the matrices after randomization (making the donors no longer match) throughout the entire library. This process was performed 401 times, resulting in a total of 2.94 million random correlations. This number was considered sufficient because repeated calculations demonstrated high precision.

[0381] Figure 15 The cumulative distribution function (CDF) plot of the Pearson coefficient distribution generated by comparing random plasma / serum values ​​with DBS dried for seven days is shown. The dashed line represents the cutoff value (0.45) for the 95% random Pearson coefficient to fall within. The solid line represents the cutoff value (0.56) for the 98% random Pearson coefficient to fall within. For DBS precision studies, randomly assigning DBS and plasma to 6 individuals would result in a much higher probability of donor random alignment than to 16 individuals; therefore, the 95% and 98% thresholds are represented by larger Pearson coefficients (0.75 and 0.86, respectively). Given these limiting values, the 95% threshold was used.

[0382] For a correlation to be considered relevant, the Pearson coefficient for plasma-serum correlation must be greater than the 98% cutoff value, or for DBS precision studies, the Pearson coefficient must be greater than the 95% cutoff value (0.75). In a 16-donor study, results obtained from either the 98% or 95% cutoff values ​​showed no functional difference, thus a more stringent threshold was used. Pearson coefficient thresholds were determined individually for each matrix and condition. Correlation coefficients below the threshold were manually assigned a p-value of 1. The resulting p-values ​​for paired plasma-serum correlations were multiplied to obtain the probability that both outcomes were caused by random chance. This is illustrated in Table 5.

[0383] Table 5: Examples of Algorithms for Paired Observation Data

[0384]

[0385] A two-comparison version of the algorithm is shown in Table 5 as an example. In this representation of the paired observation data algorithm, the threshold for significant correlation is shown at the bottom of the table. Pearson coefficients below the threshold are highlighted in italics, while those above the cutoff value are highlighted in bold. Pearson coefficients below the acceptance threshold are assigned a p-value of 1 such that when multiplied by their partner values ​​in the other matrix, they do not affect the combined probability of spurious correlations. The resulting p-value is then compared with the α value corrected by Bonferroni-Holme. 2(α = 0.1) Comparisons are made. In this algorithm, α is raised to a power of the number of combined correlations (2 in this case). In this way, a highly significant correlation in only one matrix is ​​sufficient to classify a plasma analyte. Alternatively, multiple weaker correlations can be combined to identify plasma analytes. Decreasing the α value by a power of the number of correlations prevents p-values ​​from simply grouping together until random chance makes them significant. Results indicated by * are examples of analytes for which plasma or serum correlations alone do not meet acceptance criteria. Values ​​indicated by # remain unchanged.

[0386] Correlation significance filtered by false discovery rate:

[0387] The null hypothesis can also be rejected by requiring the set of all significant correlations within the dataset to contain the maximum false discovery rate of spurious correlations. Using the random correlations described above, a Pearson coefficient threshold for adjusting the false discovery rate of spurious correlations can be selected. Taking a specific example of the random correlation between plasma and 7-day dried DBS, it can be seen that 99.9% of the Pearson coefficients are less than 0.87. If this cutoff value is used in this example, the false positive rate, or the chance that an individual correlation will be attributed to chance, can be expected to be 1 / 1000. If the analyte correlations are sorted from the largest to the smallest Pearson coefficient, the average number of spurious correlations in the group with the smallest coefficient value can be determined. Dividing by the number of analytes in that group yields the false discovery rate (FDR).

[0388] The following method determines how many significant analytes can be classified as significantly correlated between matrices at different confidence levels. The probability distribution of false positives or spurious correlations is given by Equation 1.

[0389] Equation 1: Binomial probability distribution of spurious correlation

[0390]

[0391] n = the number of analytes being compared (7335 human analytes)

[0392] k = the number of false positives (FP) or spurious correlations.

[0393] p = False positive rate (FPR) of intermatrix correlation.

[0394] The false positive rate was determined by selecting a threshold of 2.9 million random Pearson coefficients generated by random decoupling of the donor matrix and finding the percentage of random coefficients above the cutoff value.

[0395] Figure 16The diagram shows probability curves generated using Pearson cutoff values ​​of 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, and 0.99, illustrating different probability density functions of the possible number of spuriously correlated analytes when comparing plasma with DBS samples dried for 7 days. Higher allowable Pearson cutoff values ​​result in fewer false positives. These curves can be used to determine a weighted average of the expected number of false positives given a specific threshold.

[0396] By multiplying the number of false positives by their probability density function (e.g.) Figure 17 As shown in the figure, the area under the curve (Equation 2) can be used to obtain the weighted average of the pseudocorrelation.

[0397] Equation 2: Weighted average of pseudocorrelation

[0398]

[0399] FPR = False Positive Rate of Intermatrix Correlation

[0400] FP = the number of false positives for a given FDR

[0401] The integral can only be estimated using integer values ​​of FP, and the area under the curve is calculated using the trapezoidal rule. In fact, the precise measure of the integral can be determined by restricting the integral to the region where the curve is significantly above the x-axis.

[0402] For a given threshold for the Pearson coefficient, the weighted average of spurious correlations is given by hollow circles and derived from the number of spurious correlations multiplied by the area under the curve of the probability density function (Equation 2 and ). Figure 18 The number of significant correlations (solid circles) is the sum of all correlations between the matrix and the DBS sample (in this case, 7-day dried DBS) with a Pearson coefficient higher than the cutoff value, minus the number of spurious correlations. The number of significant correlations appears to increase linearly.

[0403] Equation 3: False detection rate of spurious correlations

[0404]

[0405] Total correlation = The number of correlations between the matrix and the DBS condition whose Pearson correlation coefficient is higher than a given threshold.

[0406] Average false positives = Weighted average of false positives given by Equation 2.

[0407] The false detection rate of spurious correlations (calculated by Equation 3) is shown in... Figure 19The study clearly demonstrates the danger of arbitrarily high cutoff values ​​for the Pearson coefficient when attempting to eliminate spurious correlations. Any Pearson threshold that causes the FDR to exceed its minimum value reduces the number of analytes and increases the rate of false correlations.

[0408] like Figure 20 As shown, each FDR (x-axis) can be mapped to the number of significant analytes (total correlation - false correlation). Figure 20 The curves shown are derived from the correlation between plasma and DBS that has been allowed to dry for 7 days. The values ​​in Table 4 are calculated in this manner.

[0409] Scheme for DBS accuracy study

[0410] Whole blood samples were dried and extracted using a Whatman 903™ and Guthrie cards for close compatibility with any available equipment in the field. Whole blood spots were dried, then cut from their cards and extracted with plasma diluent and PBS. A 2.5% extract concentration was used, resulting in an extraction buffer volume exceeding 1 mL. This minimized the percentage of volume distortion caused by different sized Guthrie card cuts. After 1 hour, the extract was transferred to matrix tubes and frozen at -80°C. On the day of assay, samples were thawed and diluted to concentrations of 2.5%, 0.05%, and 0.005%. By the end of extraction, the Guthrie cuts appeared 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 a biological sample for multiplex assays, the method comprising: The biological sample containing multiple target molecules is deposited onto the collection device; The biological sample on the collection device is dried for a period of time to stabilize the dried sample before any temperature fluctuations occur. The biological sample is dried at approximately room temperature or below room temperature for at least 4 hours, so that the target molecules in the dried biological sample can be detected in the multiplex assay.

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

3. The method of claim 1 or claim 2, wherein the dried biological sample is stored at approximately room temperature or below room temperature prior to detection in the multiple assay.

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

5. The method according to any one of claims 1-4, 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, one day, at least two days, or at least three days.

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

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

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

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

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

11. A method for detecting multiple target molecules, the method comprising: Target molecules are extracted from dried biological samples using a collection device; The extracted target molecules were diluted into a first diluent and a second diluent. If the target molecule is present in the first diluent, the first diluent is contacted with the first capturing reagent to form a first capturing reagent affinity complex with its target molecule; If the target molecule is present in the second diluent, the second diluent is contacted with the second capture reagent to form a second capture reagent affinity complex with its target molecule; The first and second diluent samples were incubated separately to allow the formation of the capture reagent affinity complex; wherein each of the first and second capture reagent affinity complexes was immobilized on a separate first solid support; The first capturing reagent affinity complex is released and captured on the second solid support; After releasing the first capturing reagent affinity complex, the second capturing reagent affinity complex is released and captured on the second solid support; and The presence or level of the first or second capture reagent in the first or second capture reagent affinity complex is detected or determined.

12. The method of claim 11, wherein the extraction of the target molecule from the collection device in the formulation continues for at least 5 minutes.

13. The method of claim 11 or claim 12, wherein the extraction of the target molecule from the collection device in the formulation lasts 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 of claim 12 or claim 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 of claim 14, wherein the one or more salts are each independently selected from sodium salts, potassium salts, and magnesium salts.

16. The method of claim 14, wherein, The one or more salts include sodium salts, potassium salts, and magnesium salts.

17. The method of claim 15 or 16, wherein the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2.

18. The method of claim 17, wherein 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 mM-125 mM or about 100 mM.

19. The method of claim 17 or claim 18, 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.

20. The method of any one of claims 17-19, wherein the concentration of MgCl2 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 8 mM.

21. The method of any one of claims 14-20, wherein the buffer is selected from HEPES, IVIES, Bistris methane, ADA, ACES, Bistris propane, PIPES, MOPSO, choline chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, glycamide, glycylglycine, HEPBS, Bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

22. The method of any one of claims 14-21, wherein the concentration of the buffer in the formulation is 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 of any one of claims 14-22, wherein the chelating agent is selected from EGTA, EDTA, DTPA, BAPTA, DMPS and ALA.

24. The method of any one of claims 14-23, wherein 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.

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

26. The method of any one of claims 14-25, wherein the nonionic surfactant is, on a volume-to-volume basis, 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% or about 1.5% of the formulation, or about 1.2% of the formulation.

27. The method of any one of claims 14-26, wherein the pH of the preparation is from about pH 5 to about pH 9, or from about pH 6 to about pH 8, or from about pH 7 to about pH 7.9 or about pH 7.

5.

28. The method of any one of claims 14-28, wherein the formulation comprises 50 mM HEPES, 100 mM NaCl, 5 mM KCl, 8 mM MgCl2, 1.25 mM EGTA and 1.2% Tween-20.

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

5.

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

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

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

33. The method of any one of claims 14-32, wherein the protease inhibitor is benzoamidine.

34. The method of any one of claims 14-33, wherein the concentration of the protease inhibitor in the formulation is 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 of any one of claims 14-34, wherein the oligonucleotide is a single-stranded oligonucleotide.

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

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

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

39. The method of any one of claims 14-38, wherein the oligonucleotide comprises the sequence [(ACXX)7-AC], where X is BndU.

40. The method of any one of claims 14-39, wherein 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.

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

42. The method of any one of claims 11-41, wherein the biological sample has been dried at approximately room temperature, 4°C-8°C, or -20°C.

43. The method of any one of claims 11-42, wherein the dried biological sample has been stored at or below room temperature prior to detection.

44. The method of any one of claims 11-43, wherein the dried biological sample has been stored at about 4°C-8°C or -20°C prior to detection.

45. The method of any one of claims 11-44, wherein the biological sample has been 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 one day, at least two days, or at least three days.

46. ​​The method of any one of claims 11-45, wherein the biological sample is selected from plasma, serum, urine, whole blood, leukocytes, peripheral blood mononuclear cells, erythrocyte sedimentation rate (ESR), sputum, tears, mucus, nasal wash, nasal aspirate, semen, saliva, peritoneal lavage fluid, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph, papillary aspirate, bronchial aspirate, bronchial brushing, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid.

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

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

49. The method of any one of claims 11-48, wherein the dried biological sample has been homogenized by a drying process.

50. The method of any one of claims 11-49, 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 of any one of claims 11-50, wherein the first diluent is a diluent of 0.001% to 0.1% of the eluted target molecule sample, and the second diluent is a diluent of 0.1% to 10% of the eluted target molecule sample.

52. The method of any one of claims 11-50, wherein 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%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005% of the test sample, and the second diluent is 0.01% to 1% 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% dilution.

53. The method of any one of claims 11-50, wherein 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%), 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005% of the test sample; and the second diluent is 5% to 39% of the test sample. (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% dilution.

54. The method of any one of claims 11-50, wherein the first 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%, 0.25%, 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% of the test sample; and the second diluent is the test sample... Diluted at 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% of the sample.

55. The method of any one of claims 11-50, wherein the first 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%, 0.25%, 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% of the test sample; and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 method of any one of claims 11-50, wherein the first 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%, or about 20% of the test sample, and the second diluent is... The test sample was diluted in the form 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% of the sample.

57. The method of any one of claims 11-50, wherein the first 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%, or about 20% of the test sample, and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 method of any one of claims 11-50, wherein 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%), 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005% of the test sample, and the second diluent is 0.01% to 1% 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 a dilution of 0.01% to 0.5%, or 0.02% to 0.1% or about 0.05%.

59. The method of any one of claims 11-50, wherein 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%), 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005% of the test sample, and the second diluent is 0.5% to 5% of the test sample. (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% dilution.

60. The method of any one of claims 11-50, wherein the first 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%, 0.25%, 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% of the test sample, and the second diluent is 0.5% to 5% of the test sample. (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% dilution.

61. The method of any one of claims 11-50, wherein the first 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%, 0.25%, 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% of the test sample, and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 method of any one of claims 11-50, wherein the first diluent is 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% of the test sample, and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 method of any one of claims 11-50, wherein the first diluent is 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% of the test sample, and the second diluent is 0.01% to 1% 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 a dilution of 0.01% to 0.5%, or 0.02% to 0.1% or about 0.05%.

64. The method of any one of claims 11-63, further comprising contacting a third diluent sample with a third capture reagent, wherein if the target molecule is present in the third diluent sample, a third capture reagent affinity complex is formed through the interaction of the third capture reagent with its target molecule; wherein the third diluent sample is incubated separately from the first diluent sample and the second diluent sample to allow the formation of a capture reagent affinity complex between the third aptamer and its target molecule.

65. The method of claim 64, further comprising, after releasing the second capturing reagent affinity complex, releasing and capturing the third capturing reagent affinity complex on the second solid support.

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

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

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

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

70. The method of claim 66, wherein 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%, or 0.01% to 1% 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%), 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% dilutions.

71. The method of claim 66, wherein the third diluent is 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% of the test sample; 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%, 0.25%, 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 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 of any one of claims 11-71, wherein the first capture reagent and the second capture reagent are independently selected from aptamers or antibodies.

73. The method of any one of claims 64-72, wherein the third capture reagent is selected from aptamers or antibodies.

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

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

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

77. The method of claim 76, wherein the connector is selected from amide connectors, carbonyl connectors, propynyl connectors, alkyne connectors, ester connectors, urea connectors, urethane connectors, guanidine connectors, amidine connectors, sulfoxide connectors, and sulfone connectors.

78. The method of claim 76 or 77, wherein the portion is a hydrophobic portion.

79. The method of claim 78, wherein the portion is selected from naphthyl, benzyl, fluorobenzyl, tyrosinyl, indole, morpholino, isobutyl, 3,4-methylenedioxybenzyl, benzo[a]phenylthio, benzo[a]furanyl, phenylbenzyl, 4-phenoxybenzyl, diphenylpropyl, and diphenylmethyl.

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

81. The method of any one of claims 11-80, wherein the presence of the dissociated first and second capture reagents or the level of the dissociated first and second capture reagents are detected by PCR, mass spectrometry, nucleic acid sequencing, next-generation sequencing (NGS), or hybridization.

82. A method for preparing a liquid sample, the method comprising: The sample was dried at a constant temperature from -20°C to room temperature for at least four (4) hours to produce a dried sample; as well as The dried sample was reconstituted using a formulation containing buffers, one or more salts, chelating agents, protease inhibitors, nonionic surfactants, and oligonucleotides.

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

84. The method of claim 82 or claim 83, wherein, The one or more salts include sodium salts, potassium salts, and magnesium salts.

85. The method of claim 83 or claim 84, wherein the sodium salt is NaCl, the potassium salt is KCl, and the magnesium salt is MgCl2.

86. The method of claim 85, wherein 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 mM-125 mM or about 100 mM.

87. The method of 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 method of any one of claims 85-87, wherein the concentration of MgCl2 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 8 mM.

89. The method of any one of claims 82-88, wherein the buffer is selected from HEPES, IVIES, Bistris methane, ADA, ACES, Bistris propane, PIPES, MOPSO, choline chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, glycamide, glycylglycine, HEPBS, Bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS.

90. The method of any one of claims 82-89, wherein the concentration of the buffer in the formulation is 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 of any one of claims 82-90, wherein the chelating agent is selected from EGTA, EDTA, DTPA, BAPTA, DMPS and ALA.

92. The method of any one of claims 82-91, wherein 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.

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

94. The method of claim 93, wherein the nonionic surfactant comprises, on a volume-to-volume basis, 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% or about 1.5% of the formulation, or about 1.2% of the formulation.

95. The method of any one of claims 82-94, wherein the pH of the preparation is from about pH 5 to about pH 9, or from about pH 6 to about pH 8, or from about pH 7 to about pH 7.9 or about pH 7.

5.

96. The method of any one of claims 82-95, wherein the formulation comprises 50 mM HEPES, 100 mM NaCl, 5 mM KCl, 8 mM MgCl2, 1.25 mM EGTA and 1.2% Tween-20.

97. The method of claim 96, wherein the formulation has a pH of about 7.

5.

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

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

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

101. The method of any one of claims 82-100, wherein the protease inhibitor is benzoamidine.

102. The method of any one of claims 82-101, wherein the concentration of the protease inhibitor in the formulation is 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 of any one of claims 82-102, wherein the oligonucleotide is a single-stranded oligonucleotide.

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

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

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

107. The method of any one of claims 82-106, wherein the oligonucleotide comprises the sequence [(ACXX)7-AC], where X is BndU.

108. The method of any one of claims 82-107, wherein 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 37 μM.

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

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

111. The method of any one of claims 82-110, wherein the sample is diluted into a first diluent and a second diluent, wherein the first diluent is a diluent 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% of the test sample, and the second diluent is 0.01% to 1% 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% dilution.

112. The method of any one of claims 82-110, wherein the sample is diluted into a first diluent and a second diluent, wherein 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%), or 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005% of the test sample; and the second diluent is 5% to 39% of the test sample. (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% dilution.

113. The method of any one of claims 82-110, wherein the sample is diluted into a first diluent and a second diluent, wherein the first 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%, 0.25%, 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% of the test sample; and the second diluent is 5% to 39% of the test sample. (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% dilution.

114. The method of any one of claims 82-110, wherein the sample is diluted into a first diluent and a second diluent, wherein the first 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%, 0.25%, 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% of the test sample; and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 method of any one of claims 82-110, wherein the sample is diluted into a first diluent and a second diluent, wherein the first 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%, or about 20% of the test sample, and the second diluent is 0.01% to 1% 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% dilution.

116. The method of any one of claims 82-110, wherein the sample is diluted into a first diluent and a second diluent, wherein the first 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%, or about 20% of the test sample, and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 method of any one of claims 82-110, wherein the sample is diluted to a third diluent, wherein the third diluent is a fraction of the test sample selected from 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%, or 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%), 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% dilutions.

118. The method of any one of claims 82-110, wherein 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%), 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005% of the test sample, and the second diluent is 0.01% to 1% 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 a dilution of 0.01% to 0.5%, or 0.02% to 0.1% or about 0.05%.

119. The method of any one of claims 82-110, wherein 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%), 0.002% to 0.008%, 0.003% to 0.007%, or about 0.005% of the test sample, and the second diluent is 0.5% to 5% of the test sample. (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% dilution.

120. The method of any one of claims 82-110, wherein the first 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%, 0.25%, 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% of the test sample, and the second diluent is 0.5% to 5% of the test sample. (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% dilution.

121. The method of any one of claims 82-110, wherein the first 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%, 0.25%, 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% of the test sample, and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 method of any one of claims 82-110, wherein the first diluent is 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% of the test sample, and the second diluent is 0.001% to 0.009% of the test sample. (or a dilution of 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 method of any one of claims 82-110, wherein the first diluent is 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% of the test sample, and the second diluent is 0.01% to 1% 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 a dilution of 0.01% to 0.5%, or 0.02% to 0.1% or about 0.05%.

124. The method of any one of claims 111-123, further comprising a third diluent of the test sample, wherein the third diluent is a diluent of the test sample 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%), 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%, 0.25%, 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 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 comprising: Dry samples and formulations, the formulations comprising buffers, one or more salts, chelating agents, protease inhibitors, nonionic surfactants, and oligonucleotides; in, The dried sample was derived from a liquid sample that had been dried at a constant temperature of about -20°C to room temperature for at least four (4) hours.

126. A method for detecting an analyte in a sample, the method comprising: The sample was dried at a constant temperature of about -20°C to room temperature for at least four (4) hours to produce a dried sample; The dried sample was reconstituted using a formulation comprising buffers, one or more salts, chelating agents, protease inhibitors, nonionic surfactants, and oligonucleotides; as well as The analyte was detected from the reconstructed sample.

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

128. The method of claim 126 or claim 127, wherein the protein binding agent is selected from aptamers or antibodies.

129. The method of any one of claims 126-128, wherein the detection is performed in multiple assays.

130. The method of claim 129, wherein the multiplex determination 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 analytes.

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