Multiplexed analyses of test samples

Abstract

The present disclosure describes methods, devices, reagents, and kits for the detection of one or more target molecules that may be present in a test sample. In one embodiment, a test sample is contacted with an aptamer that includes a tag and has a specific affinity for a target molecule. An aptamer affinity complex that includes an aptamer bound to its target molecule is allowed to form. If the test sample contains the target molecule, an aptamer affinity complex will generally form in the test sample. The aptamer affinity complex is optionally converted to an aptamer covalent complex that includes an aptamer covalently bound to its target molecule. The aptamer affinity complex (or optional aptamer covalent complex) can then be detected and/or quantified using any of a variety of methods known to one skilled in the art, including using a solid support, using mass spectrometry, and using quantitative polymerase chain reaction (Q-PCR).

1 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer comprising a tag and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (b) contacting a surface of a solid support comprising a probe with said aptamer affinity complex, such that said tag is permitted to associate with said probe;    (c) at any point prior to (d), contacting said aptamer affinity complex with a labeling agent; and    (d) detecting said target molecule on said surface by detecting said labeling agent.    
   
   
       2 . The method of  claim 1 , wherein said aptamer is a single-stranded nucleic acid or a double-stranded nucleic acid.  
   
   
       3 . The method of  claim 2 , wherein said aptamer comprises DNA or RNA  
   
   
       4 . The  claim 3 , wherein said aptamer comprises at least one chemical modification.  
   
   
       5 . The method of  claim 4 , wherein said at least one chemical modification is a chemical substitution at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.  
   
   
       6 . The method of  claim 4 , wherein said at least one chemical modification is independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH2), a 2′-fluoro (2′-F), a 2′-O-methyl (2′-OMe), a 5-position pyrimidine modification, an 8-position purine modification, a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap, and a 5′ cap.  
   
   
       7 . The method of  claim 4 , wherein said at least one chemical modification is independently selected from 5-(N-benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N-[2-(1H-indole-3yl)ethyl]carboxyamide)-2′-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxyamide)-2′-deoxyuridine chloride, 5-(N-napthylcarboxyamide)-2′-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine.  
   
   
       8 . The method of  claim 2  wherein said tag is a 5′-end of said aptamer.  
   
   
       9 . The method of  claim 2  wherein said tag is a 3′-end of said aptamer.  
   
   
       10 . The method of  claim 2  wherein said tag is a nucleotide sequence that is located between a 5′-end and a 3′-end of said aptamer.  
   
   
       11 . The method of  claim 10  wherein said aptamer comprises a 5′-fixed end separated from a 3′-fixed end by a variable sequence, and wherein said tag is included within either the 5′-fixed end or the 3′-fixed end of said aptamer.  
   
   
       12 . The method of  claim 10  wherein said aptamer comprises a 5′-fixed end separated from a 3′-fixed end by a variable sequence, and wherein said tag is the variable sequence of said aptamer or any part of the variable sequence of said aptamer.  
   
   
       13 . The method of  claim 10  wherein said aptamer comprises a 5′-fixed end separated from a 3′-fixed end by a variable sequence, and wherein said tag is a nucleotide sequence that includes at least a portion of the variable sequence and at least a portion of one of said fixed ends of said aptamer.  
   
   
       14 . The method of  claim 10  wherein said aptamer comprises a 5′-fixed end separated from a 3′-fixed end by a variable sequence, and wherein said tag is a nucleotide sequence that includes the variable sequence and a portion of at least one of said fixed ends of said aptamer.  
   
   
       15 . The method of  claim 10  wherein said aptamer comprises a 5′-fixed end separated from a 3′-fixed end by a variable sequence, and wherein said tag is a nucleotide sequence that includes the variable sequence and both fixed ends of said aptamer.  
   
   
       16 . The method of  claim 1  wherein said target molecule is selected from the group consisting of a protein, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, an antigen, an antibody, a virus, a substrate, a metabolite, a transition state analog, a cofactor, an inhibitor, a drug, a dye, a nutrient, a growth factor, a tissue, and a controlled substance.  
   
   
       17 . The method of  claim 16 , wherein said target molecule is a protein.  
   
   
       18 . The method of  claim 1 , wherein said test sample is a biological sample.  
   
   
       19 . The method  claim 18 , wherein said biological sample is selected from whole blood, leukocytes, peripheral blood mononuclear cells, plasma, serum, sputum, breath, urine, semen, saliva, meningial fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, a cellular extract, stool, tissue, a tissue extract, a tissue biopsy, and cerebrospinal fluid.  
   
   
       20 . The method of  claim 19 , wherein said biological sample is plasma or serum.  
   
   
       21 . The method of  claim 1 , wherein said probe comprises at least one component independently selected from a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, and any portion of any of these structures.  
   
   
       22 . The method of  claim 1 , wherein said tag comprises at least one component independently selected from a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, and any portion of any of these structures.  
   
   
       23 . The method of  claim 1 , wherein said tag associates with said probe through hybridization.  
   
   
       24 . The method of  claim 23 , wherein said tag comprises a first nucleotide sequence and said probe comprises a second nucleotide sequence, and wherein said first nucleotide sequence is complementary to said second nucleotide sequence.  
   
   
       25 . The method of any of  claim 1 , further comprising contacting said tag or said probe with a linker molecule, such that said tag is permitted to associate with said probe through said linker molecule.  
   
   
       26 . The method of  claim 25 , wherein said linker molecule comprises one or more molecules independently selected from a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, and any portion of any of these structures.  
   
   
       27 . The method of  claim 26 , wherein said linker molecule comprises at least one single-stranded polynucleotide, said polynucleotide comprising DNA, RNA, or both.  
   
   
       28 . The method of  claim 27 , wherein said tag comprises a first nucleotide sequence and said probe comprises a second nucleotide sequence, wherein said at least one single-stranded polynucleotide comprises a tag association component that is complementary to said first nucleotide sequence and a probe association component that is complementary to said second nucleotide sequence, and wherein said tag association component hybridizes to said first nucleotide sequence and said probe association component hybridizes to said second nucleotide sequence.  
   
   
       29 . The method of  claim 1 , wherein said solid support is selected from a microtitre well, a microscope slide, a cyclo-olefin copolymer substrate, a membrane, a plastic substrate, a paramagnetic bead, charged paper, nylon, a Langmuir-Bodgett film, glass, a germanium substrate, a silicon substrate, a silicon wafer chip, a flow through chip, a microbead, a polytetrafluoroethylene substrate, a polystyrene substrate, a gallium arsenide substrate, a gold substrate, and a silver substrate.  
   
   
       30 . The method of  claim 29 , wherein said surface comprises at least one surface modification layer independently selected from a metal, a metal oxide, a polymer, a small organic molecule, a methacrylate copolymer, a polyacrylamide, a polysaccharide, a phospholipid, a polyurethane, a polyester, a polycarbonate, a polyurea, a polyamide, a polyethylene amine, a polyarylene sulfide, a polysiloxane, a polyimide, a polyacetate, and a three-dimensional polymer matrix.  
   
   
       31 . The method of  claim 1 , wherein said surface comprises a plurality of spatially defined addresses, and wherein each of a plurality of said addresses comprises at least one probe disposed thereon.  
   
   
       32 . The method of  claim 31 , wherein detecting said target molecule comprises detecting said labeling agent at an address on said surface.  
   
   
       33 . The method of  claim 1 , wherein said labeling agent comprises one or more reagents that can label said target molecule with a detectable moiety.  
   
   
       34 . The method of  claim 33 , wherein at least one of said reagents reacts with a functional group on said target molecule.  
   
   
       35 . The method of  claim 34 , wherein said functional group is selected from the group consisting of primary amines, thiols, alcohols, and carboxylates.  
   
   
       36 . The method of  claim 33 , wherein said detectable moiety is selected from the group consisting of a dye, a radiolabel, an enzyme, and an enzyme substrate.  
   
   
       37 . The method of  claim 36 , wherein said dye is a fluorophore.  
   
   
       38 . The method of  claim 36 , wherein said enzyme is alkaline phosphatase or horseradish peroxidase.  
   
   
       39 . The method of  claim 1 , further comprising quantifying said target molecule.  
   
   
       40 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer comprising a tag and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (b) contacting a surface of a solid support comprising a probe with said aptamer affinity complex, such that said tag is permitted to associate with said probe;    (c) preparing said aptamer affinity complex for detection; and    (d) detecting said target molecule using mass spectrometry.    
   
   
       41 . The method of  claim 40 , further comprising quantifying said target molecule.  
   
   
       42 . The method of  claim 40  wherein said target is detected using electrospray ionization, matrix-assisted laser desorption ionization (MALDI), surface enhanced laser desorption ionization (SELDI), or electron impact ionization.  
   
   
       43 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (b) partitioning any free aptamer present in said test sample from the aptamer affinity complex; and    (c) detecting said aptamer affinity complex using quantitative-PCR (Q-PCR) and thereby detecting said target molecule.    
   
   
       44 . The method of  claim 43 , further comprising quantifying said target molecule.  
   
   
       45 . The method of  claim 43  wherein said Q-PCR is performed using TaqMan® PCR, an intercalating fluorescent dye during the PCR process, or a molecular beacon during the PCR process.  
   
   
       46 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer comprising a tag and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (b) converting said aptamer affinity complex into an aptamer covalent complex;    (c) contacting a surface of a solid support comprising a probe with said aptamer covalent complex, such that said tag is permitted to associate with said probe;    (d) at any point prior to (e), contacting said aptamer covalent complex with a labeling agent; and    (e) detecting said target molecule on said surface by detecting said labeling agent.    
   
   
       47 . The method of  claim 46 , wherein said aptamer is a single-stranded nucleic acid or a double-stranded nucleic acid.  
   
   
       48 . The method of  claim 47 , wherein said aptamer comprises DNA or RNA  
   
   
       49 . The  claim 48 , wherein said aptamer comprises at least one chemical modification.  
   
   
       50 . The method of  claim 49 , wherein said at least one chemical modification is a chemical substitution at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.  
   
   
       51 . The method of  claim 49 , wherein said at least one chemical modification is independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH2), a 2′-fluoro (2′-F), a 2′-O-methyl (2′-OMe), a 5-position pyrimidine modification, an 8-position purine modification, a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap, and a 5′ cap.  
   
   
       52 . The method of  claim 49 , wherein said at least one chemical modification is independently selected from 5-(N-benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N-[2-(1H-indole-3yl)ethyl]carboxyamide)-2′-deoxyuridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxyamide)-2′-deoxyuridine chloride, 5-(N-napthylcarboxyamide)-2′-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine.  
   
   
       53 . The method of  claim 46 , wherein said aptamer is a photoaptamer.  
   
   
       54 . The method of  claim 53 , wherein said photoaptamer comprises one or more photoreactive functional groups independently selected from 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-thiouracil, 4-thiocytosine, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-aziodoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, 7-deaza-7-bromoguanine, benzophenone, anthraquinone, 4-azido-2-nitro-aniline, psoralen, and derivatives of any of these.  
   
   
       55 . The method of  claim 54 , wherein converting said photoaptamer affinity complex into a photoaptamer covalent complex comprises exposing said photoaptamer affinity complex to a radiation source.  
   
   
       56 . The method of  claim 47  wherein said tag is a 5′-end of said aptamer.  
   
   
       57 . The method of  claim 47  wherein said tag is a 3′-end of said aptamer.  
   
   
       58 . The method of  claim 47  wherein said tag is a nucleotide sequence that is located between a 5′-end and a 3′-end of said aptamer.  
   
   
       59 . The method of  claim 58  wherein said aptamer comprises a 5′-fixed end separated from a 3′-fixed end by a variable sequence, and wherein said tag is included within either the 5′-fixed end or the 3′-fixed end of said aptamer.  
   
   
       60 . The method of  claim 58  wherein said aptamer comprises a 5′-fixed end separated from a 3′-fixed end by a variable sequence, and wherein said tag is the variable sequence of said aptamer or any part of the variable sequence of said aptamer.  
   
   
       61 . The method of  claim 58  wherein said aptamer comprises a 5′-fixed end separated from a 3′-fixed end by a variable sequence, and wherein said tag is a nucleotide sequence that includes at least a portion of the variable sequence and at least a portion of one of said fixed ends of said aptamer.  
   
   
       62 . The method of  claim 58  wherein said aptamer comprises a 5′-fixed end separated from a 3′-fixed end by a variable sequence, and wherein said tag is a nucleotide sequence that includes the variable sequence and a portion of at least one of said fixed ends of said aptamer.  
   
   
       63 . The method of  claim 58  wherein said aptamer comprises a 5′-fixed end separated from a 3′-fixed end by a variable sequence, and wherein said tag is a nucleotide sequence that includes the variable sequence and both fixed ends of said aptamer.  
   
   
       64 . The method of  claim 46  wherein said target molecule is selected from the group consisting of a protein, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, an antigen, an antibody, a virus, a substrate, a metabolite, a transition state analog, a cofactor, an inhibitor, a drug, a dye, a nutrient, a growth factor, a tissue, and a controlled substance.  
   
   
       65 . The method of  claim 64 , wherein said target molecule is a protein.  
   
   
       66 . The method of  claim 46 , wherein said test sample is a biological sample.  
   
   
       67 . The method  claim 66 , wherein said biological sample is selected from whole blood, leukocytes, peripheral blood mononuclear cells, plasma, serum, sputum, breath, urine, semen, saliva, meningial fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, a cellular extract, stool, tissue, a tissue extract, a tissue biopsy, and cerebrospinal fluid.  
   
   
       68 . The method of  claim 66 , wherein said biological sample is plasma or serum.  
   
   
       69 . The method of  claim 46 , wherein said probe comprises at least one component independently selected from a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, and any portion of any of these structures.  
   
   
       70 . The method of  claim 46 , wherein said tag comprises at least one component independently selected from a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, and any portion of any of these structures.  
   
   
       71 . The method of  claim 46 , wherein said tag associates with said probe through hybridization.  
   
   
       72 . The method of  claim 71 , wherein said tag comprises a first nucleotide sequence and said probe comprises a second nucleotide sequence, and wherein said first nucleotide sequence is complementary to said second nucleotide sequence.  
   
   
       73 . The method of any of  claim 46 , further comprising contacting said tag or said probe with a linker molecule, such that said tag is permitted to associate with said probe through said linker molecule.  
   
   
       74 . The method of  claim 73 , wherein said linker molecule comprises one or more molecules independently selected from a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, and any portion of any of these structures.  
   
   
       75 . The method of  claim 74 , wherein said linker molecule comprises at least one single-stranded polynucleotide, said polynucleotide comprising DNA, RNA, or both.  
   
   
       76 . The method of  claim 75 , wherein said tag comprises a first nucleotide sequence and said probe comprises a second nucleotide sequence, wherein said at least one single-stranded polynucleotide comprises a tag association component that is complementary to said first nucleotide sequence and a probe association component that is complementary to said second nucleotide sequence, and wherein said tag association component hybridizes to said first nucleotide sequence and said probe association component hybridizes to said second nucleotide sequence.  
   
   
       77 . The method of  claim 46 , wherein said solid support is selected from a microtitre well, a microscope slide, a cyclo-olefin copolymer substrate, a membrane, a plastic substrate, a paramagnetic bead, charged paper, nylon, a Langmuir-Bodgett film, glass, a germanium substrate, a silicon substrate, a silicon wafer chip, a flow through chip, a microbead, a polytetrafluoroethylene substrate, a polystyrene substrate, a gallium arsenide substrate, a gold substrate, and a silver substrate.  
   
   
       78 . The method of  claim 77 , wherein said surface comprises at least one surface modification layer independently selected from a metal, a metal oxide, a polymer, a small organic molecule, a methacrylate copolymer, a polyacrylamide, a polysaccharide, a phospholipid, a polyurethane, a polyester, a polycarbonate, a polyurea, a polyamide, a polyethylene amine, a polyarylene sulfide, a polysiloxane, a polyimide, a polyacetate, and a three-dimensional polymer matrix.  
   
   
       79 . The method of  claim 46 , wherein said surface comprises a plurality of spatially defined addresses, and wherein each of a plurality of said addresses comprises at least one probe disposed thereon.  
   
   
       80 . The method of  claim 79 , wherein detecting said target molecule comprises detecting said labeling agent at an address on said surface.  
   
   
       81 . The method of  claim 46 , wherein said labeling agent comprises one or more reagents that can label said target molecule with a detectable moiety.  
   
   
       82 . The method of  claim 81 , wherein at least one of said reagents reacts with a functional group on said target molecule.  
   
   
       83 . The method of  claim 82 , wherein said functional group is selected from the group consisting of primary amines, thiols, alcohols, and carboxylates.  
   
   
       84 . The method of  claim 81 , wherein said detectable moiety is selected from the group consisting of a dye, a radiolabel, an enzyme, and an enzyme substrate.  
   
   
       85 . The method of  claim 84 , wherein said dye is a fluorophore.  
   
   
       86 . The method of  claim 84 , wherein said enzyme is alkaline phosphatase or horseradish peroxidase.  
   
   
       87 . The method of  claim 46 , further comprising quantifying said target molecule.  
   
   
       88 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer comprising a tag and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (b) converting said aptamer affinity complex into an aptamer covalent complex;    (c) contacting a surface of a solid support comprising a probe with said aptamer covalent complex, such that said tag is permitted to associate with said probe;    (d) preparing said aptamer covalent complex for detection; and    (e) detecting said target molecule using mass spectrometry.    
   
   
       89 . The method of  claim 88 , further comprising quantifying said target molecule.  
   
   
       90 . The method of  claim 88  wherein said target is detected using electrospray ionization, matrix-assisted laser desorption ionization (MALDI), surface enhanced laser desorption ionization (SELDI), or electron impact ionization.  
   
   
       91 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (b) converting said aptamer affinity complex into an aptamer covalent complex;    (c) partitioning any free aptamer present in said test sample from the aptamer covalent complex; and    (d) detecting said aptamer covalent complex using quantitative-PCR (Q-PCR) and thereby detecting said target molecule.    
   
   
       92 . The method of  claim 91 , further comprising quantifying said target molecule.  
   
   
       93 . The method of  claim 91  wherein said Q-PCR is performed using TaqMan® PCR, an intercalating fluorescent dye during the PCR process, or a molecular beacon during the PCR process.  
   
   
       94 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with at least one competitor molecule, wherein at least one non-specific complex is formed if said test sample comprises a target molecule, a non-target molecule, or both a target molecule and a non-target molecule;    (b) contacting said test sample with an aptamer comprising a tag and having a specific affinity for said target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (c) converting said aptamer affinity complex into an aptamer covalent complex;    (d) contacting a surface of a solid support comprising a probe with said aptamer covalent complex, such that said tag is permitted to associate with said probe;    (e) at any point prior to (f), contacting said aptamer covalent complex with a labeling agent; and    (f) detecting said target molecule on said surface by detecting said labeling agent.    
   
   
       95 . The method of  claim 94  wherein said at least one competitor molecule is independently selected from an oligonucleotide, a polyanion, an abasic phosphodiester polymer, a dNTP, and a pyrophosphate.  
   
   
       96 . The method of  claim 95 , wherein said at least one competitor molecule is heparin or a polydextran.  
   
   
       97 . The method of  claim 95 , wherein said at least one competitor molecule is dextran sulphate.  
   
   
       98 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer comprising a tag and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (b) exposing said test sample to a condition or treatment that kinetically challenges said aptamer, said target molecule, and any non-target molecules present in said test sample;    (c) converting said aptamer affinity complex into an aptamer covalent complex;    (d) contacting a surface of a solid support comprising a probe with said aptamer covalent complex, such that said tag is permitted to associate with said probe;    (e) at any point prior to (f), contacting said aptamer covalent complex with a labeling agent; and    (f) detecting said target molecule on said surface by detecting said labeling agent.    
   
   
       99 . The method of  claim 98 , wherein said condition or treatment that kinetically challenges said aptamer comprises contacting said test sample with at least one competitor molecule.  
   
   
       100 . The method of  claim 99 , wherein said at least one competitor molecule is independently selected from an oligonucleotide, a polyanion, an abasic phosphodiester polymer, a dNTP, and a pyrophosphate.  
   
   
       101 . The method of  claim 100 , wherein said at least one competitor molecule is heparin or a polydextran.  
   
   
       102 . The method of  claim 100 , wherein said at least one competitor molecule is dextran sulphate.  
   
   
       103 . The method of  claim 98 , wherein said condition or treatment that kinetically challenges said aptamer comprises diluting said test sample.  
   
   
       104 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer comprising a tag and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (b) converting said aptamer affinity complex into an aptamer covalent complex;    (c) partitioning from said test sample any free aptamer present in said test sample;    (d) contacting a surface of a solid support comprising a probe with said aptamer covalent complex, such that said tag sequence is permitted to associate with said probe;    (e) at any point prior to (f), contacting said aptamer covalent complex with a labeling agent; and    (f) detecting said target molecule on said surface by detecting said labeling agent.    
   
   
       105 . The method of  claim 104 , wherein partitioning from said test sample any free aptamer present in said test sample comprises precipitating said aptamer covalent complex, any protein, and any protein complex in said test sample.  
   
   
       106 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer comprising a tag and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (b) converting said aptamer affinity complex into an aptamer covalent complex;    (c) partitioning from said test sample said aptamer covalent complex;    (d) contacting a surface of a solid support comprising a probe with said aptamer covalent complex, such that said tag is permitted to associate with said probe;    (e) at any point prior to (f), contacting said aptamer covalent complex with a labeling agent; and    (f) detecting said target molecule on said surface by detecting said labeling agent.    
   
   
       107 . The method of  claim 106 , wherein partitioning from said test sample said aptamer covalent complex comprises precipitating said aptamer covalent complex from said test sample or capturing said aptamer covalent complex from said test sample.  
   
   
       108 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer comprising a tag and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (b) converting said aptamer affinity complex into an aptamer covalent complex;    (c) partitioning from said test sample any free aptamer present in said test sample;    (d) partitioning from said test sample said aptamer covalent complex;    (e) contacting a surface of a solid support comprising a probe with said aptamer covalent complex, such that said tag is permitted to associate with said probe;    (f) at any point prior to (g), contacting said aptamer covalent complex with a labeling agent; and    (g) detecting said target molecule on said surface by detecting said labeling agent.    
   
   
       109 . The method of  claim 108 , wherein partitioning from said test sample any free aptamer present in said test sample comprises precipitating said aptamer covalent complex, any protein, and any protein complex in said test sample.  
   
   
       110 . The method of  claim 108 , wherein partitioning from said test sample said aptamer covalent complex comprises precipitating said aptamer covalent complex from said test sample or capturing said aptamer covalent complex from said test sample.  
   
   
       111 . A method for detecting a target molecule that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer comprising a tag and having a specific affinity for a target molecule, wherein an aptamer affinity complex is formed if said target molecule is present in said test sample;    (b) exposing said test sample to a condition or treatment that kinetically challenges said aptamer, said target molecule, and any non-target molecules present in said test sample;    (c) converting said aptamer affinity complex into an aptamer covalent complex;    (d) partitioning from said test sample any free aptamer present in said test sample;    (e) partitioning from said test sample said aptamer covalent complex;    (f) contacting a surface of a solid support comprising a probe with said aptamer covalent complex, such that said tag is permitted to associate with said probe;    (g) at any point prior to (h), contacting said aptamer covalent complex with a labeling agent; and    (h) detecting said target molecule on said surface by detecting said labeling agent.    
   
   
       112 . The method of  claim 111 , wherein said condition or treatment that kinetically challenges said aptamer comprises contacting said test sample with at least one competitor molecule.  
   
   
       113 . The method of  claim 112 , wherein said at least one competitor molecule is independently selected from an oligonucleotide, a polyanion, an abasic phosphodiester polymer, a dNTP, and a pyrophosphate.  
   
   
       114 . The method of  claim 113 , wherein said at least one competitor molecule is heparin or a polydextran.  
   
   
       115 . The method of  claim 113 , wherein said at least one competitor molecule is dextran sulphate.  
   
   
       116 . The method of  claim 111 , wherein said condition or treatment that kinetically challenges said aptamer comprises diluting said test sample.  
   
   
       117 . The method of  claim 111 , wherein partitioning from said test sample any free aptamer present in said test sample comprises precipitating said aptamer covalent complex, any protein, and any protein complex in said test sample.  
   
   
       118 . The method of  claim 111 , wherein partitioning from said test sample said aptamer covalent complex comprises precipitating said aptamer covalent complex from said test sample or capturing said aptamer covalent complex from said test sample.  
   
   
       119 . A method for detecting one or more target molecules that may be present in a test sample, the method comprising: 
 (a) contacting a test sample with a plurality of aptamers, each of said aptamers comprising a tag and having a specific affinity for a target molecule, wherein one or more aptamer affinity complexes are formed if a target molecule corresponding to any of said aptamers is present in said test sample;    (b) converting said aptamer affinity complexes into aptamer covalent complexes;    (c) contacting a surface of a solid support comprising at least one probe with said aptamer covalent complexes, such that said tag on each of said aptamers is permitted to associate with its corresponding probe;    (d) at any point prior to (e), contacting said aptamer covalent complexes with a labeling agent; and    (e) detecting one or more target molecules on said surface by detecting said labeling agent.    
   
   
       120 . A method for determining an amount or concentration of a target molecule in a test sample, the method comprising: 
 (a) contacting a test sample with an aptamer comprising a tag and having a specific affinity for a target molecule, wherein sample;    (b) converting said aptamer affinity complex into an aptamer covalent complex;    (c) contacting a surface of a solid support comprising a probe with said aptamer covalent complex, such that said tag is permitted to associate with said probe;    (d) at any point prior to (e), contacting said aptamer covalent complex with a labeling agent; and    (e) determining an amount or concentration of said target molecule by quantitatively detecting said labeling agent.    
   
   
       121 . A kit for detecting at least one target molecule that may be present in a test sample, the kit comprising: at least one aptamer comprising a tag and having specific affinity for a target molecule; a labeling agent; and a solid support comprising at least one probe capable of associating with said tag.