Multiplexed analyses of test samples
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 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 labelling agent;
a solid support comprising at least one probe;
a buffer comprising HEPES, NaCl, KCl, MgCl 2 , and EDTA, and
a competitor molecule, wherein the competitor molecule is selected from dNTPs, abasic phosphodiester polymers, pyrophosphate, dextran sulphate, heparin and polydextran.
2. The kit of claim 1 , wherein the aptamer comprises a 5-position pyrimidine modification.
3. The kit of claim 2 , wherein the 5-position pyrimidine modification is selected from the group consisting 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.
4. The kit of claim 1 , wherein the probe is capable of associating with the tag.
5. The kit of claim 1 , further comprising washing solution for sample dilution.
6. The kit of claim 1 , wherein the labelling agent includes a label and a binding partner that is specific for the target molecule.
7. The kit of claim 6 , wherein the binding partner is an antibody, antibody fragment, synthetic antibody mimetic, biomimetic, aptamer or molecular imprinted ligand.
8. The kit of claim 6 , wherein the label is selected from reporter molecules, specific binding pair members, mass tags, oligonucleotide primers and specific polynucleotide sequences.
9. The kit of claim 8 , wherein the label is reporter molecule is a catalyst, enzyme, polynucleotide encoding the catalyst, promoter, dye, fluorescent molecule, quantum dot, chemiluminescent molecule, coenzyme, enzyme substrate, radioactive group, small organic molecule, amplifiable polynucleotide sequence, particle, latex particle, carbon particle, metal sol, crystallite, liposome, cell, mass tag that alters the weight of the molecule to which it is conjugated, electromagnetic or electrochemical material or fluorescent dye.
10. The kit of claim 9 , wherein one or more of the kit components is provided as a dry powder.