Multiplexed analyses of test samples

Patent metadata

Field	Value
Publication number	US-9404919-B2
Application number	US-201414176933-A
Country	US
Kind code	B2
Filing date	Feb 10, 2014
Priority date	Jan 16, 2007
Publication date	Aug 2, 2016
Grant date	Aug 2, 2016

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. The described methods, devices, kits, and reagents facilitate the detection and quantification of a non-nucleic acid target (e.g., a protein target) in a test sample by detecting and quantifying a nucleic acid (i.e., an aptamer). The methods described create a nucleic acid surrogate for a non-nucleic acid target, thus allowing the wide variety of nucleic acid technologies, including amplification, to be applied to a broader range of desired targets, especially protein targets. The disclosure further describes aptamer constructs that facilitate the use of aptamers in a variety of analytical detection applications.

First claim

What is claimed is:

1. A method for detecting for the presence of a target molecule in a test sample, the method comprising:
(a) preparing a mixture by contacting the test sample with an aptamer comprising a first tag and having specific affinity for the target molecule, wherein an aptamer affinity complex is formed if the target molecule is present in the test sample;
(b) exposing the mixture to a first solid support comprising a first capture element, and allowing the first tag to associate with the first capture element;
(c) removing components of the mixture not associated with the first solid support;
(d) releasing the aptamer affinity complex from the first solid support;
(e) attaching a second tag to the target molecule of the aptamer affinity complex;
(f) exposing the released aptamer affinity complex to a second solid support comprising a second capture element and allowing the second tag to associate with the second capture element;
(g) removing uncomplexed aptamer from the mixture by partitioning the uncomplexed aptamer from the aptamer affinity complex;
(h) detecting for the presence of the target molecule by detecting the aptamer portion of the aptamer affinity complex; and
wherein, the method is capable of detecting for the presence of the target molecule in the test sample when the target molecule is present at a sub-picomolar level.

2. The method of claim 1 , wherein the aptamer comprises at least one modified nucleotide.

3. The method of claim 2 , wherein the at least one modified nucleotide is a C-5 modified pyrimidine.

4. The method of claim 3 , wherein the C-5 modified pyrimidine is selected from the group listed in FIG. 16 .

5. The method of claim 3 , wherein the aptamer further comprises at least one chemical modification at one or more positions independently selected from the group consisting of a 2′-position sugar modification, a 2′-amino (2′—NH 2 ), 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.

6. The method of claim 1 , wherein (h) further comprises dissociating the aptamer from said aptamer affinity complex before detecting the aptamer.

7. The method of claim 1 , wherein the rate of dissociation of the aptamer affinity complex (t 1/2 ) is selected from the group consisting of ≧30 minutes, ≧60 minutes, ≧90 minutes, ≧120 minutes, ≧150 minutes, ≧180 minutes, ≧210 minutes, and ≧240 minutes.

8. The method of claim 1 , wherein the aptamer is detected and optionally quantified using a method selected from the group consisting of Q-PCR, MS, and hybridization.

9. The method of claim 1 further comprising adding a detectable moiety to the aptamer.

10. The method of claim 9 , wherein the detectable moiety is selected from the group consisting of a dye, a quantum dot, a radiolabel, a electrochemical functional group, an enzyme, and an enzyme substrate.

11. The method of claim 1 , wherein the aptamer comprises DNA, RNA or both DNA and RNA.

12. The method of claim 1 , wherein the target molecule is selected from the group consisting of a protein, a peptide, 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.

13. The method of claim 1 , wherein the test sample is selected from the group consisting of a biological sample, an environmental sample, a chemical sample, a pharmaceutical sample, a food sample, an agricultural sample, and a veterinary sample.

14. The method of claim 13 , wherein the biological sample is selected from the group consisting of 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.

15. The method of claim 1 , wherein the first tag and the second tag each comprise at least one component independently selected from the group consisting of 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, biotin, avidin, streptavidin, Extravidin, neutravidin, a metal, histidine, and any portion of any of these structures.

16. The method of claim 1 , wherein the first capture element and said second capture element each 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, biotin, avidin, streptavidin, Extravidin, neutravidin, a metal, histidine, and any portion of any of these structures.

17. The method of claim 1 , wherein the first tag comprises a releasable moiety.

18. The method of claim 1 , wherein the first solid support and the second solid support each is independently selected from the group consisting of a polymer bead, an agarose bead, a polystyrene bead, an acrylamide bead, a solid core bead, a porous bead, a paramagnetic bead, glass bead, controlled pore bead, a microtitre well, a cyclo-olefin copolymer substrate, a membrane, a plastic substrate, nylon, a Langmuir-Blodgett 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.

19. The method of claim 1 , wherein the method has a limit of detection (LOD) selected from the group consisting of 630 fM, 530 fM and 90 fM.

20. The method of claim 1 , wherein the method is capable of detecting the target molecule FGF7 in a test sample with an LOD of 90 fM.

Assignees

• Somalogic Inc

Inventors

• Schneider Daniel J
• Nieuwlandt Dan
• Eaton Bruce
• Stanton Martin
• Gupta Shashi
• Kraemer Stephan
• Zichi Dominic
• Gold Larry

Classifications

• G01N33/5308 Primary

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• G01N33/74

  involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors} · CPC title

• G01N33/54306

  Solid-phase reaction mechanisms · CPC title

• G01N33/6863

  Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors · CPC title