Methods and arrays for target analyte detection and determination of target analyte concentration in solution

Patent metadata

Field	Value
Publication number	US-9395359-B2
Application number	US-201514638245-A
Country	US
Kind code	B2
Filing date	Mar 4, 2015
Priority date	Feb 21, 2006
Publication date	Jul 19, 2016
Grant date	Jul 19, 2016

Abstract

Arrays of single molecules and methods of producing an array of single molecules are described. Arrays with defined volumes between 10 attoliters and 50 picoliters enable single molecule detection and quantitation.

First claim

The invention claimed is:

1. A method of measuring the concentration of a target analyte in a sample, comprising:
(a) exposing the sample to a plurality of reaction vessels under conditions such that analyte is captured in reaction vessels, each reaction vessel comprising a capture component and having a defined volume between 10 attoliters and 50 picoliters, wherein the analyte is captured in the reaction vessels via immobilization to the capture component, and wherein the ratio of molecules of target analyte to the number of reaction vessels is less than 1:1; and
(b) determining the percentage of said reaction vessels that contain a captured target analyte molecule to determine a measure of the target analyte concentration.

2. The method of claim 1 , further comprising sealing the reaction vessels with a sealing component such that the contents of each reaction vessel are prevented from escaping from said reaction vessel.

3. The method of claim 2 , wherein the molecules of target analyte are captured prior to sealing the reaction vessels.

4. The method of claim 1 , wherein the target analyte comprises a first type of target analyte and a second type of target analyte.

5. The method of claim 1 , wherein said target analyte are selected from the group consisting of proteins, nucleic acids, lipids, carbohydrates, hormones, cytokines, cellular antigens, receptors, and cells.

6. The method of claim 1 , wherein a wall of the reaction vessel defines a binding surface that has the capture component immobilized thereon.

7. The method of claim 1 , further comprising contacting the plurality of reaction vessels with an enzymatic component, and optionally, further contacting the plurality of reaction vessels with an enzymatic substrate.

8. The method of claim 1 , further comprising attaching or otherwise immobilizing nanoparticles with respect to the analyte, optionally wherein the nanoparticles are a gold nanoparticles, or optionally, wherein the nanoparticles are each functionalized with a fluorescent species.

9. The method of claim 1 , wherein said ratio is (i) less than 1:5; or (ii) between 1:5 and 1:500; or (iii) 1:10.

10. The method of claim 1 , wherein said defined volume of each site ranges from 30 femtoliters to 60 femtoliters.

11. The method of claim 1 , wherein said the plurality of reaction vessels comprises (i) at least about 1000 reaction vessels; or (ii) between 20,000 and 10,000,000 reaction vessels; or (iii) between 20,000 and 30,000 reaction vessels; or (iv) between 100,000 and 10,000,000 reaction vessels; or (v) between 10,000 and 50,000 reaction vessels.

12. The method of claim 1 , wherein said determining comprises detecting a change in an optical property at said sites as an indication of the presence of said target analyte molecule.

Assignees

• Tufts College

Inventors

• Walt David R
• Rissin David M

Classifications

• G01N33/543 Primary

  with an insoluble carrier for immobilising immunochemicals · CPC title

• G01N33/54366

  Apparatus specially adapted for solid-phase testing · CPC title

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• G01N33/54346

  Nanoparticles · CPC title

• G01N2333/938

  acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase · CPC title