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

• US 9,395,359 B2
• Filed: 03/04/2015
• Issued: 07/19/2016
• Est. Priority Date: 02/21/2006
• Status: Active Grant

• Current Assignee: Trustees of Tufts College
• Original Assignee: Trustees of Tufts College
• Inventors: Walt, David R., Rissin, David M.
• Primary Examiner(s): Hines, Ja'Na
• Application Number:
  US14/638,245
• Publication Number:
  US 20150233905A1
• Time in Patent Office: 503 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: G01N 2333/938 acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54346 Nanoparticles
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

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.

Claims

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.