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

Patent metadata

Field	Value
Publication number	US-11237171-B2
Application number	US-201916286811-A
Country	US
Kind code	B2
Filing date	Feb 27, 2019
Priority date	Feb 21, 2006
Publication date	Feb 1, 2022
Grant date	Feb 1, 2022

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 using a competitive assay, comprising:
contacting labelled molecules and target analyte from the sample with a plurality of capture components capable of binding to the labelled molecule or the target analyte, wherein the target analyte competes with the labelled molecules for binding to the capture components, and wherein a captured labelled molecule generates an optical signal and a captured target analyte does not generate an optical signal;
immobilizing the plurality of capture components with respect to a plurality of reaction vessels comprising the capture components, wherein a portion of the plurality of reaction vessels contains capture components with immobilized labelled molecules, and another portion of the plurality of reaction vessels contains capture components that are not immobilized to labelled molecules;
determining a total percentage of the reaction vessels that do not contain a captured labelled molecule; and
determining the concentration of the target analyte in the sample from the total percentage.

2. The method of claim 1 , wherein the plurality of reaction vessels comprise the capture components prior to the step of contacting the labelled molecules and the target analyte from the sample with the plurality of capture components.

3. The method of claim 1 , further comprising removing any non-bound labelled molecules from the plurality of reaction vessels prior to the step of determining a total percentage of the reaction vessels that do not contain a captured labelled molecule.

4. The method of claim 3 , further comprising determining a total percentage of the reaction vessels that contain target analyte from the total percentage of the reaction vessels that do not contain a captured labelled molecule.

5. The method of claim 3 , wherein the step of determining a total percentage of the reaction vessels that do not contain a labelled molecule comprises determining the percentage of reaction vessels in which the optical signal is not generated.

6. The method of claim 3 , wherein each capture component is capable of forming a binding complex in which it is bound to a single molecule that is either a labelled molecule or a target analyte.

7. The method of claim 1 , wherein the optical signal is generated by exposing the captured labelled molecules to a chromogenic substrate and forming a chromogenic product.

8. The method of claim 3 , wherein the labelled molecule is an enzyme or enzymatic component.

9. The method of claim 3 , wherein the plurality of reaction vessels are an array of microwells.

10. The method of claim 3 , wherein each reaction vessel has a defined volume between 10 attoliters and 50 picoliters.

11. The method of claim 3 , wherein the ratio of molecules of target analyte to the number of reaction vessels is less than 1:1.

12. The method of claim 3 , wherein the ratio of molecules of target analyte to the number of reaction vessels is less than 1:5.

13. The method of claim 3 , wherein the reaction vessels are present on the surface of an array substrate.

14. The method of claim 3 , wherein the step of removing any non-bound labelled molecules from the plurality of reaction vessels comprises performing a wash step.

15. The method of claim 3 , 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.

16. The method of claim 15 , wherein molecules of the target analyte or the labelled molecules are captured prior to sealing the reaction vessels.

17. The method of claim 3 , wherein the target analyte is selected from the group consisting of proteins, nucleic acids, lipids, carbohydrates, hormones, cytokines, cellular antigens, receptors, and cells.

18. The method of claim 3 , wherein each reaction vessel comprises a wall that defines a binding surface that has any of the capture components that are contained in a particular reaction vessel immobilized thereon.

19. The method of claim 8 , wherein the target analyte does not have enzymatic properties.

Assignees

• Tufts College

Inventors

• Walt David R
• Rissin David M

Classifications

• C12Q1/6837 Primary

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

• G01N33/581 Primary

  with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates) · CPC title

• G01N33/543

  with an insoluble carrier for immobilising immunochemicals · CPC title

• G01N33/54386

  Analytical elements · CPC title

• G01N33/54366

  Apparatus specially adapted for solid-phase testing · CPC title