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

• US 10,261,089 B2
• Filed: 07/08/2016
• Issued: 04/16/2019
• 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, Jana A
• Application Number:
  US15/205,497
• Publication Number:
  US 20170038390A1
• Time in Patent Office: 1,012 Days
• 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 target analytes in a sample comprising more than one type of target analyte, comprising:

(a) exposing the sample to a plurality of reaction vessels under conditions such that the target analytes are captured and immobilized in the plurality of reaction vessels, the plurality of reaction vessels comprising a plurality of capture components that are immobilized within the reaction vessels and are capable of capturing the target analytes within the reaction vessels, the plurality of capture components comprising more than one type of capture component, and each reaction vessel having a defined volume between 10 attoliters and 50 picoliters, wherein the target analytes are immobilized in the reaction vessels via binding to the immobilized capture components; and

(b) determining a total percentage of the reaction vessels for which each type of captured target analyte molecule is present and determining the concentration of each type of target analyte from said percentage.

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 molecules of the target analytes are captured prior to sealing the reaction vessels.

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

5. The method of claim 1, 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.

6. The method of claim 1, further comprising contacting the plurality of reaction vessels with an enzymatic component.

7. The method of claim 6, further comprising 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 target analytes.

9. The method of claim 8, wherein the nanoparticles are each functionalized with a fluorescent species.

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

5.

11. The method of claim 1, wherein said defined volume of each reaction vessel ranges from 30 femtoliters to 60 femtoliters.

12. The method of claim 1, wherein 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.

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

14. 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 the target analyte is captured and immobilized in the plurality of reaction vessels, each reaction vessel comprising a capture component that is immobilized within the reaction vessels and is capable of capturing the target analyte within the reaction vessels, and each reaction vessel having a defined volume between 10 attoliters and 50 picoliters, wherein the analyte is immobilized in the reaction vessels via binding to the immobilized capture component, and wherein a secondary binding ligand is associated or bound to at least a portion of the captured target analyte; and

(b) determining a total percentage of the reaction vessels for which a secondary binding ligand is present and determining the target analyte concentration from said percentage.

15. The method of claim 14, wherein the secondary binding ligand comprises an enzymatic component.

16. The method of claim 15, further comprising contacting the plurality of reaction vessels with a chromogenic enzymatic substrate, wherein the chromogenic enzymatic substrate is modified to a chromophore product upon exposure to a secondary binding ligand comprising an enzymatic component.

17. The method of claim 14, wherein the ratio of the number of molecules of target analyte to the number of reaction vessels is less than 1:

5.

18. The method of claim 14, wherein said defined volume of each reaction vessel ranges from 30 femtoliters to 60 femtoliters.

19. The method of claim 14, wherein 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.