Methods and arrays for target analyte detection and determination of reaction components that affect a reaction

• US 8,492,098 B2
• Filed: 02/16/2007
• Issued: 07/23/2013
• 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., Gorris, Hans-Heiner
• Primary Examiner(s): Hines, Ja'na
• Application Number:
  US11/707,384
• Publication Number:
  US 20070259381A1
• Time in Patent Office: 2,349 Days
• Field of Search: None
• US Class Current: 435/7.1
• 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

Methods are described for detecting reaction components with affect a reaction. Biomolecules such as enzymes can be addressed at the single molecule level in order to discover function, detect binding partners or inhibitors, and/or measure rate constants.

Claims

1. A method of detecting a reaction component that affects a reaction by inducing, activating, altering, or inhibiting the reaction, the method comprising:

a) providing a reaction component, a sample and a solid support, said sample comprising a target analyte in a first concentration, said solid support comprising at least 1000 assay sites, each assay site having a defined volume between 10 attoliters and 50 picoliters;

b) contacting said solid support with said sample such that the ratio of a total number of target analyte molecules contained within said assay sites to the total number of assay sites contacted with said sample is less than 1;

5;

c) introducing said reaction component to each assay site of said solid support; and

d) determining a parameter indicative of whether the reaction component has affected a reaction by inducing, activating, altering, or inhibiting the reaction.

2. The method of claim 1, wherein said target analyte is an enzyme and an enzymatic substrate for said enzyme is present in each assay site of said solid support prior to step d).

3. The method of claim 2, wherein said reaction component is an enzyme inhibitor.

4. The method of claim 3, wherein said determining of step d) comprises determining whether said enzyme inhibitor inhibits the reaction of said enzyme with said enzymatic substrate.

5. The method of claim 4, wherein said enzymatic substrate is a chromogenic enzymatic substrate.

6. The method of claim 1, wherein said ratio is 1:

10.

7. The method of claim 1, wherein said ratio is between 1:

5 and 1;

500.

8. The method of claim 1, further comprising, after step c), sealing the assay sites such that the contents of each assay site cannot escape said assay site.

9. The method of claim 1, wherein said defined volume is measured in femtoliters.

10. The method of claim 9, wherein said defined femtoliter volume is the same at each assay site and ranges from about 30 femtoliters to about 60 femtoliters.

11. The method of claim 10, wherein said defined femtoliter volume is 46 femtoliters.

12. The method of claim 1, wherein said solid support comprises between 20,000 and 30,000 assay sites.

13. The method of claim 1, wherein said solid support comprises between 100,000 and 10,000,000 assay sites.

14. The method of claim 1, wherein each assay site of said solid support comprises a capture component.

15. The method of claim 1, wherein the target analyte comprises an enzymatic component and an enzymatic substrate for said enzymatic component is present in each assay site of said solid support prior to step d).

16. The method of claim 15, wherein the method further comprises determining a reaction velocity.

17. The method of claim 16, wherein the reaction velocity is determine by determining the amount of chromophore product detected over time.

18. The method of claim 1, wherein said solid support comprises between 10,000 and 50,000 assay sites.