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

• US 20070259381A1
• Filed: 02/16/2007
• Published: 11/08/2007
• Est. Priority Date: 02/21/2006
• Status: Active Grant

• Current Assignee: Trustees of Tufts College
• Original Assignee: Trustees of Tufts College
• Inventors: Rissin, David, Walt, David, Gorris, Hans-Heiner
• Granted Patent: US 8,492,098 B2
• Time in Patent Office: Days
• US Class Current: 435/7.400
• 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, the method comprising:

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

b) contacting said array with said sample such that the ratio of target analyte to each site of said array is less than 1;

1;

c) introducing said reaction component to each site of said array; and

d) determining whether the reaction component affects a reaction.

2. The method of claim 1, wherein said target analyte is an enzyme and substrate for said enzyme is present in each site of said array 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 substrate.

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

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

5.

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

10.

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

5 and 1;

500

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

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

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

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

13. The method of claim 1, wherein said array comprises between 20,000 and 30,000 sites.

14. The method of claim 1, wherein said array comprises between 100,000 and 10,000,000 sites.

15. The method of claim 1, wherein each site of said array comprises a capture component.

16. A method of detecting a reaction component that affects a reaction, the method comprising:

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

b) diluting said sample to create a diluted sample, said diluted sample comprising a biomolecule target analyte in a second concentration;

c) contacting said array with said diluted sample such that the ratio of biomolecule target analyte to each site of said array is less than 1;

1;

d) introducing said reaction component to each site of said array; and

e) determining whether the reaction component affects a reaction.

17. The method of claim 16, wherein said biomolecule target analyte is an enzyme and substrate for said enzyme is present in each site of said array prior to step e).

18. The method of claim 17, wherein said reaction component is an enzyme inhibitor.

19. The method of claim 18, wherein said determining of step e) comprises determining whether said enzyme inhibitor inhibits the reaction of said enzyme with said substrate.

20. The method of claim 19, wherein said substrate is a chromogenic substrate.

21. The method of claim 16, wherein said ratio is less than 1:

5.

22. The method of claim 16, wherein said ratio is 1:

10.

23. The method of claim 16, wherein said ratio is between 1:

5 and 1;

500

24. The method of claim 16, further comprising, after step d), sealing the array such that the contents of each site cannot escape said site.

25. The method of claim 16, wherein said defined volume is measured in femtoliters

26. The method of claim 25, wherein said defined femtoliter volume is the same at each site and ranges from about 30 femtoliters to about 60 femtoliters.

27. The method of claim 26, wherein said defined femtoliter volume is 46 femtoliters.

28. The method of claim 16, wherein said array comprises between 20,000 and 30,000 sites.

29. The method of claim 16, wherein said array comprises between 100,000 and 10,000,000 sites.

30. The method of claim 16, wherein each site of said array comprises a capture component.

31. The method of claim 30, wherein said capture component immobilizes said enzyme.

32. A method of detecting enzyme inhibition, the method comprising:

a) providing i) a reaction component, ii) a sample, said sample comprising a enzymatic target analyte in a first concentration, iii) a substrate for said enzymatic target analyte, and iv) an array, said array comprising at least 10,000 sites, each site having a defined femtoliter volume;

b) diluting said sample to create a diluted sample, said diluted sample comprising a enzymatic target analyte in a second concentration;

c) contacting said array, in any order, with said substrate, said reaction component, and said diluted sample such that the ratio of enzymatic target analyte to each site of said array is between 1;

5 and 1;

500; and

d) determining whether the reaction component inhibits the reaction of said enzyme with said substrate.

33. The method of claim 32, wherein said substrate is a chromogenic substrate.

34. The method of claim 32, further comprising, after step c), sealing the array such that the contents of each site cannot escape said site.

35. The method of claim 32, wherein said defined femtoliter volume is the same at each site and ranges from about 30 femtoliters to about 60 femtoliters.

36. The method of claim 35, wherein said defined femtoliter volume is 46 femtoliters.

37. The method of claim 32, wherein said ratio is 1:

10.

38. The method of claim 32, wherein said array comprises between 20,000 and 30,000 sites.

39. The method of claim 32, wherein said array comprises between 100,000 and 10,000,000 sites.

40. The method of claim 32, wherein said substrate is introduced first into each site of said array.

41. The method of claim 40, wherein said substrate is immobilized in said site as a capture component.