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

• US 20070259448A1
• 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
• Granted Patent: US 8,460,879 B2
• Time in Patent Office: Days
• US Class Current: 436/501
• 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 producing an array of single molecules, comprising:

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

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

1, so as to produce an array comprising sites containing single molecules.

2. The method of claim 1, wherein said target analyte comprises biomolecules.

3. The method of claim 2, wherein said biomolecule is selected from the group consisting of proteins, nucleic acids, lipids, and carbohydrates.

4. The method of claim 2, wherein said biomolecule is selected from the group consisting of hormones, cytokines and cellular antigens.

5. The method of claim 2, wherein said biomolecule is a receptor.

6. The method of claim 5, wherein said receptor is selected from the group consisting of neural receptors, hormonal receptors, and nutrient receptors.

7. The method of claim 2, wherein said biomolecules are labeled.

8. The method of claim 1, wherein said target analyte is an environmental pollutant.

9. The method of claim 1, wherein said target analyte is selected from the group consisting of pesticides, insecticides and toxins.

10. The method of claim 1, wherein said target analyte is a drug.

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

5.

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

10.

13. The method of claim 11, wherein said ratio is between 1:

5 and 1;

500.

14. The method of claim 1, wherein the percentage of sites containing a single molecule provide a measure of the concentration of molecules in said sample.

15. An array produced according to the method of claim 1.

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

17. An array produced according to the method of claim 16.

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

19. An array produced according to the method of claim 18.

20. The method of claim 18, wherein said defined volume is the same at each site and ranges from about 30 femtoliters to about 60 femtoliters.

21. The method of claim 20, wherein said defined femtoliter volume is 46 femtoliters.

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

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

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

25. The method of claim 24, wherein said target analyte comprises a single-stranded nucleic acid, and said capture component comprises a complementary nucleic acid.

26. A method of detecting a target analyte in a sample, the method comprising:

a) providing a sample and an array, said sample comprising a biomolecule target analyte, 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 biomolecule target analyte to each site of said array is less than 1;

1; and

c) determining the number of sites of said array which contain a single biomolecule.

27. The method of claim 26, wherein said ratio is less than 1:

5.

28. The method of claim 26, wherein said ratio is 1:

10.

29. The method of claim 27, wherein said ratio is between 1:

5 and 1;

500.

30. The method of claim 26, wherein said biomolecules are labeled.

31. The method of claim 26, wherein said determining comprises detecting a change in an optical property at said sites as an indication of the presence of said target analyte.

32. The method of claim 26, wherein the percentage of sites containing a single biomolecule provides a measure of the concentration of biomolecules in said sample.

33. The method of claim 26, further comprising, after step b), sealing the array such that the contents of each site cannot escape said site.

34. The method of claim 26, wherein said defined volume is measured in femtoliters.

35. The method of claim 34, 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 26, wherein said array comprises between 20,000 and 30,000 sites.

38. The method of claim 26, wherein said array comprises between 100,000 and 10,000,000 sites.

39. The method of claim 26, wherein each site of said array comprises a capture component.

40. The method of claim 39, wherein said target analyte comprises a single-stranded nucleic acid, and said capture component comprises a complementary nucleic acid.

41. The method of claim 26, wherein said biomolecule is selected from the group consisting of proteins, nucleic acids, lipids, and carbohydrates.

42. The method of claim 26, wherein said biomolecule is selected from the group consisting of hormones, cytokines and cellular antigens.

43. The method of claim 26, wherein said biomolecule is a receptor.

44. The method of claim 43, wherein said receptor is selected from the group consisting of neural receptors, hormonal receptors, and nutrient receptors.

45. A method of detecting a target analyte in a sample, the method comprising:

a) providing a sample and an array, said sample comprising a biomolecule target analyte in a first concentration, 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 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 between 1;

5 and 1;

500; and

d) determining the number of sites of said array which contain a single biomolecule.

46. The method of claim 45, wherein said biomolecules are labeled.

47. The method of claim 45, wherein said determining comprises detecting a change in an optical property at said sites as an indication of the presence of said target analyte.

48. The method of claim 45, wherein the percentage of sites containing a single biomolecule provides a measure of the concentration of biomolecules in said diluted sample.

49. The method of claim 45, further comprising, after step c), sealing the array such that the contents of each site cannot escape said site.

50. The method of claim 45, wherein said defined volume is measured in femtoliters.

51. The method of claim 50, wherein said defined femtoliter volume is the same at each site and ranges from about 30 femtoliters to about 60 femtoliters.

52. The method of claim 51, wherein said defined femtoliter volume is 46 femtoliters.

53. The method of claim 45, wherein said ratio is 1:

10.

54. The method of claim 45, wherein said array comprises between 20,000 and 30,000 sites.

55. The method of claim 45, wherein said array comprises between 100,000 and 10,000,000 sites.

56. The method of claim 45, wherein each site of said array comprises a capture component.

57. The method of claim 56, wherein said target analyte comprises a single-stranded nucleic acid, and said capture component comprises a complementary nucleic acid.

58. The method of claim 45, wherein said biomolecule is selected from the group consisting of proteins, nucleic acids, lipids, and carbohydrates.

59. The method of claim 45, wherein said biomolecule is selected from the group consisting of hormones, cytokines and cellular antigens.

60. The method of claim 45, wherein said biomolecule is a receptor.

61. The method of claim 60, wherein said receptor is selected from the group consisting of neural receptors, hormonal receptors, and nutrient receptors.