Methods and arrays for detecting cells and cellular components in small defined volumes

• US 20070259385A1
• 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: Walt, David, Rissin, David
• Granted Patent: US 8,460,878 B2
• Time in Patent Office: Days
• US Class Current: 435/7.230
• 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 cells and methods of producing an array of single cells are described. Arrays with defined volumes between 10 attoliters and 50 picoliters enable single cell capture, detection and quantitation.

Claims

1. A method of producing an array of single cells, the method comprising:

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

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

2. The array produced according to the method of claim 1.

3. The method of claim 1, further comprising d) determining the number of sites of said array which contain a single cell.

4. The method of claim 3, wherein the percentage of sites containing a single cell provide a measure of the concentration of cells in said sample.

5. The method of claim 1, wherein said cells are prokaryotic.

6. The method of claim 1, wherein said cells are eukaryotic.

7. The method of claim 6, wherein said cells are mammalian tumor cells.

8. The method of claim 6, wherein said cells are nerve cells.

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

10. The method of claim 9, wherein said cells are immobilized by said capture component after step b).

11. The method of claim 10, further comprising, after step b), sealing the array such that the contents of each site cannot escape said site.

12. The array produced according to the method of claim 11.

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

14. The array produced according to the method of claim 13.

15. The method of claim 13, wherein said defined femtoliter volume is the same at each site and ranges from about 30 femtoliters to about 60 femtoliters.

16. The method of claim 15, wherein said defined femtoliter volume is 46 femtoliters.

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

10.

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

5 and 1;

500.

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

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

21. The method of claim 9, wherein said capture component is a receptor.

22. The method of claim 9, wherein said capture component is fibronectin.

23. The method of claim 9, wherein said capture component is an antibody.

24. A method of detecting cells in a sample, the method comprising:

a) providing a sample and an array, said sample comprising cells 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 cells in a second concentration;

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

1; and

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

25. The method of claim 24, wherein said cells are prokaryotic.

26. The method of claim 24, wherein said cells are eukaryotic.

27. The method of claim 26, wherein said cells are mammalian tumor cells.

28. The method of claim 26, wherein said cells are nerve cells.

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

30. The method of claim 24, wherein said defined volume is measured in femtoliters.

31. The method of claim 24, wherein said ratio is 1:

10.

32. The method of claim 24, wherein said array comprises between 20,000 and 30,000 sites.

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

34. The method of claim 33, wherein said capture component is a receptor.

35. The method of claim 33, wherein said capture component is fibronectin.

36. The method of claim 33, wherein said capture component is an antibody.

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

a) providing a sample and an array, said sample comprising cells in a first concentration, each cell comprising a target analyte, said array comprising at least 1000 sites, each site having a defined femtoliter volume;

b) diluting said sample to create a diluted sample, said diluted sample comprising cells in a second concentration;

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

1;

d) treating said cells in said array under conditions such that said cells are lysed; and

e) determining the number of sites of said array which contain a target analyte of said cells.

38. The method of claim 37, wherein said cells are prokaryotic.

39. The method of claim 37, wherein said cells are eukaryotic.

40. The method of claim 37, further comprising, after step d), sealing the array such that the contents of each site cannot escape said site.

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

10.

42. The method of claim 37, wherein said array comprises between 20,000 and 30,000 sites.

43. The method of claim 37, wherein each site of said array comprises a capture component.

44. The method of claim 43, wherein said capture component immobilizes said cells.

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

a) providing a sample and an array, said sample comprising cells in a concentration, said array comprising at least 1000 sites, each site having a defined femtoliter volume;

b) treating said cells under conditions such that said cells are lysed so as to create a treated sample;

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

1; and

d) determining the number of sites of said array which contain a target analyte.

46. The method of claim 45, wherein said cells are prokaryotic.

47. The method of claim 46 wherein said cells are bacterial.

48. The method of claim 47, wherein said target analyte is a bacterial enzyme.

49. The method of claim 45, wherein said cells are eukaryotic.

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

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

10.

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

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