Enzymatic Assays for a Droplet Actuator

• US 20100151439A1
• Filed: 03/23/2008
• Published: 06/17/2010
• Est. Priority Date: 03/22/2007
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee K., Eckhardt, Allen E.
• Granted Patent: US 8,440,392 B2
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: B01F 33/451 wherein the mixture is directly exposed to an electromagnetic field without use of a stirrer, e.g. for material comprising ferromagnetic particles or for molten metal
• CPC Class Codes: C07H 15/04 attached to an oxygen atom of the saccharide radical
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2333/928 acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
• CPC Class Codes: G01N 2800/042 Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Abstract

A method of conducting a droplet-based enzymatic assay is provided. The method generally makes use of a droplet actuator. A droplet comprising an enzyme of interest is provided on the droplet actuator along with a droplet comprising a substrate which is potentially modified in the presence of the enzyme. The method involves executing droplet operations on the droplet actuator to combine the droplets, thereby yielding an assay droplet, and detecting modification of the substrate by the enzyme in the assay droplet on the droplet actuator. The enzyme of interest may, for example, be a potentially mutated or improperly folded enzyme exhibiting altered enzyme activity as compared to a corresponding normal enzyme.

Claims

1. A method of conducting a droplet-based enzymatic assay, the method comprising:

(a) providing a droplet actuator;

(b) providing on the droplet actuator;

(i) a droplet comprising an enzyme of interest;

(ii) a droplet comprising a substrate which is potentially modified in the presence of the enzyme;

(c) executing droplet operations on the droplet actuator to combine the droplet of (b)(i) with the droplet of (b)(ii), thereby yielding an assay droplet;

(d) detecting modification of the substrate by the enzyme in the assay droplet on the droplet actuator.

2. The method of claim 1 wherein the droplet comprising an enzyme of interest has a volume which is less than about 500 μ

L.

3. The method of claim 1 wherein the droplet comprising an enzyme of interest has a volume which is less than about 100 μ

L.

4. The method of claim 1 wherein the droplet comprising an enzyme of interest has a volume which is less than about 50 μ

L.

5. The method of claim 1 wherein the droplet comprising an enzyme of interest has a volume which is less than about 25 μ

L.

6. The method of claim 1 wherein the droplet comprising a substrate has a volume which is less than about 500 μ

L.

7. The method of claim 1 wherein the droplet comprising a substrate has a volume which is less than about 100 μ

L.

8. The method of claim 1 wherein the droplet comprising a substrate has a volume which is less than about 50 μ

L.

9. The method of claim 1 wherein the droplet comprising a substrate has a volume which is less than about 25 μ

L.

10. The method of claim 1 wherein the enzyme of interest is a potentially mutated or improperly folded enzyme exhibiting altered enzyme activity as compared to a corresponding normal enzyme.

11. A method of providing diagnostic information, the method comprising:

(a) conducting an assay according to the method of claim 1, wherein the droplet of l(b)(i) comprises a clinical sample from a subject, the sample comprising the enzyme of interest;

(b) providing diagnostic information based on the activity of the enzyme of interest from the human clinical sample relative to the expected activity of a corresponding normal enzyme.

12. The method of claim 11 wherein the diagnostic information comprises information diagnostically relevant to a glycogen storage disease.

13. The method of claim 12 wherein the glycogen storage disease comprises a lysosomal storage diseases involving a defect in a glycosidase.

14. The method of claim 12 wherein the glycogen storage disease comprises a deficiency in α

-glucosidase activity.

15. The method of claim 12 wherein the glycogen storage disease comprises a deficiency in α

-galactosidase activity.

16. The method of claim 11 wherein the assay is conducted and the diagnostic information is provided at a point of sample collection.

17. The method of claim 16 wherein the point of sample collection is in the presence of the subject.

18. The method of claim 11 wherein the clinical sample comprises a sample substance selected from the group consisting of:

blood, plasma, serum, tears, saliva, and urine.

19. The method of claim 11 wherein the clinical sample comprises a dried blood sample.

20. The method of claim 11 wherein the clinical sample comprises a fresh blood sample.

21. The method of claim 20 wherein the fresh blood sample is collected from the subject and immediately loaded onto a droplet actuator for conducting the assay.

22. The method of claim 21 wherein time from collection of the blood sample to providing diagnostic information is less than about 5 hours.

23. The method of claim 21 wherein time from collection of the blood sample to providing diagnostic information is less than about 1 hour.

24. The method of claim 21 wherein time from collection of the blood sample to providing diagnostic information is less than about 30 minutes.

25. The method of claim 21 wherein time from collection of the blood sample to providing diagnostic information is less than about 15 minutes.

26. The method of claim 21 wherein time from collection of the blood sample to providing diagnostic information is less than about 5 minutes.

27. The method of claim 11 wherein the clinical sample is a human clinical sample.

28. The method of claim 11 wherein the clinical sample is a non-human animal clinical sample.

29. The method of claim 1 wherein the substrate comprises a glycoside substrate.

30. The method of claim 1 wherein the substrate releases a detectable upon contact with the enzyme of interest.

31. The method of claim 30 wherein two or more assays are conducted simultaneously using different fluorophores for each enzyme tested.

32. The method of claim 1 wherein the substrate releases a fluorophore upon contact with the enzyme of interest.

33. The method of claim 32 wherein two or more assays are conducted simultaneously using different fluorophores for each enzyme tested.

34. The method of claim 32 wherein the fluorophore comprises 4-methylumbelliferyl.

35. The method of claim 1 wherein the substrate comprises a glycoside substrate which releases a fluorophore upon contact with the enzyme of interest.

36. The method of claim 1 wherein the substrate comprises a glycoside substrate comprising glucose, galactose, fucose, mannose, sialic acid, hexose, hexosamine and/or N-acetylated hexosamine.

37. The method of claim 35 wherein the substrate comprises a 4-methylumbelliferyl glycoside.

38. The method of claim 29 further comprising reducing or eliminating reaction contaminants associated with the substrate prior to yielding the assay droplet.

39. The method of claim 38 wherein the reducing or eliminating reaction contaminants comprises photobleaching the substrate prior to yielding the assay droplet.

40. The method of claim 39 wherein the photobleaching is effected prior to providing the droplet comprising the substrate on the droplet actuator.

41. The method of claim 39 wherein the photobleaching is effected after to providing the droplet comprising the substrate on the droplet actuator.

42. The method of claim 1 wherein the substrate comprises a 4-methylumbelliferyl glycoside substrate.

43. The method of claim 42 further comprising photobleaching the substrate prior to yielding the assay droplet.

44. The method of claim 1 wherein each of the droplet of l(b)(i), the droplet of l(b)(ii) and the assay droplet is at least partially surrounded by a filler fluid comprising an oil.

45. The method of claim 44 wherein the filler fluid comprises a silicone oil.

46. The method of claim 44 wherein the filler fluid comprises a surfactant.

47. The method of claim 46 wherein the surfactant comprises nonionic low hydrophile-lipophile balanced (HLB) surfactant.

48. The method of claim 47 wherein the HLB is less than about 10.

49. The method of claim 47 wherein the HLB is less than about 5.

50. The method of claim 46 wherein the surfactant is selected from the group consisting of Triton X-15;

Span 85;

Span 65;

Span 83;

Span 80;

Span 60; and

fluorinated surfactants.

51. The method of claim 1 wherein the droplet operations comprise electrode-mediated droplet operations.

52. The method of claim 1 wherein the droplet operations are electrowetting mediated.

53. The method of claim 1 wherein the droplet operations are dielectrophoresis mediated.

54. The method of claim 1 wherein step l(d) is effected during and/or following an incubation period during which the assay droplet is incubated for a period not longer than about 15 hours.

55. The method of claim 1 wherein step l(d) is effected during and/or following an incubation period during which the assay droplet is incubated for a period not longer than about 10 hours.

56. The method of claim 1 wherein step l(d) is effected during and/or following an incubation period during which the assay droplet is incubated for a period not longer than about 5 hours.

57. The method of claim 1 wherein step l(d) is effected during and/or following an incubation period during which the assay droplet is incubated for a period not longer than about 1 hour.

58. The method of claim 1 wherein step l(d) is effected during and/or following an incubation period during which the assay droplet is incubated for a period not longer than about 45 minutes.

59. The method of claim 1 wherein step l(d) is effected during and/or following an incubation period during which the assay droplet is incubated for a period not longer than about 30 minutes.

60. The method of claim 1 wherein step l(d) is effected during and/or following an incubation period during which the assay droplet is incubated for a period not longer than about 15 minutes.

61. The method of claim 1 wherein step l(d) is effected during and/or following an incubation period during which the assay droplet is incubated for a period not longer than about 10 minutes.

62. The method of claim 1 wherein step l(d) is effected during and/or following an incubation period during which the assay droplet is incubated for a period not longer than about 5 minutes.

63. A photobleached 4-methylumbelliferyl-enzyme substrate preparation.

64. The preparation of claim 63 wherein the 4-methylumbelliferyl-enzyme substrate comprises a 4-methylumbelliferyl glycoside substrate.

65. The preparation of claim 63 wherein the 4-methylumbelliferyl-enzyme substrate comprises 4-methylumbelliferyl coupled through an α

or β

linkage to glucose, galactose, fucose, mannose, sialic acid, hexose, hexosamine and/or N-acetylated hexosamine.

66. The preparation of claim 63 wherein the 4-methylumbelliferyl-enzyme substrate is selected from the group consisting of 4-methylumbelliferyl α

-D-glucopyranoside and/or 4-methylumbelliferyl α

-D-galactopyranoside.

67. The preparation of claim 63 wherein the composition comprises multiple different 4-methylumbelliferyl glycoside substrates.

68. A method of conducting an enzymatic assay, the method comprising:

(a) combining a droplet comprising an enzyme of interest with a droplet comprising a substrate which is potentially modified in the presence of the enzyme, wherein the substrate comprises a photobleached 4-methylumbelliferyl-enzyme substrate preparation; and

(b) detecting modification of the substrate by the enzyme in the assay droplet on the droplet actuator.