Method of conducting a droplet based enzymatic assay

• US 8,440,392 B2
• Filed: 03/23/2008
• Issued: 05/14/2013
• 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, Rouse, Jeremy, Srinivasan, Vijay
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US12/531,844
• Publication Number:
  US 20100151439A1
• Time in Patent Office: 1,878 Days
• Field of Search: 435/4, 347/68
• 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, wherein the droplet operations are electrowetting mediated;

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

wherein each of the droplet of (b)(i), the droplet of (b)(ii) and the assay droplet is at least partially surrounded by a silicone oil filler fluid comprising an nonionic low hydrophile-lipophile balanced (HLB) surfactant.

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

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

13. The method of claim 12 wherein two or more assays are conducted simultaneously using different fluorophores for each enzyme tested.

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

15. The method of claim 14 wherein two or more assays are conducted simultaneously using different fluorophores for each enzyme tested.

16. The method of claim 14 wherein the fluorophore comprises 4-methylumbelliferyl.

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

18. 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.

19. The method of claim 17 wherein the substrate comprises a 4-methylumbelliferyl glycoside.

20. The method of claim 11 further comprising reducing or eliminating reaction contaminants associated with the substrate prior to yielding the assay droplet.

21. The method of claim 20 wherein the reducing or eliminating reaction contaminants comprises photobleaching the substrate prior to yielding the assay droplet.

22. The method of claim 21 wherein the photobleaching is effected prior to providing the droplet comprising the substrate on the droplet actuator.

23. The method of claim 21 wherein the photobleaching is effected after to providing the droplet comprising the substrate on the droplet actuator.

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

25. The method of claim 24 further comprising photobleaching the substrate prior to yielding the assay droplet.

26. The method of claim 1 wherein the HLB is less than about 10.

27. The method of claim 1 wherein the HLB is less than about 5.

28. The method of claim 1 wherein the surfactant is selected from the group consisting of octylphenol ethoxylate;

sorbitan trioleate;

sorbitan tristearate;

sorbitan sesquioleate;

sorbitan monooleate;

sorbitan monostearate; and

fluorinated surfactants.

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

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

31. The method of claim 1 wherein step 1(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.

32. The method of claim 1 wherein step 1(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.

33. The method of claim 1 wherein step 1(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.

34. The method of claim 1 wherein step 1(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.

35. The method of claim 1 wherein step 1(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.

36. The method of claim 1 wherein step 1(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.

37. The method of claim 1 wherein step 1(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.

38. The method of claim 1 wherein step 1(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.

39. The method of claim 1 wherein step 1(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.