Droplet actuators and techniques for droplet-based enzymatic assays

1. A method of conducting an assay, the method comprising:

(a) incubating a droplet in oil, the droplet comprising an umbelliferone substrate, a sample potentially comprising an enzyme which cleaves the umbelliferone substrate, and a zwitterionic surfactant; and

(b) detecting a signal emitted from the droplet.

2. The method of claim 1 wherein the droplet further comprises a cyclodextrin compound.

3. The method of claim 2 wherein the cyclodextrin compound is selected from the group consisting of α-cyclodextrins, β-cyclodextrins, and γ-cyclodextrins, and analogs and derivatives of the foregoing.

4. The method of claim 2 wherein the umbilliferone substrate is selected from the group consisting of alkylumbelliferyl-α-L-iduronides, 4-methylumbelliferyl-α-L-iduronide, 4-methylumbelliferyl-α-L-iduronide-2-sulfate, 4-methylumbelliferyl-α-L-idopyranosiduronic acid, 4-methylumbelliferyl-α-L-fucoside, 4-methylumbelliferyl-α-L-mannoside, 4-methylumbelliferyl-β-D-mannoside, 4-methylumbelliferyl-β-D-N-acetylglucosaminide, 4-methylumbelliferyl-β-D-N-acetylglucosaminide sulfate, alkylumbelliferyl-β-D-glycosides, methylumbelliferyl-β-D-glycosides, 4-methylumbelliferyl-α-D-galactoside, 4-methylumbelliferyl-β-D-galactoside, 4-methylumbelliferyl-β-D-glucouronic acid, phenolphthalein-β-D-glucuronic acid, ethylumbelliferyl-β-D-glycosides, multifluoroethylumbelliferyl-β-D-glycosides, pentafluoroethylumbelliferyl-β-D-glycosides, pentafluoroethylumbelliferyl-β-D-glucoside, umbelliferylchiotriosides, 4-alkyumbelliferylchiotrioside, 4-methylumbelliferylchiotrioside, 4-methylumbelliferyl-β-galactose, 4-alkyumbeliferrone phosphates, 4-methylumbeliferrone phosphate, 6-alkanoylamido-4-methylumbelliferones, substrates comprising a 4-methyllumbelliferyl group, 6-hexadecanoylamido-4-methylumbelliferone, 4-methyllumbelliferyl-β-D-glucosaminide, 4-methylumbelliferyl-α-neuraminic acid, 4-methylumbelliferyl-α-D-N-acetylgalactosaminide, and their functional analogs and derivatives.

5. The method of claim 1 , wherein the steps are performed within droplets controlled by a droplet actuator.

6. The method of claim 5 , wherein the droplet actuator controls the steps using electrode mediated droplet operations.

7. The method of claim 5 , wherein the droplet actuator controls the steps using electrowetting mediated droplet operations.

8. The method of claim 5 , wherein the droplet actuator controls the steps using dielectrophoresis mediated droplet operations.

9. A method of conducting a droplet-based enzyme assay, the method comprising:

(a) providing an immiscible fluid comprising:

(i) a sample droplet comprising an enzyme of interest; and

(ii) one or more reagent droplets comprising:

(1) a substrate which is potentially modified in the presence of the enzyme yielding one or more signal-producing products;

(2) a zwitterionic surfactant; and

(3) optionally, other reagents sufficient to produce activity of the target enzyme;

(b) combining the sample droplet and the one or more reagent droplets in the immiscible fluid to yield a reaction droplet effecting an enzyme reaction in the immiscible fluid; and

(c) measuring any signal produced by the one or more signal producing products.

10. The method of claim 1 , wherein the zwitterionic surfactant is selected form the group consisting of n-Octylphosphocholine, n-Nonylphosphocholine, n-decylphosphocholine, n-dodecylphosphocholine, 3-cyclohexyl-1-propylphosphocholine, decylphospho-N-methylethanolamine, n-decyl-N,N-dimethylglycine, n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, dimethylbenzylammonium propane sulfonate, 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate, and 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate.

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

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

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 12 wherein the fluorophore comprises 4-methylumbelliferyl.

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

16. 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.

17. The method of claim 16 wherein the substrate comprises a 4-methylumbelliferyl glycoside.

18. The method of claim 1 , further comprising reducing or eliminating reaction contaminants associated with the substrate.

19. The method of claim 18 wherein the reducing or eliminating reaction contaminants comprises photobleaching the substrate.

20. The method of claim 19 wherein the photobleaching is effected prior to providing the droplet comprising the substrate on the droplet actuator.

21. The method of claim 19 wherein the photobleaching is effected after providing the droplet comprising the substrate on the droplet actuator.

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

23. The method of claim 22 further comprising photobleaching the substrate.

24. The method of claim 9 , wherein the immiscible fluid comprises a filler fluid.

25. The method of claim 9 , wherein the immiscible fluid comprises a silicone oil.

26. The method of claim 24 wherein the filler fluid comprises a surfactant.

27. The method of claim 26 wherein the surfactant comprises nonionic low hydrophile-lipophile balanced (HLB) surfactant.

28. The method of claim 27 wherein the HLB is less than about 10.

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

30. The method of claim 27 wherein the surfactant is selected from the group consisting of

wherein R is octyl (C8) and X is 1.5 (avg);

and fluorinated surfactants.

31. The method of claim 9 , wherein:

(a) the sample droplet comprises a reconstituted blood sample;

(b) the blood sample is reconstituted using a single universal reconstitution solution;

(c) the blood sample is divided to yield two or more reaction droplets; and

(d) two or more of the reaction droplets are each combined with one or more sets of one or more reagent droplets, each such set comprising reagents selected for establishing reaction conditions for a different enzyme assay.

32. The method of claim 31 wherein the universal reconstitution solution comprises a saline solution.

33. The method of claim 31 wherein the universal reconstitution solution comprises water.

34. The method of claim 9 , wherein the enzyme assay is selected to provide diagnostic information about an enzyme deficiency.

35. The method of claim 34 wherein the enzyme deficiency is selected from lysosomal storage diseases.

36. The method of claim 34 wherein the enzyme deficiency is selected from the group consisting of Pompe, Niemann-Pick, Fabry, Krabbe, and Gaucher.

37. The method of claim 34 further comprising providing therapeutic treatment to a subject based on the diagnostic information.

38. The method of claim 9 , wherein the sample droplet comprising an enzyme of interest comprises cultured cells and/or supernatant from a cell culture.

39. The method of claim 9 , wherein the substrate is selected from the group consisting of 4-methylumbelliferyl-α-L-iduronide, 4-methylumbelliferyl-β-D-galactoside, 4-methylumbelliferyl-β-D-glucuronic acid, 4-methylumbelliferyl-α-L-fucoside, 4-methylumbelliferyl-α-mannoside, 4-methylumbelliferyl-β-D-mannoside, 4-nitrocathecol sulfate, 4-methylumbelliferyl-β-D-N-acetylglucosaminide, 4-methylumbelliferyl-β-D-N-acetylglucosaminide sulfate, 4-methylumbelliferyl-β-D-glucosaminide, 4-methylumbelliferyl-α-D-galactoside, 4-methylumbelliferyl-α-D-neuraminic acid, 4-methylumbelliferyl-α-D-N-acetylgalactosaminide, phenolphthalein β-D-glucuronic acid, and mixtures and derivatives thereof.

40. The method of claim 9 , wherein the substrate comprises a fluorophoric moiety.

41. The method of claim 40 wherein the fluorophoric moiety comprises 4-methyllumbelliferyl.

42. The method of claim 9 , wherein the substrate comprises a chromophoric moiety.

43. The method of claim 42 wherein the chromophoric moiety comprises 4-nitrocathecol or phenolphthalein.

44. The method of claim 9 , wherein the substrate comprises a radioactive moiety.

45. The method of claim 44 wherein the radioactive moiety comprises 14 C sphingomyelin or 3 H galactosylceramide.

46. The method of claim 9 , comprising incubating the reaction droplet for a period of less than about 12 hours.