EMULSION CHEMISTRY FOR ENCAPSULATED DROPLETS

• US 20110217712A1
• Filed: 12/22/2010
• Published: 09/08/2011
• Est. Priority Date: 03/02/2010
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: QuantaLife, Inc. (Bio-Rad Laboratories Incorporated)
• Inventors: Hindson, Benjamin J., Hiddessen, Amy L.
• Granted Patent: US 9,598,725 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components

Abstract

System, including methods, apparatus, compositions, and kits, for making and using a stabilized emulsion. A method of generating a stabilized emulsion is provided. In the method, an aqueous phase may be provided. The aqueous phase may include an effective concentration of one or more skin-forming proteins. An emulsion may be formed. The emulsion may include droplets of a dispersed phase disposed in a continuous phase, with the aqueous phase being the continuous phase or the dispersed phase. The emulsion may be heated to create an interfacial skin between each droplet and the continuous phase, to transform the droplets into capsules.

Claims

1. A method of generating a stabilized emulsion, comprising:

providing an aqueous phase including an effective concentration of one or more skin-forming proteins;

forming an emulsion including droplets of a dispersed phase disposed in a continuous phase, the aqueous phase being the continuous phase or the dispersed phase; and

heating the emulsion to create an interfacial skin between each droplet and the continuous phase, to transform the droplets into capsules.

2. The method of claim 1, wherein the step of heating includes a step of heating the emulsion to a temperature of at least about 55°

C.

3. The method claim 1, further comprising a step of thermally cycling the capsules through multiple rounds of heating and cooling after the step of heating.

4. The method of claim 1, wherein the step of heating is part of a thermal cycling process that includes multiple rounds of heating and cooling.

5. The method of claim 1, further comprising a step of amplifying a nucleic acid target in one or more of the capsules.

6. The method of claim 1, wherein the step of heating the emulsion includes a step of heating the emulsion to at least a threshold temperature.

7. The method of claim 6, wherein the threshold temperature is a denaturation temperature of the skin-forming protein.

8. The method of claim 1, wherein the aqueous phase provided includes the skin-forming proteins at a concentration of at least about 0.01% by weight.

9. The method of claim 1, wherein the aqueous phase provided includes the skin-forming proteins at a concentration of at least about 0.03% by weight.

10. The method of claim 1, wherein the aqueous phase provided includes the skin-forming proteins at a concentration of at least about 0.1% by weight.

11. The method of claim 1, wherein the aqueous phase provided includes a surfactant including a block copolymer of polypropylene oxide and polyethylene oxide.

12. The method of claim 11, wherein the aqueous phase provided includes the surfactant at a concentration of about 0.01% to 10% by weight.

13. The method of claim 1, wherein the aqueous phase comprises nucleic acid.

14. The method of claim 1, wherein the step of providing an aqueous phase includes a step of providing an aqueous phase including a surfactant and the skin-forming proteins, and wherein the surfactant is not required for creation of the interfacial skin.

15. The method of claim 1, further comprising a step of providing an oil phase that is used as a continuous phase for the step of forming an emulsion, and wherein the oil phase includes a fluorinated surfactant that is negatively charged.

16. The method of claim 15, wherein the fluorinated surfactant is a carboxylate.

17. The method of claim 15, wherein the continuous phase also includes a fluorinated alcohol.

18. The method of claim 1, further comprising a step of providing an oil phase that is used as a continuous phase for the step of forming an emulsion, and wherein the oil phase includes at least one fluorinated surfactant at a concentration of about 0.02% to 0.5% by weight.

19. The method of claim 1, wherein the skin-forming proteins are required for formation of the interfacial skin.

20. The method of claim 1, wherein the skin-forming proteins are selected from the group consisting of albumin, casein, gelatin, and globulin.

21. The method of claim 1, wherein the step of heating the emulsion is performed with the droplets disposed in a three-dimensional arrangement having a high packing density.

22. The method of claim 1, further comprising a step of selectively removing a portion of the continuous phase after the step of forming an emulsion and before the step of heating the emulsion.

23. The method of claim 1, wherein the step of forming an emulsion includes a step of generating droplets serially using a droplet generator.

24. The method of claim 1, wherein the step of forming an emulsion includes a step of generating monodisperse droplets of the aqueous phase.

25. The method of claim 24, wherein the step of generating monodisperse droplets includes a step of generating monodisperse droplets having a diameter of about 1 μ

m to 500 μ

m.

27. The method of claim 1, further comprising a step of placing an overlay onto the emulsion before the step of heating the emulsion.

28. The method of claim 1, wherein the step of heating is performed without an overlay on the emulsion such that the emulsion is in contact with air.

26. The method of 1, wherein the capsules are buoyant in the continuous phase.

29. A composition for generating a stabilized emulsion, comprising:

a continuous phase formed with an oil composition including a fluorinated oil and at least one fluorinated surfactant; and

a plurality of aqueous droplets disposed in the continuous phase and including an effective concentration of one or more skin-forming proteins,

wherein heating the continuous phase and the aqueous droplets above a threshold temperature creates an interfacial skin between each droplet and the continuous phase, to transform the droplets into capsules.

30. The composition of claim 29, where the capsules provide individual reaction mixtures for performing a reaction in the capsules.

31. The composition of claim 30, wherein the capsules provide individual reaction mixtures for amplification of a nucleic acid target.

32. The composition of claim 29, wherein the aqueous droplets include the one or more skin-forming proteins at a concentration of about 0.01% to 10% by weight.

33. A stabilized emulsion, comprising:

a continuous phase formed with an oil composition including a fluorinated oil and at least one fluorinated surfactant; and

a plurality of capsules disposed in the continuous phase, each capsule including a proteinaceous, interfacial skin encapsulating an aqueous phase.

34. The stabilized emulsion of claim 33, wherein the aqueous phase provides a reaction mixture for performing a reaction in individual capsules.

35. The stabilized emulsion of claim 34, wherein the aqueous phase provides a reaction mixture for performing amplification of a nucleic acid target in individual capsules.

36. The stabilized emulsion of claim 33, wherein the proteinaceous skin includes at least one protein selected from the group consisting of albumin, casein, gelatin, and globulin.

37. The stabilized emulsion of claim 33, wherein the at least one fluorinated surfactant includes a first fluorinated surfactant that is negatively charged and a second fluorinated surfactant that is an alcohol.

38. A stabilized emulsion, comprising:

a continuous phase formed with an oil composition including a fluorinated oil and at least one fluorinated surfactant; and

a plurality of capsules disposed in the continuous phase and each containing an aqueous phase,

wherein the capsules are resistant to coalescence if disposed at a high packing density and incubated at 90°

C. for at least one minute.

39. The stabilized emulsion of claim 38, wherein the capsules are resistant to coalescence if incubated at 90°

C. for at least ten minutes.

40. A method of emulsion preparation, comprising:

generating aqueous droplets in a continuous phase that includes a fluorinated oil;

transforming the droplets to capsules each including an aqueous phase encapsulated by a proteinaceous, interfacial skin; and

adding a spacing fluid to the continuous phase, the spacing fluid being miscible with the continuous phase and having a different composition than the continuous phase.

41. The method of claim 40, wherein the step of transforming is performed with the capsules disposed in a three-dimensional arrangement having a high packing density.

42. The method of claim 40, wherein the step of transforming includes a step of heating the continuous phase.

43. The method of claim 42, wherein the step of heating the continuous phase includes a step of heating the continuous phase to a temperature of at least about 55°

C.

44. The method of claim 40, further comprising a step of selectively removing a portion of the continuous phase after the step of generating aqueous droplets and before the step of transforming the droplets.

45. The method of claim 40, further comprising a step of amplifying a nucleic acid target in individual capsules.

46. The method of claim 45, further comprising a step of thermally cycling the capsules to promote amplification of the nucleic acid target.

47. The method of claim 40, further comprising a step of driving flow of capsules through a detection region, and a step of collecting assay data from capsules as such capsules travel through the detection region.

48. The method of claim 40, further comprising a step of imaging capsules to detect assay data from capsules.

49. The method of claim 40, wherein the spacing fluid contains no surfactant that is present at a concentration substantially above the critical micelle concentration of the surfactant.

50. The method of claim 40, wherein the step of generating is performed with an aqueous phase and an oil phase, wherein each of the oil phase and the spacing fluid has a percent by weight of surfactant, and wherein the percent by weight of surfactant in the oil phase is substantially higher than in the spacing fluid.

51. The method of claim 50, wherein the percent by weight of surfactant in the oil phase is at least about ten-fold higher than in the spacing fluid.

52. The method of claim 40, wherein the step of generating is performed with an aqueous phase and an oil phase, wherein each of the oil phase and the spacing fluid has a percent by weight of ionic surfactant, and wherein the percent by weight of ionic surfactant in the oil phase is substantially higher than in the spacing fluid.

53. The method of claim 40, wherein the step of generating is performed with an aqueous phase and an oil phase, and wherein the oil phase includes an ionic surfactant, and wherein the spacing fluid includes a nonionic surfactant.

54. The method of claim 53, wherein each of the ionic surfactant and the nonionic surfactant is a fluorinated polyether.

55. The method of claim 53, wherein the nonionic surfactant has a concentration in the spacing fluid that is about the same as or greater than a concentration of the ionic surfactant in the oil phase.

56. The method of claim 40, wherein the step of generating is performed with an aqueous phase and an oil phase, and wherein each of the oil phase and the spacing fluid includes a different primary or exclusive surfactant.

57. The method of claim 40, further comprising a step of selectively removing a portion of the continuous phase before the step of transforming.

58. The method of claim 40, wherein the step of adding a spacing fluid does not substantially wrinkle or break the skin of more than a minority of the capsules.

59. A kit for emulsion preparation, comprising:

an aqueous phase including an effective concentration of one or more skin-forming proteins;

a nonaqueous continuous phase including a fluorinated oil and at least one fluorinated surfactant; and

a droplet generator capable of forming an emulsion including droplets of the aqueous phase disposed in the nonaqueous continuous phase,

wherein heating the emulsion above a threshold temperature creates an interfacial skin between each droplet and the continuous phase, to transform the droplets into capsules.

60. The kit of claim 59, wherein the aqueous phase includes one or more reaction components for amplification of a nucleic acid target.

61. The kit of claim 59, wherein the step of heating the emulsion reduces a solubility of the one or more skin-forming proteins in the aqueous phase.

62. A method of generating a stabilized emulsion, comprising:

providing an oil phase including a fluorinated oil and at least one ionic surfactant that is fluorinated and negatively-charged;

forming an emulsion including droplets of an aqueous phase disposed in the oil phase, wherein the aqueous phase provides a reaction mixture for amplification of a nucleic acid target; and

heating the emulsion to a temperature of at least about 50°

C.

63. The method of claim 62, wherein the at least one ionic surfactant is a fluorinated polyether.

64. The method of claim 62, wherein the reaction mixture includes at least one magnesium-dependent enzyme.

65. A method of generating a stabilized emulsion, comprising:

providing an oil phase including a fluorinated oil, a fluorinated alcohol, and a fluorinated surfactant;

forming an emulsion including droplets of an aqueous phase disposed in the oil phase; and

heating the emulsion to a temperature of at least about 50°

C.

66. The method of claim 65, wherein the fluorinated alcohol has no more than two hydroxyl groups.

67. The method of claim 65, wherein the fluorinated alcohol has no more than twenty carbons.

68. The method of claim 65, wherein the fluorinated alcohol is perfluorodecanol.

69. The method of claim 65, wherein the fluorinated surfactant is a fluorinated polyether.

70. The method of claim 69, wherein the fluorinated polyether is negatively charged.