Assays and other reactions involving droplets

• US 10,221,437 B2
• Filed: 01/30/2018
• Issued: 03/05/2019
• Est. Priority Date: 03/07/2007
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Weitz, David A., Agresti, Jeremy, Chu, Liang-Yin, Kim, Jin-Woong, Rowat, Amy, Sommer, Morten, Dantas, Gautam, Church, George
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/884,215
• Publication Number:
  US 20180171373A1
• Time in Patent Office: 399 Days
• CPC Class Codes: B01F 2101/23 Mixing of laboratory samples e.g. in preparation of analysing or testing properties of materials
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 23/4105 Methods of emulsifying
• CPC Class Codes: B01F 33/3011 using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01J 13/0052 Preparation of gels
• CPC Class Codes: B01J 13/0065 containing an organic phase
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• 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 2563/149 Particles, e.g. beads
• 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
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 2015/1006 for cytology

Abstract

The present invention generally relates to droplets and/or emulsions, such as multiple emulsions. In some cases, the droplets and/or emulsions may be used in assays, and in certain embodiments, the droplet or emulsion may be hardened to form a gel. In some aspects, a heterogeneous assay can be performed using a gel. For example, a droplet may be hardened to form a gel, where the droplet contains a cell, DNA, or other suitable species. The gel may be exposed to a reactant, and the reactant may interact with the gel and/or with the cell, DNA, etc., in some fashion. For example, the reactant may diffuse through the gel, or the hardened particle may liquefy to form a liquid state, allowing the reactant to interact with the cell. As a specific example, DNA contained within a gel particle may be subjected to PCR (polymerase chain reaction) amplification, e.g., by using PCR primers able to bind to the gel as it forms. As the DNA is amplified using PCR, some of the DNA will be bound to the gel via the PCR primer. After the PCR reaction, unbound DNA may be removed from the gel, e.g., via diffusion or washing. Thus, a gel particle having bound DNA may be formed in one embodiment of the invention.

Claims

1. A method, comprising:

(a) providing a fluidic droplet in a carrying fluid, wherein said fluidic droplet comprises a first fluid that is substantially immiscible in said carrying fluid, and wherein said carrying fluid is substantially immiscible in water;

(b) hardening said fluidic droplet to form a gel;

(c) removing said carrying fluid;

(d) placing said hardened fluidic droplet in a second fluid; and

(e) subsequent to (d), containing said hardened fluidic droplet in a surrounding droplet.

2. The method of claim 1, wherein said second fluid is immiscible in water.

3. The method of claim 1, wherein said fluidic droplet comprises a cell.

4. The method of claim 1, wherein said fluidic droplet comprises a nucleic acid.

5. The method of claim 1, wherein said fluidic droplet comprises a pre-polymer and said fluidic droplet is hardened by polymerizing said pre-polymer to form said gel.

6. The method of claim 5, wherein said fluidic droplet further comprises a polymerization initiator.

7. The method of claim 6, wherein said fluidic droplet further comprises a polymerization accelerator.

8. The method of claim 7, wherein said pre-polymer comprises acrylamide, said initiator comprises ammonium persulfate, or said accelerator comprises TEMED.

9. The method of claim 1, wherein said surrounding droplet comprises a nucleic acid molecule that attaches to said gel of said hardened droplet.

10. A method, comprising:

(a) providing a plurality of fluidic droplets of a first fluid in a second fluid, wherein said first fluid comprises water and said second fluid comprises a carrying fluid that is substantially immiscible with water;

(b) hardening said plurality of fluidic droplets to form a plurality of hardened droplets;

(c) placing said plurality of hardened droplets in a third fluid comprising a nucleic acid;

(d) causing said nucleic acid in said third fluid to be contained within a hardened droplet of said plurality of hardened droplets; and

(e) containing said hardened droplet within a surrounding droplet.

11. The method of claim 10, wherein (a) comprises forming said plurality of fluidic droplets using a microfluidic device.

12. The method of claim 10, wherein (b) comprises polymerizing said plurality of fluidic droplets into said hardened droplets.

13. The method of claim 12, wherein said plurality of fluidic droplets comprises a pre-polymer.

14. The method of claim 13, wherein said polymerizing comprises crosslinking said pre-polymer within said plurality of fluidic droplets.

15. The method of claim 13, wherein said pre-polymer comprises acrylamide.

16. The method of claim 10, further comprising separating said carrying fluid from said plurality of hardened droplets prior to (c).

17. The method of claim 10, wherein (d) comprises attaching said nucleic acid to a polymeric component of said hardened droplet.

18. The method of claim 17, wherein said attaching comprising covalently attaching said nucleic acid to said polymeric component of said hardened droplet.

19. The method of claim 18, wherein said covalently attaching comprises covalently attaching said nucleic acid to said polymeric component of said hardened droplet using an acrydite moiety.

20. The method of claim 10, wherein said nucleic acid comprises a primer sequence for conducting a nucleic acid reaction.

21. The method of claim 10, wherein said nucleic acid comprises a sequence for conducting a polymerase chain reaction (PCR).

22. The method of claim 10, further comprising containing said hardened droplet within said surrounding droplet using a microfluidic device.

23. The method of claim 10, further comprising liquefying said hardened droplet within said surrounding droplet.

24. The method of claim 10, wherein said plurality of hardened droplets is monodisperse.

25. The method of claim 24, wherein said plurality of hardened droplets has a distribution of diameters such that no more than about 10% of said plurality of hardened droplets have an average diameter greater than about 10% of an average diameter of said plurality of hardened droplets.

26. The method of claim 10, wherein said plurality of hardened droplets comprises a first group of hardened droplets and a second group of hardened droplets, wherein hardened droplets in said first group of hardened droplets comprise a first species and hardened droplets in said second group of hardened droplets comprise a second species, wherein said second species is different from said first species.

27. The method of claim 26, wherein said first species is a first nucleic acid, said second species is a second nucleic acid, and said first nucleic acid has a different sequence from said second nucleic acid.

40. The method of claim 10, wherein said plurality of hardened droplets comprises a library of distinguishable entities.

41. The method of claim 40, wherein said library of distinguishable entities are distinguishable through a species encapsulated in said plurality of hardened droplets.

42. The method of claim 10, wherein said plurality of hardened droplets comprises a plurality of first species, wherein said third fluid comprises a plurality of second species, including said nucleic acid, and wherein (e) comprises partitioning said plurality of hardened droplets into a plurality of droplets, wherein at least a portion of said plurality of droplets, including said surrounding droplet, comprises at least one of said plurality of hardened droplets and at least one of said plurality of second species.

43. The method of claim 42, further comprising liquefying said hardened droplet within said surrounding droplet to release a first species of said plurality of first species into said surrounding droplet, wherein said first species contacts said at least one of said plurality of second species.

44. The method of claim 42, wherein one or both of said plurality of first species and said plurality of second species comprises nucleic acids.

45. The method of claim 42, wherein said plurality of hardened droplets comprises a library of distinguishable entities.

46. The method of claim 45, wherein said library of distinguishable entities are distinguishable through a species encapsulated in said plurality of hardened droplets.

47. The method of claim 10, further comprising sequencing said nucleic acid or derivative thereof.

48. The method of claim 10, wherein said plurality of hardened droplets are capable of undergoing a phase change.

49. The method of claim 26, wherein (e) comprises partitioning said plurality of hardened droplets into a plurality of droplets, wherein a first droplet of said plurality of droplets comprises a first hardened droplet of said first group of hardened droplets and wherein a second droplet of said plurality of droplets comprises a second hardened droplet of said second group of hardened droplets.

50. The method of claim 49, wherein at least some droplets of said plurality of droplets further comprise another species.

51. The method of claim 50, wherein said another species comprise nucleic acids.

52. The method of claim 49, wherein said plurality of droplets are formed using a microfluidic device.

53. The method of claim 49, wherein (d) comprises attaching said nucleic acid to a polymeric component of said hardened droplet.

54. The method of claim 53, wherein said attaching comprising covalently attaching said nucleic acid to said polymeric component of said hardened droplet.

55. The method of claim 49, wherein said plurality of hardened droplets comprises a library of distinguishable entities.

56. The method of claim 55, wherein said library of distinguishable entities are distinguishable through a species encapsulated in said plurality of hardened droplets.

57. The method of claim 49, further comprising fusing at least some of said plurality of droplets.

58. The method of claim 26, further comprising exposing said hardened droplet to an environmental change to liquefy said hardened droplet to release said nucleic acid into said surrounding droplet.

59. The method of claim 26, wherein in (c), both said first group of hardened droplets and said second group of hardened droplets are suspended in said third fluid.

60. The method of claim 26, wherein said plurality of fluidic droplets each comprises one or more of a plurality of distinguishable nucleic acid species and a gel precursor.

61. The method of claim 60, wherein said plurality of hardened droplets comprises said one or more of said plurality of distinguishable nucleic acid species, wherein said plurality of hardened droplets are capable of liquefaction via exposure to an environmental change.

62. The method of claim 61, wherein said environmental change comprises one or more of a change in temperature, pH, or osmotic pressure.

28. A method, comprising:

(a) providing a fluidic droplet in a carrying fluid, wherein said fluidic droplet comprises a first fluid that is substantially immiscible in said carrying fluid, and wherein said carrying fluid is substantially immiscible in water;

(b) exposing said fluidic droplet to an environmental change to harden said fluidic droplet to form a gel;

(c) removing said carrying fluid;

(d) placing said hardened fluidic droplet in a second fluid; and

(e) subsequent to (d), containing said hardened fluidic droplet in a surrounding droplet.

29. The method of claim 28, wherein said environmental change comprises a change in temperature.

30. The method of claim 28, wherein said fluidic droplet comprises a nucleic acid, and wherein subsequent to (d), said hardened fluidic droplet is exposed to an additional environmental change to liquefy said gel to release said nucleic acid into said surrounding droplet.

31. The method of claim 30, wherein said additional environmental change comprises one or more of a change in temperature, pH, or osmotic pressure.

32. The method of claim 30, wherein said nucleic acid comprises a primer.

33. The method of claim 30, further comprising attaching said nucleic acid to said gel.

34. The method of claim 33, wherein said attaching comprises covalently attaching said nucleic acid to said gel.

35. The method of claim 28, wherein (a) comprises forming said fluidic droplet using a microfluidic device.

36. The method of claim 28, wherein said second fluid is aqueous.

37. The method of claim 28, wherein said fluidic droplet comprises a pre-polymer and (b) comprises polymerizing said pre-polymer to form said gel.

38. The method of claim 37, wherein said fluidic droplet further comprises a polymerization initiator.

39. The method of claim 38, wherein said fluidic droplet further comprises a crosslinker.