Generating microdroplet emulsions with microfluidic chambers containing pores

Abstract

Provided herein are devices, assemblies, systems, kits, and methods for generating an emulsion with a microfluidic chamber composed of impediments (e.g., posts) regularly or irregularly spaced apart to create pores (e.g., 10-1000 microns in diameter). In certain embodiments, a non-emulsion mixture composed of sample fluid (e.g., containing cells and / or beads) and an immiscible carrier fluid are flown through the microfluidic chamber once or repeatedly such that an emulsion is generated comprising sample microdroplets (e.g., about the same size as the pores, or of irregular shape). In some embodiments, the microfluidic chamber is housed in a pipette tip and a micropipette is used to push or draw the non-emulsion mixture through the microfluidic chamber.

Description

Abstract This document provides apparatus, components, systems, kits, and methods for generating emulsions using a microfluidic chamber composed of barriers (e.g., columns) spaced regularly or irregularly to create pores (e.g., diameters of 10 to 1000 micrometers). In certain embodiments, a non-emulsion mixture consisting of a sample fluid (e.g., containing cells and / or microbeads) and an immiscible carrier fluid is passed through the microfluidic chamber once or multiple times to generate an emulsion containing sample microdroplets (e.g., approximately the same size as the pores or of an irregular shape). In some embodiments, the microfluidic chamber is housed in a pipette tip, and a micropipette is used to push or aspirate the non-emulsion mixture through the microfluidic chamber.

Claims

CLAIMSI claim:

1. A system, kit, or device for generating an emulsion from a non-emulsion mixture that is composed of sample fluid, an immiscible carrier fluid, and optionally a surfactant comprising: a) a housing component; b) at least one microfluidic chamber housed in, integral with, or configured to be housed in said housing component, wherein said at least one microfluidic chamber comprises: i) at least 2, and optionally at 100, impediments regularly or irregularly spaced apart to create at least 2, and optionally at least 100, pores therebetween, wherein said at least 2, and optionally 100, pores are about 5-2000 microns in diameter, and ii) a first open end and a second open end which are in fluid communication with each other such that, when said non-emulsion mixture is pushed or drawn once or multiple times through said microfluidic chamber from said first open end to said second open end, and / or vice versa, it passes through said at least 2, and optionally said at least 100 pores, and generates an emulsion comprising a plurality of sample microdroplets in said immiscible carrier liquid, wherein optionally at least some, or most, of said plurality of microdroplets are about the same size as said at least 2, and optionally said 100 pores, or wherein optionally at least some, or most, of said plurality of microdroplets are of non-uniform size.

2. The system or kit of claim 1, further comprising: c) said non-emulsion mixture.

3. The system or kit of claim 2, wherein said surfactant is present in said non-emulsion mixture.

4. The system or kit of claim 1, further comprising: c) said sample fluid, and / or said immiscible carrier fluid, and / or said surfactant.

5. The system, kit, or device of claim 1 , wherein housing component comprises a micropipcttc tip or container configured to be attached to said micropipcttc tip, and / or wherein said microfluidic chamber further comprises a semi-permeable membrane.

6. The system, kit, or device of claim 1, wherein housing component comprises at least a portion of a microfluidic device.

7. The system or kit of claim 1, further comprising; said sample fluid, and wherein said sample fluid comprises water and at least one of the following: i) a liquid gel precursor, ii) a plurality of cells, iii) a plurality of capture beads, optionally barcoded and / or composed of hydrogel, and iv) a plurality of core-shell particles.

8. The system or kit of claim 7, wherein said capture beads are configured to hybridize to RNA, DNA, and / or protein targets.

9. The system or kit of claim 1, further comprising: c) said emulsion.

10. The system, kit, or device of claim 1, wherein said at least one microfluidic chamber comprises at least 2, at least 10, or at least 20 of said microfluidic chambers.

11. The system, kit, or device of claim 1, wherein said at least one microfluidic chamber is located in said housing component.

12. The system, kit, or device of claim 1, wherein said about 5-2000 microns in diameter for said pores is about 10-1000 microns, or about 20-100 microns, or about 50-90 microns.

13. The system, kit, or device of claim 1, wherein said at least 2 or at least 100 impediments, is at least 1000, at least 10,000, or at least 100,000 impediments.

14. The system, kit, or device of claim 1, wherein said at least 2 or at least 100 pores, is at least 1000, at least 10,000, or at least 100,000 pores.

15. The system, kit, or device of claim 1, wherein said housing component is configured to mate with a pressure-providing device such that said pressure -providing device can apply pressure to said microfluidic chamber.

16. The system, kit, or device of claim 15, wherein said pressure-providing device comprises a micro-pipettor or an air-pressure pump.

17. The system, kit, or device of claim 1, further comprising: c) a micro-pipettor.

18. The system, kit, or device of claim 1, wherein said pushed through comprises positive pressure and said drawn through comprises negative pressure.

19. The system, kit, or device of claim 1, wherein said emulsion comprises at least 1000, 10,000, 100,000, or 1 million of said sample microdroplets in said immiscible carrier liquid.

20. The system, kit, or device of claim 1, wherein said immiscible carrier liquid comprises oil, and / or wherein said immiscible carrier liquid comprises oleogel and / or oleogel.

21. The system, kit, or device of claim 1, wherein said plurality of sample microdroplets are substantially monodisperse in said immiscible carrier fluid.

22. The system, kit, or device of claim 1, wherein said at least 2 or at least 100 impediments have a shape selected from: square, round, rectangle, posts, octagon, star shaped, and combinations thereof.

23. The system, kit, or device of claim 1, i) wherein said at least 2, and optionally 100, impediments comprises at least 20 impediments and at least 20 pores therebetween, and ii) wherein said at least 20 impediments are arranged in first and second regularly spaced apart groups that are separate from each other, andiii) wherein said first regularly spaced apart group has first impediments that create a first pore size therebetween, and said second regularly spaced apart group has second impediments that create a second pore size therebetween that is different from said first pore size, and optionally wherein said first and second impediments have different shapes.

23. The system or kit of claim 1, further comprising; c) said sample fluid, and wherein said sample fluid comprises water and particles, wherein said impediment, said pores, and said particles are sized such that said plurality of sample droplets on average contain one and only one particle.

24. The system or kit of claim 1, further comprising: c) said immiscible fluid, wherein said immiscible fluid is present in said microfluidic chamber, and said sample fluid is not present in said microfluidic chamber.

25. The system, kit, or device of claim 1, wherein said microfluidic chamber comprises at least one additional opening configured to introduce said sample fluid, or said immiscible carrier fluid, or said non-emulsion mixture, wherein said configured to introduce is under laminar flow conditions.

26. The system, kit, or device of claim 1, wherein said at least one microfluidic chamber and said housing component are integral with each other such that they form a single combined component.

27. A method of generating an emulsion with a microfluidic chamber comprising: a) attaching an emulsion generating assembly to a pressure-providing device, wherein said emulsion generating assembly comprises: i) a housing component; ii) at least one microfluidic chamber housed in, or integral with, said housing component, wherein said at least one microfluidic chamber comprises:A) at least 2, and optionally 100, impediments regularly or irregularly spaced apart to create at least 2, and optionally 100 pores, therebetween, wherein said at least 2, and optionally 100 pores, are about 5-2000 microns in diameter, andB) a first open end and a second open end which are in fluid communication with each other, and iii) wherein optionally an immiscible carrier fluid is present in said microfluidic chamber; and b) activating said pressure-providing device such that a first liquid is pushed or drawn through said microfluidic chamber once or multiple times, from said first open end to said second open end, or from said second open end to said first open end, or both, wherein said first liquid comprises: i) a sample fluid if said immiscible carrier fluid is present in said microfluidic chamber, and / or ii) a non-emulsion mixture comprising: said sample fluid, said immiscible carrier fluid, and optionally a surfactant, wherein when said first liquid is pushed or drawn through said microfluidic chamber once or multiple times, this generates an emulsion comprising a plurality of sample microdroplets in said immiscible carrier liquid, wherein optionally at least some, or most, of said plurality of microdroplets are about the same size as said at least 2 or at least 100 pores, or wherein optionally at least some, or most, of said plurality of microdroplets are of non- uniform size.

28. The method of claim 27, wherein said activating in repeated at least once.

29. The method of claim 27, wherein said activating is repeated at least five times.

30. The method of claim 27, wherein said pressure-providing device is configured to provide negative pressure, positive pressure, or negative and positive pressure.

31. The method of claim 27, wherein said surfactant is present in said non-emulsion mixture.

32. The method of claim 27, wherein housing component comprises a micropipette tip or container configured to be attached to said micropipcttc tip, and / or wherein said microfluidic chamber further comprises a semi-permeable membrane.

33. The method of claim 27, wherein housing component comprises at least a portion of a microfluidic device.

34. The method of claim 27, wherein said sample fluid comprises water and at least one of the following: i) a liquid gel precursor, ii) a plurality of cells, iii) a plurality of capture beads, optionally barcoded and / or composed of hydrogel, and iv) a plurality of core-shell particles.

35. The method of claim 34, wherein said capture beads are configured to hybridize to RNA, DNA, and / or protein targets.

36. The method of claim 27, wherein said plurality of microdroplets comprise a plurality of nuclei acid molecules, and the method further comprises: c) processing said plurality of microdroplets such that said plurality of nucleic acid molecules are sequenced.

37. The method of claim 27, wherein said at least one microfluidic chamber comprises at least 2, at least 10, or at least 20 of said microfluidic chambers.

38. The method of claim 27, wherein said at least one microfluidic chamber is integral with said housing component.

39. The method of claim 27, wherein said about 5-2000 microns in diameter for said pores is about 10-1000 microns, or about 20-100 microns, or about 50-90 microns.

40. The method of claim 27, wherein said at least 2 or at least 100 impediments, is at least 1000, at least 10,000, or at least 100,000 impediments.41 . The method of claim 27, wherein said at least 2 or at least 100 pores, is at least 1000, at least 10,000, or at least 100,000 pores.

42. The method of claim 27, wherein said attaching comprises push-fitting said emulsion generating assembly to said pressure -providing device.

43. The method of claim 27, wherein said pressure-providing device comprises a micropipettor or an air-pressure pump.

44. The method of claim 27, wherein said activating said pressure -providing device pushes said first liquid through said microfluidic chamber with positive air pressure.

45. The method of claim 27, wherein said activating said pressure -providing device draws said first liquid through said microfluidic chamber with negative air pressure.

46. The method of claim 27, wherein said emulsion comprises at least 1000, 10,000, 100,000, or 1 million of said sample microdroplets in said immiscible carrier liquid.

47. The method of claim 27, wherein said immiscible carrier liquid comprises oil, and / or wherein said immiscible carrier liquid comprises oleogel and / or oleogel.

48. The method of claim 27, wherein said plurality of sample microdroplets are substantially monodisperse in said immiscible carrier fluid.

49. The method of claim 27, wherein said at least 100 impediments have a shape selected from: square, round, octagon, star shaped, rectangle, posts, and combinations thereof.

50. The method of claim 27, i) wherein said at least 2, and optionally 100, impediments comprises at least 20 impediments and at least 20 pores therebetween, and ii) wherein said at least 20 impediments are arranged in first and second regularly spaced apart groups that are separate from each other, andiii) wherein said first regularly spaced apart group has first impediments that create a first pore size therebetween, and said second regularly spaced apart group has second impediments that create a second pore size therebetween that is different from said first pore size, and optionally wherein said first and second impediments have different shapes.

51. The method of claim 27, wherein said sample fluid comprises water and particles, wherein said impediments, said pores, and said particles are sized such that said plurality of sample microdroplets on average contain one and only one particle.

52. The method of claim 27, wherein said immiscible fluid is present in said microfluidic chamber and said first liquid is said sample fluid.

53. The method of claim 27, wherein said microfluidic chamber further comprises a third and / or fourth opening configured to introduce said sample fluid, or said immiscible carrier fluid, or said non-emulsion mixture into said microfluidic chamber.

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