Microfluidic droplet generators

Patent metadata

Field	Value
Publication number	US-11602722-B2
Application number	US-202016780104-A
Country	US
Kind code	B2
Filing date	Feb 3, 2020
Priority date	Feb 4, 2019
Publication date	Mar 14, 2023
Grant date	Mar 14, 2023

Abstract

Disclosed herein is a novel method of producing monodisperse aqueous droplets, as well as a novel microfluidic droplet generator. In some examples, the method comprises flowing an aqueous solution through a microchannel and into a sample reservoir of the microfluidic droplet generator, wherein monodisperse droplets of the aqueous solution form by step-emulsification at a step change in height at an intersection of a reservoir end of the microchannel and a sidewall of the sample reservoir. In some examples, the aqueous solution is a hydrogel precursor solution and monodisperse droplets of the hydrogel precursor solution form by step-emulsification at the step change in height at the intersection of the reservoir end of the microchannel and the sidewall of the sample reservoir. In some examples, the monodisperse droplets of the hydrogel precursor solution are incubated under conditions suitable for gelation to form hydrogel beads.

First claim

It is claimed:

1. A method, comprising:
providing a microfluidic droplet generator comprising:
a body having a single inlet fluidly connected to a microchannel fluidly connected to a sample reservoir, wherein:
the sample reservoir comprises a floor and a sidewall coupled to and extending outward from the floor, and contains a reservoir fluid that is immiscible in water;
the microchannel comprises an inlet end and a reservoir end; and
the reservoir end of the microchannel intersects the sidewall of the sample reservoir at a location that is submerged beneath the reservoir fluid and is spaced from the floor; and

flowing an aqueous solution into the single inlet, through the microchannel, and into the sample reservoir by applying pressure at the single inlet, wherein monodisperse droplets of the aqueous solution form by step-emulsification at a step change in height at the intersection of the reservoir end of the microchannel and the sidewall of the sample reservoir.

2. The method of claim 1 , wherein the pressure at the single inlet is applied with a manual or electric air-displacement micropipette.

3. The method of claim 1 , wherein the pressure at the single inlet is constant or varying pressure of up to about 2000 Pa.

4. The method of claim 1 , wherein the flow rate of the aqueous solution at the reservoir end of the microchannel is from about 0.5 μL/min to about 5 μL/min.

5. The method of claim 1 , wherein the microchannel and the sample reservoir are a single microchannel and a single sample reservoir, respectively.

6. The method of claim 1 , wherein a flow axis of the aqueous solution through the reservoir end of the microchannel is parallel to the floor and perpendicular to the sidewall of the sample reservoir.

7. The method of claim 1 , wherein the reservoir end of the microchannel comprises a cross-sectional area of from about 100 μm 2 to about 10000 μm 2 .

8. The method of claim 1 , wherein the microchannel comprises a length of at least about 100 μm from the reservoir end to the inlet end.

9. The method of claim 1 , wherein a fluidic resistance of the microchannel prevents jetting of the aqueous solution into the sample reservoir at the reservoir end of the microchannel when the pressure is applied at the single inlet.

10. The method of claim 1 , wherein the reservoir fluid comprises:
an oil, a viscous aqueous solution, or a combination thereof, and
a surfactant.

11. The method of claim 10 , wherein:
the oil is mineral oil, silicone oil, fluorinated oil, or a combination of two or more thereof;
the viscous aqueous solution is a solution containing poly-ethylene glycol (PEG), poly-vinyl-pyrrolidone, pluronic dextran, or sucrose, or a combination of two or more thereof.

12. The method of claim 1 , wherein the aqueous solution comprises genetic material, and the genetic material is encapsulated within the monodisperse droplets of the aqueous solution.

13. The method of claim 1 , wherein the aqueous solution is less dense than the reservoir fluid, wherein a buoyancy force on the aqueous solution promotes formation of the monodisperse droplets at the step change in height at the intersection of the reservoir end of the microchannel and the sidewall of the sample reservoir.

14. The method of claim 1 , wherein the aqueous solution is a hydrogel precursor solution, the monodisperse droplets of the aqueous solution are monodisperse droplets of the hydrogel precursor solution, and the method further comprises incubating the monodisperse droplets of the hydrogel precursor solution under conditions suitable for gelation to form hydrogel beads.

15. The method of claim 14 , wherein the hydrogel precursor solution comprises hydrogel polymer and crosslinker and does not comprise a gelation catalyst for the hydrogel polymer and crosslinker; and
incubating the monodisperse droplets of the hydrogel precursor solution under conditions suitable for gelation comprises incubating the monodisperse droplets of the hydrogel precursor solution with the gelation catalyst to initiate gelation of the hydrogel polymer and crosslinker to form the hydrogel beads.

16. The method of claim 15 , wherein the sample reservoir fluid comprises the gelation catalyst and the monodisperse droplets of the hydrogel precursor solution undergo gelation in the sample reservoir to form the hydrogel beads.

17. The method of claim 15 , wherein:
the hydrogel precursor solution comprises acrylamide, bis-acrylamide, and potassium persulfate; and
the gelation catalyst is tetramethylethylenediamine.

18. The method of claim 14 , wherein the hydrogel beads comprise pores having a diameter of sufficient size to allow diffusion of reagents through the hydrogel beads while retaining encapsulated genetic material.

19. The method of claim 14 , wherein the hydrogel beads have a diameter of from about 10 μm to about 200 μm.

20. The method of claim 14 , further comprising linking an outer surface of the hydrogel beads to a transposome complex.

21. The method of claim 14 , wherein the hydrogel beads are degradable hydrogel beads that are degraded by:
contacting the hydrogel beads with a reagent that cleaves a reversible hydrogel crosslinker that crosslinks polymers of the hydrogel;
heating the hydrogel beads to about 90° C.;
exposing the hydrogel beads to a wavelength of light that cleaves a photo-cleavable hydrogel crosslinker that crosslinks polymer of the hydrogel; or
any combination thereof.

22. The method of claim 1 , wherein the flow of the aqueous solution through the microfluidic droplet generator is not due to capillary action.

23. The method of claim 1 , wherein the sidewall is a vertical sidewall and the floor of the sample reservoir is disposed vertically below the reservoir end.

24. The method of claim 1 , wherein the reservoir end is disposed vertically between the floor of the sample reservoir and the single inlet.

25. The method of claim 1 , wherein the sample reservoir has a constant height.

26. A method, comprising:
providing a microfluidic droplet generator comprising:
a body having a single inlet fluidly connected to a microchannel fluidly connected to a sample reservoir, wherein:
the sample reservoir comprises a floor and a sidewall, and contains a reservoir fluid that is immiscible in water;
the microchannel comprises an inlet end and a reservoir end; and
the reservoir end of the microchannel intersects the sidewall of the sample reservoir at a location submerged beneath the reservoir fluid; and

flowing an aqueous solution into the inlet, through the microchannel, and into the sample reservoir by applying pressure at the inlet, wherein monodisperse droplets of the aqueous solution form by step-emulsification at a step change in height at the intersection of the reservoir end of the microchannel and the sidewall of the sample reservoir;
wherein the reservoir fluid comprises:
an oil, a viscous aqueous solution, or a combination thereof, and
a surfactant;
wherein:
the oil is mineral oil, silicone oil, fluorinated oil, or a combination of two or more thereof; and
the viscous aqueous solution is a solution containing poly-ethylene glycol (PEG), poly-vinyl-pyrrolidone, pluronic dextran, or sucrose, or a combination of two or more thereof; and
wherein the reservoir fluid comprises:
1,1,2,2,3,3,4,4,4-Nonafluoro-N-(nonafluorobutyl)-N-(1,1,2,2-tetrafluoroethyl)-1-butanamine;
3-ethoxyperfluoro(2-m

Assignees

• Illumina Inc

Inventors

• Khurana Tarun Kumar
• Mashayekhi Foad
• Tam Hei Ka Patrick

Classifications

• B01L2400/0403

  specific forces · CPC title

• B01L3/502784 Primary

  specially adapted for droplet or plug flow, e.g. digital microfluidics · CPC title

• B01L2200/0673

  Handling of plugs of fluid surrounded by immiscible fluid · CPC title

• B01L2400/084

  Passive control of flow resistance · CPC title

• B01L2400/0487

  fluid pressure, pneumatics · CPC title