Granted Patent B2

US 12,708,886 · App. 16/649,068 · Granted Aug 18, 2026

Inventors: Adam R. Abate (Daly City, CA); Makiko N. Hatori (Oakland, CA); Leqian Liu (Oakland, CA); Samuel Kim (Palo Alto, CA); Cyrus Modavi (San Francisco, CA)

Assignee: The Regents of the University of California

B01J13/08C09K23/42C12Q1/6844C12Q1/6869C09K23/00

Abstract

Claims (24)

1 . A method for generating an emulsion, the method comprising:

(i) providing a first fluid comprising a plurality of target particles, water that surrounds each target particle, and a surfactant selected from the group consisting of octylphenol ethoxylate and octylphenoxypolyethoxyethanol (IGEPAL);

(ii) combining a plurality of monodisperse template particles with the first fluid to provide a first mixture comprising the plurality of target particles, the surfactant, the plurality of monodisperse template particles, and water that is in contact with and surrounds each target particle and each monodisperse template particle, wherein 99% or more of the plurality of monodisperse template particles vary in diameter by less than a factor of 10, wherein the monodisperse template particles comprise polyethylene glycol (PEG) or agarose;

(iii) combining the first mixture with a second fluid in the absence of a microfluidic channel to provide a second mixture, wherein the second fluid is immiscible with the first fluid; and

(iv) shearing the second mixture by vortexing in the absence of a microfluidic channel such that a plurality of the monodisperse template particles are encapsulated in a plurality of monodisperse droplets ranging from 0.1 to 1000 microns in diameter in the second fluid, thereby providing a plurality of monodisperse droplets comprising one of the monodisperse template particles, one of the plurality of target particles, and water that is in contact with and surrounds both the one monodisperse template particle and the one target particle,

wherein each monodisperse droplet further comprises a surfactant membrane comprising the surfactant, wherein the surfactant membrane is in contact with both the water inside the monodisperse droplet and the second fluid outside the droplet, wherein the surfactant membrane is in contact with and surrounds the water that is in contact with and surrounds both the one monodisperse template particle and the one target particle,

wherein the plurality of monodisperse droplets have a variation in diameter of less than a factor of 10,

wherein the one target particle is not immobilized on the one monodisperse template particle,

wherein the second fluid is 2-(trifluoromethyl)-3-ethoxydodecafluorohexane fluorinated oil.

2 . The method of claim 1 , wherein combining the plurality of monodisperse template particles with the first fluid to provide the first mixture comprises causing a portion of the first fluid to be absorbed by the monodisperse template particles.

3 . The method of claim 1 , comprising removing excess first fluid from the first mixture after causing the portion of the first fluid to be absorbed by the monodisperse template particles.

4 . The method of claim 1 , wherein combining the plurality of monodisperse template particles with the first fluid to provide the first mixture comprises flowing a portion of the first fluid into the monodisperse template particles.

5 . The method of claim 1 , further comprising removing one or more droplets that do not comprise one of the monodisperse template particles by filtration or centrifugation.

6 . The method of claim 5 , wherein the monodisperse droplets have an average diameter and the one or more droplets that do not comprise one of the monodisperse template particles have an average diameter which is smaller than the average diameter of the monodisperse template particles.

7 . The method of claim 1 , wherein the target particles are one or more of DNA molecules, RNA molecules, or cells.

8 . The method of claim 7 , wherein the target particles are cells, and wherein a majority of the monodisperse droplets do not comprise more than one cell per droplet.

9 . The method of claim 7 , wherein the target particles are cells, further comprising incorporating a cell lysis reagent into the monodisperse droplets, wherein the cell lysis reagent is present in the first mixture prior to encapsulation of the plurality of the monodisperse template particles in the plurality of monodisperse droplets.

10 . The method of claim 1 , further comprising sorting the monodisperse droplets.

11 . The method of claim 10 , wherein the sorting comprises one or more steps selected from the group consisting of dielectrophoretic deflection, selective coalescence, fluorescence activated cell sorting (FACS), electrophoresis, acoustic separation, magnetic activated cell sorting (MACS), flow control, or other stimulus used to selectively deflect monodisperse droplets.

12 . The method of claim 1 , wherein the target particles are nucleic acids, wherein the first fluid further comprises nucleic acid synthesis reagents, wherein the monodisperse droplets further comprise the nucleic acid synthesis reagents, and wherein the method further comprising subjecting one or more of the monodisperse droplets to nucleic acid synthesis conditions.

13 . The method of claim 12 , further comprising sequencing nucleic acids and/or nucleic acid synthesis and/or amplification products from one or more of the plurality of monodisperse droplets.

14 . The method of claim 13 , further comprising detecting one or more of the target molecules, a portion thereof, a nucleic acid synthesis product thereof, and/or a nucleic acid amplification product thereof by detecting one or more of detection reagents encapsulated in the monodisperse droplets.

15 . The method of claim 1 , wherein the monodisperse template particles are a first type of particle, and wherein the method further comprises encapsulating one or more of a second type of particle in a droplet with one or more of the first type of particle.

16 . The method of claim 1 , wherein the surfactant is octylphenoxypolyethoxyethanol (IGEPAL).

Continuity (2)

References Cited (400)