# US 10,766,032 B2 · Devices having a plurality of droplet formation regions

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- Number: US 10,766,032 B2
- Document ID: US10766032B2
- Status: Granted
- Kind: B2
- Application number: 16/706,313
- Filed: 2019-12-06
- Published: 2020-04-16
- Granted: 2020-09-08
- Earliest claimed: 2017-08-22
- Assignees: 10x Genomics, Inc.
- Applicants: 10X Genomics, Inc.
- Inventors: Rajiv Bharadwaj; Anthony Makarewicz; Bill Kengli Lin
- Web page: https://patentgrep.com/patent/US10766032B2

## CPC

- B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics}
- B01F 23/41: Emulsifying
- B01F 23/4105: {Methods of emulsifying}
- B01F 25/3141: {with additional mixing means other than injector mixers}
- B01F 25/31422: {with a plurality of perforations in the axial direction only}
- B01F 25/31423: {with a plurality of perforations in the circumferential direction only and covering the whole circumference}
- B01F 31/84: {for material continuously moving through a tube, e.g. by deforming the tube}
- 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
- 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}
- B01L 3/0241: {Drop counters; Drop formers}
- B01L 3/50273: {characterised by the means or forces applied to move the fluids}
- B01L 3/502761: {specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 33/54313: {the carrier being characterised by its particulate form}
- G01N 33/58: involving labelled substances (G01N33/53 takes precedence)
- B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L 2200/0652: Sorting or classification of particles or molecules
- B01L 2200/0673: Handling of plugs of fluid surrounded by immiscible fluid
- B01L 2300/0851: Bottom walls
- B01L 2300/161: Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
- B01L 2400/0439: ultrasonic vibrations, vibrating piezo elements
- B01L 2400/086: using baffles or other fixed flow obstructions
- C12N 2533/50: Proteins
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2563/185: Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

Devices, systems, and their methods of use, for generating droplets are provided. One or more geometric parameters of a microfluidic channel can be selected to generate droplets of a desired and predictable droplet size.

## Claims

### Claim 1

A system of producing droplets, the system comprising:
(i) a device comprising:
a) a first channel having a first depth, a first width, a first proximal end, and a first distal end;
b) a second channel having a second depth, a second width, a second proximal end, and a second distal end, wherein the second channel intersects the first channel between the first proximal and first distal ends;
c) a shelf region, wherein the shelf region is in fluid communication with the first distal end, wherein the shelf region had a width greater than the first width; and
d) a collection reservoir to collect a population of droplets and comprising at least one wall forming a step, wherein the step is fluidically connected to the shelf region, and wherein the step has a fourth depth that is greater than the first depth;
(ii) a first liquid in the first channel;
(iii) a second liquid in the collection reservoir and the shelf region, wherein, during droplet formation, the second liquid required to form droplets is present only in the collection reservoir and the shelf region; and
(iv) a third liquid in the second channel;
wherein the first and third liquids combine at the intersection of the first and second channels and form droplets in the second liquid; and
wherein the first and second liquids are immiscible, and the first and third liquids are miscible.

### Claim 2 (depends on 1)

The system of claim 1, wherein the first liquid comprises particles.

### Claim 3 (depends on 2)

The system of claim 2, wherein the third liquid is aqueous or miscible with water.

### Claim 4 (depends on 3)

The system of claim 3, wherein the first liquid is aqueous or miscible with water.

### Claim 5

A method of producing droplets, the method comprising:
providing a system comprising:
(A) a device comprising:
a) a first channel having a first depth, a first width, a first proximal end, and a first distal end;
b) a second channel having a second depth, a second width, a second proximal end, and a second distal end, wherein the second channel intersects the first channel between the first proximal and first distal ends;
c) a shelf region, wherein the shelf region is in fluid communication with the first distal end, wherein the shelf region had a width greater than the first width; and
d) a collection reservoir to collect a population of droplets and comprising at least one wall forming a step, wherein the step is fluidically connected to the shelf region, and wherein the step has a fourth depth that is greater than the first depth;
(B) a first liquid in the first channel;
(C) a second liquid in the collection reservoir and the shelf region, wherein, during droplet formation, the second liquid required to form droplets is present only in the collection reservoir and the shelf region; and
(D) a third liquid in the second channel;
wherein the first and second liquids are immiscible, and the first and third liquids are miscible;
(ii) combining the first liquid from the first channel and the third liquid from the second channel at the intersection; and
(iii) flowing the combined first and third liquids to the step region to produce droplets of the combined first and third liquids, wherein the droplets are dispersed in the second liquid.

### Claim 6 (depends on 5)

The method of claim 5, wherein the first liquid comprises particles, and wherein the droplets comprise particles.

### Claim 7 (depends on 5)

The method of claim 5, wherein the third liquid is aqueous or miscible with water.

### Claim 8 (depends on 5)

The method of claim 5, wherein the first liquid is aqueous or miscible with water.

### Claim 9

A device for producing droplets, the device comprising:
a) a first channel having a first depth, a first width, a first proximal end, and a first distal end;
b) a second channel having a second depth, a second width, a second proximal end, and a second distal end, wherein the second channel intersects the first channel between the first proximal and first distal ends;
c) a shelf region, wherein the shelf region is in fluid communication with the first distal end, wherein the shelf region had a width greater than the first width;
d) a collection reservoir to collect a population of droplets and comprising at least one wall forming a step, wherein the step is fluidically connected to the shelf region, and wherein the step has a fourth depth that is greater than the first depth;
e) a first liquid in the first channel;
f) a second liquid in the collection reservoir and the shelf region, wherein, during droplet formation, the second liquid required to form droplets is present only in the collection reservoir and the shelf region; and
g) a third liquid in the second channel;
wherein the first and third liquids combine at the intersection of the first and second channels and form droplets in the second liquid; and
wherein the first and second liquids are immiscible, and the first and third liquids are miscible.

### Claim 10 (depends on 9)

The device of claim 9, wherein the first liquid comprises particles.

### Claim 11 (depends on 9)

The device of claim 9, wherein the third liquid is aqueous or miscible with water.

### Claim 12 (depends on 9)

The device of claim 9, wherein the first liquid is aqueous or miscible with water.