METHODS OF PRODUCING DROPLETS INCLUDING A PARTICLE AND AN ANALYTE
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.
1 . A method of producing an analyte detection droplet, the method comprising:
(a) providing a device comprising: (i) a plurality of particles in a liquid carrier, the particles comprising an analyte detection moiety; (ii) a sample liquid comprising an analyte; (iii) a particle channel;
(iv) a sample channel that intersects with the particle channel; (v) a droplet formation region distal to the particle channel and the sample channel, wherein the droplet formation region is configured to allow the liquid carrier to expand in at least one dimension and comprises a step; and (vi) a droplet collection region;
(b) allowing the particles in the liquid carrier to flow proximal-to-distal through the particle channel; and
(c) allowing the sample liquid to flow proximal-to-distal through the sample channel, wherein the sample liquid combines with the particles in the liquid carrier to form an analyte detection liquid at the intersection, wherein the analyte detection liquid meets a partitioning liquid at the droplet formation region under droplet forming conditions, thereby forming a plurality of analyte detection droplets comprising one or more of the particles in the analyte detection liquid.
2 . The method of claim 1 , wherein the particle channel is one of a plurality of particle channels and the sample channel is one of a plurality of sample channels, and wherein the device further comprises a particle reservoir connected proximally to the plurality of particle channels and a sample reservoir connected proximally to the plurality of sample channels.
3 . The method of claim 1 , wherein the sample liquid and the liquid carrier are aqueous liquids and the partitioning liquid is immiscible with the sample liquid and the liquid carrier.
4 . The method of claim 1 , wherein the analyte is a bioanalyte.
5 . The method of claim 4 , wherein the bioanalyte is selected from the group consisting of a nucleic acid, an intracellular protein, a glycan, and a surface protein.
6 . The method of claim 1 , wherein the analyte detection moiety comprises a nucleic acid or an antigen-binding protein.
7 . The method of claim 1 , wherein the sample comprises a cell, or a component or product thereof.
8 . The method of claim 1 , wherein the plurality of analyte detection droplets accumulate as a population in the droplet collection region.
9 . The method of claim 7 , wherein the plurality of analyte detection droplets accumulate as a substantially stationary population in the droplet collection region.
10 . A method of producing a bioanalyte detection droplet, the method comprising:
(a) providing a device comprising: (i) a plurality of particles in an aqueous carrier, the particles comprising a bioanalyte detection moiety; (ii) a particle channel; (iii) a droplet formation region configured to allow the aqueous carrier to expand in at least one dimension; and (iv) a droplet collection region, wherein the particle channel is proximal to the droplet formation region and the droplet formation region is proximal to the droplet collection region; and
(b) allowing the particles in the aqueous carrier to flow proximal-to-distal through the particle channel and droplet formation region, wherein the aqueous carrier meets a partitioning liquid at the droplet formation region under droplet forming conditions, thereby forming a plurality of bioanalyte detection droplets comprising one or more of the particles in the aqueous carrier, wherein the plurality of bioanalyte detection droplets accumulate in the droplet collection region.
11 . The method of claim 10 , wherein the device further comprises a sample channel that intersects with the particle channel proximal to the droplet formation region, wherein an aqueous sample comprising a bioanalyte flows proximal-to-distal through the sample channel and combines with particles in the aqueous carrier at the intersection, wherein the plurality of bioanalyte detection droplets comprises the aqueous sample and one or more particles in the aqueous carrier.
12 . The method of claim 10 , wherein the droplet formation region comprises a step.
13 . The method of claim 11 , wherein the particle channel is one of a plurality of particle channels and the sample channel is one of a plurality of sample channels, and wherein the device further comprises a particle reservoir connected proximally to the plurality of particle channels and a sample reservoir connected proximally to the plurality of sample channels.
14 . The method of claim 10 , wherein the bioanalyte detection moiety comprises a nucleic acid.
15 . The method of claim 10 , wherein the bioanalyte detection moiety comprises a barcode.
16 . The method of claim 10 , wherein the bioanalyte is selected from the group consisting of a surface-expressed protein, an intracellular protein, a glycan, and a nucleic acid.
17 . The method of claim 10 , wherein the aqueous sample comprises a cell, or a component or product thereof.
18 . The method of claim 17 , wherein the aqueous carrier comprises one or more enzymes and/or lysis agents.
19 . The method of claim 10 , further comprising: (c), after step (b) incubating the droplets under conditions sufficient to allow the bioanalyte detection moiety to label the bioanalyte.
20 . The method of claim 19 , wherein the bioanalyte is a nucleic acid, and wherein after step (e), incubating the reaction droplets under conditions sufficient to amplify the barcoded nucleic acids.
21 . The method of claim 20 , wherein the aqueous carrier comprises one or more enzymes, wherein the one or more enzymes is reverse transcriptase.
22 . A method of barcoding a population of cells, the method comprising:
(a) providing a device comprising: (i) a plurality of particles in an aqueous carrier, the particles comprising a nucleic acid primer sequence and a barcode; (ii) an aqueous sample comprising a population of cells; (iii) a particle channel; (iv) a sample channel; (v) a droplet formation region configured to allow the aqueous carrier to expand in at least one dimension; and (vi) a droplet collection region, wherein the particle channel intersects the sample channel proximal to the droplet formation region and the droplet formation region is proximal to the droplet collection region;
(b) allowing the particles in the aqueous carrier to flow proximal-to-distal through the particle channel;
(c) allowing the aqueous sample to flow proximal-to-distal through the sample channel, wherein the aqueous sample combines with the particles in the aqueous carrier to form a reaction liquid at the intersection, and wherein the reaction liquid meets a partitioning liquid at the droplet formation region under droplet forming conditions, thereby forming a plurality of reaction droplets comprising one or more of the particles in the reaction liquid, wherein the plurality of reaction droplets accumulate in the droplet collection region; and
(d) incubating the reaction droplets under conditions sufficient to allow for barcoding nucleic acids in the population of cells.
23 . The method of claim 22 , wherein the aqueous carrier comprises a lysis reagent configured to lyse the cells before or during step (d).
24 . The method of claim 22 , wherein the aqueous carrier or the aqueous sample comprises reverse transcriptase.
25 . A method of single-cell nucleic acid sequencing, the method comprising:
(a) providing a device comprising: (i) a plurality of particles in an aqueous carrier, the particles comprising a nucleic acid primer sequence and a barcode; (ii) an aqueous sample comprising a population of cells; (iii) a particle channel; (iv) a sample channel; (v) a droplet formation region configured to allow the aqueous carrier to expand in at least one dimension; and (vi) a droplet collection region, wherein the particle channel intersects the sample channel proximal to the droplet formation region and the droplet formation region is proximal to the droplet collection region;
(b) allowing the particles in the aqueous carrier to flow proximal-to-distal through the particle channel;
(c) allowing the aqueous sample to flow proximal-to-distal through the sample channel, wherein the aqueous sample combines with the particles in the liquid carrier to form a reaction liquid at the intersection, and wherein the reaction liquid meets a partitioning liquid at the droplet formation region under droplet forming conditions, thereby forming a plurality of reaction droplets comprising one or more of the particles and a single cell or lysate thereof, wherein the plurality of droplets accumulate in the droplet collection region;
(d) incubating the reaction droplets under conditions sufficient to generate barcoded nucleic acids; and
(e) sequencing the barcoded nucleic acid transcripts to obtain nucleic acid sequences associated with single cells.
26 . The method of claim 25 , wherein one or more of the plurality of reaction droplets comprises a single particle and a single cell.
27 . The method of claim 25 , wherein the aqueous carrier comprises a lysis reagent configured to lyse the cells before or during step (d).
28 . The method of claim 27 , wherein the aqueous carrier or the aqueous sample comprises reverse transcriptase.
29 . The method of claim 25 , further comprising compiling the nucleic acid sequences associated with single cells into a genome library.