# US 12,000,842 B2 · Systems and methods for generating droplets and performing digital analyses > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,000,842 B2 - Document ID: US12000842B2 - Status: Granted - Kind: B2 - Application number: 17/711,417 - Filed: 2022-04-01 - Published: 2022-09-08 - Granted: 2024-06-04 - Earliest claimed: 2021-03-05 - Assignees: Enumerix, Inc. - Applicants: Enumerix, Inc. - Inventors: Hei Mun Christina Fan; Janice Hoiyi Lai; Sixing Li; Stephen P. A. Fodor; Eleen Yee Lam Shum - Web page: https://patentgrep.com/patent/US12000842B2 ## CPC - B01L 3/50255: {Multi-well filtration} - B01L 3/502753: {characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation} - B01F 25/4522: {the components being pressed through porous bodies, e.g. flat plates, blocks or cylinders, which obstruct the whole diameter of the tube (B01F25/45243 takes precedence)} - B01F 25/4523: {the components being pressed through sieves, screens or meshes which obstruct the whole diameter of the tube} - B01F 31/10: with a mixing receptacle rotating alternately in opposite directions - B01F 33/305: {using mixing means not otherwise provided for (B01F25/00, B01F27/00, B01F29/00, B01F31/00, B01F33/301 - B01F33/3045, B01F33/40 and B01F33/45 take precedence)} - B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces} - B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics} - B01L 3/50855: {using modular assemblies of strips or of individual wells} - B01L 7/52: {with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples} - C12Q 1/6844: Nucleic acid amplification reactions - G01N 21/645: {Specially adapted constructive features of fluorimeters} - G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)} - G01N 33/689: {related to pregnancy or the gonads} - B01F 2101/23: Mixing of laboratory samples e.g. in preparation of analysing or testing properties of materials - B01L 2200/026: Fluid interfacing between devices or objects, e.g. connectors, inlet details - B01L 2200/027: for microfluidic devices - B01L 2200/028: Modular arrangements - B01L 2200/0673: Handling of plugs of fluid surrounded by immiscible fluid - B01L 2200/0689: Sealing - B01L 2300/0681: Filter - B01L 2300/0832: cylindrical, tube shaped - B01L 2400/0409: centrifugal forces - B01L 2400/0487: fluid pressure, pneumatics - G01N 2021/6482: {Sample cells, cuvettes} - 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) ## Abstract This disclosure provides for devices, methods, and systems for generating a plurality of droplets within a collecting container at an extremely high rate (e.g., of at least 1 million droplets per minute, etc.), each of the plurality of droplets comprising an aqueous mixture for a digital analysis, wherein upon generation, the plurality of droplets is stabilized in position within a region of the collecting container. The inventions enable partitioning of samples for digital analyses at unprecedented rates, where readout of signals from targets within such partitions can still be achieved in accordance with various assays. ## Claims ### Claim 1 A method comprising: performing a digital analysis of target nucleic acid material from a sample, wherein performing the digital analysis comprises: (a) generating an emulsion within a collection container, wherein the emulsion comprises a plurality of droplets, wherein each droplet of the plurality of droplets is surrounded by an immiscible film, and wherein the plurality of droplets is in a continuous phase immiscible with the immiscible film, and wherein the plurality of droplets comprises at least 20 million droplets, wherein the at least 20 million droplets comprise portions of the sample and amplification reaction reagents, and wherein a droplet of the at least 20 million droplets comprises the target nucleic acid material; (b) amplifying the target nucleic acid material in the droplet; and (c) optically interrogating via light sheet imaging the emulsion within the collection container comprising the at least 20 million droplets across a plurality of cross-sections of the emulsion, wherein (a)-(c) are performed in less than or equal to 2.5 hours. ### Claim 2 (depends on 1) The method of claim 1, wherein the sample comprises a mixture of fetal genetic material and maternal genetic material. ### Claim 3 (depends on 1) The method of claim 1, wherein generating the emulsion comprising the plurality of droplets comprises generating the plurality of droplets at an average rate of at least 1 million droplets per minute. ### Claim 4 (depends on 1) The method of claim 1, wherein the emulsion is a gel. ### Claim 5 (depends on 1) The method of claim 1, wherein the emulsion is characterized by a transmissivity greater than 60% transmissivity of light without refractive index matching of components of the emulsion. ### Claim 6 (depends on 1) The method of claim 1, wherein the plurality of cross-sections comprises at least 500 cross-sections. ### Claim 7 (depends on 1) The method of claim 1, wherein (b) comprises heating the emulsion. ### Claim 8 (depends on 1) The method of claim 1, wherein (c) comprises detecting optical signals from only a subset of droplets of the plurality of droplets. ### Claim 9 (depends on 1) The method of claim 1, further comprising using said optically interrogating of (c) to diagnose a disorder. ### Claim 10 (depends on 1) The method of claim 1, wherein the plurality of droplets comprises a plurality of cells, and wherein the target nucleic acid material is derived from the plurality of cells. ### Claim 11 A method comprising: performing a digital analysis of target nucleic acid material from a sample, wherein performing the digital analysis comprises: generating an emulsion within a collection container, wherein the emulsion comprises a plurality of droplets, wherein each droplet of the plurality of droplets is surrounded by an immiscible film, and wherein the plurality of droplets is in a continuous phase immiscible with the immiscible film, and wherein the plurality of droplets comprises at least 10 million droplets, wherein the at least 20 million droplets comprise portions of the sample and amplification reaction reagents, and wherein a droplet of the at least 20 million droplets comprises the target nucleic acid material; amplifying the target nucleic acid material in the droplet; and optically interrogating via light sheet imaging the emulsion within the collection container comprising the at least 10 million droplets across a plurality of cross-sections of the emulsion using a first channel and a second channel of a detection system interacting with the emulsion, wherein the optically interrogating for each of the first channel and the second channel is performed within 50 seconds. ### Claim 12 (depends on 11) The method of claim 11, wherein generating the plurality of droplets comprises generating at least 20 million droplets. ### Claim 13 (depends on 11) The method of claim 11, wherein generating the emulsion comprises driving the sample through a distribution of holes of a membrane under centrifugal force. ### Claim 14 (depends on 13) The method of claim 13, wherein the membrane comprises a track-etched membrane and wherein the distribution of holes comprises a hole-to-hole spacing greater than 30 micrometers. ### Claim 15 (depends on 11) The method of claim 11, further comprising optically interrogating the at least 10 million droplets using, a third channel and a fourth channel, wherein readout for each of the first channel, the second channel, the third channel, and the fourth channel, individually, is performed within a duration of at most 50 seconds. ### Claim 16 (depends on 11) The method of claim 11, wherein the plurality of cross-sections comprises at least 400 cross-sections.