# US 11,123,733 B2 · Inertial droplet generation and particle encapsulation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,123,733 B2 - Document ID: US11123733B2 - Status: Granted - Kind: B2 - Application number: 16/691,405 - Filed: 2019-11-21 - Published: 2020-03-19 - Granted: 2021-09-21 - Earliest claimed: 2015-11-10 - Assignees: Illumina, Inc. - Applicants: Illumina, Inc. - Inventors: Hamed Amini; Arash Jamshidi; Tarun Kumar Khurana; Foad Mashayekhi; Yir-Shyuan Wu - Web page: https://patentgrep.com/patent/US11123733B2 ## CPC - 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} - B01F 23/41: Emulsifying - 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 - B01L 3/502776: {specially adapted for focusing or laminating flows} - B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics} - B01J 2219/00468: by manipulation of individual beads - B01J 2219/005: Beads - B01J 2219/00722: Nucleotides - B01L 2200/0636: Focussing flows, e.g. to laminate flows - B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles - B01L 2200/0652: Sorting or classification of particles or molecules - B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions - B01L 2300/0858: Side walls - B01L 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution - B01L 2300/0883: Serpentine channels - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE - B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06) - B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS ## Abstract Described are microfluidic devices and methods for providing a predetermined number of microspheres or beads, together with a cell, within a fluid droplet being processed. The system may provide each droplet with a single bead and a single cell, and the bead may contain DNA or other reagents for later identifying the specific cell associated with that bead. ## Claims ### Claim 1 A method of generating liquid droplets containing beads and cells, the method comprising: inertial focusing a bead fluid having beads suspended therein into a first ordered stream of beads within a first inertial focusing microchannel; inertial focusing a cell fluid having cells suspended therein into a second ordered stream of cells within a second inertial focusing microchannel; and merging the first ordered stream with the second ordered stream at a convergence channel at a combined flow rate and forming a plurality of droplets at a junction, wherein each droplet comprises a first predetermined number of one or more cells and a second predetermined number of one or more beads within each droplet, wherein the first inertial focusing microchannel and the second inertial focusing microchannel are connected to the convergence channel and the convergence channel is connected to the junction, and wherein the combined flow rate is selected so that a width of the convergence channel divided by a width of the first inertial focusing microchannel is proportional to the combined flow rate of the cell fluid and the bead fluid divided by a flow rate of the bead fluid to control the number of beads per droplet. ### Claim 2 (depends on 1) The method of claim 1, wherein forming the plurality of droplets at the junction comprises contacting the merged first ordered stream and second ordered stream with a third fluid immiscible in the bead fluid and the cell fluid. ### Claim 3 (depends on 1) The method of claim 1, wherein inertial focusing the beads comprises flowing the beads through a first inertial focusing portion of the first inertial focusing microchannel. ### Claim 4 (depends on 3) The method of claim 3, further comprising adjusting of the spacing between beads in the first ordered stream by flowing the bead fluid through an expansion or contraction region of the first inertial focusing microchannel after the first inertial focusing portion. ### Claim 5 (depends on 1) The method of claim 1, wherein inertial focusing the cells comprises flowing the cells through a second inertial focusing portion of the second inertial focusing microchannel. ### Claim 6 (depends on 5) The method of claim 5, further comprising adjusting of the spacing between cells in the second ordered stream by flowing the cell fluid through an expansion or contraction region of the second inertial focusing microchannel after the second inertial focusing portion. ### Claim 7 (depends on 1) The method of claim 1, wherein at least one of the first inertial focusing portion of the first inertial focusing microchannel and the second inertial focusing portion of the second inertial focusing microchannel has a curved region. ### Claim 8 (depends on 7) The method of claim 7, wherein each curved region is independently S-shaped, sigmoidal, sinusoidal, or spiral shaped. ### Claim 9 (depends on 1) The method of claim 1, wherein the beads comprise nucleotide fragments. ### Claim 10 (depends on 9) The method of claim 9, wherein the nucleotide fragments comprise a barcode region, an index region, and a capture region. ### Claim 11 (depends on 10) The method of claim 10, wherein the barcode region of each nucleotide fragment is at least about six nucleotides in length. ### Claim 12 (depends on 10) The method of claim 10, wherein the index region of each nucleotide fragment is at least about four nucleotides in length. ### Claim 13 (depends on 10) The method of claim 10, wherein the capture region comprises poly-T nucleotides and is at least about ten nucleotides in length. ### Claim 14 (depends on 1) The method of claim 1, wherein the first predetermined number of one or more cells is one and the second predetermined number of one or more beads is one. ### Claim 15 (depends on 1) The method of claim 1, wherein the flow rate of the first ordered stream is at least about 10 μL/min. ### Claim 16 (depends on 1) The method of claim 1, wherein the flow rate of the second ordered stream is at least about 10 μL/min. ### Claim 17 (depends on 1) The method of claim 1, wherein inertial focusing the beads further comprises co-flowing the bead fluid with a bead-free fluid. ### Claim 18 (depends on 1) The method of claim 1, wherein inertial focusing the cells further comprises co-flowing the cell fluid with a cell-free fluid. ### Claim 19 (depends on 1) The method of claim 1, further comprising: determining a first distribution of the number of beads in a droplet and a second distribution of the number of cells in a droplet; and adjusting the first ordered stream based on the determined first distribution and second distribution such that the first distribution and the second distribution achieve underdispersed-Poisson statistics. ### Claim 20 (depends on 19) The method of claim 19, wherein adjusting the first ordered stream achieves one bead and one cell within a single droplet. ### Claim 21 (depends on 19) The method of claim 19, wherein adjusting the first ordered stream comprises adjusting the concentration of the bead fluid by adjusting a first diluent introduced to the bead fluid. ### Claim 22 (depends on 19) The method of claim 19, further comprising: adjusting the second ordered stream based on the determined first distribution and second distribution, such that the first distribution and the second distribution achieve underdispersed-Poisson statistics. ### Claim 23 (depends on 22) The method of claim 22, wherein adjusting the second ordered stream achieves one bead and one cell within a single droplet. ### Claim 24 (depends on 22) The method of claim 22, wherein adjusting the second ordered stream comprises adjusting the concentration of the cell fluid by adjusting a second diluent introduced to the cell fluid.