Inertial droplet generation and particle encapsulation

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.

What is claimed is: 
     
         1 . A method of generating liquid droplets containing two or more types of particles, the method comprising:
 focusing a bead fluid having beads suspended therein into a first ordered stream of beads within a first microchannel;   focusing a cell fluid having cells suspended therein into a second ordered stream of cells within a second microchannel; and   merging the first ordered stream with the second ordered stream to form a plurality of droplets having a predetermined number of cells and beads within each droplet.   
     
     
         2 . The method of  claim 1 , wherein the first microchannel has a minimum cross-sectional dimension D and the beads have a cross-sectional dimension that is at least about 0.1 D. 
     
     
         3 . The method of  claim 2 , wherein the cells have a cross-sectional dimension that is at least about 0.1 D. 
     
     
         4 . The method of  claim 2 , wherein merging of the first ordered stream and the second ordered stream comprises contacting the first ordered stream and the second ordered stream with a third fluid immiscible in the first fluid and the second fluid. 
     
     
         5 . The method of  claim 1 , wherein focusing the beads comprises passing the beads through a first inertial focusing portion of the first microchannel. 
     
     
         6 . The method of  claim 1 , wherein focusing the cells comprises passing the cells through a second inertial focusing portion of the second microchannel. 
     
     
         7 . The method of  claim 1 , wherein focusing the beads comprises passing the beads through a first inertial focusing portion of the first microchannel, and wherein focusing the cells comprises passing the cells through a second inertial focusing portion of the second microchannel. 
     
     
         8 . The method of  claim 7 , wherein at least one of the first inertial focusing portion of the first microchannel and the second inertial focusing portion of the second microchannel has a curved region. 
     
     
         9 . The method of  claim 8 , wherein each curved region is independently S-shaped, sigmoidal, sinusoidal, or spiral shaped. 
     
     
         10 . The method of  claim 1 , wherein the beads comprise nucleotide fragments. 
     
     
         11 . The method of  claim 10 , wherein the nucleotide fragments comprise a barcode region, an index region, and a capture region. 
     
     
         12 . The method of  claim 11 , wherein the barcode region of each nucleotide fragment is at least about six nucleotides in length. 
     
     
         13 . The method of  claim 11 , wherein the index region of each nucleotide fragment is at least about four nucleotides in length. 
     
     
         14 . The method of  claim 11 , wherein the capture region comprises poly-T nucleotides and is at least about ten nucleotides in length. 
     
     
         15 . The method of  claim 1 , wherein the predetermined number of cells is one and the predetermined number of beads is one. 
     
     
         16 . The method of  claim 15 ,
 wherein each bead has a Reynolds number of at least about 1,   wherein the Reynolds number of a bead is defined as   
       
         
           
             
               Re 
               = 
               
                 
                   ρ 
                    
                   
                       
                   
                    
                   
                     U 
                     m 
                   
                    
                   H 
                 
                 μ 
               
             
           
         
         where ρ is the density of the bead fluid, U m  is the maximum flow speed of the bead fluid, H is the hydraulic diameter of the bead fluid, and μ is the dynamic viscosity of the bead fluid, 
         wherein each cell has a Reynolds number of at least about 1, and 
         wherein the Reynolds number of a cell is defined as 
       
       
         
           
             
               Re 
               = 
               
                 
                   ρ 
                    
                   
                       
                   
                    
                   
                     U 
                     m 
                   
                    
                   H 
                 
                 μ 
               
             
           
         
         where ρ is the density of the cell fluid, U m  is the maximum flow speed of the cell fluid, H is the hydraulic diameter of the cell fluid, and μ is the dynamic viscosity of the cell fluid. 
       
     
     
         17 . The method of  claim 16 , wherein the proportion of the plurality of droplets containing k 1  beads and k 2  cells is greater than (λ 1   k1 exp)(−λ 1 !)) (λ 2   k2 exp(−λ 2 )/(k 2 !)), where λ 1  is the average number of the beads per droplet and λ 2  is the average number of the cells per droplet. 
     
     
         18 . The method of  claim 1 , wherein the flow rate of the first ordered stream is at least about 10 μL/min. 
     
     
         19 . The method of  claim 1 , wherein the flow rate of the second ordered stream is at least about 10 μL/min. 
     
     
         20 . A droplet generation system, comprising:
 a first inlet connected to a first inertial focusing microchannel disposed in a substrate;   a first flow source configured to drive a bead fluid containing beads through the first inertial focusing microchannel;   a second inlet connected to a second inertial focusing microchannel disposed in the substrate, wherein the first inertial focusing microchannel is connected to the second inertial focusing microchannel for forming the bead fluid and the cell fluid into a plurality of droplets;   a second flow source configured to drive a cell fluid containing cells through the second inertial focusing microchannel.   
     
     
         21 . The system of  claim 20 , wherein the first inertial focusing microchannel comprises a side wall having an irregular shape. 
     
     
         22 . The system of  claim 21 , wherein the irregular shape comprises a first irregularity protruding from a baseline surface away from a longitudinal axis of the inertial focusing microchannel with the irregular shape. 
     
     
         23 . The system of  claim 22 , wherein each irregular shape is selected from the group consisting of trapezoidal, triangular, rounded, and rectangular. 
     
     
         24 . The system of  claim 20 , wherein the second inertial focusing microchannel comprises a side wall having an irregular shape. 
     
     
         25 . The system of  claim 20 , wherein at least one of the first inertial focusing microchannel and the second inertial focusing microchannel has an expansion/contraction region having a side wall, wherein the side wall has a stepped surface. 
     
     
         26 . The system of  claim 20 , wherein at least one of the first inertial focusing microchannel and the second inertial focusing microchannel has an expansion/contraction region having a side wall, wherein the side wall has a curved surface. 
     
     
         27 . The system of  claim 20 , wherein at least one of the first inertial focusing microchannel and the second inertial focusing microchannel has a curved region. 
     
     
         28 . The system of  claim 27 , wherein each curved region is independently S-shaped, sinusoidal, sigmoidal, or spiral shaped. 
     
     
         29 . The system of  claim 27 , wherein the curved region has a Dean number of up to about 30.