Systems and methods for generating droplets and performing digital analyses

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.

What is claimed is: 
     
         1 . A method comprising:
 generating a plurality of droplets within a collecting container at a rate of at least 1 million droplets per minute, each of the plurality of droplets comprising an aqueous mixture for a digital analysis of nucleic acid material.   
     
     
         2 . The method of  claim 1 , wherein upon generation, the plurality of droplets is stabilized in position in a close-packed format within a continuous phase, within a region of the collecting container. 
     
     
         3 . The method of  claim 1 , wherein generating the plurality of droplets comprises driving a sample fluid through a membrane comprising a distribution of holes, the membrane coupled to a reservoir outlet of a reservoir for the sample fluid, and the reservoir aligned with the collecting container. 
     
     
         4 . The method of  claim 3 , wherein the distribution of holes has a density less than 5000 holes per cm 2  and a hole-to-hole spacing greater than 30 micrometers. 
     
     
         5 . The method of  claim 3 , wherein each hole in the distribution of holes has a diameter from 1 through 3 micrometers. 
     
     
         6 . The method of  claim 3 , wherein driving the sample fluid through the membrane comprises spinning the sample fluid, the membrane, and the collecting container within a centrifuge in a first direction of rotation, and reversing the direction of rotation, thereby adjusting an equilibrium surface profile of an emulsion comprising the plurality of droplets within the collecting container. 
     
     
         7 . The method of  claim 3 , wherein driving the sample fluid through the membrane comprises spinning the sample fluid, the membrane, and the collecting container within a centrifuge at a first rotational velocity and at a second rotational velocity less than the first rotational velocity, thereby adjusting an equilibrium surface profile of an emulsion comprising the plurality of droplets within the collecting container. 
     
     
         8 . The method of  claim 1 , wherein the collecting container has a volumetric capacity from 10 through 300 microliters, and wherein each of the plurality of droplets has a characteristic diameter from 10 through 30 micrometers. 
     
     
         9 . The method of  claim 1 , further comprising transmitting heat to and from the plurality of droplets, within the collecting container, during a heat transmission operation, wherein the temperature varies within a temperature range from 4° C. to 95° C. during the heat transmission operation, and wherein individual droplets of the plurality of droplets remain unmerged with adjacent droplets in a close-packed format during the heat transmission operation. 
     
     
         10 . The method of  claim 1 , wherein generating the plurality of droplets comprises generating greater than 25 million droplets within the collecting container. 
     
     
         11 . The method of  claim 1 , wherein generating the plurality of droplets comprises transmitting two dimensional arrays of droplets toward a closed end of the collecting container, thereby stabilizing the plurality of droplets in a three dimensional close-packed format toward the closed end of the collecting container. 
     
     
         12 . A system for generating droplets, the system comprising:
 a first substrate defining a reservoir comprising a reservoir inlet and a reservoir outlet;   a membrane coupled to the reservoir outlet and comprising a distribution of holes; and   a second substrate comprising an opening configured to retain a collecting container in alignment with the reservoir outlet,
 wherein the system comprises:
 a first operation mode wherein the first substrate is coupled with the second substrate and encloses the collecting container, with the reservoir outlet seated within the collecting container, 
 a second operation mode wherein the reservoir contains a sample fluid comprising an aqueous mixture for a digital analysis of nucleic acid material, and 
 a third operation mode wherein the membrane generates a plurality of droplets within the collecting container at a rate of at least 1 million droplets per minute in response to a force applied to the sample fluid, and 
 a fourth operation mode wherein the plurality of droplets is stabilized in position in a close-packed format within a region of the collecting container. 
 
   
     
     
         13 . The system of  claim 12 , wherein the membrane is bonded to the reservoir outlet at a perimeter of the reservoir outlet. 
     
     
         14 . The system of  claim 12 , wherein the distribution of holes has a density less than 10,000 holes per cm 2  and a hole-to-hole spacing greater than 10 micrometers. 
     
     
         15 . The system of  claim 12 ,
 wherein the first substrate comprises a set of reservoirs comprising the reservoir;   wherein the system further comprises a set of membranes comprising the membrane, the set of membranes paired with and bonded to outlets of the set of reservoirs; and   wherein the second substrate comprises a set of openings comprising the opening, wherein the set of openings is configured to retain a set of collecting containers in alignment with the set of reservoirs.   
     
     
         16 . The system of  claim 15 , wherein a reservoir number of the set of reservoirs is different from a collecting container number of the set of collecting containers, wherein the first substrate comprises two or more fluidic pathways from the set of reservoirs to the set of membranes. 
     
     
         17 . The system of  claim 12 , wherein the second substrate is configured as a spacer separating the reservoir outlet from a base surface of the collecting container. 
     
     
         18 . A method comprising:
 generating a plurality of droplets within a collecting container, each of the plurality of droplets comprising an aqueous mixture for a digital analysis of nucleic acid material,   wherein generating the plurality of droplets comprises driving the aqueous mixture through a distribution of holes of a track-etched membrane to stabilized positions, toward a closed end of the collecting container, and   wherein the plurality of droplets is characterized by less than 13% coefficient of variation for polydispersity.   
     
     
         19 . The method of  claim 18 , wherein generating the plurality of droplets comprises generating the plurality of droplets at a rate of at least 600,000 droplets per minute. 
     
     
         20 . The method of  claim 18 , wherein the plurality of droplets is characterized by less than 10% occupancy of droplets by said nucleic acid material.