Systems and methods for generating droplets and performing digital analyses
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.
1. A method comprising:
(a) providing a device, wherein said device comprises:
(i) a reservoir comprising sample fluid, wherein said sample fluid comprises a plurality of nucleic acid molecules, wherein said plurality of nucleic acid molecules comprise a plurality of target nucleic acid molecules;
(ii) a collecting container comprising a continuous phase; and
(iii) a membrane comprising a plurality of holes, wherein said membrane is in fluid communication with said collecting container; and
(b) subjecting said sample fluid to flow through said plurality of holes of said membrane to generate a plurality of droplets within said continuous phase of said collecting container at a rate of at least 500,000 droplets per minute,
wherein a droplet of said plurality of droplets comprises a target nucleic acid molecule of said plurality of target nucleic acid molecules, 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.
2. The method of claim 1 , wherein a distribution of said plurality of holes across said membrane has a density of less than 5,000 holes per cm 2 .
3. The method of claim 1 , wherein a hole of said plurality of holes has a diameter of 1 micrometer to 3 micrometers.
4. The method of claim 1 , wherein (b) comprises spinning said device within a centrifuge in a first direction of rotation.
5. The method of claim 4 , further comprising spinning said device within a centrifuge in a second direction of rotation, wherein said second direction of rotation is a reverse direction relative to said first direction.
6. The method of claim 1 , wherein said collecting container has a volumetric capacity from 10 microliters to 300 microliters.
7. The method of claim 1 , wherein a droplet of said plurality of droplets has a diameter of 10 micrometers to 30 micrometers.
8. The method of claim 1 , further comprising heating said plurality of droplets.
9. The method of claim 1 , wherein said plurality of droplets comprises at least 25 million droplets.
10. The method of claim 1 , wherein said membrane has a thickness of 25 micrometers to 125 micrometers.
11. The method of claim 1 , wherein said plurality of droplets is stabilized within said collecting container as a gel.
12. The method of claim 1 , wherein (b) comprises generating said plurality of droplets within said continuous phase of said collecting container at a rate of at least 1 million droplets per minute.
13. The method of claim 12 , wherein said rate is at least 2 million droplets per minute.
14. The method of claim 1 , wherein said sample fluid is subjected to flow using pressure applied to said sample fluid in said reservoir.
15. The method of claim 1 , wherein a first subset of droplets of said plurality of droplets comprises a target nucleic acid molecule of said plurality of target nucleic acid molecules.
16. The method of claim 15 , wherein a second subset of droplets of said plurality of droplets comprises no target nucleic acid molecule of said plurality of target nucleic acid molecules.
17. A method comprising:
(a) providing a device, wherein said device comprises:
(i) a reservoir comprising sample fluid, wherein said sample fluid comprises a plurality of nucleic acid molecules, wherein said plurality of nucleic acid molecules comprise a plurality of target nucleic acid molecules;
(ii) a collecting container comprising a continuous phase; and
(iii) a membrane comprising a plurality of holes, wherein said membrane is disposed between said reservoir and said collecting container; and
(b) subjecting said sample fluid to flow through said plurality of holes of said membrane to generate a plurality of droplets within said continuous phase of said collecting container,
wherein a droplet of said plurality of droplets comprises a target nucleic acid molecule of said plurality of target nucleic acid molecules, and
wherein said plurality of droplets is characterized by a coefficient of variation for polydispersity of less than 13%, 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.
18. The method of claim 17 , wherein a distribution of said plurality of holes across said membrane has a density of less than 5000 holes per cm 2 .
19. The method of claim 17 , wherein a hole of said plurality of holes has a diameter of 1 micrometer to 3 micrometers.
20. The method of claim 17 , wherein (b) comprises spinning said device within a centrifuge in a first direction of rotation.
21. The method of claim 20 , further comprising spinning said device within a centrifuge in a second direction of rotation, wherein said second direction of rotation is a reverse direction relative to said first direction.
22. The method of claim 17 , wherein said collecting container has a volumetric capacity of 10 microliters to 300 microliters.
23. The method of claim 17 , wherein a droplet of said plurality of droplets has a diameter from 10 micrometers to 30 micrometers.
24. The method of claim 17 , further comprising heating said plurality of droplets.
25. The method of claim 17 , wherein said plurality of droplets comprises at least 25 million droplets.
26. The method of claim 17 , wherein said membrane comprises a thickness of 25 micrometers to 125 micrometers.
27. The method of claim 17 , wherein said plurality of droplets is stabilized within said collecting container as a gel.
28. The method of claim 17 , wherein said sample fluid is subjected to flow using pressure applied to said sample fluid in said reservoir.
29. The method of claim 17 , wherein a first subset of droplets of said plurality of droplets comprises a target nucleic acid molecule of said plurality of target nucleic acid molecules.
30. The method of claim 29 , wherein a second subset of droplets of said plurality of droplets comprises no target nucleic acid molecule of said plurality of target nucleic acid molecules.