Assays and other reactions involving droplets
The present invention generally relates to droplets and/or emulsions, such as multiple emulsions. In some cases, the droplets and/or emulsions may be used in assays, and in certain embodiments, the droplet or emulsion may be hardened to form a gel. In some aspects, a heterogeneous assay can be performed using a gel. For example, a droplet may be hardened to form a gel, where the droplet contains a cell, DNA, or other suitable species. The gel may be exposed to a reactant, and the reactant may interact with the gel and/or with the cell, DNA, etc., in some fashion. For example, the reactant may diffuse through the gel, or the hardened particle may liquefy to form a liquid state, allowing the reactant to interact with the cell. As a specific example, DNA contained within a gel particle may be subjected to PCR (polymerase chain reaction) amplification, e.g., by using PCR primers able to bind to the gel as it forms. As the DNA is amplified using PCR, some of the DNA will be bound to the gel via the PCR primer. After the PCR reaction, unbound DNA may be removed from the gel, e.g., via diffusion or washing. Thus, a gel particle having bound DNA may be formed in one embodiment of the invention.
1. A composition, comprising:
a collection of hardened droplets comprising a library of distinguishable nucleic acid species, wherein a hardened droplet of said collection of hardened droplets comprises a nucleic acid species distinguishable from other nucleic acid species in other hardened droplets of said collection of hardened droplets, and wherein said collection of hardened droplets is contained within a plurality of aqueous droplets in a water-in-oil emulsion.
2. The composition of claim 1 , wherein said collection of hardened droplets is a collection of gel particles.
3. The composition of claim 2 , wherein said collection of gel particles comprises a polyacrylamide polymer.
4. The composition of claim 3 , wherein said polyacrylamide polymer comprises a cross-linked polyacrylamide polymer.
5. The composition of claim 1 , wherein said nucleic acids species is covalently coupled to said hardened droplet.
6. The composition of claim 1 , wherein said nucleic acid species is coupled to said hardened droplet by an acrydite linkage.
7. The composition of claim 1 , wherein said plurality of aqueous droplets further comprises one or more reagents for facilitating a reaction on a nucleic acid molecule.
8. The composition of claim 7 , wherein said one or more reagents comprise a buffer, a salt, dNTPs, a primer, a protein, or an enzyme.
9. The composition of claim 8 , wherein said enzyme is a DNA polymerase or a reverse transcriptase.
10. The composition of claim 1 , wherein at least some of said plurality of aqueous droplets do not comprise a hardened droplet.
11. The composition of claim 1 , wherein at least 75% of said plurality of aqueous droplets each comprise at least one hardened droplet of said collection of hardened droplets.
12. The composition of claim 1 , wherein said nucleic acid species comprises a primer sequence.
13. The composition of claim 1 , wherein said hardened droplet is dissolvable such that, when dissolved, said nucleic acid species is released into an aqueous droplet of said plurality of aqueous droplets.
14. The composition of claim 1 , wherein said collection of hardened droplets is monodisperse.
15. The composition of claim 1 , wherein no more than about 10% of said hardened droplets of said collection of hardened droplets have an average diameter that is greater than about 10% of said average hardened droplet diameter of said collection of hardened droplets.
16. The composition of claim 1 , wherein said collection of hardened droplets comprises a library of at least about 100,000 distinguishable species.
17. The composition of claim 1 , wherein said water-in-oil emulsion comprises a fluorocarbon oil.
18. The composition of claim 1 , wherein said water-in-oil emulsion comprises a surfactant that prevents coalescence of aqueous droplets of said plurality of aqueous droplets.
19. The composition of claim 1 , wherein said water-in-oil emulsion comprises a fluorosurfactant.
20. The composition of claim 1 , wherein said plurality of aqueous droplets further comprises a plurality of cells.
21. The composition of claim 20 , wherein an aqueous droplet of said plurality of aqueous droplets comprises a hardened droplet of said collection of hardened droplets and a cell of said plurality of cells.
22. A composition, comprising:
a collection of gel particles comprising a plurality of nucleic acid primers, wherein said collection of gel particles is contained within a plurality of aqueous droplets in a water-in-oil emulsion.
23. The composition of claim 22 , wherein said plurality of aqueous droplets further comprises a plurality of cells.
24. The composition of claim 22 , wherein said plurality of aqueous droplets further comprises one or more reagents for facilitating a reaction on a nucleic acid molecule.
25. The composition of claim 24 , wherein said one or more reagents comprise a buffer, a salt, dNTPs, a primer, a protein, or an enzyme.
26. The composition of claim 25 , wherein said enzyme is a DNA polymerase or a reverse transcriptase.
27. The composition of claim 22 , wherein nucleic acid primers of said plurality of nucleic acid primers are covalently coupled to said collection of gel particles.
28. The composition of claim 22 , wherein an aqueous droplet of said plurality of aqueous droplets comprises a gel particle of said collection of gel particles and a cell.
29. A composition, comprising:
a plurality of gel particles contained within a plurality of aqueous droplets in a water-in-oil emulsion, wherein a gel particle of said plurality of gel particles comprises a nucleic acid species distinguishable from other nucleic acid species in other gel particles of said plurality of gel particles, and wherein said plurality of gel particles comprises at least 100,000 distinguishable species.
30. The composition of claim 29 , wherein an aqueous droplet of said plurality of aqueous droplets comprises a gel particle of said collection of gel particles and a cell.