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 method, comprising:
providing a gel droplet containing a species, wherein the gel droplet is within a surrounding droplet; and
exposing the species to a reactant which is reactive with the species.
2. The method of claim 1 , wherein providing the gel droplet containing the species comprises:
providing a fluid droplet containing the species; and
causing the fluid droplet to form the gel droplet containing the species, wherein the gel droplet is within the surrounding droplet.
3. The method of claim 1 , wherein exposing the species to the reactant occurs after releasing the species from the gel droplet.
4. The method of claim 1 , wherein the species is a first nucleic acid, a small interfering ribonucleic acid (siRNA), a ribonucleic acid interference (RNAi) nucleic acid, or a deoxyribonucleic acid (DNA).
5. The method of claim 4 , wherein the reactant is a second nucleic acid.
6. The method of claim 1 , wherein the reactant is a nucleic acid.
7. The method of claim 1 , wherein the gel droplet comprises alginate, polyacrylamide or poly N-isopropylpolyacrylamide.
8. The method of claim 1 , wherein the surrounding droplet forms part of an emulsion.
9. The method of claim 1 , wherein the gel droplet comprises alginate.
10. The method of claim 5 , wherein the second nucleic acid is a primer and the first nucleic acid is a target nucleic acid molecule, and wherein the method further comprises using the primer and the target nucleic acid molecule to perform one or more reactions selected from the group consisting of a polymerase chain reaction (PCR), a reverse transcription polymerase chain reaction (rtPCR), and a whole genome amplification.
11. The method of claim 10 , wherein a library of nucleic acid molecules is formed.
12. The method of claim 1 , wherein the species is a nucleic acid and the reactant is a fluorescent entity configured to bind to at least a portion of the nucleic acid.
13. The method of claim 1 , wherein exposing the species to the reactant comprises causing the reactant to diffuse into the gel droplet.
14. The method of claim 1 , wherein exposing the species to the reactant comprises causing the gel droplet to form a liquid droplet.
15. The method of claim 14 , wherein causing the gel droplet to form the liquid droplet comprises exposing the gel droplet to an environmental change.
16. The method of claim 15 , wherein the environmental change comprises one or more members selected from the group consisting of a change in temperature, a change in pH, and a change of osmotic pressure.
17. The method of claim 10 , further comprising thermally cycling the gel droplet.
18. The method of claim 1 , wherein the reactant is a polynucleotide template.
19. The method of claim 1 , wherein the gel droplet further comprises amplification reagents.
20. The method of claim 19 , wherein the amplification reagents comprise a polymerase and nucleotides.
21. The method of claim 2 , wherein causing the fluid droplet to form the gel droplet comprises use of a gelation initiator.
22. The method of claim 21 , wherein the gelation initiator comprises a reducing agent.
23. The method of claim 21 , wherein the gelation initiator is selected from the group consisting of ammonium persulfate, tetramethylethylenediamine (TEMED), and Ca2+.
24. The method of claim 1 , wherein the gel droplet comprises polyacrylamide.
25. The method of claim 2 , wherein the fluid droplet comprises a fluoro surfactant.
26. The method of claim 2 , wherein the fluid droplet comprises an ammonium carboxylate salt surfactant.
27. The method of claim 2 , wherein the fluid droplet comprises a surfactant selected from the group consisting of Span80, Span80/Tween-20, Span80/Triton X-100, Abil EM90, Abil we09, polyglycerol polyricinoleate, Tween-85, 749 Fluid, and an ammonium carboxylate salt of Krytox.
28. The method of claim 10 , wherein the one or more reactions is performed using a polymerase.
29. The method of claim 28 , wherein the polymerase is a Phi29 polymerase.
30. The method of claim 19 , wherein the first nucleic acid is a polynucleotide of a plurality of polynucleotides from a whole genome.
31. The method of claim 30 , wherein the one or more reactions comprise amplifying a plurality of different regions of the plurality of polynucleotides.
32. The method of claim 30 , wherein the plurality of polynucleotides comprises deoxyribonucleic acid molecules.
33. The method of claim 30 , wherein the whole genome comprises a polymorphism.
34. The method of claim 30 , wherein the whole genome is a mammalian genome.
35. The method of claim 30 , wherein the whole genome is a primate genome.
36. The method of claim 30 , wherein the plurality of polynucleotides comprises ribonucleic acid molecules.
37. The method of claim 2 , wherein the fluid droplet comprises a surfactant that prevents droplet coalescence.
38. The method of claim 37 , wherein the surfactant comprises perfluorinated polyether.
39. A method, comprising:
providing a fluid droplet containing a species; causing the fluid droplet to form a gel droplet containing the species, wherein the gel droplet is within a surrounding droplet; and
exposing the species to a reactant which is reactive with the species.