Microfluidic devices and methods of use in the formation and control of nanoreactors
The present invention provides novel microfluidic devices and methods that are useful for performing high-throughput screening assays and combinatorial chemistry. The invention provides for aqueous based emulsions containing uniquely labeled cells, enzymes, nucleic acids, etc., wherein the emulsions further comprise primers, labels, probes, and other reactants. An oil based carrier-fluid envelopes the emulsion library on a microfluidic device, such that a continuous channel provides for flow of the immiscible fluids, to accomplish pooling, coalescing, mixing, sorting, detection, etc., of the emulsion library.
1. A method for detecting a polymerase chain reaction (PCR) product in an aqueous droplet, the method comprising:
forming a plurality of aqueous droplets in a microfluidic chip;
moving the plurality of aqueous droplets to a separate off-chip reservoir outside of the microfluidic chip, wherein at least one of the aqueous droplets comprises a nucleic acid, a polymerase, and a primer;
conducting a plurality of primer-extending reactions while the aqueous droplets are in the off-chip reservoir;
moving the plurality of aqueous droplets that have been subject to the plurality of primer-extending reactions from the off-chip reservoir and into a channel of a second microfluidic device that is operably associated with a detector; and
detecting a product of the PCR reaction via the detector in one or more of the aqueous droplets in the channel.
2. The method of claim 1 , wherein the aqueous droplets are surrounded by immiscible carrier fluid.
3. The method of claim 2 , wherein the immiscible carrier fluid is injected from a direction substantially perpendicular to the channel.
4. The method of claim 1 , wherein the aqueous droplets have the same composition.
5. The method of claim 1 , wherein the aqueous droplets have different compositions.
6. The method of claim 2 , wherein the immiscible carrier fluid is an oil.
7. The method of claim 6 , wherein the oil comprises a surfactant.
8. The method of claim 7 , wherein the surfactant is a fluorosurfactant.
9. The method of claim 2 , wherein the immiscible carrier fluid is a fluorinated oil.
10. The method of claim 2 , wherein the immiscible carrier fluid is injected using a syringe or pump.
11. The method of claim 2 , wherein the immiscible carrier fluid is injected using positive or negative pressure source.
12. The method of claim 1 , wherein the product of the PCR reaction detected in the one or more aqueous droplets comprises a plurality of fluorescent reporter molecules.
13. The method of claim 12 , wherein the fluorescent reporter molecules are separated by a polymerase from quencher molecules during the PCR reaction.
14. The method of claim 1 , wherein the nucleic acid is an antibiotic resistant gene.
15. The method of claim 1 , wherein the at least one of the aqueous droplets further comprises a label.
16. The method of claim 15 , wherein the label is an organic dye.
17. The method of claim 15 , wherein the product of the PCR reaction comprises the label.
18. The method of claim 1 , further comprising cloning the PCR product.
19. The method of claim 18 , wherein the PCR product is cloned into a vector.
20. The method of claim 19 , wherein the vector comprises a second antibiotic resistant gene.
21. The method of claim 18 , wherein the PCR product is cloned into a host.
22. The method of claim 21 , wherein the host is a bacterium.