Microfluidic devices and methods of use in the formation and control of nanoreactors

Patent metadata

Field	Value
Publication number	US-10633652-B2
Application number	US-201715694108-A
Country	US
Kind code	B2
Filing date	Sep 1, 2017
Priority date	Jan 11, 2006
Publication date	Apr 28, 2020
Grant date	Apr 28, 2020

Abstract

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.

First claim

The invention claimed is:

1. A method for detecting a polymerase chain reaction (PCR) product in an aqueous droplet, the method comprising:
providing a repertoire of barcoded compounds attached to beads in an aqueous fluid;
emulsifying the aqueous fluid to generate a plurality of aqueous droplets, each aqueous droplet comprising one of the beads;
conducting inside the plurality of aqueous droplets an amplification reaction involving one or more of the repertoire of barcoded compounds, wherein the conducting step results in barcoded amplicons that are released from the beads;
breaking the plurality of aqueous droplets to pool the barcoded amplicons; and
analyzing the barcoded amplicons.

2. The method of claim 1 , wherein the aqueous droplets are surrounded by the immiscible carrier fluid.

3. The method of claim 2 , wherein emulsifying comprises flowing the aqueous fluid through a channel into an immiscible carrier fluid that is injected from a direction substantially perpendicular to the channel.

4. The method of claim 2 , wherein the immiscible carrier fluid is an oil.

5. The method of claim 1 , wherein analyzing comprises sorting the barcoded amplicons based on a presence of a plurality of fluorescent reporter molecules.

6. The method of claim 1 , wherein the at least one of the aqueous droplets further comprises a label.

7. The method of claim 6 , wherein the label is an organic dye.

8. The method of claim 6 , wherein the barcoded amplicons of the amplification reaction comprise the label.

9. The method of claim 1 , wherein the amplicons are released from the beads by heating.

10. The method of claim 9 , wherein heating occurs during the amplification reaction.

11. The method of claim 1 , wherein the amplification reaction involves thermocycling.

12. The method of claim 1 , wherein the repertoire of compounds comprise nucleic acids.

13. The method of claim 12 , wherein the nucleic acids comprise genomic DNA, cDNA, or RNA.

14. The method of claim 1 , wherein the amplicons comprise DNA or cDNA.

15. The method of claim 1 , wherein each aqueous droplet comprises a primer pair.

16. The method of claim 15 , wherein the primer pairs are different between each aqueous droplet.

17. The method of claim 1 , wherein the aqueous fluid further comprises a polymerase and a plurality of deoxynucleotides.

18. The method of claim 6 , wherein the label is a fluorescent label.

Assignees

• Bio Rad Laboratories

Inventors

• Link Darren Roy
• Boitard Laurent
• Branciforte Jeffrey
• Charles Yves
• Feke Gilbert
• Lu John Q
• Marran David
• Tabatabai Ahmadali
• Weiner Michael
• Hinz Wolfgang
• Rothberg Jonathan M

Classifications

• B01J19/0046

  Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays (synthesis methods per se C40B50/00) · CPC title

• G01N2500/10

  involving cells · CPC title

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• C40B70/00

  Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes · CPC title

• B01J2219/00459

  Beads · CPC title