Assays and other reactions involving droplets

Patent metadata

Field	Value
Publication number	US-10508294-B2
Application number	US-201715792218-A
Country	US
Kind code	B2
Filing date	Oct 24, 2017
Priority date	Mar 7, 2007
Publication date	Dec 17, 2019
Grant date	Dec 17, 2019

Abstract

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.

First claim

What is claimed is:

1. A composition comprising a plurality of droplets in an emulsion, wherein a droplet of the plurality of droplets comprises a cell and a particle, wherein the particle comprises one or more reagents for facilitating a reaction on a nucleic acid molecule of the cell.

2. The composition of claim 1 , wherein some of the plurality of droplets are unoccupied by a particle.

3. The composition of claim 1 , wherein the particle is a gel particle, and wherein the gel particle comprises the one or more reagents for facilitating the reaction on the nucleic acid molecule.

4. The composition of claim 3 , wherein the gel particle comprises a hydrogel.

5. The composition of claim 3 , wherein the gel particle comprises a polyacrylamide polymer.

6. The composition of claim 1 , wherein the one or more reagents comprise a protein or a polypeptide.

7. The composition of claim 1 , wherein the one or more reagents comprise a nucleic acid.

8. The composition of claim 1 wherein the one or more reagents comprise a nucleic acid primer.

9. The composition of claim 7 , wherein the nucleic acid is coupled to the particle.

10. The composition of claim 9 , wherein the nucleic acid is coupled to the particle by an acrydite linkage.

11. The composition of claim 1 , wherein the particle is dissolvable such that, when dissolved, the one or more reagents are released into the droplet to carry out the reaction on the nucleic acid molecule from the cell.

12. The composition of claim 1 , wherein the reaction is not a polymerase chain reaction.

13. The composition of claim 1 , wherein each of at least 75% of the plurality of droplets comprises a particle.

14. The composition of claim 1 , wherein the plurality of droplets is monodisperse.

15. The composition of claim 1 , wherein the reaction is a nucleic acid extension reaction.

16. The composition of claim 1 , wherein the reaction is a reverse transcription reaction.

17. The composition of claim 1 , wherein the reaction is a polymerase chain reaction.

18. The composition of claim 1 , wherein the droplet further comprises one or more additional reagents selected from the group consisting of a buffer, a salt, dNTPs, a primer, a DNA polymerase, and a reverse transcriptase.

19. The composition of claim 1 , wherein at least 90% of the plurality of droplets comprise a particle.

20. The composition of claim 14 , wherein the plurality of aqueous droplets is monodisperse with standard deviations less than 20%.

21. The composition of claim 14 , wherein the plurality of aqueous droplets is monodisperse with standard deviations less than 15%.

22. The composition of claim 14 , wherein the plurality of aqueous droplets is monodisperse with standard deviations less than 10%.

23. The composition of claim 14 , wherein the plurality of aqueous droplets is monodispersed with standard deviations less than 2%.

24. The composition of claim 14 , wherein the plurality of aqueous droplets is monodispersed with standard deviations less than 1%.

25. The composition of claim 5 , wherein the polyacrylamide polymer comprises a cross-linked polyacrylamide polymer.

26. The composition of claim 1 , wherein the reaction is whole genome amplification.

Assignees

• Harvard College

Inventors

• Weitz David A
• Agresti Jeremy
• Chu Liang-Yin
• Kim Jin-Woong
• Rowat Amy
• Sommer Morten
• Dantas Gautam
• Church George

Classifications

• G01N2015/1006

  for cytology · CPC title

• B01L3/502784

  specially adapted for droplet or plug flow, e.g. digital microfluidics · CPC title

• B01J13/0065

  containing an organic phase · CPC title

• C12Q1/6848

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• G01N15/1404

  Handling flow, e.g. hydrodynamic focusing · CPC title