ASSAY AND OTHER REACTIONS INVOLVING DROPLETS

• US 20180023109A1
• Filed: 08/07/2017
• Published: 01/25/2018
• Est. Priority Date: 03/07/2007
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Weitz, David A., Agresti, Jeremy, Chu, Liang-Yin, Kim, Jin-Woong, Rowat, Amy, Sommer, Morten, Dantas, Gautam, Church, George
• Granted Patent: US 10,683,524 B2
• Time in Patent Office: Days
• CPC Class Codes: B01F 2101/23 Mixing of laboratory samples e.g. in preparation of analysing or testing properties of materials
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 23/4105 Methods of emulsifying
• CPC Class Codes: B01F 33/3011 using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01J 13/0052 Preparation of gels
• CPC Class Codes: B01J 13/0065 containing an organic phase
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 2015/1006 for cytology

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.

Claims

1-20. -20. (canceled)

21. A method, comprising:

providing a gel droplet containing a species, wherein the gel droplet is within a surrounding droplet; and

exposing the species within the gel droplet to a reactant which is reactive with the species.

22. The method of claim 21, wherein providing a gel droplet containing a species comprises:

providing a fluidic droplet containing a species; and

causing the fluidic droplet to form a gel droplet containing the species, wherein the gel droplet is within a surrounding droplet.

23. The method of claim 21, wherein exposing the species occurs after releasing the species from the gel droplet.

24. The method of claim 21, wherein the species is a first nucleic acid, siRNA, RNAi or DNA.

25. The method of claim 24, wherein the reactant is a second nucleic acid.

26. The method of claim 21, wherein the reactant is a second nucleic acid.

27. The method of claim 21, wherein the gel comprises alginate, polyacrylamide or poly N-isopropylpolyacrylamide.

28. The method of claim 21, wherein the surrounding droplet forms part of an emulsion.

29. The method of claim 21, wherein the gel droplet comprises alginate.

30. The method of claim 21, wherein the nucleic acid is a primer, and the second nucleic acid is a target and one of the following reactions is performed, a PCR, an rtPCR or whole genome amplification.

31. The method of claim 30, wherein a library is formed.

32. A method, comprising:

providing a gel droplet comprising a first nucleic acid and a second nucleic acid different from the first nucleic acid, the first nucleic acid being bound to the gel and being a primer; and

growing the first nucleic acid within the gel, using the second nucleic acid as a template.

33. The method of claim 32, wherein growing the first nucleic acid occurs after releasing the first nucleic acid from the gel.

34. The method of claim 32, wherein the gel comprises alginate.

35. The method of claim 32, wherein the gel droplet has an average diameter of less than about 1 mm.

36. A method, comprising:

providing a fluidic droplet in a carrying fluid, the fluidic droplet substantially immiscible in water and the carrying fluid substantially immiscible in water;

hardening the fluidic droplet to form a gel;

removing the carrying fluid; and

placing the hardened fluidic droplet in a third fluid which is immiscible in water.