ASSAY AND OTHER REACTIONS INVOLVING DROPLETS

• US 20150353999A1
• Filed: 05/26/2015
• Published: 12/10/2015
• 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: Agresti, Jeremy, Chu, Liang-Yin, Weitz, David A., Kim, Jin-Woong, Rowat, Amy, Sommer, Morten, Dantas, Gautam, Church, George
• Granted Patent: US 9,850,526 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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

1-58. -58. (canceled)

59. A composition comprising a plurality of aqueous droplets in a water-in-oil emulsion, wherein at least about 75% of the plurality of aqueous droplets comprises a single particle within a given droplet in the plurality of aqueous droplets, wherein the single particle comprises one or more reagents for carrying out a reaction.

60. The composition of claim 59, wherein the single particle comprises a gel particle.

61. The composition of claim 60, wherein the gel particle comprises a hydrogel.

62. The composition of claim 61, wherein the hydrogel comprises a polyacrylamide polymer.

63. The composition of claim 62, wherein the polyacrylamide polymer comprises a cross-linked polyacrylamide polymer.

64. The composition of claim 59, wherein the one or more reagents are coupled to the single particle.

65. The composition of claim 59, wherein the one or more reagents are encapsulated within the single particle.

66. The composition of claim 59, wherein the one or more reagents are selected from the group consisting of nucleic acids, proteins, and peptides.

67. The composition of claim 59, wherein the one or more reagents comprise nucleic acids.

68. The composition of claim 67, wherein the nucleic acids comprise nucleic acid primers.

69. The composition of claim 67, wherein the nucleic acids are coupled to the single particle through an acrydite linkage.

70. The composition of claim 59, wherein the particle is dissolvable.

71. The composition of claim 59, wherein at least 80% of the plurality of aqueous droplets comprises the single particle.

72. The composition of claim 71, wherein at least 90% of the droplets in the plurality of droplets contain the single particle.