Granted Patent
B2
US 12,416,033 · App. 18/892,086 · Granted Sep 16, 2025
Assays and other reactions involving droplets
Inventors:
David A. Weitz (Cambridge, MA); Jeremy Agresti (Cambridge, MA); Liang-Yin Chu (Cambridge, MA); Jin-Woong Kim (Cambridge, MA); Amy Rowat (Cambridge, MA); Morten Sommer (Cambridge, MA); Gautam Dantas (Cambridge, MA); George M. Church (Cambridge, MA)
Assignee:
President and Fellows of Harvard College
C12P19/34B01F23/4105B01F33/3011B01F33/3021B01J13/0052B01J13/0065C12Q1/6806C12Q1/6834C12Q1/6848C12Q1/686G01N15/1404B01F23/41B01F2101/23B01L3/502784C12Q2563/149C12Q2563/159G01N2015/1006
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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 subject 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 (25)
1. A method, comprising:
(a) providing:
(i) a plurality of hardened gel droplets comprising nucleic acid molecules;
(ii) a plurality of droplets, wherein a droplet of the plurality of droplets comprises a hardened gel droplet of the plurality of hardened gel droplets, and wherein the hardened gel droplet comprises a nucleic acid molecule of the nucleic acid molecules, and
(iii) a plurality of cells, wherein a cell of the plurality of cells, comprises a sample nucleic acid molecule; and
(b) using the nucleic acid molecule and the sample nucleic acid molecule to generate a nucleic acid product.
2. The method according to claim 1 , wherein the droplet of the plurality of droplets comprises the cell of the plurality of cells.
3. The method according to claim 1 , wherein the sample nucleic acid molecule is a ribonucleic acid (RNA) molecule.
4. The method according to claim 1 , wherein the hardened gel droplet is comprises a hydrogel.
5. The method according to claim 4 , wherein the hydrogel comprises polyacrylamide.
6. The method according to claim 1 , wherein the nucleic acid molecule comprises an acrydite moiety.
7. The method according to claim 1 , wherein the nucleic acid molecule is coupled to the hardened gel droplet via an acrydite linkage.
8. The method according to claim 1 , wherein the nucleic acid molecule is covalently coupled to the hardened gel droplet.
9. The method according to claim 1 , wherein (b) comprises generating the nucleic acid product via reverse transcription.
10. The method according to claim 1 , wherein (b) comprises generating the nucleic acid product via amplification.
11. The method according to claim 1 , wherein the droplet of the plurality of droplets comprises a reagent selected from an enzyme, a salt, a buffer, a primer, a deoxynucleotide triphosphate (dNTP), or a preservative.
12. The method according to claim 11 , wherein the enzyme is selected from a polymerase or a reverse transcriptase.
13. The method according to claim 1 , wherein droplets of the plurality of droplets are monodisperse.
14. The method according to claim 1 , wherein hardened gel droplets of the plurality of hardened gel droplets are monodisperse.
15. The method according to claim 1 , wherein the plurality of droplets is dispersed in an oil-based fluid.
16. The method according to claim 15 , wherein the oil-based fluid comprises a surfactant.
17. The method according to claim 15 , wherein the oil-based fluid comprises a fluorocarbon.
18. The method according to claim 1 , wherein the plurality of droplets is dispersed in an aqueous fluid.
19. The method according to claim 1 , further comprising: providing the droplet of the plurality of droplets in a first fluid; removing the droplet of the plurality of droplets from the first fluid; and placing the droplet of the plurality of droplets in a second fluid.
20. The method according to claim 1 , wherein (a) comprises providing the cell of the plurality of cells in a polymerosome.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Oct 4, 2024
From: AGRESTI, JEREMY; CHU, LIANG-YIN; WEITZ, DAVID A.; ROWAT, AMY; SOMMER, MORTEN; DANTAS, GAUTAM; KIM, JIN-WOONG; CHURCH, GEORGE
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 069115/0610 →
Continuity (9)
Continuation
12529926
Provisional Application
60905567
· Mar 7, 2007
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