System and method to synthesize a target molecule within a droplet

Patent metadata

Field	Value
Publication number	US-11001896-B2
Application number	US-201916382080-A
Country	US
Kind code	B2
Filing date	Apr 11, 2019
Priority date	Aug 13, 2012
Publication date	May 11, 2021
Grant date	May 11, 2021

Abstract

The disclosed embodiments generally relate to a method and system to synthesize a target molecule within a droplet. In an exemplary embodiment, a first microfluidic device configured to contact a polynucleotide-containing component from a sample with lysis reagents to form a first droplet. The lysis reagents include an enzyme having protease activity. The first droplet is encapsulated with an immiscible carrier fluid. A collection reservoir is provided to receive and incubate the first droplet for a first duration of time. The first duration of time is sufficient to inactivate the enzyme of the lysis reagent. A second microfluidic device is provided to receive the first droplet and add nucleic acid synthesis reagent to thereby form a second nucleic acid synthesis droplet in the immiscible carrier fluid. Finally, a reaction chamber is provided to synthesize the target polynucleotide within the second nucleic acid synthesis droplet.

First claim

What is claimed is:

1. A system to synthesize a target polynucleotide, comprising:
a first microfluidic device configured to contact a polynucleotide-containing component from a sample with lysis reagents to form a first droplet, the lysis reagents comprising an enzyme having protease activity, wherein the first droplet is encapsulated with an immiscible carrier fluid;
a collection reservoir interposed between the first microfluidic device and a second microfluidic device, the collection reservoir positioned to receive the first droplet from the first microfluidic device and incubate the first droplet for a first duration sufficient to inactivate the enzyme, the second microfluidic device fluidically coupled to the collection reservoir, the second microfluidic device configured to receive the first droplet and add nucleic acid synthesis reagent to form a second nucleic acid synthesis droplet in the immiscible carrier fluid; and
a reaction chamber fluidically coupled to the second microfluidic device, the reaction chamber configured to synthesize the target polynucleotide within the second nucleic acid synthesis droplet;
wherein the first and the second device are discrete.

2. The system of claim 1 , wherein each of the first microfluidic device and the second microfluidic device is formed as separate entities on a microfluidic chip.

3. The system of claim 2 , wherein the collection reservoir is segregated from the microfluidic chip.

4. The system of claim 3 , wherein the collection reservoir receives the first droplet for the first duration to inactivate the enzyme.

5. The system of claim 2 , wherein the reaction chamber is segregated from the microfluidic chip and configured to synthesize the target polynucleotide within the second nucleic acid synthesis droplet.

6. The system of claim 1 , wherein the collection reservoir is integrated with the first microfluidic device and wherein the reaction chamber is integrated with the second microfluidic device.

7. The system of claim 1 , wherein the first duration is a period longer than necessary for cell lysis.

8. The system of claim 1 , wherein the collection reservoir is configured to incubate the first droplet and the enzyme having protease activity at a temperature to inactivate the enzyme having protease activity.

9. The system of claim 1 , wherein the first microfluidic device is further configured to receive and add a plurality of nucleic acid synthesis reagents to the first droplet.

10. The system of claim 1 , wherein the second nucleic acid synthesis droplet has a volume of 0.001 to 1000 picoliters.

11. The system of claim 1 , wherein the second nucleic acid synthesis droplet has a diameter of between 0.1 microns to 1000 microns.

12. The system of claim 1 , wherein the second microfluidic device further comprises: one or more channels configured to contact the lysis reagents with a continuous stream of fluid comprising the nucleic acid synthesis reagent and form the second nucleic acid synthesis droplet from a portion of the continuous stream of fluid.

13. The system of claim 1 , further comprising a detector to detect the target polynucleotide by determining a sequence of a nucleic acid synthesis product of the nucleic acid synthesis droplet.

14. The system of claim 1 , further comprising a detector to detect the target polynucleotide, wherein the system is configured to form a double-emulsion, the double emulsion including the nucleic acid synthesis droplet within an outer droplet, and sort the double-emulsion based on at least one of droplet size and fluorescence.

Assignees

• Univ California

Inventors

• Abate Adam R
• Eastburn Dennis Jay
• Sciambi Adam R

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• B01F33/3011

  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 title

• B01F25/4335

  Mixers with a converging-diverging cross-section · CPC title

• B01F23/41

  Emulsifying · CPC title

• B01F33/3031

  using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids · CPC title