MICROFABRICATED DROPLET GENERATOR FOR SINGLE MOLECULE/CELL GENETIC ANALYSIS IN ENGINEERED MONODISPERSED EMULSIONS

• US 20100285975A1
• Filed: 07/24/2008
• Published: 11/11/2010
• Est. Priority Date: 07/24/2007
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Kumaresan, Palani, Yang, Chaoyang, Mathies, Richard A., Blazej, Robert G.
• Granted Patent: US 8,454,906 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: B01F 23/41 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 33/81 Combinations of similar mixers, e.g. with rotary stirring devices in two or more receptacles
• CPC Class Codes: B01F 33/813 mixing simultaneously in two or more mixing receptacles
• CPC Class Codes: B01J 2219/00466 in a slurry
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0803 Disc shape
• CPC Class Codes: B01L 2400/0439 ultrasonic vibrations, vibrating piezo elements
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 3/0268 using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
• CPC Class Codes: G01N 2035/1034 Transferring microquantities of liquid
• CPC Class Codes: G01N 35/08 using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
• CPC Class Codes: Y10T 436/2575 Volumetric liquid transfer

Abstract

Provided are microfluidic designs and methods for rapid generation of monodisperse nanoliter volume droplets of reagent/target (e.g., molecule or cell) mix in emulsion oil. The designs and methods enable high-throughput encapsulation of a single target (e.g., DNA/RNA molecules or cells) in controlled size droplets of reagent mix. According to various embodiments, a microfabricated, 3-valve pump is used to precisely meter the volume of reagent/target mix in each droplet and also to effectively route microparticles such as beads and cells into the device, which are encapsulated within droplets at the intersection of the reagent channel and an oil channel. The pulsatile flow profile of the microfabricated pumps provides active control over droplet generation, thereby enabling droplet formation with oils that are compatible with biological reactions but are otherwise difficult to form emulsions with.

Claims

1. A microdroplet generator comprising:

a reagent inlet to a reagent channel;

a plurality of oil inlets to a plurality of oil channels;

a pneumatic layer comprising a plurality of pneumatic channels;

a first fluidic layer comprising the reagent channel;

an elastomeric layer sandwiched between the pneumatic layer and the first fluidic layer;

a three-valve pump comprising valve displacements in the plurality of pneumatic channels aligned with discontinuities in the reagent channel and configured to provide monodisperse pulsatile flow of reagent from the reagent inlet to a nozzle via the reagent channel, the nozzle comprising a junction of the reagent channel and the oil channels and configured to form monodisperse emulsion droplets; and

a droplet outlet.

2. The microdroplet generator of claim 1 further comprising a second fluidic layer, said second fluidic layer comprising the nozzle.

3. The microdroplet generator of claim 2 further comprising a via connecting the reagent channel to the second fluidic layer.

4. The microdroplet generator of claim 1 wherein the first fluidic layer further comprises the nozzle and the oil channel.

5. The microdroplet generator of claim 1 wherein the three-valve pump is configured to provide monodisperse pulses having volumes ranging from 100 pL to 10 nL.

6. The microdroplet generator of claim 1 wherein the reagent channel is a hybrid glass-elastomer channel.

7. The microdroplet generator of claim 1 wherein the generator is configured to form monodisperse emulsion droplets at a frequency of 0.01-100 Hz.

8. (canceled)

9. A microdroplet generator comprising

a reagent inlet connected to a plurality of glass-elastomeric hybrid reagent channels;

a plurality of three-valve elastomeric valves configured to produce pulsatile flow of reagent mix in the plurality of glass-elastomeric hybrid channels and pump the reagent mix to a plurality of nozzles;

a pneumatic layer comprising a plurality of pneumatic lines configured to simultaneously control the three-valve pumps; and

a plurality of oil channels connected to a plurality of injectors, the plurality of injectors configured to produce monodisperse emulsion droplets and flow said emulsion droplets to a plurality of outputs.

10. A method of generating microdroplets, the method comprising:

providing a reagent and target mixture to a reagent inlet channel on a microfabricated structure;

forming droplets of the mixture in the channel via an in-channel three-valve elastomeric pump, wherein the pumping frequency and the droplet formation frequency have a 1;

1 correspondence; and

routing the droplets to an injector to form monodisperse emulsion droplets encapsulating the reagent and target mixture, wherein no more than a single target is encapsulated in the majority of emulsion droplets.

11. The method of claim 10 wherein the volume of the droplets is between about 100 pL-100 nL.

12. The method of claim 10 wherein the volume of the droplets is between 2 and 5 nL.

13. The method of claim 10 wherein the reagent and target mixture further comprises a microcarrier element.

14. The method of claim 10 wherein the reagent and target mixture comprises PCR reagent, primer functionalized microcarrier elements, and target.

15. The method of claim 14 wherein forming monodisperse emulsion droplets comprises forming emulsion droplets encapsulating PCR reagent, a single primer functionalized microcarrier element and a single target.

16. The method of claim 10 wherein the target is a cell or molecule.

17-18. -18. (canceled)

19. A microdroplet generator comprising:

a reagent inlet to a reagent channel;

a plurality of oil inlets to a plurality of oil channels;

a pneumatic layer comprising a plurality of pneumatic channels;

a first fluidic layer comprising the reagent channel;

an elastomeric layer sandwiched between the pneumatic layer and the first fluidic layer;

a micro-pump comprising valve displacements in the plurality of pneumatic channels aligned with discontinuities in the reagent channel and configured to provide pulsatile flow of reagent from the reagent inlet to an injector via the reagent channel, the injector configured to form emulsion droplets; and

a droplet outlet.

20. The microdroplet generator of claim 19 wherein the generator is configured to form monodisperse emulsion droplets at a frequency of 0.01-100 Hz.

21. The microdroplet generator of claim 19 wherein the generator is configured to form monodisperse emulsion droplets at a frequency of 1-50 Hz.

22. The microdroplet generator of claim 19 wherein the generator is configured to form monodisperse emulsion droplets at a frequency of 1-10 Hz.

23-26. -26. (canceled)