DROPLET GENERATION FOR DROPLET-BASED ASSAYS

• US 20120190032A1
• Filed: 12/30/2011
• Published: 07/26/2012
• Est. Priority Date: 03/25/2010
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Ness, Kevin D., Masquelier, Donald A., Kelly, Christopher F.
• Granted Patent: US 9,500,664 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/0275 Interchangeable or disposable dispensing tips
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502723 characterised by venting arrangements
• 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: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: G01N 2035/00148 Test cards, e.g. Biomerieux or McDonnel multiwell test cards
• CPC Class Codes: G01N 2035/1034 Transferring microquantities of liquid
• CPC Class Codes: G01N 35/085 Flow Injection Analysis
• CPC Class Codes: G01N 35/1065 Multiple transfer devices
• CPC Class Codes: Y10T 29/494 Fluidic or fluid actuated device making

Abstract

A system, including method and apparatus, for generating droplets suitable for droplet-based assays. The disclosed systems may include either one-piece or multi-piece droplet generation components configured to form sample-containing droplets by merging aqueous, sample-containing fluid with a background emulsion fluid such as oil, to form an emulsion of sample-containing droplets suspended in the background fluid. In some cases, the disclosed systems may include channels or other suitable mechanisms configured to transport the sample-containing droplets to an outlet region, so that subsequent assay steps may be performed.

Claims

1. A system for forming a plurality of sample-containing droplets suspended in a background fluid, comprising:

a substrate;

a sample well, a background fluid well, and a droplet outlet region selected from the group consisting of a pipette tip and a droplet well, wherein at least one of the sample well, the background fluid well, and the droplet outlet region is integrally formed with the substrate;

a network of channels formed in the substrate and fluidically interconnecting the sample well, the background fluid well, and the droplet outlet region; and

a droplet generation region defined by the network of channels and configured to generate sample-containing droplets suspended in the background fluid;

wherein the droplet generation region is defined by the intersection of a first channel, a second channel, and a third channel; and

wherein the first channel is configured to transport sample-containing fluid from the sample well to the droplet generation region, the second channel is configured to transport background fluid from the background fluid well to the droplet generation region, and the third channel is configured to transport sample-containing droplets from the droplet generation region to the droplet outlet region.

2. The system of claim 1, wherein the substrate is substantially planar, and further comprising a substantially planar sealing member configured to be disposed adjacent to a bottom surface of the substrate and to form a substantially fluid tight seal with the bottom surface of the substrate.

3. The system of claim 1, wherein the droplet outlet region is a droplet well.

4. The system of claim 1, wherein the droplet outlet region is a pipette tip.

5. The system of claim 1, wherein the first channel includes an air trap configured to prevent sample-containing fluid from being inadvertently drawn through the first channel by capillary action.

6. The system of claim 1, wherein the second channel includes two background fluid sub-channels that intersect the first channel from two different directions to form a cross-shaped intersection region with the first channel and the third channel.

7. The system of claim 6, wherein the two background fluid sub-channels have substantially equal hydraulic resistances.

8. The system of claim 1, wherein the substrate, the sample well, the background fluid well, the droplet outlet region, and the network of channels are all formed in a single injection molding process.

9. The system of claim 1, wherein the droplet generation region is configured to generate sample-containing droplets with volumes in the range of 0.1 nanoliters to 10 nanoliters.

10. The system of claim 1, further comprising a sealing member configured to be disposed adjacent to a bottom surface of the substrate and to form a substantially fluid tight seal with the bottom surface of the substrate, and wherein at least one of the sample well, the background fluid well, and the droplet outlet region is integrally formed with the sealing member.

11. A method of generating sample-containing droplets suspended in a background fluid, comprising:

transporting sample-containing fluid into a sample well attached to a substrate;

transporting background fluid into a background fluid well attached to the substrate;

transporting sample-containing fluid through a first channel formed in the substrate, from the sample well to a droplet generation region;

transporting background fluid through a second channel formed in the substrate, from the background fluid well to the droplet generation region;

generating sample-containing droplets suspended in the background fluid at the droplet generation region; and

transporting the sample-containing droplets through a third channel formed in the substrate, from the droplet generation region to a droplet outlet region integrally attached to the substrate;

wherein at least one of the sample well, the background fluid well, and the droplet outlet region are integrally formed with the substrate, and wherein the droplet outlet region is selected from the group consisting of a pipette tip and a droplet well.

12. The method of claim 11, wherein the droplet outlet region is a droplet well.

13. The method of claim 11, wherein the droplet outlet region is a pipette tip.

14. The method of claim 11, wherein transporting sample-containing fluid through the first channel includes transporting the sample-containing fluid through an air trap region configured to prevent inadvertent transport of the sample-containing fluid to the droplet generation region.

15. The method of claim 11, wherein transporting background fluid through the second channel includes transporting the background fluid through two background fluid sub-channels that intersect the first channel from two different directions to form a cross-shaped intersection region with the first channel and the third channel.

16. The method of claim 15, wherein the two background fluid sub-channels are configured to have substantially the same hydraulic resistance.

17. The method of claim 11, wherein generating sample-containing droplets includes generating droplets having volumes in the range of 0.1 nanoliters to 10 nanoliters.

18. A method of manufacturing a droplet generation system, comprising:

(i) forming a substrate;

(ii) forming a sample well;

(iii) forming a background fluid well;

(iv) forming a droplet outlet region;

(v) forming a network of channels fluidically interconnecting the sample well, the background fluid well, and the droplet outlet region; and

(vi) forming a droplet generation region defined by the intersection of a first channel fluidically connected with the sample well, a second channel fluidically connected with the background fluid well, and a third channel fluidically connected with the droplet outlet region;

wherein the substrate, the network of channels, and at least one of the sample well, the background fluid well, and the droplet outlet region are integrally formed, and wherein the droplet outlet region is selected from the group consisting of a pipette tip and a droplet well.

19. The method of claim 18, wherein the substrate is substantially planar, and further comprising forming a substantially planar sealing member configured to form a substantially fluid tight seal with a bottom surface of the substrate.

20. The method of claim 19, further comprising attaching the sealing member to the bottom surface of the substrate.

21. The method of claim 18, wherein forming the substrate, the sample well, the background fluid well, the droplet outlet region, and the network of channels is accomplished by injection molding.