Method of generating droplets

• US 10,279,350 B2
• Filed: 11/30/2016
• Issued: 05/07/2019
• Est. Priority Date: 09/23/2008
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Hindson, Benjamin J., Ness, Kevin D., Colston, Jr., Billy W., Milanovich, Fred P., Masquelier, Donald A.
• Primary Examiner(s): Hobbs, Michael L
• Application Number:
  US15/365,894
• Publication Number:
  US 20170144161A1
• Time in Patent Office: 888 Days
• 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 23/4143 Microemulsions
• CPC Class Codes: B01F 23/4145 Emulsions of oils, e.g. fuel, and water
• 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 35/10 Maintenance of mixers
• CPC Class Codes: B01F 35/165 Making mixers or parts thereof
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/041 Connecting closures to device or container
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0858 Side walls
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2400/0478 pistons
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/049 : vacuum
• CPC Class Codes: B01L 2400/0622 : distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• CPC Class Codes: B01L 3/0241 : Drop counters; Drop formers making arrays for combinatorial libraries B01J19/0046; automation of dispensing for analysis G01N35/10
• CPC Class Codes: B01L 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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: B01L 7/525 : with physical movement of samples between temperature zones
• CPC Class Codes: B29C 2045/0079 : applying a coating or covering
• CPC Class Codes: B29C 45/0053 : combined with a final operation, e.g. shaping injection-compression moulding B29C45/561
• CPC Class Codes: B29C 45/006 : Joining parts moulded in separate cavities
• CPC Class Codes: B29L 2031/752 : Measuring equipment
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: G01N 2021/6439 : with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/3563 : for analysing solids; Preparation of samples therefor
• CPC Class Codes: G01N 21/49 : within a body or fluid
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6486 : Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: Y02A 90/10 : Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Methods of generating droplets. In an exemplary method, a device including a sample well, a carrier well, a droplet well, and a plurality of microfluidic channels is selected. The microfluidic channels include a first channel, a second channel, and a third channel. A discrete volume of sample-containing fluid is placed into the sample well, and a discrete volume of carrier fluid is placed into the carrier well. A pressure differential is created after placing the discrete volumes, to cause fluid flow. Sample-containing fluid flows from the sample well to a droplet-generation region of the device via the first channel. Carrier fluid flows from the carrier well to the droplet-generation region via the second channel. Sample-containing droplets and carrier fluid flow from the droplet-generation region to the droplet well via the third channel.

Claims

1. A method of generating droplets, the method comprising:

selecting a device including a sample well, a carrier well, a droplet well, and a plurality of microfluidic channels, the microfluidic channels including a first channel, a second channel, and a third channel that meet one another at a droplet-generation region, wherein the device includes an upper member and a lower member, wherein the upper member has a base portion and a plurality of protrusions, wherein the protrusions are formed integrally with the base portion and project from a top surface of the base portion, wherein a rim of each well is formed by one of the protrusions, wherein the microfluidic channels are formed in a bottom surface of the base portion, and wherein the lower member is attached to the bottom surface of the base portion and seals a bottom side of each of the microfluidic channels;

placing a discrete volume of sample-containing fluid into the sample well, and a discrete volume of carrier fluid into the carrier well; and

creating a pressure differential after the step of placing, such that (a) sample-containing fluid flows from the sample well to the droplet-generation region of the device via the first channel, (b) carrier fluid flows from the carrier well to the droplet-generation region via the second channel, and (c) sample-containing droplets and carrier fluid flow from the droplet-generation region to the droplet well via the third channel.

2. The method of claim 1, wherein the step of creating a pressure differential includes a step of applying vacuum to the droplet well.

3. The method of claim 1, wherein the step of step of creating a pressure differential includes a step of engaging a rim of at least one of the wells with a gasket.

4. The method of claim 3, wherein the pressure differential is created via a manifold that is engaged with the gasket.

5. The method of claim 1, wherein the step of creating a pressure differential causes carrier fluid to flow separately to the droplet-generation region via a pair of the microfluidic channels.

6. The method of claim 1, wherein the device has a plurality of droplet-generation regions each connected to a different sample well, a different carrier well, and a different droplet well, and wherein the step of creating a pressure differential causes droplet generation to occur in parallel in the plurality of droplet-generation regions.

7. The method of claim 1, wherein the upper member is injection molded as a single piece.

8. A method of generating droplets, the method comprising:

selecting a device including a plurality of emulsion-generation units, each emulsion-generation unit including a sample well, a carrier well, a droplet well, and a plurality of microfluidic channels connecting the wells to a droplet-generation region, wherein the microfluidic channels include at least three channels that meet one another at the droplet-generation region, wherein the device includes an upper member and a lower member, wherein the upper member has a base portion and a plurality of protrusions, wherein the protrusions are formed integrally with the base portion and project from a top surface of the base portion, wherein each well has an upper portion formed by one of the protrusions, wherein the microfluidic channels are formed in a bottom surface of the base portion, and wherein the lower member is attached to the bottom surface of the base portion and seals a bottom side of each of the microfluidic channels;

placing sample-containing fluid into the sample well of each emulsion-generation unit, and carrier fluid into the carrier well of each emulsion-generation unit; and

creating a pressure differential in each emulsion-generation unit after the step of placing, such that (a) sample-containing fluid flows from the sample well to the droplet-generation region, (b) carrier fluid flows from the carrier well to the droplet-generation region, and (c) sample-containing droplets and carrier fluid flow from the droplet-generation region to the droplet well;

wherein the step of creating a pressure differential is performed via a manifold, and wherein the manifold is sealed to each droplet well with a gasket that is engaged with a rim of the droplet well.

9. The method of claim 8, wherein the step of creating a pressure differential includes a step of applying vacuum to each droplet well via the manifold.

10. The method of claim 8, wherein the step of creating a pressure differential causes all of the emulsion-generation units to generate droplets in parallel.

11. The method of claim 8, wherein the upper member is injection molded as a single piece.

12. A method of generating droplets, the method comprising:

selecting a device including a body and cover layer, the body being injection molded as a single piece and including a base portion and a plurality of protrusions projecting from a top surface of the base portion, a bottom surface of the base portion having a plurality of microfluidic channels formed therein, the cover layer being attached to the bottom surface of the base portion and sealing a bottom side of each microfluidic channel, the device providing a sample well, a carrier well, and a droplet well, wherein an upper portion of each well is formed by one of the protrusions, and wherein the microfluidic channels include a first channel, a second channel, and a third channel that meet one another at a droplet-generation region;

placing a discrete volume of sample-containing fluid into the sample well, and a discrete volume of carrier fluid into the carrier well; and

creating a pressure differential after the step of placing, such that (a) sample-containing fluid flows from the sample well to the droplet-generation region of the device via the first channel, (b) carrier fluid flows from the carrier well to the droplet-generation region via the second channel, and (c) sample-containing droplets and carrier fluid flow from the droplet-generation region to the droplet well via the third channel.

13. The method of claim 12, further comprising a step of engaging a rim of the droplet well with a gasket.

14. The method of claim 12, wherein the step of creating a pressure differential includes a step of applying vacuum to the droplet well via a manifold, and wherein the manifold is sealed to the droplet well using the gasket.

15. The method of claim 12, wherein the device includes a plurality of droplet wells, and wherein the step of creating a pressure differential includes a step of applying vacuum to the plurality of droplet wells via a manifold.