System for generating droplets with pressure monitoring

• US 9,764,322 B2
• Filed: 11/14/2016
• Issued: 09/19/2017
• Est. Priority Date: 09/23/2008
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Hiddessen, Amy L., Ness, Kevin D., Hindson, Benjamin J., Masquelier, Donald A., Chia, Erin R.
• Primary Examiner(s): Wecker, Jennifer
• Application Number:
  US15/351,354
• Publication Number:
  US 20170151558A1
• Time in Patent Office: 309 Days
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 25/14 Mixing drops, droplets or bodies of liquid which flow together or contact each other micromixers using where the materials to be mixed are in the form of droplets B01F33/302
• CPC Class Codes: B01F 25/23 Mixing by intersecting jets
• 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 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/813 mixing simultaneously in two or more mixing receptacles
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0636 Focussing flows, e.g. to laminate flows
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/14 Process control and prevention of errors
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0609 Holders integrated in container to position an object
• CPC Class Codes: B01L 2300/0681 Filter
• 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 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2400/049 vacuum
• CPC Class Codes: B01L 3/502 with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: B01L 3/50273 : characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• 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 3/52 : Containers specially adapted for storing or dispensing a reagent B01L3/02 takes precedence; containers for medical or pharmaceutical purposes A61J1/00; containers in general B65D; storing or dispensing test elements G01N33/4875; automated reagent dispensing G01N35/1002
• CPC Class Codes: B01L 9/527 : for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis

Abstract

System, including methods, apparatus, and kits, for forming emulsions. In an exemplary method of generating droplets, a device may be selected that includes a plurality of emulsion-formation units each including a sample well, a continuous-phase well, a droplet well, and a channel network that fluidically interconnects the wells and creates a droplet-generation region. A discrete volume of sample-containing fluid may be placed into the sample well of each emulsion-formation unit, and a discrete volume of continuous-phase fluid into the continuous-phase well of each emulsion-formation unit. Pressure may be applied to the device with a fluidics assembly after the step of placing, such that the plurality of emulsion-formation units generate droplets in parallel with one another. A pressure signal may be detected from the fluidics assembly. Application of the pressure may be stopped when the pressure signal indicates that a sample well is empty.

Claims

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

selecting a device including a plurality of emulsion-formation units each including a sample well, a continuous-phase well, a droplet well, and a channel network that fluidically interconnects the wells and creates a droplet-generation region;

placing a discrete volume of sample-containing fluid into the sample well of each emulsion-formation unit, and a discrete volume of continuous-phase fluid into the continuous-phase well of each emulsion-formation unit;

applying pressure to the device with a fluidics assembly after the step of placing, such that in each emulsion-formation unit (a) sample-containing fluid flows from the sample well to the droplet-generation region, (b) continuous-phase fluid flows from the continuous-phase well to the droplet-generation region, and (c) sample-containing droplets and continuous-phase fluid flow from the droplet-generation region to the droplet well, wherein the plurality of emulsion-formation units generate droplets in parallel with one another;

detecting a pressure signal from the fluidics assembly; and

stopping application of the pressure when the pressure signal indicates that a sample well is empty.

2. The method of claim 1, wherein the device has a row of sample wells, a row of continuous-phase wells, and a row of droplet wells, and wherein the step of applying pressure includes a step of applying pressure in parallel to each well of at least one of the rows of wells, and wherein the step of stopping application of the pressure includes a step of stopping application of pressure in parallel to each well of the at least one row of wells.

3. The method of claim 1, wherein the step of stopping application of the pressure includes a step of adjusting a valve to vent a portion of the fluidics assembly that is applying the pressure.

4. The method of claim 1, wherein the step of applying pressure includes a step of applying negative pressure in parallel to each of the droplet wells, and wherein the step of stopping application of the pressure includes a step stopping application of the negative pressure to each of the droplet wells.

5. The method of claim 4, wherein the fluidics assembly includes a manifold, wherein the negative pressure is applied via the manifold, and wherein the manifold is sealed to each of the droplet wells via a same gasket.

6. The method of claim 1, wherein the fluidics assembly includes a pressure reservoir that is operatively connected to a pump, wherein the pump is configured to create negative or positive pressure in the pressure reservoir, and wherein the step of applying pressure includes a step of applying negative or positive pressure via the pressure reservoir while the pressure reservoir remains isolated from the pump.

7. The method of claim 1, the pressure being first pressure, further comprising a step of applying second pressure after the step of stopping application, such that continuous-phase fluid is removed selectively, relative to droplets, from an emulsion present in each droplet well.

8. The method of claim 7, wherein the second pressure includes positive pressure applied to each of the droplet wells.

9. The method of claim 7, wherein the second pressure causes continuous-phase fluid to flow from the droplet well of each emulsion-formation unit via a channel that connects the droplet well to the corresponding droplet-generation region.

10. The method of claim 7, wherein the step of applying first pressure is performed for a first length of time, and wherein the step of applying second pressure is performed for a second length of time that is based on the first length of time.

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

selecting a device including a plurality of separate emulsion-formation units each including a sample well, a continuous-phase well, a droplet well, and a channel network that fluidically interconnects the wells and creates a droplet-generation region;

placing a discrete volume of sample-containing fluid into the sample well of each emulsion-formation unit, and a discrete volume of continuous-phase fluid into the continuous-phase well of each emulsion-formation unit;

applying negative pressure to the droplet well of each of the emulsion-formation units via a manifold after the step of placing, with the manifold sealed to each droplet well via a same gasket, such that in each emulsion-formation unit (a) sample-containing fluid flows from the sample well to the droplet-generation region, (b) continuous-phase fluid flows from the continuous-phase well to the droplet-generation region, and (c) sample-containing droplets and continuous-phase fluid flow from the droplet-generation region to the droplet well, wherein the plurality of emulsion-formation units generate droplets in parallel with one another;

detecting a pressure signal corresponding to the negative pressure; and

stopping application of negative pressure to all of the droplet wells in parallel when the pressure signal indicates that air has entered the channel network of an emulsion-formation unit.

12. The method of claim 11, wherein the step of stopping application includes a step of adjusting a valve to vent each droplet well.

13. The method of claim 11, wherein the manifold is connected to a pressure reservoir that is operatively connected to a pump, wherein the pump is configured to create negative pressure in the pressure reservoir, and wherein the step of applying negative pressure is performed via the pressure reservoir while the pressure reservoir remains isolated from the pump.

14. The method of claim 11, further comprising a step of applying positive pressure in parallel to each of the droplet wells after the step of stopping application, such that continuous-phase fluid is removed selectively, relative to droplets, from an emulsion present in each droplet well.

15. The method of claim 14, wherein the positive pressure causes continuous-phase fluid to flow from the droplet well of each emulsion-formation unit via a channel that connects the droplet well to the corresponding droplet-generation region.

16. The method of claim 14, wherein the step of applying negative pressure is performed for a first length of time, and wherein the step of applying positive pressure is performed for a second length of time that is based on the first length of time.