Droplet-based analysis method

• US 10,512,910 B2
• Filed: 09/18/2017
• Issued: 12/24/2019
• Est. Priority Date: 09/23/2008
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Colston, Jr., Billy W., Hindson, Benjamin J., Ness, Kevin D., Masquelier, Donald A.
• Primary Examiner(s): Wecker, Jennifer
• Application Number:
  US15/707,908
• Publication Number:
  US 20180147573A1
• Time in Patent Office: 827 Days
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 23/4145 Emulsions of oils, e.g. fuel, and water
• 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 33/3017 Mixing chamber
• 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 33/3022 the components being formed by independent droplets which are alternated, the mixing of the components being achieved by diffusion between 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 2200/0636 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 2300/1822 : using Peltier elements
• CPC Class Codes: B01L 2400/0487 : fluid pressure, pneumatics
• 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 7/525 : with physical movement of samples between temperature zones
• 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 15/01 : specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/06 : Investigating concentration of particle suspensions by weighing G01N5/00; investigating sedimentation of particle suspensions G01N15/04; investigating individual particles G01N15/10
• CPC Class Codes: G01N 15/075 : by optical means
• CPC Class Codes: G01N 2015/0687 : in solutions, e.g. non volatile residue
• CPC Class Codes: G01N 2035/00237 : Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/00465 : Separating and mixing arrangements
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis

Abstract

Droplet-based methods of analysis. In an exemplary method, a device having a port connected to a chamber may be selected. A sample-containing fluid may be placed into the port. A pressure differential may be created that drives the sample-containing fluid from the port to the chamber and separates the sample-containing fluid into droplets. A two-dimensional monolayer of the droplets may be formed in the chamber. At least a portion of the monolayer may be imaged.

Claims

1. A method of analysis, the method comprising:

selecting a device having a port connected to a chamber;

placing a sample-containing fluid into the port;

creating a pressure differential that drives the sample-containing fluid from the port to the chamber and separates the sample-containing fluid into droplets;

forming a two-dimensional monolayer of the droplets in the chamber; and

imaging at least a portion of the monolayer.

2. The method of claim 1, wherein the step of placing includes a step of placing the sample-containing fluid as a continuous phase into a well of the device.

3. The method of claim 1, wherein the droplets are aqueous droplets that are separated from one another by an immiscible carrier liquid.

4. The method of claim 1, wherein the step of forming includes a step of collecting the droplets in a chamber having a height corresponding to a diameter of the droplets.

5. The method of claim 1, wherein the step of creating a pressure differential includes a step of applying positive gas pressure or negative gas pressure to a port of the device.

6. The method of claim 1, further comprising a step of thermally cycling the monolayer to promote nucleic acid amplification in a subset of droplets of the monolayer.

7. The method of claim 1, wherein the step of imaging includes a step of detecting fluorescence from the monolayer.

8. The method of claim 1, wherein the step of imaging creates one or more images of droplets of the monolayer, wherein only a subset of the droplets contain an analyte, and wherein the one or more images indicate whether the analyte is present in individual droplets.

9. The method of claim 8, further comprising a step of determining a number of droplets containing the analyte using the one or more images.

10. The method of claim 8, wherein the analyte is a nucleic acid target, further comprising a step of amplifying the nucleic acid target within the monolayer.

11. The method of claim 10, wherein the step of amplifying includes a step of thermally cycling the monolayer to promote nucleic acid amplification in droplets thereof.

12. The method of claim 10, wherein the droplets contain an intercalating dye or a probe, wherein the probe includes an oligonucleotide labeled with a fluorophore, and wherein the step of imaging includes a step of detecting fluorescence from the intercalating dye or the fluorophore.

13. The method of claim 1, wherein the chamber lies in a plane and defines an area of the plane, and wherein the step of forming includes a step of covering a majority of the area with droplets.

14. A method of analysis, the method comprising:

selecting a device having a chamber connected separately to a port and a vent;

placing an aqueous fluid into the port;

applying gas pressure to the device to drive the aqueous fluid from the port to the chamber and form partitions of the aqueous fluid, the partitions being separated from one another by a carrier liquid;

amplifying a nucleic acid target in only of a subset of the partitions while the partitions are arranged in a two-dimensional monolayer in the chamber;

imaging at least a portion of the monolayer to create one or more images; and

determining whether individual partitions of the monolayer contain the nucleic acid target using the one or more images.

15. The method of claim 14, wherein the chamber has a top wall facing a bottom wall, and wherein the chamber has a height measured between the top and bottom walls corresponding to a diameter of the partitions.

16. The method of claim 14, wherein the chamber lies in a plane and defines an area of the plane, and wherein the step of amplifying is performed while a majority of the area is covered with partitions.

17. The method of claim 14, wherein the port includes a well, and wherein the step of placing an aqueous fluid includes a step of placing the aqueous fluid into the well.

18. The method of claim 14, wherein the step of imaging includes a step of detecting fluorescence from the monolayer.

19. The method of claim 14, wherein the step of amplifying includes a step of thermally cycling the monolayer.

20. The method of claim 14, wherein the step of applying gas pressure includes a step of applying positive gas pressure or negative gas pressure to the device.