System for detection of spaced droplets

• US 9,492,797 B2
• Filed: 01/20/2014
• Issued: 11/15/2016
• Est. Priority Date: 09/23/2008
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Makarewicz, Anthony J., Hiddessen, Amy L.
• Primary Examiner(s): HOBBS, MICHAEL L
• Application Number:
  US14/159,410
• Publication Number:
  US 20140200164A1
• Time in Patent Office: 1,030 Days
• Field of Search: 435/303.1
• US Class Current: 1/1
• 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 33/3021 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: 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 2200/0684 Venting, avoiding backpressure, avoid gas bubbles
• 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 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/088 Channel loops
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• 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 3/502 : 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: G01N 15/1404 : Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G01N 15/1434 : Optical arrangements
• CPC Class Codes: G01N 15/1436 : the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
• CPC Class Codes: G01N 15/1459 : the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1484 : microstructural devices
• CPC Class Codes: G01N 2015/1006 : for cytology
• CPC Class Codes: G01N 2015/1415 : Control of particle position

Abstract

Method of detection for droplet-based assays. In an exemplary method, an open end of a channel network may be placed into an emulsion. Droplets of the emulsion may be driven along a flow path from the open end, through a confluence region where a dilution fluid is introduced into the flow path from at least one dilution inlet channel to increase an average distance between droplets, and through an examination region disposed downstream of the confluence region. Light may be detected from the examination region as droplets pass through. The channel network may include a droplet inlet channel that meets the at least one dilution inlet channel at the confluence region. The droplet inlet channel may form a tapered region that is sized such that droplets leave the tapered region in single file.

Claims

1. A method of detection for droplet-based assays, the method comprising:

placing an open end of a channel network into an emulsion;

driving droplets of the emulsion along a flow path from the open end, through a confluence region where a dilution fluid is introduced into the flow path from at least one dilution inlet channel to increase an average distance between droplets, and through an examination region disposed downstream of the confluence region; and

detecting light from the examination region as droplets pass through;

wherein the channel network includes a droplet inlet channel that meets the at least one dilution inlet channel at the confluence region, and wherein the droplet inlet channel forms a tapered region that is sized such that droplets leave the tapered region in single file.

2. The method of claim 1, wherein the emulsion is held by a container, and wherein the step of placing an open end of a channel network is performed by moving the open end, the container, or both the open end and the container.

3. The method of claim 1, wherein the step of driving droplets includes a step of aspirating droplets of the emulsion into the channel network via the open end.

4. The method of claim 1, wherein the step of driving droplets includes a step of operating one or more syringe pumps.

5. The method of claim 1, wherein the step of detecting light is performed as droplets that are in single file pass serially through the examination region.

6. The method of claim 1, wherein the flow path has a minimum diameter between the tapered region and the confluence region.

7. The method of claim 6, further comprising a droplet outlet channel that extends from the confluence region, and wherein the droplet outlet channel has about a same diameter as the minimum diameter of the flow path.

8. The method of claim 6, wherein the minimum diameter is less than or approximately equal to the average diameter of the droplets.

9. The method of claim 8, wherein the minimum diameter is between about 90% and 100% of the average diameter of the droplets.

10. The method of claim 6, wherein the flow path is defined as least in part by at least two pieces of tubing and a discrete connector that provides fluid communication between at least a pair of ends of the at least two pieces of tubing, and wherein the minimum diameter is defined by the connector.

11. The method of claim 1, wherein the confluence region is formed at a junction where the droplet inlet channel, a droplet outlet channel, and the at least one dilution inlet channel meet one another.

12. The method of claim 1, wherein a portion of the flow path extends upward from the open end.

13. The method of claim 1, wherein the flow path has a smaller diameter in a region of the droplet inlet channel than in the examination region.

14. The method of claim 1, wherein the at least one dilution inlet channel includes a pair of dilution inlet channels.

15. A method of detection for droplet-based assays, the method comprising:

placing an open end of a conduit into an emulsion;

driving droplets of the emulsion along a flow path from the open end to an examination region; and

detecting light from the examination region as droplets pass through;

wherein the flow path is defined in part by a droplet inlet channel that forms a tapered region, and wherein the tapered region is sized such that droplets leave the tapered region in single file.

16. The method of claim 15, wherein the flow path has a smaller diameter in a region of the droplet inlet channel than in the examination region.

17. The method of claim 15, wherein a region of the droplet inlet channel downstream from the tapered region has a minimum diameter that is approximately equal to an average diameter of the droplets.

18. The method of claim 15, further comprising a step of introducing a dilution fluid into the flow path downstream of the tapered region to increase an average distance between droplets.

19. The method of claim 15, wherein the emulsion is held by a container, and wherein the step of placing an open end is performed by moving the open end, the container, or both the open end and the container.

20. A method of detection for droplet-based assays, the method comprising:

placing an open end of a conduit into an emulsion;

driving droplets of the emulsion into the conduit and along a flow path extending continuously from the open end to an examination region;

introducing a dilution fluid into the flow path at a position upstream of the examination region to increase an average distance between droplets; and

detecting light from the examination region as droplets pass through.