Cartridge with lysis chamber and droplet generator

• US 9,243,288 B2
• Filed: 09/30/2011
• Issued: 01/26/2016
• Est. Priority Date: 09/23/2008
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Ness, Kevin Dean, Burd, Samuel, Hindson, Benjamin Joseph, Belgrader, Phillip, Colston, Billy W. Jr.
• Primary Examiner(s): HOBBS, MICHAEL L
• Application Number:
  US13/250,815
• Publication Number:
  US 20120028311A1
• Time in Patent Office: 1,579 Days
• Field of Search: 435/91.2, 435/287.2, 435/294.1
• US Class Current: 1/1
• 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

Systems, including apparatus, methods, compositions, kits, and software, for preparing, reacting, detecting, and/or analyzing samples in droplet-based assay systems, among others. The disclosure emphasizes, but is not limited to, a disposable cartridge with lysis chamber and droplet chamber, particularly for use in droplet-based assays.

Claims

1. A method of nucleic acid amplification, comprising:

purifying a fluid sample in a portion of a disposable, single-use cartridge;

lysing the sample in a portion of the cartridge;

combining the purified, lysed sample with a reagent mixture in a portion of the cartridge;

driving flow of at least one carrier fluid stream and a sample stream to a channel intersection at which droplets containing the sample are generated to form an emulsion, wherein the sample stream includes the purified, lysed sample combined with the reagent mixture; and

cycling the emulsion thermally in a thermocycling instrument to which the emulsion is transferred from the cartridge,

wherein the step of combining includes a step of advancing a first plunger that is operatively connected to a first reservoir containing one of the sample and the reagent mixture, while retracting a second plunger that is operatively connected to a second reservoir containing the other of the sample and the reagent mixture, to move fluid out of the first reservoir and into the second reservoir.

2. The method of claim 1, wherein the step of purifying includes a step of extracting the sample from a sample collector within the cartridge.

3. The method of claim 1, wherein the step of purifying includes a step of concentrating the sample within the cartridge.

4. The method of claim 1, wherein the step of purifying includes a step of separating sample components based on size before the step of lysing.

5. The method of claim 1, wherein the step of purifying includes a step of separating nucleic acids from other sample components after the step of lysing.

6. The method of claim 1, wherein the step of lysing includes a step of agitating the sample mechanically.

7. The method of claim 6, wherein the step of agitating the sample mechanically includes a step of applying magnetic force to a stir element.

8. The method of claim 1, where the step of cycling includes a step of driving flow of the emulsion along a channel that extends through a plurality of temperature zones.

9. The method of claim 1, wherein the step of cycling includes a step of holding the emulsion in a container while a temperature of the container is changed over time.

10. The method of claim 1, wherein the cartridge is a first cartridge, wherein the steps of purifying, lysing, combining, and driving are performed with the first cartridge operatively connected to an instrument that controls the steps of purifying, lysing, combining, and driving, further comprising a step of disconnecting the first cartridge and a step of repeating the steps of purifying, lysing, combining, and driving within a second cartridge operatively connected to the instrument after the first cartridge is disconnected.

11. The method of claim 10, wherein the instrument supplies fluid to the first and second cartridges during at least one of the steps of purifying, lysing, combining, and driving within each cartridge.

12. The method of claim 11, wherein the instrument supplies a continuous phase for the emulsion.

13. The method of claim 10, wherein the instrument performs the step of cycling the emulsion thermally.

14. The method of claim 1, further comprising a step of providing a cartridge including

a sample chamber configured to receive a sample,

one or more reagent chambers each fluidically connected to the sample chamber and each configured to receive sample-containing fluid from the sample chamber and to combine the sample-containing fluid with a reagent mixture, and

one or more droplet generation regions, each including a channel intersection at which droplets containing the sample are generated to form an emulsion and each fluidically connected to one of the reagent chambers and each configured to receive sample/reagent mixture fluid from one of the reagent chambers and to combine the sample/reagent mixture fluid with a continuous phase to form an emulsion of sample-containing droplets, and

wherein the cartridge is configured to be connected to a reusable instrument that drives operation of the cartridge.

15. The method of claim 14, wherein the step of providing includes a step of providing a cartridge including a reservoir chamber fluidically connected to the sample chamber and configured to supply a reconstitution fluid to the sample chamber.

16. The method of claim 14, wherein the step of providing includes a step of providing a cartridge in which the one or more reagent chambers are a plurality of reagent chambers.

17. The method of claim 14, wherein the step of providing includes a step of providing a cartridge in which the one or more droplet generation regions are a plurality of droplet generation regions.

18. The method of claim 14, wherein the step of providing includes a step of providing a cartridge in which the sample chamber, the reagent chambers, and the droplet generation regions are fluidically connected to each other by fluid channels defined collectively by a first body portion and a second body portion that are attached to each other, and wherein the fluid channels are created by grooves that are formed in a surface of the first body portion and that are covered by a substantially planar surface of the second body portion.

19. The method of claim 14, wherein the step of providing includes a step of providing a cartridge including an input chamber configured to receive a continuous phase to be transferred to the droplet generation regions.

20. The method of claim 19, wherein the step of providing includes a step of providing a cartridge including an outlet chamber configured to receive a portion of the continuous phase that has been transferred out of the input chamber, but that has not been utilized in one of the emulsions.

21. The method of claim 14, wherein the step of providing includes a step of providing a cartridge including a plurality of droplet chambers each configured to receive one of the generated emulsions.

22. The method of claim 14, wherein the step of providing includes a step of providing a cartridge including a plurality of plungers configured to be moved to transfer fluid into and out of the chambers.

23. The method of claim 22, wherein the step of providing includes a step of providing a cartridge in which each plunger is configured to act as a valve by selectively closing an entrance to at least one fluid channel when such plunger is in its most advanced position.

24. The method of claim 14, wherein the step of providing includes a step of providing a cartridge in which a plurality of connectors carry fluid between at least one chamber of the cartridge and the instrument.

25. The method of claim 14, wherein the step of providing includes a step of providing a cartridge in which the sample chamber includes an agitation element configured to be agitated by magnetic forces.

26. The method of claim 14, wherein the step of providing includes a step of providing a cartridge in which the one or more reagent chambers include a plurality of reagent chambers that are fluidically connected to the sample chamber in parallel.

27. The method of claim 14, wherein the continuous phase includes oil, wherein the step of providing includes a step of providing a cartridge in which the cartridge further comprises at least one oil reservoir fluidically connected to at least one of the reagent chambers and configured to supply the oil used to form a corresponding emulsion.

28. The method of claim 27, wherein the step of providing includes a step of providing a cartridge in which the one or more reagent chambers are a plurality of reagent chambers and in which the at least one oil reservoir includes a distinct oil reservoir for each reagent chamber and configured to supply the oil used to form the corresponding emulsion.

29. The method of claim 14, wherein the step of providing includes a step of providing a cartridge in which at least one of the reagent chambers has been pre-loaded with reagents.

30. The method of claim 29, wherein the step of providing includes a step of providing a cartridge in which at least one of the reagent chambers has been pre-loaded with primers for amplification of a nucleic acid target.

31. The method of claim 18, wherein the step of providing includes a step of providing a cartridge in which the droplet generation regions are defined collectively by the surface of the first body portion and the substantially planar surface of the second body portion.

32. The method of claim 1, wherein a same amount of fluid moves out of the first reservoir and moves into the second reservoir during the step of advancing.

33. The method of claim 1, wherein a third plunger is operatively connected to a third reservoir, and wherein the second plunger and the third plunger are retracted during the step of advancing.