Droplet-based assay system

• US 20100173394A1
• Filed: 09/23/2009
• Published: 07/08/2010
• Est. Priority Date: 09/23/2008
• Status: Active Grant

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Inventors: Modlin, Douglas N., Burd, Samuel, Belgrader, Phillip, Ness, Kevin Dean, Masquelier, Donald Arthur, Hindson, Benjamin Joseph, Makarewicz, Anthony Joseph JR., Colston, Billy Wayne JR., Milanovich, Fred Paul, Riot, Vincent
• Granted Patent: US 9,156,010 B2
• Time in Patent Office: Days
• US Class Current: 435/287.200
• 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 and methods, for performing assays. These systems may involve separating sample components by partitioning them into droplets or other partitions, amplifying or otherwise reacting the components within the droplets, detecting the amplified components, or characteristics thereof, and/or analyzing the resulting data, among others.

Claims

28. A system for sample analysis, comprising:

at least one droplet generator that forms a plurality of emulsions including droplets that each contain a sample partition prepared as a reaction mixture for amplification of a nucleic acid target;

a plate defining an array of cavities to hold the emulsions;

a heating and cooling device to heat the emulsions disposed in the cavities to induce nucleic acid amplification in droplets;

a detection assembly to detect signals from intact droplets of the emulsions; and

a controller in communication with the detection assembly and programmed to estimate a presence, if any, of the nucleic acid target in a sample based on signals detected from the intact droplets.

1. A system for analyzing a sample, comprising

a droplet generator configured to generate droplets containing portions of a sample to be analyzed, the droplets being disposed in an immiscible fluid forming a sample emulsion,

a heating and cooling station having a fluid inlet and a fluid outlet,

a detection station downstream from the heating and cooling station,

a channel forming a single-pass continuous fluid route from the fluid inlet to the fluid outlet of the heating and cooling station,

a pump for moving the sample emulsion through the channel,

a controller programmed to operate fluid transport through the channel, and

an analyzer configured to process data collected at the detection station.

2. The system of claim 1, wherein the detection system is situated to detect presence of target in the sample emulsion after passing through the heating and cooling system.

3. The system of claim 1 further comprising a droplet reservoir, a first fluid conduit connecting the droplet generator to the reservoir, and a second fluid conduit connecting the reservoir to the fluid inlet of the heating and cooling station.

4. The system of claim 1, wherein the droplet generator is adapted for single-use detachable connection to the heating and cooling station without exposing the heating and cooling station to contamination from sample contained in the sample emulsion.

5. The system of claim 1, wherein the droplet generator is configured to generate the sample emulsion external to the heating and cooling station.

6. The system of claim 1, wherein the heating and cooling station includes multiple heating zones along the fluid route configured for performing a polymerase chain reaction on a nucleic acid target contained in a droplet.

7. The system of claim 1, wherein the heating and cooling station includes at least one thermoelectric cooler.

8. The system of claim 1, wherein the controller is programmed to adjust the droplet generator to alter droplet size based on data received from the detection station.

9. The system of claim 1, wherein the controller is programmed to alter sample concentration prior to droplet generation based on data received from the detection station.

10. The system of claim 1, wherein the controller is programmed to alter a sample preparation procedure prior to droplet generation in the droplet generator based on data received from the detection station.

11. The system of claim 1, wherein the analyzer is programmed to determine a concentration of a target molecule in the sample based at least partially on the frequency of droplets containing the target out of a population of droplets containing sample portions.

12. The system of claim 1, wherein the droplet generator includes a sample reservoir, an oil source, an oil/sample intersection, and an emulsion outlet, the emulsion outlet having a distal end portion adapted for detachable sealed engagement with a receiving port on the heating and cooling station.

13. The system of claim 1, wherein the droplet generator is contained in a cartridge having at least one piston for driving emulsification.

14. The system of claim 1, wherein the droplet generator is contained in a cartridge having at least one piston for pumping sample emulsion through the channel network.

15. The system of claim 1, wherein the channel includes a helical capillary tube portion passing through the heating and cooling station.

16. The system of claim 15, wherein the capillary tube portion has a diameter approximately equal to the diameter of droplets generated by the droplet generator.

17. The system of claim 1, wherein the capillary tube portion includes a hot-start segment passing through a hot-start zone prior to a denaturation zone in the heating and cooling station.

18. The system of claim 1, wherein the heating and cooling station includes thermoelectric coolers configured for controlling temperatures in heating and cooling zones by transferring heat between a thermal core and the heating and cooling zones.

19. The system of claim 15, wherein the helical capillary tube portion defines a helical path that decreases in length over successive cycles.

20. The system of claim 1, wherein the heating and cooling station includes:

a core defining a central longitudinal axis,

a plurality of segments attached to the core and defining a plurality of temperature regions; and

a plurality of heating elements configured to maintain each temperature region approximately at a desired temperature, a portion of the channel configured to transport a sample emulsion cyclically through the temperature regions.

21. The system of claim 20, wherein the plurality of segments includes a plurality of inner segments defining the plurality of temperature regions and a plurality of outer segments attached to the inner segments, and wherein the portion of the channel is disposed between the inner and outer segments.

22. The system of claim 21, wherein the portion of channel includes fluidic tubing that wraps around the inner segments.

23. The system of claim 21, wherein the fluidic tubing is disposed in grooves of the inner segments that wrap substantially helically around the inner segments.

24. The system of claim 1, wherein the droplet generator is contained in a disposable cartridge.

25. The system of claim 24, wherein the cartridge includes a cell lysing region, a separating region, a reagent mixing region, and a droplet generation region for extracting nucleic acid from a sample and formation of droplets into a heat stable sample emulsion.

26. The system of claim 1, wherein the channel has open ends for permitting continuous flow of a sample emulsion.

27. The system of claim 1, wherein the droplet generator is capable of generating a heat stable sample emulsion.

29. The system of claim 28, wherein the droplet generator is integrated with the plate.

30. The system of claim 29, wherein each cavity is supplied by a separate droplet generator.

31. The system of claim 29, wherein each cavity is supplied by the same droplet generator.

32. The system of claim 28, wherein the droplet generator is not part of the plate.

33. The system of claim 28, wherein the droplet generator includes at least one oil reservoir, a sample reservoir, and a fluid path from each reservoir to at least one cavity.

34. The system of claim 28, further comprising a pressure source that drives droplet generation.

35. The system of claim 28, wherein the detection assembly is configured to detect signals from droplets while disposed in the cavities.

36. The system of claim 28, further comprising a fluid transfer device configured to transfer droplets from the cavities to a detection site of the detection assembly.

37. The system of claim 36, wherein the detection site is separate from the plate.

38. The system of claim 36, wherein the detection assembly is configured to detect droplets serially.

39. The system of claim 36, wherein the detection assembly is configured to image batches of droplets.

40. The system of claim 39, wherein the detection assembly is configured to image droplet batches serially, each droplet batch corresponding to a different emulsion.

41. The system of claim 28, wherein each cavity is bounded above and below by walls of the plate.

42. The system of claim 28, wherein each cavity is bounded by a transparent wall of the plate that permits detection of droplets in such cavity through the transparent wall.

43. The system of claim 28, wherein the droplet generator includes a sample reservoir that opens upwardly to permit sample loading from above the plate.

44. The system of claim 28, wherein the cavity is a well, further comprising a sealing member to seal the well.

45. The system of claim 28, wherein the droplet generator includes one or more orifices from which the droplets are generated serially.

46. The system of claim 28, wherein the droplet generator is configured to form droplets that are monodisperse.

47. The system of claim 28, wherein the controller is configured to estimate the presence of the nucleic acid target based on a percentage of droplets that are determined to be positive for amplification of the nucleic acid target.

40-1. The system of claim 28, wherein the detection assembly includes confocal optics.

48. A system for sample analysis, comprising:

a droplet generator including an oil reservoir, a sample reservoir, a cavity, and a channel intersection that receives a sample from the sample reservoir and a carrier fluid from the oil reservoir and generates droplets that flow to the cavity as an emulsion; and

a heating device to heat the droplet generator to induce nucleic acid amplification in droplets of the emulsion in the cavity.

49. The system of claim 48, further comprising a plate that includes the droplet generator and a plurality of other droplet generators.

50. The system of claim 48, further comprising a pressure source that drives droplet generation.

51. The system of claim 50, wherein the pressure source includes a manifold that forms a sealed relation with the droplet generator.

52. The system of claim 48, further comprising a detection assembly to detect signals from droplets of the emulsion.

53. The system of claim 52, wherein the detection assembly is configured to detect signals from droplets while the droplets are disposed in the cavity.

54. The system of claim 52, wherein the detection assembly is configured to detect signals from the droplets while the droplet generator is thermally coupled to the heating device.

55. The system of claim 52, wherein the detection assembly is configured to image a batch of droplets.

56. The system of claim 55, wherein the detection assembly includes confocal optics.

57. The system of claim 52, further comprising a controller in communication with the detection assembly and programmed to estimate a presence, if any, of a nucleic acid target in the sample based on the signals detected.

58. The system of claim 48, wherein the heating device includes a temperature-controlled chamber that receives the droplet generator.

59. The system of claim 48, wherein the heating device is a heating and cooling device that thermally cycles the droplet generator to induce PCR amplification in the droplets of the emulsion in the cavity.

60. The system of claim 48, wherein the cavity is bounded above and below by walls of the droplet generator.

61. The system of claim 48, wherein the cavity is bounded by a transparent wall of the droplet generator that permits detection of droplets in the cavity through the transparent wall.

62. The system of claim 48, wherein the cavity is a well, further comprising a sealing member to seal the well.