SYSTEM FOR DROPLET-BASED ASSAYS USING AN ARRAY OF EMULSIONS

• US 20110092376A1
• Filed: 12/07/2010
• Published: 04/21/2011
• 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: QuantaLife, Inc. (Bio-Rad Laboratories Incorporated)
• Inventors: Ness, Kevin Dean, Masquelier, Donald Arthur, Hindson, Benjamin Joseph, Makarewicz, Anthony Joseph JR., Colston, Billy Wayne JR., Milanovich, Fred Paul
• Granted Patent: US 9,248,417 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• 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

System, including method and apparatus, for performing droplet-based assays. In the method, an emulsion may be obtained that includes droplets packed closely together in a three-dimensional packing arrangement. Data related to an analyte may be collected from individual droplets of the reacted emulsion as such droplets travel serially through a detection region.

Claims

1. A method of performing a droplet-based assay, comprising:

obtaining an emulsion including droplets packed closely together in a three-dimensional packing arrangement;

reacting the emulsion with the droplets packed closely together; and

collecting data related to an analyte from individual droplets of the reacted emulsion as such droplets travel serially through a detection region.

2. The method of claim 1, wherein the step of obtaining an emulsion includes a step of forming an emulsion by driving a first fluid and a second fluid through a droplet generator to form droplets of the second fluid disposed in the first fluid.

3. The method of claim 2, wherein the step of forming an emulsion includes a step of regulating air pressure adjacent the first fluid, the second fluid, or both of the first and second fluids.

4. The method of claim 3, wherein the step of regulating air pressure includes a step of applying a vacuum such that the first fluid and the second fluid are drawn through the droplet generator.

5. The method of claim 3, wherein the step of regulating air pressure includes a step of applying pressure to the first fluid via air that is in contact with the first fluid.

6. The method of claim 2, wherein the step of forming an emulsion includes a step of creating a pressure drop between input wells and an output well of an emulsion production unit.

7. The method of claim 6, wherein the step of forming an emulsion includes a step of forming a plurality of emulsions with a plate including a plurality of emulsion production units.

8. The method of claim 7, further comprising a step of transferring the emulsions from the plate to a sample holder that holds the emulsions in an array during the step of reacting.

9. The method of claim 7, wherein the step of forming a plurality of emulsions includes a step of forming a plurality of emulsions in parallel.

10. The method of claim 1, wherein the step of obtaining an emulsion includes a step of obtaining an emulsion of monodisperse droplets.

11. The method of claim 1, wherein the step of obtaining an emulsion includes a step of obtaining droplets disposed in a continuous phase, and wherein the droplets are buoyant in the continuous phase such that the droplets collect in an upper region of the continuous phase to achieve the three-dimensional packing arrangement.

12. The method of claim 1, wherein the step of reacting the emulsion includes a step of heating the emulsion.

13. The method of claim 12, wherein the step of heating the emulsion includes a step of thermally cycling the emulsion.

14. The method of claim 13, wherein the step of thermally cycling the emulsion includes a step of amplifying one or more nucleic acid targets in droplets of the emulsion, and wherein the step of collecting data includes a step of collecting data related to amplification of the targets in individual droplets of the emulsion.

15. The method of claim 1, further comprising a step of processing the data to determine whether the analyte is present or absent in individual droplets of the emulsion.

16. The method of claim 1, further comprising a step of picking up droplets of the emulsion after the step of reacting, and a step of driving flow of picked-up droplets through the detection region.

17. The method of claim 16, wherein the step of picking up droplets includes a step of operating a pump to draw droplets of the emulsion into an intake conduit.

18. The method of claim 16, further comprising a step of spatially separating droplets of the emulsion after such droplets are picked up and before such droplets reach the detection region.

19. The method of claim 18, wherein the step of spatially separating droplets includes a step of adding fluid to a continuous phase in which such droplets are disposed.

20. The method of claim 16, wherein the step of picking up droplets includes a step of drawing droplets from the emulsion into an intake conduit having an end disposed in the emulsion.

21. The method of claim 16, wherein the step of picking up droplets includes a step of picking up droplets from each emulsion of an array of reacted emulsions, further comprising a step of driving flow of picked-up droplets from each emulsion through the detection region.

22. The method of claim 21, wherein the detection region is connected to an intake conduit, and wherein the step of picking up includes a step of disposing an end of the intake conduit serially in a plurality of emulsions while such emulsions are held in wells of a plate.

23. The method of claim 22, wherein the step of picking up includes a step of successively picking up a first emulsion and a second emulsion, further comprising a step of drawing a fluid into the intake conduit after picking up the first emulsion and before picking up the second emulsion.

24. The method of claim 23, wherein the step of drawing a fluid includes a step of drawing air.

25. The method of claim 23, wherein the step of drawing a fluid includes a step of drawing a liquid comprising an oil.

26. The method of claim 1, wherein the step of reacting includes a step of reacting an array of emulsions while such emulsions are held by wells of a plate.

27. The method of claim 1, wherein the step of obtaining an emulsion includes a step of forming a plurality of separate emulsions using a plate including a plurality of emulsion production units, wherein the step of reacting includes a step of reacting each of the emulsions, and wherein the step of collecting data is performed with droplets of each of the emulsions.

28. The method of claim 27, wherein each emulsion production unit is configured to produce a separate emulsion and each unit includes a set of wells interconnected by channels that intersect to form a site of droplet generation, each set of wells including

at least one first input well to receive a first fluid,

a second input well to receive a second fluid, and

an output well configured to receive from the site of droplet generation an emulsion of droplets of the second fluid disposed in the first fluid.

29. A method of performing a droplet-based assay, comprising:

producing a plurality of emulsions;

thermocycling the emulsions in parallel while such emulsions are held in an array, to amplify one or more nucleic acid targets in droplets of the emulsions;

driving flow of droplets of each emulsion through a detection region; and

collecting data related to amplification of the nucleic acid targets from individual droplets of each emulsion as such droplets travel through the detection region.

30. The method of claim 29, further comprising a step of picking up emulsions from the array serially using an intake conduit connected to the detection region.

31. The method of claim 30, wherein the step of picking up includes a step of successively picking up a first emulsion and a second emulsion, further comprising a step of introducing a fluid into the intake conduit after picking up the first emulsion and before picking up the second emulsion.

32. The method of claim 31, wherein the step of introducing a fluid includes a step of drawing air into the intake conduit.

33. The method of claim 31, wherein the step of introducing a fluid includes a step of drawing a liquid comprising an oil into the intake conduit.

34. The method of claim 29, wherein the step of producing includes a step of producing a plurality of emulsions in parallel by application of a vacuum in parallel to a plurality of emulsion production units.

35. The method of claim 29, wherein the step of driving flow includes a step of driving flow of each emulsion through a fluid channel that intersects the detection region.

36. The method of claim 35, further comprising a step of loading the emulsions serially into the fluid channel.

37. The method of claim 36, wherein the step of loading is performed using an autosampler operatively connected to the detection region.

38. The method of claim 29, further comprising a step of separating droplets of each emulsion from one another after such droplets are removed from the array and before such droplets reach the detection region.

39. The method of claim 29, wherein the step of collecting data includes a step of collecting fluorescence data from droplets.

40. The method of claim 29, further comprising a step of determining, based on the data collected, whether each nucleic acid target is present or absent in individual droplets of the emulsions.

41. A method of performing a droplet-based assay, comprising:

producing a plurality of emulsions;

thermocycling the emulsions to amplify one or more nucleic acid targets in droplets of the emulsions;

picking up droplets from the emulsions disposed in an array using an intake conduit disposed serially in each of the emulsions;

driving flow of each emulsion through a detection region connected to the intake conduit; and

collecting data related to amplification of the nucleic acid targets from individual droplets of each emulsion traveling through the detection region.

42. The method of claim 41, wherein the step of picking up includes a step of successively picking up a first emulsion and a second emulsion, further comprising a step of introducing a fluid into the intake conduit after picking up the first emulsion and before picking up the second emulsion.

43. The method of claim 42, wherein the fluid is air or a liquid comprising an oil.

44. A system for performing a droplet-based assay, comprising:

a plate including a plurality of emulsion production units capable of producing a plurality of emulsions in parallel;

a thermocycler configured to thermally cycle the emulsions held in an array, to amplify one or more nucleic acid targets in the emulsions;

a droplet transporter configured to pick up droplets of each emulsion from the array and to drive flow of the droplets through a detection region; and

a detector configured to collect data related to amplification of the nucleic acid targets from individual droplets of the emulsions traveling through the detection region.

45. The system of claim 44, wherein the droplet transporter includes an intake conduit that is connected to the detection region and having an inlet end, and wherein the droplet transporter is configured to dispose the inlet end of the intake conduit serially in the emulsions while such emulsions are held in wells of a sample holder, to pick up droplets of each emulsion.

46. The system of claim 45, wherein the droplet transporter is configured to draw each emulsion into the intake conduit.

47. The system of claim 46, wherein the droplet transporter is configured to pick up a first emulsion and a second emulsion successively and to draw a fluid into the intake conduit after picking up the first emulsion and before picking up the second emulsion.

48. The system of claim 47, wherein the droplet transporter is configured to draw air or a liquid comprising an oil as the fluid.

49. The system of claim 44, wherein the droplet transporter includes an intake conduit with an inlet end configured be disposed in contact with each emulsion, and wherein the droplet transporter is configured to draw air or a liquid comprising an oil into the intake conduit between contact of the inlet end with different emulsions.

50. The system of claim 44, wherein the droplet transporter is configured to spatially separate droplets of an emulsion from one another after such droplets are picked up and before such droplets reach the detection region.

51. The system of claim 44, wherein the detector includes a fluorescence detector.

52. The system of claim 44, further comprising a sample holder capable of holding the emulsions in the array.

53. The system of claim 44, further comprising a controller programmed to determine, based on the data collected, whether each nucleic acid target is present or absent in individual droplets of the emulsions.

54. A system for performing a droplet-based assay, comprising:

a plate including a plurality of emulsion production units capable of producing a plurality of emulsions in parallel;

a droplet transporter configured to pick up droplets from the emulsions held in an array and to drive flow of the droplets through a detection region;

a detector configured to collect data related to amplification of one or more nucleic acid targets from individual droplets of emulsions as such individual droplets travel through the detection region; and

a controller programmed to determine, based on the data collected, whether each nucleic acid target is present or absent in individual droplets of the emulsions.

55. The system of claim 54, wherein the droplet transporter includes an intake conduit that is connected to the detection region and having an inlet end, and wherein the droplet transporter is configured to dispose the inlet end of the intake conduit serially in the emulsions while such emulsions are held in wells of a sample holder, to pick up droplets of each emulsion.

56. The system of claim 54, wherein the droplet transporter is configured to draw each emulsion into the intake conduit.

57. The system of claim 56, wherein the droplet transporter is configured to pick up a first emulsion and a second emulsion successively and to draw a fluid into the intake conduit after picking up the first emulsion and before picking up the second emulsion.

58. The system of claim 57, wherein the droplet transporter is configured to draw air as the fluid.

59. The system of claim 57, wherein the droplet transporter is configured to draw a liquid comprising an oil as the fluid.

60. The system of claim 54, wherein the droplet transporter is configured to spatially separate droplets of an emulsion after such droplets are picked up and before such droplets reach the detection region.

61. The system of claim 54, wherein the detector includes a fluorescence detector.

62. The system of claim 54, further comprising a sample holder capable of holding the emulsions in the array.

63. The system of claim 54, wherein each emulsion production unit includes a set of wells interconnected by channels that intersect to form a site of droplet generation, each set of wells including

at least one first input well to receive a continuous phase,

a second input well to receive an aqueous sample, and

an output well configured to receive from the site of droplet generation an emulsion of aqueous droplets disposed in the continuous phase.

64. The system of claim 63, wherein the at least one first input well of each emulsion production unit is not shared with other emulsion production units of the plate.

65. The system of claim 54, wherein the controller is programmed to determine a presence/absence or a concentration of each nucleic acid target in one or more samples included in the emulsions.

66. A system for performing a droplet-based assay, comprising:

a plate including a plurality of emulsion production units;

an instrument configured to be operatively connected to the plate to drive production of emulsions collected in wells of the plate;

a droplet transporter configured to pick up droplets from the emulsions held in an array and to drive flow of the droplets through a detection region;

a detector configured to collect data related to one or more analytes from individual droplets of the emulsions as such individual droplets travel through the detection region; and

a controller programmed to determine, based on the data collected, an aspect of the one or more analytes in one or more samples included in droplets of the emulsions.

67. The system of claim 66, wherein the instrument is configured to be disconnected from the plate and then connected to another plate to drive production of additional emulsions.

68. The system of claim 66, wherein the instrument is a first instrument, further comprising a second instrument including the droplet transporter, the detector, and the controller.