Droplet-based assay system

• US 9,156,010 B2
• Filed: 09/23/2009
• Issued: 10/13/2015
• 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: Bio-Rad Laboratories Incorporated
• Inventors: Colston, Billy Wayne Jr., Hindson, Benjamin Joseph, Ness, Kevin Dean, Masquelier, Donald Arthur, Milanovich, Fred Paul, Modlin, Douglas N., Riot, Vincent, Burd, Samuel, Belgrader, Phillip, Makarewicz, Anthony Joseph Jr.
• Primary Examiner(s): HOBBS, MICHAEL L
• Application Number:
  US12/586,626
• Publication Number:
  US 20100173394A1
• Time in Patent Office: 2,211 Days
• Field of Search: 435/287.2
• 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 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

1. A system for sample analysis, comprising:

a plurality of droplet generators that form a plurality of separate emulsions including droplets of partitioned samples, each droplet generator including a channel intersection at which one of the emulsions is formed from at least one carrier fluid stream and a sample stream, the samples being prepared as reaction mixtures for amplification of at least one nucleic acid target;

containers defining cavities to hold the emulsions;

a transport structure to transfer the emulsions from the droplet generators to the containers;

a heating and cooling device to heat the emulsions in the cavities, with the emulsions held in an array, to induce nucleic acid amplification in droplets of the emulsions;

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

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

at least one pressure source that drives flow of the at least one carrier fluid stream and the sample stream to the channel intersection of each droplet generator; and

a transport structure to transfer droplets of the emulsions from the containers to the detection assembly.

2. The system of claim 1, wherein a plate provides the containers, and wherein each droplet generator is integrated with the plate.

3. The system of claim 2, wherein each cavity is supplied by a separate droplet generator.

4. The system of claim 1, wherein each droplet generator is provided by a device that does not include the containers.

5. The system of claim 1, wherein each droplet generator includes at least one oil reservoir, a sample reservoir, and a fluid path from each reservoir to an emulsion well.

6. The system of claim 1, wherein the transport structure to transfer droplets of the emulsions from the containers to the detection assembly includes a fluid transfer device connected to the detection assembly and configured to transfer droplets from the cavities to a detection site of the detection assembly, the fluid transfer device having a tip with an open end to serially contact emulsions held in the cavities.

7. The system of claim 6, wherein the detection assembly is configured to detect droplets serially.

8. The system of claim 6, wherein the detection assembly is configured to image batches of droplets.

9. The system of claim 8, wherein the detection assembly is configured to image droplet batches serially, each droplet batch corresponding to a different emulsion.

10. The system of claim 1, further comprising a plate, wherein each cavity is bounded above and below by walls of the plate.

11. The system of claim 1, further comprising a plate, wherein each cavity is bounded by a transparent wall of the plate.

12. The system of claim 1, wherein each droplet generator is fluidically connected to a sample well and a carrier well that each open upwardly to permit loading of sample and carrier fluid from above the wells.

13. The system of claim 1, wherein each cavity is a well, further comprising a sealing member to seal the well.

14. The system of claim 1, wherein each droplet generator includes one or more orifices from which droplets are generated serially, and wherein each droplet generator is configured to form droplets that are monodisperse.

15. The system of claim 1, wherein the controller is configured to estimate the concentration of the nucleic acid target based on a percentage of droplets that are determined to be positive for amplification of the nucleic acid target.

16. The system of claim 1, wherein the pressure source creates a vacuum that drives flow of the at least one carrier fluid stream and the sample stream to the channel intersection of each droplet generator.

21. The system of claim 1, wherein a same at least one pressure source drives flow of the at least one carrier fluid stream and the sample stream to the channel intersection of each droplet generator.

22. The system of claim 21, wherein a same single pressure source drives flow of the at least one carrier fluid stream and the sample stream to the channel intersection of each droplet generator.

23. The system of claim 22, wherein the same single pressure source creates a vacuum that drives flow of the at least one carrier fluid stream and the sample stream to the channel intersection of each droplet generator.

28. The system of claim 1, wherein the transport structure to transfer the emulsions from the droplet generators to the containers includes a plurality of channels providing fluid communication between the plurality of droplet generators and the plurality of containers.

17. A system for sample analysis, comprising:

a plurality of droplet generators that form a plurality of separate emulsions, each droplet generator including a carrier reservoir holding a carrier fluid, a sample reservoir holding a reaction mixture capable of amplification of a target, if present, in the reaction mixture, and an emulsion reservoir fluidically connected to the sample reservoir and the carrier reservoir, each droplet generator including a channel intersection at which one of the emulsions is formed from at least one carrier fluid stream received from the carrier reservoir and a sample stream received from the sample reservoir;

at least one pressure source operatively connected to the droplet generators;

containers defining cavities to hold the emulsions;

a transport structure to transfer the emulsions from the emulsion reservoirs to the containers;

a heating and cooling device to heat the emulsions in the containers, with the emulsions held in an array, to induce nucleic acid amplification in droplets of the emulsions;

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

a transport structure to transfer droplets of the emulsions from the containers to the detection assembly; and

a controller in communication with the detection assembly and programmed to determine a level of the target based on signals detected from intact droplets,

wherein application of pressure to one or more of the reservoirs of each droplet generator with the pressure source drives flow of the at least one carrier fluid stream and the sample stream to the channel intersection of each droplet generator to form droplets of the reaction mixture disposed in the carrier fluid and to move the droplets in the carrier fluid to the emulsion reservoir.

18. The system of claim 17, wherein each cavity is a well, further comprising a sealing member to seal the well.

19. The system of claim 17, wherein each of the reservoirs is a well.

20. The system of claim 17, wherein the pressure source creates a vacuum that drives flow of the at least one carrier fluid stream and the sample stream to each channel intersection and movement of the droplets in the carrier fluid to the emulsion reservoir.

24. The system of claim 17, wherein the transport structure to transfer droplets of the emulsions from the containers to the detection assembly includes a fluid transfer device connected to the detection assembly and configured to transfer droplets from the cavities to a detection site of the detection assembly, the fluid transfer device having a tip with an open end to serially contact emulsions held in the cavities.

25. The system of claim 17, wherein a same at least one pressure source drives flow of the at least one carrier fluid stream and the sample stream to the channel intersection of each droplet generator.

26. The system of claim 25, wherein a same single pressure source drives flow of the at least one carrier fluid stream and the sample stream to the channel intersection of each droplet generator.

27. The system of claim 26, wherein the same single pressure source creates a vacuum that drives flow of the at least one carrier fluid stream and the sample stream to the channel intersection of each droplet generator.