Droplet-based nucleic acid amplification device, system, and method

• US 20080038810A1
• Filed: 12/15/2006
• Published: 02/14/2008
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Paik, Philip, Pamula, Vamsee, Pollack, Michael
• Granted Patent: US 7,816,121 B2
• Time in Patent Office: Days
• US Class Current: 435/283.1
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• 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 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0442 thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 2400/0448 Marangoni flow; Thermocapillary effect
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• 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 3/502792 : for moving individual droplets on a plate, e.g. by locally altering surface tension
• 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: G01N 15/1484 : microstructural devices
• CPC Class Codes: G01N 2015/1006 : for cytology
• CPC Class Codes: G01N 2035/1046 : Levitated, suspended drops
• CPC Class Codes: Y10T 137/0318 : Processes
• CPC Class Codes: Y10T 29/49002 : Electrical device making

Abstract

The present invention relates to a droplet-based nucleic acid amplification device, system, and method. According to one embodiment, a droplet microactuator is provided and includes: (a) a substrate comprising electrodes for conducting droplet operations; and (b) one or more temperature control means arranged in proximity with one or more of the electrodes for heating and/or cooling a region of the droplet microactuator and arranged such that a droplet can be transported on the electrodes into the region for heating.

Claims

1. A droplet microactuator comprising:

(a) a substrate comprising electrodes for conducting droplet operations; and

(b) one or more temperature control means arranged in proximity with one or more of the electrodes for heating and/or cooling a region of the droplet microactuator and arranged such that a droplet can be transported on the electrodes into the region for heating.

2. The droplet microactuator of claim 1 wherein the temperature control means comprises a heater.

3. The droplet microactuator of claim 2 wherein the temperature control means comprises a thin film heater.

4. The droplet microactuator of claim 1 wherein the temperature control means comprises one or more cooling elements.

5. The droplet microactuator of claim 1 wherein the cooling element comprises a thermoelectric cooler.

6. The droplet microactuator of claim 1 wherein the cooling element comprises a Peltier device.

7. The droplet microactuator of claim 1 wherein a region of the droplet microactuator is set to a temperature sufficient for annealing.

8. The droplet microactuator of claim 1 wherein a region of the droplet microactuator is set to a temperature sufficient for denaturation.

9. The droplet microactuator of claim 1 wherein a region of the droplet microactuator is set to a temperature sufficient for extension.

10. The droplet microactuator of claim 1 wherein one or more of the droplet operations is electrowetting-mediated.

11. The droplet microactuator of claim 1 wherein one or more of the droplet operations is dielectrophoresis-mediated.

12. The droplet microactuator of claim 2 wherein the temperature control means comprises at least two heaters:

(a) at least one heater set to a temperature sufficient to effect denaturation of a nucleic acid in a nearby droplet on the droplet microactuator; and

(b) at least one heater set to a temperature sufficient to effect extension and/or annealing of a nucleic acid in a nearby droplet on the droplet microactuator.

13. The droplet microactuator of claim 1 further comprising a means for effecting a magnetic field in proximity to one or more of the electrodes, wherein the strength and proximity of the magnetic field is sufficient to immobilize magnetically responsive beads in a droplet during execution of a droplet operation.

14. The droplet microactuator of claim 13 wherein the means for effecting a magnetic field comprises an electromagnetic and a means for controlling power supply to the electromagnet.

15. The droplet microactuator of claim 13 wherein the means for effecting a magnetic field comprises a magnet and a means for moving the magnet into and out of proximity with the one or more of the electrodes.

16. The droplet microactuator of claim 1 comprising one or more droplet microactuator reservoirs in proximity to one or more of the electrodes arranged such that electrodes can dispense droplets from fluid in the droplet microactuator reservoirs, wherein the reservoirs comprise reagents therein each comprising one or more components selected from the group consisting of:

buffers, primers, nucleotides, polymerases, reverse transcriptases and other nucleic acid amplification reagents.

17. The droplet microactuator of claim 1 further comprising an amplification-ready droplet loaded thereon.

18. The droplet microactuator of claim 17 wherein the amplification-ready droplet has a volume which ranges from about 1 mL to about 10 μ

L.

19. The droplet microactuator of claim 17 wherein the amplification-ready droplet has a volume which ranges from about 10 mL to about 1 μ

L.

20. The droplet microactuator of claim 17 wherein the amplification-ready droplet has a volume which is less than about 100 mL.

21. A system comprising the droplet microactuator of claim 1 comprising a processor electronically coupled to and configured to control:

(a) the electrodes; and

(b) the temperature of one or more of the one or more temperature control means.

22. The system of claim 21 programmed to execute steps in a nucleic acid amplification thermal cycling protocol on a droplet positioned in proximity to a temperature control means by controlling the temperature of the temperature control means.

23. The system of claim 22 wherein the nucleic acid amplification thermal cycling protocol comprises a PCR thermal cycling protocol.

24. The system of claim 22 wherein the nucleic acid amplification thermal cycling protocol comprises an RT-PCR thermal cycling protocol.

25. The system of claim 21 programmed to execute steps in a nucleic acid amplification thermal cycling protocol by transporting a droplet into and out of proximity with one or more temperature control means.

26. The system of claim 25 wherein the nucleic acid amplification thermal cycling protocol comprises a PCR thermal cycling protocol.

27. The system of claim 21 programmed to execute droplet operations to combine one or more sample droplets with one or more amplification reagent droplets on the droplet microactuator in appropriate ratios to constitute one or more amplification-ready droplets.

28. A system comprising the droplet microactuator of claim 13 programmed to execute droplet operations for a droplet-based washing protocol in which magnetically responsive beads are washed on the droplet microactuator.

29. A system comprising the droplet microactuator of claim 13 programmed to execute droplet operations for eluting one or more substances from the magnetically responsive beads.

30. A system comprising the droplet microactuator of claim 16 and programmed to:

(a) dispense reagent droplets from each of the one or more reservoirs;

(b) transport the dispensed reagent droplets;

(c) combine dispensed reagent droplets with one or more sample droplets potentially comprising a target nucleic acid to yield one or more amplification-ready droplets; and

(d) execute a thermal cycling protocol on the one or more amplification-ready droplets sufficient to amplify the target nucleic acid when present in the sample droplet.

31. The system of claim 30 wherein the thermal cycling protocol comprises a measurement step in which amplified target nucleic acid is quantified after a predetermined number of cycles.

32. The system of claim 31 wherein the system is programmed to stop the thermal cycling when a predetermined quantity of target nucleic acid is detected.

33. The system of claim 31 wherein the system is programmed to stop the thermal cycling when a predetermined quantity of target nucleic acid is not detected after a predetermined number of cycles.

34. The system of claim 30 wherein the amplification-ready droplet, having been subjected to the thermal cycling protocol 30(d) further programmed to transport the amplification-ready droplet for further processing during or after thermal cycling.

35. The system of claim 34 wherein the further processing comprises detection of one or more target nucleic acids.

36. The system of claim 35 wherein the system is programmed to provide a user output indicating the presence or absence of one or more target nucleic acids.

37. The system of claim 35 wherein the target nucleic acid comprises a diagnostic nucleic acid.

38. The system of claim 35 wherein the target nucleic acid comprises a diagnostic nucleic acid the presence of which is indicative of the presence of a pathogenic organism.

39. A cartridge comprising the droplet microactuator of claim 16, the cartridge comprising one or more cartridge reservoirs and a means for establishing a fluid path from one or more cartridge reservoirs to one or more droplet microactuator reservoirs.

40. The cartridge of claim 39 comprising one or more nucleic acid amplification reagents pre-loaded in the one or more cartridge reservoirs.

41. The cartridge of claim 40 wherein the reagent(s) are selected from the group consisting of:

buffers, primers, nucleotides, polymerases and other nucleic acid amplification reagents.

42. A method of assembling a droplet microactuator cartridge for operation, the method comprising coupling the cartridge of claim 39 to a droplet microactuator.

43. A method of loading a droplet microactuator cartridge for operation, the method comprising flowing a fluid from the cartridge of claim 39 to a droplet microactuator reservoir via the fluid path.