Integrated droplet actuator for gel; electrophoresis and molecular analysis

• US 9,091,649 B2
• Filed: 11/08/2010
• Issued: 07/28/2015
• Est. Priority Date: 11/06/2009
• Status: Expired due to Fees

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pollack, Michael G., Srinivasan, Vijay, Hua, Zhishan, Chu, Hon Lung, Hauser, Michael, Eckhardt, Allen
• Primary Examiner(s): Noguerola, Alex
• Application Number:
  US13/508,300
• Publication Number:
  US 20130059366A1
• Time in Patent Office: 1,723 Days
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2300/088 Channel loops
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/161 Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0457 passive flow or gravitation
• CPC Class Codes: B01L 2400/046 Chemical or electrochemical formation of bubbles
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: G01N 27/44743 Introducing samples
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

The invention is directed to a droplet actuator device and methods for integrating gel electrophoresis analysis with pre or post-analytical sample handling as well as other molecular analysis processes. Using digital microfluidics technology, the droplet actuator device and methods of the invention provide the ability to perform gel electrophoresis and liquid handling operations on a single integrated device. The integrated liquid handling operations may be used to prepare and deliver samples to the electrophoresis gel, capture and subsequently process products of the electrophoresis gel or perform additional assays on the same sample materials which are analyzed by gel electrophoresis. In one embodiment, one or more molecular assays, such as nucleic acid (e.g., DNA) quantification by real-time PCR, and one or more sample processing operations such as sample dilution is performed on a droplet actuator integrated with an electrophoresis gel. In one embodiment, an electrophoresis gel may be integrated on the top substrate of the droplet actuator.

Claims

1. An integrated droplet actuator device for conducting molecular assays comprising:

(a) a top substrate and a bottom substrate, the two substrates configured to form a droplet operations gap;

(b) electrodes associated with one or both of the bottom substrate and the top substrate, and configured for conducting droplet operations in the gap;

(c) a gel;

(d) a pair of electrodes in electrical contact with the gel, the electrodes arranged at first and second locations in relation to the gel;

(e) one or more fluid paths extending from inside the gap to the gel; and

(f) at least a first reaction zone and a second reaction zone.

2. The integrated droplet actuator device of claim 1, further comprising one or more loading slots formed in the gel and arranged in fluid connection with one or more of the fluid paths.

3. The integrated droplet actuator device of claim 1, wherein the gel comprises a gel slab.

4. The integrated droplet actuator device of claim 1, wherein the gel slab comprises an electrophoresis gel.

5. The integrated droplet actuator device of claim 1, further comprising at least a first heat source and a second heat source corresponding to the at least first reaction zone and a second reaction zone respectively.

6. The integrated droplet actuator of claim 5, wherein the at least first heat source and second heat source each comprise a heater bar.

7. The integrated droplet actuator of claim 6, wherein each heater bar comprises an aluminum heater bar, the aluminum heater bar further comprising one or more heating resistors and thermistors.

8. The integrated droplet actuator of claim 1, wherein one of the at least first or second reaction zones is heated to a melting temperature.

9. The integrated droplet actuator of claim 8, wherein the melting temperature comprises a temperature sufficient for denaturing of DNA template and primers.

10. The integrated droplet actuator of claim 8, wherein the melting temperature comprises about 95°

C.

11. The integrated droplet actuator of claim 1, wherein one of the at least first or second reaction zones is heated to a temperature sufficient for annealing of DNA template and primer extension.

12. The integrated droplet actuator of claim 11, wherein the temperature sufficient for annealing of DNA template and primer extension comprises a temperature in a range of about 55°

C. to about 65°

C.

13. The integrated droplet actuator of claim 1, wherein the at least first reaction zone and second reaction zone are positioned at a sufficient distance from the gel so as to not affect the gel internal temperature.

14. The integrated droplet actuator device of claim 1, wherein the gel comprises an electrophoresis gel.

15. The integrated droplet actuator device of claim 1, wherein the gel comprises one or more loading slots formed in the gel.

16. The integrated droplet actuator device of claim 15, wherein the one or more loading slots are aligned with and in fluid connection with the one or more fluid paths.

17. The integrated droplet actuator device of claim 15, wherein the one or more loading slots comprise nucleic acid loading slots.

18. The integrated droplet actuator device of claim 15, wherein the one or more loading slots are loaded with a loading buffer.

19. The integrated droplet actuator device of claim 15, wherein the loading buffer is retained in the one or more loading slots by surface tension forces.

20. The integrated droplet actuator device of claim 15, wherein the one or more fluid paths further provide a fluidic path from one or more designated electrodes to one or more corresponding loading spots.

21. The integrated droplet actuator device of claim 15, wherein a microfluidics-gel interface is formed where the one or more fluid paths and the one or more loading slots interface.

22. The integrated droplet actuator device of claim 15, wherein the gel is in electrical contact with a pair of electrodes, wherein the electrodes are arranged at first and second locations in relation to the gel and parallel to the one or more loading slots.

23. The integrated droplet actuator device of claim 1, wherein the pair of electrodes are directly attached to and integrated with the top substrate.

24. The integrated droplet actuator device of claim 1, wherein the pair of electrodes are positioned in a corresponding pair of trenches molded into the gel slab, wherein the trenches are arranged at first and second locations in relation to the gel and facilitate an electrical interface that provides a uniform electrical field in the gel.

25. The integrated droplet actuator device of claim 24, wherein the pair of trenches comprises an electrophoresis buffer.

26. The integrated droplet actuator device of claim 25, wherein electrophoresis buffer comprises Tris-Borate-Edta (TBE).

27. The integrated droplet actuator device of claim 1, wherein the pair of electrodes are arranged at first and second locations in contact with the gel.

28. The integrated droplet actuator device of claim 1, wherein the pair of electrodes comprises platinum wire.

29. The integrated droplet actuator device of claim 1, wherein one of the pair of electrodes is a negative electrode and one of the pair of electrodes is a positive electrode.

30. The integrated droplet actuator device of claim 1, wherein electrophoresis is conducted without submerging the gel in an electrophoresis buffer.

31. The integrated droplet actuator device of claim 1, wherein the gel is arranged in a protruded structure formed on the top substrate.

32. The integrated droplet actuator device of claim 1, wherein the gel is submerged in an electrophoresis buffer during electrophoresis.

33. The integrated droplet actuator device of claim 1, wherein the top substrate and the bottom substrate are attached to one another by an epoxy glue ring.

34. The integrated droplet actuator device of claim 1, wherein the electrodes in part (b) of claim 1 comprise an array of electrodes.

35. The integrated droplet actuator device of claim 1, wherein the electrodes in part (b) of claim 1 comprise a path of electrodes.

36. The integrated droplet actuator device of claim 1, wherein the electrodes in part (b) of claim 1 comprise electrowetting electrodes.

37. The integrated droplet actuator device of claim 1, wherein the bottom substrate comprises a printed circuit board.

38. The integrated droplet actuator device of claim 1, wherein the top substrate comprises a high transparency and low fluorescence plastic material.

39. The integrated droplet actuator device of claim 38, wherein the plastic material is suitable for fluorimeter operation.

40. The integrated droplet actuator device of claim 38, wherein the plastic material comprises cyclo-olefin polymer and/or copolymer.

41. The integrated droplet actuator device of claim 1, wherein the droplet operations gap comprises a filler fluid.

42. The integrated droplet actuator device of claim 41, wherein the filler fluid comprises oil.

43. The integrated droplet actuator device of claim 42, wherein the oil comprises silicone oil.

44. The integrated droplet actuator device of claim 1, wherein the bottom substrate comprises a hydrophobic layer disposed on a surface of the bottom substrate facing the gap.

45. The integrated droplet actuator device of claim 44, wherein the hydrophobic layer is formed on top of the electrodes.

46. The integrated droplet actuator device of claim 1, wherein the top substrate comprises a hydrophobic layer disposed on a surface of the top substrate facing the gap.

47. The integrated droplet actuator device of claim 1, wherein the gel is arranged on the top substrate.