Integrated droplet actuator for gel electrophoresis and molecular analysis

• US 9,952,177 B2
• Filed: 07/09/2015
• Issued: 04/24/2018
• Est. Priority Date: 11/06/2009
• Status: Active Grant

• 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, ALEXANDER STEPHAN
• Application Number:
  US14/795,304
• Publication Number:
  US 20150323496A1
• Time in Patent Office: 1,020 Days
• 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 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 arranged on the top substrate;

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

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

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 2, wherein the one or more loading slots comprise nucleic acid loading slots.

4. The integrated droplet actuator device of claim 2, wherein the one or more loading slots are loaded with a loading buffer.

5. The integrated droplet actuator device of claim 4, wherein the loading buffer is retained in the one or more loading slots by surface tension forces.

6. The integrated droplet actuator device of claim 2, 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.

7. The integrated droplet actuator device of claim 2, wherein a microfluidics-gel interface is formed where the one or more fluid paths and the one or more loading slots interface.

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

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

10. The integrated droplet actuator device of claim 1, wherein the gel is in electrical contact with the pair of electrodes, wherein the pair of electrodes are arranged at first and second locations in relation to the gel and parallel to the one or more loading slots.

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

12. 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, wherein the trenches are arranged at first and second locations in relation to the gel and facilitate an electrical interface that provides an essentially uniform electrical field in the gel.

13. The integrated droplet actuator device of claim 12, wherein the pair of trenches comprises an electrophoresis buffer.

14. The integrated droplet actuator device of claim 13, wherein electrophoresis buffer comprises Tris-Borate-Edta (TBE).

15. 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.

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

17. 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.

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

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

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

21. 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.

22. The integrated droplet actuator device of claim 1, wherein the electrodes comprise an array of electrodes.

23. The integrated droplet actuator device of claim 1, wherein the electrodes comprise a path of electrodes.

24. The integrated droplet actuator device of claim 1, wherein the electrodes comprise electrowetting electrodes.

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

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

27. The integrated droplet actuator device of claim 26, wherein the plastic material is suitable for fluorimeter operation.

28. The integrated droplet actuator device of claim 26, wherein the plastic material comprises cyclo-olefin polymer and copolymer.

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

30. The integrated droplet actuator device of claim 29, wherein the filler fluid comprises oil.

31. The integrated droplet actuator device of claim 29, wherein the oil comprises silicone oil.

32. 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.

33. The integrated droplet actuator device of claim 32, wherein the hydrophobic layer is formed on top of the electrodes.

34. 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.

35. A method of preparing and delivering a sample droplet for gel electrophoresis in an integrated droplet actuator device comprising:

(a) providing an integrated droplet actuator device comprising;

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

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

(iii) a gel arranged on the top substrate;

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

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

(b) loading a droplet containing a sample material on at least one of the one or more electrodes, wherein the at least one electrode is in proximity to the one or more fluid paths;

(c) transferring the sample material into a loading buffer; and

(d) transferring the sample material from the loading buffer into the gel.

36. The method of claim 35, wherein the sample droplet comprises a quantity of nucleic acid.

37. The method of claim 36, wherein the quantity of nucleic acid comprises DNA.

38. The method of claim 35, wherein the integrated droplet actuator device, further comprising one or more loading slots formed in the gel and arranged in fluid connection with one or more of the fluid paths.

39. The method of claim 38, wherein the loading buffer is retained in the one or more loading slots.

40. The method of claim 38, wherein the capillary action is effected by a surface energy gradient formed from a hydrophobic gap of the integrated droplet actuator through the one or more fluid paths being less hydrophobic than the gap of the integrated droplet actuator to the one or more loading slots being hydrophilic.

41. The method of claim 35, wherein the sample droplet containing the sample material is merged into the loading buffer.

42. The method of claim 41, wherein the sample droplet is merged into the loading buffer through capillary action.

43. The method of claim 35, wherein the sample material is transferred into the loading buffer through chemical diffusion.

44. The method of claim 35, wherein the sample material is transferred into the loading buffer through electrophoresis.

45. The method of claim 35, wherein the sample material is transferred into the sample droplet from the loading buffer.

46. The method of claim 45, wherein the sample material is transferred into the sample droplet from the loading buffer through chemical diffusion.

47. The method of claim 45, wherein sample material is transferred into the sample droplet from the loading buffer through electrophoresis.

48. The method of claim 35, wherein sample material is transferred into the gel from the loading buffer by electrophoresis.

49. The method of claim 35, wherein the sample material is concentrated, wherein concentrating the sample material comprises:

(a) delivering, using droplet operations, additional droplets containing the sample material to at least one of the one or more electrodes in proximity to the one or more fluid paths; and

(b) transferring the sample material, from the additional droplets, into the loading buffer until a desired concentration of sample material is achieved.

50. A method of preparing and delivering a sample droplet for gel electrophoresis in an integrated droplet actuator device comprising:

(a) providing an integrated droplet actuator device comprising;

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

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

(iii) a gel arranged on the top substrate, wherein at least a portion of the gel extends into the gap of the integrated droplet actuator;

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

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

(b) loading a droplet containing a sample material on at least one of the one or more electrodes, wherein the at least one electrode is in proximity to the one or more fluid paths;

(c) transferring the sample material into a loading buffer retained in the one or more fluid paths; and

(d) transferring the sample material from the loading buffer into the gel.