Method and Device for Conducting Biochemical or Chemical Reactions at Multiple Temperatures

• US 20080274513A1
• Filed: 05/10/2006
• Published: 11/06/2008
• Est. Priority Date: 05/11/2005
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Duke University
• Inventors: Pollack, Michael G., Shenderov, Alexander D.
• Granted Patent: US 9,517,469 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/1816 using induction heating
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1872 Infrared light
• CPC Class Codes: B01L 2400/0427 Electrowetting
• 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

Abstract

Methods and devices for conducting chemical or biochemical reactions that require multiple reaction temperatures are described. The methods involve moving one or more reaction droplets or reaction volumes through various reaction zones having different temperatures on a microfluidics apparatus. The devices comprise a microfluidics apparatus comprising appropriate actuators capable of moving reaction droplets or reaction volumes through the various reaction zones.

Claims

1. A method for conducting a nucleic acid amplification reaction requiring different temperatures, the method comprising the steps of:

(a) providing at least one reaction droplet to an electrowetting array comprising at least two reaction zones, each reaction zone having a different temperature needed for the nucleic acid amplification reaction, the reaction droplet comprising a nucleic acid of interest and reagents needed to effect amplification of the nucleic acid;

(b) conducting the nucleic acid amplification reaction by moving, using electrowetting, the at least one reaction droplet through the at least two reaction zones such that a first cycle of the nucleic acid amplification reaction is completed; and

(c) optionally repeating step (b) to conduct further cycles of the nucleic acid amplification reaction.

2. A method for amplifying a nucleic acid of interest comprising the steps of:

(a) providing at least one reaction droplet to an electrowetting array, the reaction droplet comprising a nucleic acid of interest and reagents needed to effect amplification of the nucleic acid, the reagents including nucleic acid primers;

(b) moving the droplet(s), using electrowetting, through a first reaction zone of the electrowetting array having a first temperature such that the nucleic acid of interest is denatured;

(c) moving the droplet(s), using electrowetting, through a second reaction zone of the electrowetting array having a second temperature such that the primers are annealed to the nucleic acid of interest;

(d) moving the droplet(s), using electrowetting, through a third reaction zone of the electrowetting array having a third temperature such that extension of the nucleic acid primers occurs, thus amplifying the nucleic acid of interest; and

(e) optionally repeating steps (b), (c), and (d).

3. The method of claim 2, further comprising:

(a) moving the droplet(s), using electrowetting, from the third reaction zone to a detection site; and

(b) detecting for the presence of amplified nucleic acid in the reaction droplet(s).

4. The method of claim 3, further comprising moving the reaction droplet(s) from the detection site along a return path of the electrowetting array to the first reaction zone and repeating steps (b), (c), and (d).

5. A method for amplifying a nucleic acid of interest comprising the steps of:

(a) providing at least one reaction droplet to an electrowetting array, the reaction droplet comprising a nucleic acid of interest and reagents needed to effect amplification of the nucleic acid, the reagents including nucleic acid primers;

(b) moving the droplet(s), using electrowetting, through a first reaction zone of the electrowetting array having a first temperature such that the nucleic acid of interest is denatured;

(c) moving the droplet(s), using electrowetting, through a second reaction zone of the electrowetting array having a second temperature such that the primers are annealed to the nucleic acid of interest and such that extension of the nucleic acid primers occurs, thus amplifying the nucleic acid of interest; and

(d) optionally repeating steps (b) and (c).

6. A device for conducting chemical or biochemical reactions at various temperatures, the device comprising:

(a) a microfluidics apparatus comprising at least one reaction path, at least one detection site, and at least one return path;

(b) means for actuating a reaction droplet or a reaction volume through the reaction path(s), detection zone(s), and return path(s);

(c) at least two reaction zones, each reaction zone capable of maintaining a temperature different from the other reaction zones; and

(d) wherein the reaction path travels through at least two reaction zones.

7. The device of claim 6 wherein at least one of the reaction paths, at least one of the detection sites, and at least one of the return paths form a loop that is fluidly connected.

8. The device of claim 6 wherein the means for actuating a reaction droplet or a reaction volume comprises one or more electrowetting electrodes, thermal actuators, bubble-based actuators, or microvalve-based actuators.

9. A device for conducting chemical or biochemical reactions at various temperatures, the device comprising:

(a) a microfluidics apparatus comprising a plurality of reaction paths, at least one detection site, and at least one return path;

(b) means for actuating a reaction droplet or a reaction volume through the reaction paths, detection zone(s), and return path(s);

(c) at least two reaction zones, each reaction zone capable of maintaining a temperature different from the other reaction zones;

(d) wherein each of the reaction paths travels through at least two reaction zones; and

(e) wherein at least one of the reaction paths is fluidly connected to at least one detection zone.

10. A device for conducting chemical or biochemical reactions at various temperatures, the device comprising:

(a) an electrowetting array comprising a plurality of electrowetting electrodes forming at least one reaction path, at least one detection site, and at least one return path;

(b) at least two reaction zones, each reaction zone capable of maintaining a temperature different from the other reaction zones; and

(c) wherein the reaction path travels through at least two reaction zones and the electrowetting array is capable of manipulating a reaction droplet through the reaction path(s), detection zone(s), and return path(s).

11. The device of claim 10, wherein each reaction path is adjacent to at least one detection site.

12. A method for conducting a reaction requiring different temperatures, the method comprising:

(a) providing at least one reaction droplet to an electrowetting array comprising at least two reaction zones, each reaction zone having a different temperature needed for the reaction, the reaction droplet comprising reagents needed to effect the reaction;

(b) conducting the reaction by moving, using electrowetting, the at least one reaction droplet through the at least two reaction zones such that a first cycle of the reaction is completed; and

(c) optionally repeating step (b) to conduct further cycles of the reaction.

13. A method for conducting a reaction requiring different temperatures, the method comprising:

(a) providing at least one reaction droplet or volume to a microfluidics apparatus comprising at least two reaction zones and at least one detection site, each reaction zone having a different temperature needed for the reaction, the reaction droplet comprising reagents needed to effect the reaction;

(b) conducting the reaction by moving, using actuation means, the at least one reaction droplet or volume through the at least two reaction zones such that a first cycle of the reaction is completed; and

(c) optionally repeating step (b) to conduct further cycles of the reaction.

14. A device for conducting reactions, the device comprising a droplet actuator device comprising two or more reaction zones having different temperatures wherein the droplet actuator device is configured to move a droplet through each of the temperature zones.

15. The device of claim 14 wherein the droplet actuator device comprises a device for moving droplets using electrowetting.

16. The device of claim 14 wherein the droplet actuator device comprises a device for manipulating droplets using electric fields.

17. The device of claim 14 wherein the droplet actuator device comprises a plurality of electrodes configured to move the droplet through each of the temperature zones.

18. The device of claim 14 wherein the reaction zones comprise reaction zones having temperatures suitable for amplifying a nucleic acid.

19. The device of claim 18 wherein the reaction zones comprise reaction zones having temperatures selected to effect denaturing of nucleic acids, annealing of primers to nucleic acids, and/or polymerization of nucleic acids.

20. The device of claim 14 further comprising a mechanism for keeping a reaction zone at a constant temperature.

21. The device of claim 20 wherein the mechanism comprises a resistive heating mechanism.

22. The device of claim 20 wherein the mechanism comprises an inductive heating mechanism.

23. The device of claim 20 wherein the mechanism comprises an infrared heating mechanism.

24. A device comprising an electrowetting path or array comprising two or more zones having different temperatures.

25. The device of claim 24 further comprising a means for maintaining a reaction zone at a reaction temperature.

26. A device comprising an electrowetting surface and a plurality of planar electrodes configured for moving one or more droplets on the electrowetting surface, the surface comprising two or more heating and/or cooling mechanisms for providing zones having different temperatures.

27. A device for amplifying nucleic acid, the device comprising:

(a) an electrowetting path or array comprising two or more zones having different temperatures;

(b) a droplet on the electrowetting path or array comprising a nucleic acid and amplification reagents; and

(c) a filler fluid surrounding the droplet.

28. The device of claim 27 wherein the filler fluid comprises silicone oil.

29. A method of conducting a reaction at multiple temperatures in a droplet comprising reagents for effecting the reaction, the method comprising using electric fields to move the droplet between reaction zones on a surface comprising at least two reaction zones having different temperatures.

30. The method of claim 29 wherein the droplet comprises a nucleic acid and amplification reagents.

31. The method of claim 30 wherein the reagents are from the group consisting of nucleic acid primers, nucleotides and enzymes.

32. The method of claim 29 wherein the reaction zones comprise reaction zones having temperatures selected to effect denaturing of nucleic acids, annealing of primers to nucleic acids, and/or polymerization of nucleic acids.

33. The method of claim 29 wherein the reaction comprises polymerase chain reaction.

34. The method of claim 29 wherein the reaction comprises ligase chain reaction.

35. The method of claim 29 wherein the reaction comprises transcription based amplification.

36. The method of claim 29 comprising using electric fields to cycle a droplet between reaction zones on a surface comprising at least two reaction zones having different temperatures.

37. The method of claim 36 wherein the droplet comprises reagents for effecting amplification of a nucleic acid, and each cycle results in amplification of the nucleic acid.

38. The method of claim 37 further comprising cycling the droplet through a detection site for detecting amplification.

39. The method of claim 38 wherein the detecting amplification is achieved by detecting fluorescence from the droplet.

40. The method of claim 37 further comprising cycling the droplet after each amplification cycle through a detection site for detecting amplification.

41. The method of claim 29 wherein the reagents comprise amplification reagents selected from the group consisting of nucleic acid primers, nucleotides and enzymes.

42. The method of claim 29 wherein the reagents comprise polymerase.

43. A method of amplifying a nucleic acid, the method comprising:

(a) providing a droplet, wherein the droplet;

(i) comprises the nucleic acid and reagents for amplifying the droplet; and

(ii) is surrounded by silicone oil; and

(b) moving the droplet in the silicone oil between thermal zones to effect amplification of the nucleic acid.

44. The method of claim 43 wherein step (b) comprises moving the droplet using electrodes.

45. The method of claim 43 wherein step (b) comprises moving the droplet by electrowetting.

46. The method of claim 43 wherein multiple droplets are provided in step (a) and moved in step (b) to effect amplification of multiple nucleic acids.

47. A method of conducting a reaction, the method comprising:

(a) providing a device comprising a surface and a plurality of planar electrodes configured for moving one or more droplets on the surface, the surface comprising two or more zones having different temperatures; and

(b) transporting the one or more droplets on the electrowetting surface between the two or more zones to effect the reaction.

48. A method of conducting a reaction at multiple temperatures in a droplet comprising reagents for effecting the reaction, the method comprising moving the droplet between reaction zones on a surface comprising at least two reaction zones having different temperatures.

49. The method of claim 48 comprising using electric fields to cycle a droplet between reaction zones on a surface comprising at least two reaction zones having different temperatures.

50. The method of claim 49 wherein the droplet comprises reagents for effecting amplification of a nucleic acid, and each cycle results in amplification of the nucleic acid.

51. The method of claim 50 further comprising cycling the droplet through a detection site for detecting amplification.