Method and device for conducting biochemical or chemical reactions at multiple temperatures

• US 9,517,469 B2
• Filed: 05/10/2006
• Issued: 12/13/2016
• Est. Priority Date: 05/11/2005
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Shenderov, Alexander D., Pollack, Michael G.
• Primary Examiner(s): PRIEST, AARON A
• Application Number:
  US11/912,913
• Publication Number:
  US 20080274513A1
• Time in Patent Office: 3,870 Days
• Field of Search: None
• US Class Current: 1/1
• 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 PCR amplification reaction requiring temperature cycling, the method comprising the steps of:

(a) providing a droplet actuator comprising;

(i) a first substrate and a second substrate separated to form a gap; and

(ii) an electrowetting array comprising droplet operations electrodes associated with the top substrate and/or the bottom substrate;

(b) providing at least one reaction droplet to at least two reaction zones in the electrowetting array, each reaction zone having a different temperature needed for the nucleic acid amplification reaction, the at least one reaction droplet comprising a nucleic acid of interest and reagents needed to effect amplification of the nucleic acid, wherein the reaction zones are not simultaneously at the same temperature during the reaction, and the reaction droplet is disposed within a filler fluid;

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

(d) repeating step (c) to conduct further cycles of the nucleic acid amplification reaction; and

wherein the at least one reaction droplet is disposed between the first and second substrates and maintains contact with both the first and second substrates during movement of the at least one reaction droplet.

2. A method for conducting a PCR amplification reaction requiring temperature cycling, the method comprising the steps of:

(a) providing a droplet actuator comprising;

(i) a first substrate and a second substrate separated to form a gap; and

(ii) an electrowetting array comprising droplet operations electrodes associated with the top substrate and/or the bottom substrate;

(b) providing at least one reaction droplet to the electrowetting array, the at least one reaction droplet comprising a nucleic acid of interest and reagents needed to effect amplification of the nucleic acid, the reagents including nucleic acid primers, and wherein the reaction droplet is disposed within a filler fluid;

(c) moving the at least one reaction droplet through the filler fluid, using electrowetting, through a first reaction zone of the electrowetting array having a first temperature such that the nucleic acid of interest is denatured;

(d) moving the at least one reaction droplet through the filler fluid, 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;

(e) moving the at least one reaction droplet through the filler fluid, 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, wherein the first, second, and third reaction zones are not simultaneously at the same temperature during amplification;

(f) repeating steps (c), (d), and (e); and

wherein the at least one droplet is disposed between the first and second substrates and maintains contact with both the first and second substrates during movement of the at least one droplet.

3. The method of claim 2, further comprising:

(a) moving the at least one droplet, 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 at least one reaction droplet from the detection site along a return path of the electrowetting array to the first reaction zone and repeating steps (c), (d), and (e).

5. A method for conducting a PCR amplification reaction requiring temperature cycling, the method comprising the steps of:

(a) providing a droplet actuator comprising;

(i) a first substrate and a second substrate separated to form a gap; and

(ii) an electrowetting array comprising droplet operations electrodes associated with the top substrate and/or the bottom substrate;

(b) providing reaction droplets to the electrowetting array, the reaction droplets comprising a nucleic acid of interest and reagents needed to effect amplification of the nucleic acid, the reagents including nucleic acid primers, and wherein the reaction droplets are disposed within a filler fluid;

(c) moving the droplets through the filler fluid, using electrowetting, through a first reaction zone of the electrowetting array having a first temperature such that the nucleic acid of interest is denatured;

(d) moving the droplets through the filler fluid, 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, wherein the first and second reaction zones are not simultaneously at the same temperature during amplification;

(e) repeating steps (c) and (d); and

wherein the droplets are disposed between the first and second substrates and maintains contact with both the first and second substrates during movement of the droplets.

6. A method for conducting a PCR amplification reaction requiring temperature cycling, the method comprising:

(a) providing a droplet actuator comprising;

(i) a first substrate and a second substrate separated to form a gap; and

(ii) an electrowetting array comprising droplet operations electrodes associated with the top substrate and/or the bottom substrate;

(b) providing at least one reaction droplet to the electrowetting array comprising at least two reaction zones, each reaction zone having a different temperature needed for the reaction, the at least one reaction droplet comprising reagents needed to effect the reaction, wherein the reaction zones are not simultaneously at the same temperature during the reaction, and the reaction droplet is disposed within a filler fluid;

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

(d) repeating step (c) to conduct further cycles of the reaction; and

wherein the at least one reaction droplet is disposed between the first and second substrates and maintains contact with both the first and second substrates during movement of the at least one reaction droplet.

7. A method for conducting a PCR amplification reaction requiring temperature cycling, the method comprising:

(a) providing a droplet actuator comprising;

(i) a first substrate and a second substrate separated to form a gap; and

(ii) an electrowetting array comprising droplet operations electrodes associated with the top substrate and/or the bottom substrate;

(b) providing at least one reaction droplet or volume to the droplet actuator, the droplet actuator further 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, wherein the reaction zones are not simultaneously at the same temperature during the reaction, and the reaction droplet is disposed within a filler fluid;

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

(d) repeating step (c) to conduct further cycles of the reaction; and

wherein the at least one reaction droplet or volume is disposed between the first and second substrates and maintains contact with both the first and second substrates during movement of the at least one reaction droplet or volume.

8. A method for conducting a PCR amplification reaction requiring temperature cycling, the method comprising;

(a) providing a droplet actuator comprising a first surface and a second surface separated to form a gap and at least one reaction droplet, wherein the at least one reaction droplet is disposed within a filler fluid; and

(b) using electric fields to cycle the at least one reaction droplet through the filler fluid and through reaction zones on one of the first or second surfaces comprising at least two reaction zones having different temperatures, wherein the reaction zones are not simultaneously at the same temperature during the reaction, and wherein the droplet maintains contact with both the first and second surfaces during movement of the at least one reaction droplet.

9. The method of claim 8 wherein the droplet comprises a nucleic acid and amplification reagents.

10. The method of claim 9 wherein the reagents are from the group consisting of nucleic acid primers, nucleotides and enzymes.

11. The method of claim 8 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.

12. The method of claim 8 wherein the at least one droplet comprises reagents for effecting amplification of a nucleic acid, and each cycle results in amplification of the nucleic acid.

13. The method of claim 12 further comprising cycling the droplet through a detection site for detecting amplification.

14. The method of claim 13 wherein the detecting amplification is achieved by detecting fluorescence from the droplet.

15. The method of claim 12 further comprising cycling the droplet after each amplification cycle through a detection site for detecting amplification.

16. The method of claim 12 wherein the reagents comprise amplification reagents selected from the group consisting of nucleic acid primers, nucleotides and enzymes.

17. The method of claim 12 wherein the reagents comprise a polymerase.

18. A method for conducting a PCR amplification reaction requiring temperature cycling, the method comprising:

(a) providing a droplet actuator comprising;

(i) a first substrate and a second substrate separated to form a gap; and

(ii) an electrowetting array comprising droplet operations electrodes associated with the top substrate and/or the bottom substrate;

(b) providing a droplet, wherein the droplet;

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

(ii) is surrounded by a filler fluid;

(c) cycling, using electrowetting, the droplet in the filler fluid through thermal zones to effect amplification of the nucleic acid, wherein the thermal zones are not simultaneously at the same temperature during amplification; and

wherein the droplet is disposed between the first and second substrates and maintains contact with both the first and second substrates during movement of the droplet.

19. The method of claim 18 wherein multiple droplets are provided in step (a) and moved in step (b) to effect amplification of multiple nucleic acids.

21. The method of claim 18 wherein the filler fluid comprises silicone oil.

20. A method for conducting a PCR amplification reaction requiring temperature cycling, the method comprising:

(a) providing a device comprising a first surface and a second surface and a plurality of planar electrodes configured for moving one or more droplets on at least one of the first or second surfaces comprising two or more zones having different temperatures, wherein the two or more zones are not simultaneously at the same temperature during the reaction, and the one or more droplets are disposed within a filler fluid;

(b) cycling the one or more droplets through the filler fluid on an electrowetting surface and through the two or more zones to effect the reaction; and

wherein the one or more droplets maintain contact with both the first and second surfaces during transporting of the one or more droplets.