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

Patent metadata

Field	Value
Publication number	US-9517469-B2
Application number	US-91291306-A
Country	US
Kind code	B2
Filing date	May 10, 2006
Priority date	May 11, 2005
Publication date	Dec 13, 2016
Grant date	Dec 13, 2016

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.

First claim

What is claimed is:

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 reac

Assignees

• Shenderov Alexander D
• Pollack Michael G
• Advanced Liquid Logic Inc
• Univ Duke

Inventors

• Shenderov Alexander D
• Pollack Michael G

Classifications

• B01L2300/1872

  Infrared light · CPC title

• B01L2300/1827

  using resistive heater · CPC title

• B01L2300/1816

  using induction heating · CPC title

• B01L7/525 Primary

  with physical movement of samples between temperature zones · CPC title

• B01L3/502792

  for moving individual droplets on a plate, e.g. by locally altering surface tension · CPC title