Nucleic acid amplification using microfluidic devices

• US 8,486,636 B2
• Filed: 11/16/2010
• Issued: 07/16/2013
• Est. Priority Date: 04/06/2001
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Enzelberger, Markus M., Liu, Jian, Quake, Stephen R.
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US12/947,774
• Publication Number:
  US 20120009663A1
• Time in Patent Office: 973 Days
• Field of Search: 435/6.12, 435/91.2, 435/287.2, 435/287.3
• US Class Current: 435/6.12
• CPC Class Codes: B01F 25/51 in which the mixture is circulated through a set of tubes, e.g. with gradual introduction of a component into the circulating flow
• CPC Class Codes: B01F 25/54 provided with a pump inside the receptacle to recirculate the material within the receptacle
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01F 35/90 Heating or cooling systems
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/088 Channel loops
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1838 using fluid heat transfer medium
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: B01L 9/527 for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: C12Q 3/00 : Condition responsive control processes apparatus therefor C12M1/36; controlling or regulating in general G05

Abstract

The present invention provides microfluidic devices and methods using the same in various types of thermal cycling reactions. Certain devices include a rotary microfluidic channel and a plurality of temperature regions at different locations along the rotary microfluidic channel at which temperature is regulated. Solution can be repeatedly passed through the temperature regions such that the solution is exposed to different temperatures. Other microfluidic devices include an array of reaction chambers formed by intersecting vertical and horizontal flow channels, with the ability to regulate temperature at the reaction chambers. The microfluidic devices can be used to conduct a number of different analyses, including various primer extension reactions and nucleic acid amplification reactions.

Claims

1. A microfluidic device, comprising:

(a) a substrate;

(b) a flow channel disposed within the substrate, the flow channel configured such that a sample introduced into the flow channel can be cycled around the flow channel; and

comprising a plurality of temperature regions at which temperature can be regulated, each temperature region located at a different location along the flow channel;

(c) an inlet in fluid communication with the flow channel via which the sample can be introduced into the flow channel; and

(d) a temperature controller operatively disposed to regulate temperature within at least one of the plurality of temperature regions.

2. The microfluidic device of claim 1, wherein the flow channel is substantially circular.

3. The microfluidic device of claim 2, wherein at least one of the temperature regions differs in length from the other temperature regions.

4. The microfluidic device of claim 1, further comprising a plurality of reaction chambers disposed along the flow channel and in fluid communication therewith, and wherein each reaction chamber is located within one of the temperature regions.

5. The microfluidic device of claim 1, further comprising an outlet that is in fluid communication with one of the plurality of reaction chambers, and wherein the inlet is in fluid communication with one of the plurality of reaction chambers.

6. The microfluidic device of claim 1, wherein the temperature controller is selected from the group consisting of a Peltier device, a resistive heater, a heat exchanger and an indium tin oxide element.

7. The microfluidic device of claim 1, wherein a single temperature controller regulates temperature at all of the temperature regions.

8. The microfluidic device of claim 1, wherein the temperature controller is one of a plurality of temperature controllers, each temperature controller separately regulating temperature at a different temperature region.

9. The microfluidic device of claim 8, wherein there are at least three temperature regions.

10. A method for conducting an analysis, the method comprising:

(a) providing a microfluidic device, comprising (i) a substrate;

(ii) a flow channel disposed within the substrate, the flow channel configured such that a sample introduced into the flow channel can be cycled around the flow channel; and

comprising a plurality of temperature regions at which temperature can be regulated, each temperature region located at a different location along the flow channel;

(b) introducing a sample into the flow channel; and

(c) transporting the sample between the different temperature regions.

11. The method of claim 10, wherein the flow channel is substantially circular.

12. The method of claim 10, wherein the microfluidic device further comprises a plurality of reaction chambers disposed along the flow channel and in fluid communication therewith, and wherein each reaction chamber is located within one of the temperature regions;

and the transporting step comprises repeatedly transporting the sample through the plurality of reaction chambers.

13. The method of claim 10, wherein the sample is transported through at least three temperature regions, each at a different temperature.

14. The method of claim 10, wherein the sample comprises a target nucleic acid and the introducing step comprises introducing one or more components for conducting a nucleic acid amplification reaction.

15. The method of claim 14, wherein the sample and the one or more components are repeatedly transported through the flow channel, whereby the sample and the one or more components are exposed to the temperature regions multiple times and an amplified product is formed.

16. The method of claim 15, further comprising detecting the amplified product.

17. The method of claim 10, wherein a polymerase enzyme is immobilized within one or more of the temperature regions, whereby during transport the sample and the one or more components are brought into contact with the immobilized polymerase.

18. The method of claim 10, wherein one or more nucleic acids are immobilized in the flow channel within one or more of the temperature regions, such that when the sample is transported through the temperature regions the sample and the one or more components are brought into contact with the one or more nucleic acids.

19. The method of claim 10, wherein the sample comprises a target nucleic acid and the introducing step comprises introducing one or more components for conducting a sequencing reaction.