Nucleic acid amplification utilizing microfluidic devices

• US 6,960,437 B2
• Filed: 04/05/2002
• Issued: 11/01/2005
• Est. Priority Date: 04/06/2001
• Status: Expired due to Fees

• 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:
  US10/118,466
• Publication Number:
  US 20030008308A1
• Time in Patent Office: 1,306 Days
• Field of Search: 435/6, 435/91.2, 435/287.2, 435/287.3
• US Class Current: 435/6.19
• 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. Certaom 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 comprising an elastomeric material;

(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, further comprising a pump operatively disposed to transport fluid through the flow channel.

3. The microfluidic device of claim 1, further comprising one or more control channels, and wherein the pump comprises one or more of the control channels, each of the control channels of the pump formed within an elastomeric material and separated from the flow channel by a section of an elastomeric membrane, the membrane being deflectable into or retractable from the flow channel in response to an actuation force applied to the control channel.

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

5. The microfluidic device of claim 4, wherein at least one of the temperature regions differs in length from the other temperature regions.

6. The microfluidic device of claim 4, wherein the size of the substantially circular flow channel is enlarged in the plurality of temperature regions relative to the regions of the substantially circular flow channel outside of the plurality of temperature control regions.

7. The microfluidic device of claim 4, further comprising a pump operatively disposed to transport fluid through the substantially circular flow channel, and one or more control channels, and wherein the pump comprises one or more of the control channels, each of the control channels of the pump formed within an elastomeric material and separated from the substantially circular flow channel by a section of an elastomeric membrane, the membrane being deflectable into or retractable from the substantially circular flow channel in response to an actuation force applied to the control channel.

8. 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.

9. The microfluidic device of claim 8, further comprising a plurality of control channels, each formed within an elastomeric material and separated from one of the reaction chambers by an elastomeric membrane, the membrane being deflectable into one of the reaction chambers in response to an actuation force applied to the control channel;

and

wherein the sample can be transported between the reaction chambers by actuation of the control channels.

10. The microfluidic device of claim 9, wherein the plurality of reaction chambers are in fluid communication such that substantially all of the sample within the plurality of reaction chambers is collected at one of the plurality of reaction chambers upon actuation of the control channels associated with the other reaction chambers.

11. The microfluidic device of claim 9, wherein there are three reaction chambers, each reaction chamber being in fluid communication with the other two chambers.

12. The microfluidic device of claim 8, 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.

13. The micro fluidic 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.

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

15. 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.

16. The microfluidic device of claim 1, wherein one or more nucleic acids are immobilized within one or more of the temperature regions.

17. The microfluidic device of claim 1, wherein a polymerase is immobilized within one or more of the temperature regions.

18. The microfluidic device of claim 1, wherein there at least two temperature regions.

19. The microfluidic device of claim 18, wherein there are at least three temperature regions.

20. A microfluidic device, comprising

(a) a substantially circular microfabricated flow channel in fluid communication with an inlet;

(b) a plurality of temperature regions, each region located at a different location along the substantially circular flow channel; and

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

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

(a) providing a microfluidic device, comprising

(i) a substrate comprising an elastomeric material;

(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.

22. The method of claim 21, wherein the flow channel is substantially circular.

23. The method of claim 21, 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.

24. The method of claim 21, wherein the sample is transported through two temperature regions, each at a different temperature.

25. The method of claim 21, wherein the sample is transported through at least three temperature regions, each at a different temperature.

26. The method of claim 21, 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.

27. The method of claim 26, 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.

28. The method of claim 27, further comprising detecting the amplified product.

29. The method of claim 28, wherein the amplified product bears a detectable label and the detecting step comprises detecting the label.

30. The method of claim 29, wherein the label is selected from the group consisting of a fluorophore, a chromophore, a radioisotope, a luminescent agent, a mass label, an enzyme conjugated to a nucleic acid, and a magnetic agent.

31. The method of claim 28, wherein the detecting step comprises contacting the amplified product with a label such that the amplified product becomes labeled.

32. The method of claim 31, wherein the label is a interchelating dye.

33. The method of claim 31, wherein the label is a molecular beacon.

34. The method of claim 28, wherein the amplified product is detected by conducting a quantitative PCR assay.

35. The method of claim 28, wherein the amplified product is detected by conducting capillary gel electrophoresis.

36. The method of claim 28, wherein detecting comprises measuring capacitance of a solution containing amplified product.

37. The method of claim 28, wherein the microfluidic device further comprises a separation module in fluid communication with the flow channel, and wherein the method further comprises transporting a mixture containing amplified product from the flow channel to the separation module and separating amplified product from other components in the mixture prior to the detection step.

38. The method of claim 37, wherein separating comprises performing capillary gel electrophoresis.

39. The method of claim 27, wherein the sample comprises a plurality of target nucleic acids and a plurality of amplified products are formed.

40. The method of claim 39, wherein the plurality of amplified products are detected by differentially labeling the amplified products.

41. The method of claim 40, wherein the amplified products are differentially labeled by contacting the amplified products with a plurality of molecular beacons, each of the molecular beacons being complementary to a different amplified product.

42. The method of claim 39, wherein the amplified products are detected by conducting quantitative PCR with a plurality of sets of primers and probes, each set comprising a primer and probe complementary to a segment of one of the amplified products.

43. The method of claim 39, the method further comprising separating the plurality of extension products by capillary gel electrophoresis.

44. The method of claim 21, 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.

45. The method of claim 21, wherein the flow channel one or more nucleic acids are immobilized within one 6r 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.

46. The method of claim 21, wherein the sample comprises a target nucleic acid and the introducing step comprises introducing one or more components for conducting a sequencing reaction.

47. The method of claim 21, wherein the sample comprises a target nucleic acid and the introducing step comprises introducing one or more components for conducting a quantitative PCR analysis.

48. The method of claim 21, wherein the microfluidic device further comprises a pump operatively connected to the flow channel, and the sample is repeatedly transported through the flow channel under the action of the pump.