PCR microreactor for amplifying DNA using microquantities of sample fluid

• US 6,613,560 B1
• Filed: 02/11/2000
• Issued: 09/02/2003
• Est. Priority Date: 10/19/1994
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies Europe BV (Agilent Technologies, Inc.)
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Tso, Jacqueline, Wolber, Paul K., Swedberg, Sally A.
• Primary Examiner(s): Beisner, William H.
• Application Number:
  US09/502,597
• Time in Patent Office: 1,299 Days
• Field of Search: 435/287.2, 435/288.5, 435/288.7, 435/303.1, 435/6, 435/91.2, 204/451, 204/601
• US Class Current: 435/287.2
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 2300/163 Biocompatibility
• CPC Class Codes: B01L 2300/1805 Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1838 using fluid heat transfer medium
• CPC Class Codes: B01L 2300/1872 Infrared light
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0418 electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0442 thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/54 using spatial temperature gradients
• CPC Class Codes: B29C 65/00 Joining or sealing of preformed parts , e.g. welding of plastics materials; Apparatus therefor general aspects of processes or apparatus for joining preformed parts B29C66/00; using porous material formed by internal pressure generated therein for joining preformed parts B29C44/1228, B29C44/326
• CPC Class Codes: B29C 66/549 : said hollow-preforms being interconnected during their moulding process, e.g. by a hinge
• CPC Class Codes: G01N 2030/0035 : electrical field
• CPC Class Codes: G01N 2030/285 : electrically driven carrier
• CPC Class Codes: G01N 27/44721 : by optical means
• CPC Class Codes: G01N 27/44773 : Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 30/02 : Column chromatography
• CPC Class Codes: G01N 30/60 : Construction of the column
• CPC Class Codes: G01N 30/6052 : body
• CPC Class Codes: G01N 30/606 : with fluid access or exit ports
• CPC Class Codes: G01N 30/6095 : Micromachined or nanomachined, e.g. micro- or nanosize

Abstract

A microanalytical device is provided for conducting chemical processes using small amounts of fluid. The devices include microstructures, e.g., microcavities, microchannels and the like, that are laser ablated or otherwise formed in a support substrate, and can be used in a variety of chemical and biochemical methods, including chromatographic, electrophoretic and electrochromatographic separations, screening and diagnostics, and chemical and biochemical synthesis. The devices are formed from a material that is thermally and chemically stable and resistant to biofouling, significantly reducing electroosmotic flow and unwanted adsorption of solute. Preferred materials are polymeric.

Claims

1. In a device for conducting the polymerase chain reaction (PCR) process comprising a reaction chamber defined by two or more interior surfaces, a means for introducing PCR reaction components into the chamber, a means for removing the PCR reaction product from the chamber, and a means for controlling the temperature of the reaction chamber, the improvement which comprises:

fabricating the device from a chemically inert material that does not contain silicon and is thermally, chemically and mechanically stable under the conditions at which a PCR reaction is conducted, and employing a reaction chamber adapted to contain in the range of approximately 1 μ

l to 500 μ

l of fluid, wherein the interior surfaces of the reaction chamber are coated with a sorption-reducing polyethylene oxide layer so as to enhance thermal stability, chemical stability and biofouling resistance.

2. The device of claim 1, wherein the reaction chamber is adapted to contain in the range of approximately 10 μ

l to 200 μ

l of fluid.

3. The device of claim 2, wherein the material is selected from the group consisting of polyimides, polycarbonates, polyesters, polyamides, polyethers, polyurethanes, polyfluorocarbons, polystyrenes, poly(acrylonitrile-butadiene-styrene), polymethyl methacrylate, polyolefins, and copolymers thereof.

4. The device of claim 3, wherein the material is polyimide.

5. The device of claim 1, wherein the material is polymeric.

6. The device of claim 5, wherein the material is stable at temperatures in the range of approximately 37°

C. to 94°

C.

7. The device of claim 5, wherein the material has a glass transition temperature T_g of at least about 100°

C.

8. The device of claim 7, wherein the material has a glass transition temperature T_g in the range of approximately 100°

C. to 150°

C.

9. The device of claim 1, wherein the material is such that high definition features may be fabricated therein.

10. The device of claim 9, wherein high definition features are present.

11. The device of claim 10, wherein the features comprise microchannels approximately 1 μ

m to 200 μ

m in diameter.

12. The device of claim 11, wherein the features comprise microchannels approximately 10 μ

m to 75 μ

m in diameter.

13. A microreactor for amplifying DNA using the polymerase chain reaction (PCR) process, comprising:

a substrate having first and second substantially planar opposing surfaces, said substrate having a cavity and at least one microchannel formed in the first planar surface, wherein the cavity serves as a reaction zone that is in fluid communication with each microchannel;

a cover plate arranged over the first planar surface, said cover plate in combination with the cavity defining a reaction chamber, and with each microchannel defining a microcolumn; and

at least one inlet port and at least one outlet port communicating directly or indirectly with the reaction chamber, said ports enabling the passage of fluid from an external source into and through the reaction chamber, thereby defining a fluid flow path;

wherein the substrate and the cover plate are comprised of a chemically inert material that does not contain silicon and is thermally stable and resistant to biofouling under the conditions used for conducting PCR amplification of DNA, and the interior surfaces of the reaction chamber are coated with a sorption-reducing polyethylene oxide layer so as to enhance thermal stability, chemical stability and biofouling resistance.

14. The microreactor of claim 13, wherein the substrate material provides for reduced absorption of solutes compared to a substrate formed from a silicon-containing material.

15. The microreactor of claim 13, wherein the substrate material can be modified to alter the electroosmotic flow of a flowing fluid in contact therewith.

16. The microreactor of claim 15, wherein the reaction chamber is sized to contain approximately 1 μ

l to 500 μ

l of fluid.

17. The microreactor of claim 16, wherein the reaction chamber is sized to contain approximately 10 μ

l to 200 μ

l of fluid.

18. The microreactor of claim 17, wherein the at least one microchannel is approximately 1 μ

m to 200 μ

m in diameter.

19. The microreactor of claim 18, wherein the at least one microchannel is approximately 10 μ

m 75 μ

m in diameter.

20. The microreactor of claim 16, wherein at least one microchannel is approximately 1 μ

m to 200 μ

m in diameter.

21. The microreactor of claim 20, wherein at least one microchannel is approximately 10 μ

m to 75 μ

m in diameter.

22. The microreactor of claim 13, wherein the substrate material is polymeric.

23. The microreactor of claim 22, wherein the substrate material is selected from the group consisting of polyimides, polycarbonates, polyesters, polyamides, polyethers, polyurethanes, polyfluorocarbons, polystyrenes, poly(acrylonitrile-butadiene-styrene), polymethyl methacrylate, polyolefins, and copolymers thereof.

24. The microreactor of claim 23, wherein the substrate is comprised of polyimide.

25. The microreactor of claim 13, further including an additional cavity formed in the first planar surface, which in combination with the cover plate forms an additional reaction chamber for conducting PCR amplification of DNA.

26. The microreactor of claim 13, wherein the reaction chamber has an upstream region in which fluid is introduced and a downstream region from which fluid exits, and wherein the at least one microchannel comprises an upstream microchannel in fluid communication with the upstream region of the reaction chamber and a downstream microchannel in fluid communication with the downstream region of the reaction chamber.

27. The microreactor of claim 26, wherein the upstream microchannel in combination with the cover plate forms an upstream microcolumn, and the downstream microchannel in combination with the cover plate forms a downstream microcolumn.

28. The microreactor of claim 13, further including motive means to move fluid through the fluid flow path.

29. The microreactor of claim 28, wherein the motive means comprises a means for applying a voltage differential.

30. The microreactor of claim 28, wherein the motive means comprises a means for applying a pressure differential.

31. In a method for conducting the polymerase chain reaction (PCR) process to amplify DNA in a sample which comprises heating the sample to separate double-stranded DNA into single-stranded DNA, cooling the sample so as to allow hybridization of primer oligonucleotides to the single-stranded DNA, replicating the DNA using a DNA polymerase, and repeating the aforementioned steps to achieve the desired degree of amplification, the improvement,which comprises:

conducting the PCR in a microreactor comprised of a chemically inert material that does not contains silicon and is thermally, chemically and mechanically stable under the conditions at which the PCR reaction is conducted, and employing a reaction chamber adapted to contain approximately 1μ

l to 500 μ

l of fluid, wherein the interior surfaces of the reaction chamber are coated with a sorption-reducing polyethylene oxide layer so as to enhance thermal stability, chemical stability and biofouling resistance.

32. The method of claim 31, wherein the reaction chamber is adapted to contain in the range of approximately 10 μ

l to 200 μ

l of fluid.

33. The method of claim 32, wherein the material is selected from the group consisting of polyimides, polycarbonates, polyesters, polyamides, polyethers, polyurethanes, polyfluorocarbons, polystyrenes, poly(acrylonitrile-butadiene-styrene), polymethyl methacrylate, polyolefins, and copolymers thereof.

34. The method of claim 33, wherein the material is polyimide.

35. The method of claim 31, wherein the material is polymeric.

36.The method of claim 31, wherein the material is stable at temperatures in the range of approximately 37° C. to 94°

C.

36. The method of claim 31, wherein the material has a glass transition temperature T_g of at least about 100°

C.

38. The method of claim 31, wherein the material is such that high definition features may be fabricated therein.

37. The method of claim 37, wherein the material has a glass transition temperature T_g in the range of approximately 100°

C. to 150°

C.

39. The method of claim 39, wherein high definition features are present.

40. The method of claim 40, wherein the features comprise microchannels approximately 1 μ

m to 200 μ

m in diameter.

41. The method of claim 41, wherein the features comprise microchannels approximately 10 μ

m to 75 μ

m in diameter.

42. A method for amplifying the quantity of a DNA molecule of interest contained in a small volume of sample fluid using the polymerase chain reaction process, comprising:

(a) introducing into a microreactor up to about 10 μ

l of a sample fluid containing the DNA molecule of interest in double-stranded form, a first and a second primer molecule complementary to opposing strands of the DNA molecule, a thermostable DNA polymerase, free deoxynucleoside triphosphates and PCR buffer, the microreactor comprising

a substrate having first and second substantially planar opposing surfaces, said substrate having a cavity formed in the first planar surface, wherein the cavity serves as a reaction zone,

a cover plate arranged over the first planar surface, said cover plate in combination with the cavity defining a reaction chamber, and

at least one inlet port and at least one outlet port in fluid communication with the reaction chamber, said ports enabling the passage of sample fluid from an external source into and through the reaction chamber, thereby defining a fluid flow path,

wherein the substrate and the cover plate are comprised of a chemically inert material that does not contain silicon and is thermally stable and resistant to biofouling, and wherein the interior surfaces of the reaction chamber are coated directly with a sorption-reducing polyethylene oxide layer so as to enhance thermal stability, chemical stability and biofouling resistance;

(b) applying a motive force to the device to move the sample fluid along the flow path into the reaction chamber;

(c) heating the sample fluid in the reaction chamber to separate the double-stranded DNA into single-stranded DNA;

(d) cooling the sample so as to allow hybridization of the primer molecules to opposing strands of the single-stranded DNA and replication of the single-stranded DNA by the DNA polymerase; and

(e) repeating steps (c) and (d) to achieve the desired degree of amplification.

43. The method of claim 43, wherein the substrate material can be modified to alter the electroosmotic flow of a flowing fluid in contact therewith.

47. The method of claim 43, wherein the device further includes an additional cavity formed in the first planar surface, which in combination with the cover plate forms an additional PCR reaction chamber.

48. The method of claim 43, wherein the motive means comprises a means for applying a voltage differential.

49. The method of claim 43, wherein the motive means comprises a means for applying a pressure differential.

44. The method of claim 44, wherein the substrate material is polymeric.

45. The method of claim 45, wherein the substrate material is selected from the group consisting of polyimides, polycarbonates, polyesters, polyamides, polyethers, polyurethanes, polyfluorocarbons, polystyrenes, poly(acrylonitrile-butadiene-styrene), polymethyl methacrylate, polyolefins, and copolymers thereof.

46. The method of claim 46, wherein the substrate is comprised of polyimide.

50. A method for amplifying the quantity of a DNA molecule of interest contained in a small volume of sample fluid using the polymerase chain reaction process, comprising:

(a) introducing into a microreactor up to about 10 μ

l of a sample fluid containing the DNA molecule of interest in double-stranded form, a first and a second primer molecule complementary to opposing strands of the DNA molecule, a thermostable DNA polymerase, free deoxynucleoside triphosphates and PCR buffer, the microreactor comprising

a substrate having first and second substantially planar opposing surfaces, said substrate having a cavity and at least one microchannel formed in the first planar surface, wherein the cavity serves as a reaction zone that is in fluid communication with each microchannel,

a cover plate arranged over the first planar surface, said cover plate in combination with the cavity defining a reaction chamber, and with each microchannel defining a microcolumn, and

at least one inlet port and at least one outlet port communicating directly or indirectly with the reaction chamber, said ports enabling the passage of sample fluid from an external source into and through the reaction chamber, thereby defining a fluid flow path,

wherein the substrate and the cover plate are comprised of a chemically inert material that does not contain silicon and is thermally stable and resistant to biofouling, and the interior surfaces of the reaction chamber are coated with a sorption-reducing polyethylene oxide layer so as to enhance thermal stability, chemical stability and biofouling resistance;

(b) applying a motive force to the device to move the sample fluid along the flow path into the reaction chamber;

(c) heating the sample fluid in the reaction chamber to separate the double-stranded DNA into single-stranded DNA;

(d) cooling the sample so as to allow hybridization of the primer molecules to opposing strands of the single-stranded DNA and replication of the single-stranded DNA by the DNA polymerase; and

(e) repeating steps (c) and (d) to achieve the desired degree of amplification.

51. The method of claim 51, further including collecting reaction product at the outlet port.