Nucleic acid amplification using microfluidic devices

• US 20050221373A1
• Filed: 05/19/2005
• Published: 10/06/2005
• 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., Hansen, Carl L., Ma, Chiem
• Granted Patent: US 7,833,708 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

1-48. -48. (canceled)

49. A microfluidic device, comprising

(a) a substrate comprising an elastomeric material;

(b) a first plurality of flow channels formed within the substrate;

(c) a second plurality of flow channels, each formed within the substrate and in fluid communication with an inlet, the second flow channels intersecting the first flow channels to define an array of reaction chambers;

(d) isolation valves selectively actuatable to block flow between junctions along at least one of the first and second flow channels, and to regulate solution flow to the reaction chambers; and

(e) a plurality of temperature regions located along each of the second plurality of microfabricated flow channels; and

(f) a temperature controller operatively disposed to regulate temperature at one or more of the temperature regions.

50. The microfluidic device of claim 49, wherein

the first plurality of flow channels are parallel to one another and the second plurality of flow channel are parallel to one another but orthogonal to the first plurality of flow channels, thus defining the array of reaction chambers;

and

at least one of the isolation valves is disposed within each of the first and second plurality of flow channels to regulate solution flow therethrough.

51. The microfluidic device of claim 50, wherein a set of the first plurality of flow channels are in fluid communication with a shared inlet such that sample introduced into the shared inlet flows into each of the flow channels of the set.

52. The microfluidic device of claim 51, wherein the set of flow channels that are in fluid communication with the shared inlet includes every other flow channel of the first plurality of microfabricated flow channels.

53. The microfluidic device of claim 49, further comprising one or more control channels, and wherein one or more of the isolation valves comprises an elastomeric membrane that separates the microfabricated flow channel upon which it acts from one of the control channels, the elastomeric membrane able to be deflected into or retracted from the flow channel upon which it acts in response to an actuation force.

54. The microfluidic device of claim 53, wherein the isolation valves comprise at least one first isolation valve and at least one second isolation valve, the first and second isolation valve having differing activation thresholds.

55. The microfluidic device of claim 54, wherein the elastomeric membrane of the first isolation valve is wider than the elastomeric membrane of the second isolation valve.

56. The microfluidic device of claim 54, wherein

the elastomeric membrane of each of the first and second valves is located at an intersection formed between one of the microfabricated control channels and the flow channel upon which the first or second valve acts;

and

the control channel at the intersection at which the first valve is located is wider than the control channel at the intersection at which the second valve is located.

57. The microfluidic device of claim 54, wherein each of the reaction chambers disposed along at least one of the plurality of second microfabricated flow channels are separated from one another by at least two first isolation valves or at least two second isolation valves.

58. The microfluidic device of claim 49, wherein the plurality of temperature regions each comprise one of the reaction chambers.

59. The microfluidic device of claim 49, wherein a single temperature controller regulates temperature at each of the reaction chambers.

60. The microfluidic device of claim 49, wherein the temperature controller is one of a plurality of temperature controllers, each temperature controller separately regulating temperature at a different reaction chamber.

61. The microfluidic device of claim 49, 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.

62. The microfluidic device of claim 49, wherein the temperature controller comprises a microfluidic chamber adapted to contain heated fluid and configured to be placed adjacent a face of the substrate.

63. The microfluidic device of claim 62, wherein the temperature controller comprises

(i) a pair of spaced apart plates that are joined to one another by a fluid tight separation material located along the periphery of the plates, the space between the plates defining the microfluidic chamber, and the separation material defining the sides of the temperature controller;

(ii) at least one inlet through which fluid can be introduced into the chamber; and

(iii) at least one outlet through which fluid can exit the chamber, the outlet being located at a second end region opposing the first end region.

64. The microfluidic device of claim 49, further comprising at least two pumps, one pump operatively connected to the first plurality of flow channels to transport solution therethrough and a second pump operatively connected to the second plurality of flow channels to transport solution therethrough.

65. The microfluidic device of claim 49, wherein a polymerase enzyme is immobilized within one or more of the reaction chambers.

66. The microfluidic device of claim 49, wherein one or more nucleic acids are immobilized within one or more of the reaction chambers.

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

(a) providing a microfluidic device, comprising

(i) a substrate comprising an elastomeric material;

(ii) a first plurality of flow channels formed within the substrate;

(iii) a second plurality of microfabricated flow channels formed within the substrate and in fluid communication with an inlet, the second flow channels intersecting the first flow channels to define an array of reaction chambers;

(iv) isolation valves selectively actuatable to block flow between reaction chambers along at least one of the first and second flow channels, and to regulate solution flow to the reaction chambers; and

(b) introducing a sample and one or more reactants into the reaction chambers by selective actuation of one or more of the isolation valves, whereby reaction between the sample and the one or more reactants can occur; and

(c) heating regions of the microfluidic device to promote reaction between the sample and the one or more reactants within the reaction chambers.

68. The method of claim 67, wherein introducing comprises pumping sample and/or the one or more reactants into the reaction chambers for reaction.

69. The method of claim 67, wherein introducing comprises introducing the sample and the one or more reactants into the first and second plurality of flow channels and then actuating the isolation valves to allow the sample and the one or more reactants to mix by diffusion within the reaction chambers.

70. The method of claim 67, wherein

the first plurality of flow channels are parallel to one another and the second plurality of flow channel are parallel to one another but orthogonal to the first plurality of flow channels, thus defining the array of reaction chambers;

and

at least one of the isolation valves is disposed within each of the first and second plurality of flow channels to regulate solution flow therethrough.

71. The method of claim 70, wherein

a set of the first plurality of flow channels are in fluid communication with a shared inlet, the flow channels within the set including every other flow channel of the first plurality of flow channels;

introducing comprises introducing the sample into the flow channels that are not within the set of flow channels and introducing the one or more reactants into the shared inlet, whereby flow channels containing samples alternate with flow channels containing reactant.

72. The method of claim 71, wherein introducing further comprises selectively actuating the isolation valves to allow sample and reactants in adjacent flow channels to mix by diffusion.

73. The method of claim 72, wherein the sample is a sample containing a nucleic acid and the one or more reactants is polymerase.

74. The method of claim 67, wherein

the microfluidic device is positioned in a holder that is configured to form an air tight chamber over an inlet to each of the first plurality of flow channels;

and

introducing comprises placing the sample and/or the one or more reactants into the inlets to the first plurality of flow channels and then pressurizing the airtight chamber.

75. The method of claim 67, wherein heating comprises exposing the reaction chambers to a temperature cycle to promote reaction between the sample and the one or more reactants within the reaction chambers.

76. The method of claim 67, wherein heating is controlled by a temperature controller, a single temperature controller regulating the temperature at each of the reaction chambers.

77. The method of claim 67, wherein heating is controlled by a plurality of temperature controllers, different controllers regulating the temperature at different reaction chambers.

78. The method of claim 67, wherein introducing comprises introducing a sample into each of the first plurality of flow channels and introducing a reactant into each of the second plurality of flow channels.

79. The method of claim 78, wherein the sample comprises a target nucleic acid and the one or more reactants comprise one or more components for conducting a nucleic acid amplification reaction.

80. The method of claim 70, wherein the sample introduced into the first plurality of flow channels each contain a nucleic acid and the reactant introduced into the second plurality of flow channels is a component of a nucleic acid amplification or sequencing reaction.

81. The method of claim 79, wherein heating comprises exposing the target nucleic acid and the one or more components to a temperature cycle such that an amplified product is formed within the reaction chambers.

82. The method of claim 81, further comprising detecting amplified product formed with the reaction chambers.

83. The method of claim 82, wherein the amplified product bears a detectable label and the detecting step comprises detecting the label.

84. The method of claim 83, 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.

85. The method of claim 82, wherein the detecting step comprises contacting the amplified product with a label such that the amplified product becomes labeled.

86. The method of claim 85, wherein the label is a interchelating dye.

87. The method of claim 85, wherein the label is a molecular beacon.

88. The method of claim 82, wherein the amplified product is detected by conducting a quantitative PCR assay.

89. The method of claim 82, wherein detecting comprises measuring capacitance of a solution containing amplified product.

90. The method of claim 82, wherein the sample introduced into each reaction chamber comprises a plurality of target nucleic acids and a plurality of amplified products are formed.

91. The method of claim 79, wherein a polymerase enzyme is immobilized within the reaction chambers, whereby during the introduction step, the target nucleic acid and the one or more components are brought into contact with the immobilized polymerase.

92. The method of claim 79, wherein one or more nucleic acids are immobilized within the reaction chambers, whereby during the introduction step, the sample and the one or more reactants are brought into contact with the immobilized nucleic acids.

93. The method of claim 67, wherein the sample comprises a target nucleic acid and the one or more reactants are one or more components for conducting a sequencing reaction.

94. The method of claim 67, wherein the sample comprises a target nucleic acid and the one or more reactants are one or more components for conducting a quantitative PCR analysis.

95. A temperature controller, comprising

(a) a plate assembly comprising a first plate and a second plate that are separated from one another by a separation material, the separation material forming a fluid-tight seal around the periphery of the plates, the space between the plates and bounded by the separation material defining a chamber;

(b) an inlet assembly located at a first end region of the plate assembly and in fluid communication with the chamber; and

(c) an outlet assembly located at a second end region of the plate assembly opposite the first end region and in fluid communication with the chamber such that fluid in the chamber can exit therefrom via the outlet assembly, wherein

a region located between the inlet and outlet assembly and adjacent or abutting a surface of the first plate is adapted to receive a microfluidic chip;

the first and second plate comprise a transparent region that permits optical detection of the microfluidic chip; and

the top plate is less than 100 microns thick.

96. A method for conducting a temperature controlled reaction, the method comprising

(a) providing a temperature controller as defined in claim 95;

(b) placing a microfluidic chip in the region adapted to receive the microfluidic chip; and

(c) flowing one or more solutions at a desired temperature through the chamber via the inlet and outlet assembly, whereby the microfluidic chip is heated or cooled by the one or more solutions flowing through the chamber.

97. A temperature controller, comprising

(a) a plate assembly comprising

(i) a hinged assembly that comprises a first plate and a second plate that are hingeably connected, such that the first plate can be moved toward or away from an upper face of the second plate;

(ii) a third plate

(iii) a separation material that separates a lower face of the second plate opposite the upper face from the third plate and forms a fluid-tight seal therebetween;

(iv) a chamber formed by the space between the lower face of the second plate and the third plate and being bounded by the separation material; and

(v) a pair of holes, one hole being located in the first plate and the other hole being located in the second plate, the holes positioned such that when the first plate is folded onto the second plate, the pair of holes are aligned;

(b) an inlet assembly located at a first end region of the plate assembly and in fluid communication with the chamber; and

(c) an outlet assembly located at a second end region of the plate assembly opposite the first end region and in fluid communication with the chamber such that fluid in the chamber can exit therefrom via the outlet assembly, wherein

the hinged assembly is adapted to receive a microfluidic chip between the first and second plate.

98. A method for conducting a temperature controlled reaction, the method comprising

(a) providing a temperature controller as defined in claim 97;

(b) placing a microfluidic chip into the hinged assembly such that the microfluidic chip is sandwiched between the first and second plate; and

(c) flowing one or more solutions at a desired temperature through the chamber via the inlet and outlet assembly, whereby the microfluidic chip is heated or cooled by the one or more solutions flowing through the chamber.

99. A microfluidic device, comprising

(a) a substrate comprising an elastomeric material;

(b) a first plurality of flow channels formed within the substrate;

(c) a second plurality of flow channels, each formed within the substrate and in fluid communication with an inlet, the second flow channels intersecting the first flow channels to define an array of reaction chambers; and

(c) isolation valves selectively actuatable to block flow between junctions along at least one of the first and second flow channels, and to regulate solution flow to the reaction chambers, wherein the isolation valves comprise a first and second isolation valve that have differing activation thresholds.