Nanochannel arrays and their preparation and use for high throughput macromolecular analysis

• US 20040197843A1
• Filed: 01/20/2004
• Published: 10/07/2004
• Est. Priority Date: 07/25/2001
• Status: Active Grant

• Current Assignee: The Trustees of Princeton University (Princeton University)
• Original Assignee: The Trustees of Princeton University (Princeton University)
• Inventors: Austin, Robert H., Yu, Zhaoning, Chou, Stephen Y., Cao, Han, Tegenfeldt, Jonas O.
• Granted Patent: US 7,670,770 B2
• Time in Patent Office: Days
• US Class Current: 435/7.92
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• CPC Class Codes: B01L 2200/0663 Stretching or orienting elongated molecules or particles
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0896 Nanoscaled
• 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 2400/086 using baffles or other fixed flow obstructions
• 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: B81B 1/002 Holes characterised by their shape, in either longitudinal or sectional plane
• CPC Class Codes: B81B 2201/058 Microfluidics not provided for in B81B2201/051 - B81B2201/054
• CPC Class Codes: B81B 2203/0338 Channels
• CPC Class Codes: B81C 1/00071 Channels
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/48721 : Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: Y10S 977/704 : Nucleic acids, e.g. DNA or RNA
• CPC Class Codes: Y10S 977/88 : with arrangement, process, or apparatus for testing
• CPC Class Codes: Y10S 977/883 : Fluidic self-assembly, FSA

Abstract

Nanochannel arrays that enable high-throughput macromolecular analysis are disclosed. Also disclosed are methods of preparing nanochannel arrays and nanofluidic chips. Methods of analyzing macromolecules, such as entire strands of genomic DNA, are also disclosed, as well as systems for carrying out these methods.

Claims

1. A nanochannel array comprising

a surface having a plurality of channels in the material of the surface, said channels having a trench width of less than about 150 nanometers and a trench depth of less than 200 nanometers;

at least some of the channels being surmounted by sealing material to render such channels at least substantially enclosed.

2. The nanochannel array of claim 1 wherein at least one end of at least one of the channels is in fluid communication with at least one reservoir.

3. The nanochannel array of claim 1 wherein both ends of at least some of the channels are in fluid communication with reservoirs.

4. The nanochannel array of claim 1 wherein the plurality of channels is in fluid communication with at least one sample reservoir common to at least some of the channels.

5. The nanochannel array of claim 4 wherein the plurality of channels is in fluid communication with at least one waste reservoir.

6. The nanochannel array of claim 1 wherein the channels are substantially parallel.

7. The nanochannel array of claim 1 wherein the ends of the channels are capable of admitting a macromolecule in a fluid.

8. The nanochannel array of claim 1 wherein the channels are capable of transporting a macromolecule across their length.

9. The nanochannel array of claim 1 wherein the channels are capable of transporting at least one macromolecule across the length of said channels, said macromolecule being in an elongated form,

10. The nanochannel array of claim 1 wherein the channels are capable of transporting at least one biopolymer across the length of said channels.

11. The nanochannel array of claim 10 wherein the channels are capable of transporting at least one unfolded biopolymer across the length of said channels.

12. The nanochannel array of claim 10 wherein the biopolymer is a nucleic acid.

13. The nanochannel array of claim 10 wherein the biopolymer is an unfolded nucleic acid.

14. The nanochannel array of claim 1 wherein the trench width is less than 100 nanometers.

15. The nanochannel array of claim 1 wherein the trench width is less than 75 nanometers.

16. The nanochannel array of claim 1 wherein the trench width is less than 50 nanometers.

17. The nanochannel array of claim 1 wherein the trench width is less than 25 nanometers.

18. The nanochannel array of claim 1 wherein the trench width is less than 15 nanometers.

19. The nanochannel array of claim 1 wherein the trench width is about 10 nanometers.

20. The nanochannel array of claim 1 wherein the trench width is greater than 5 nanometers.

21. The nanochannel array of claim 1 wherein the trench width is greater than 2 nanometers.

22. The nanochannel array of claim 1 wherein the trench depth is less than 175 nanometers.

23. The nanochannel array of claim 1 wherein the trench depth is less than 125 nanometers.

24. The nanochannel array of claim 1 wherein the trench depth is less than 75 nanometers.

25. The nanochannel array of claim 1 wherein the trench depth is less than 50 nanometers.

26. The nanochannel array of claim 1 wherein the trench depth is less than 25 nanometers.

27. The nanochannel array of claim 1 wherein the trench depth is about 15 nanometers.

28. The nanochannel array of claim 1 wherein the trench depth is greater than 5 nanometers.

29. The nanochannel array of claim 1 wherein the trench depth is greater than 2 nanometers.

30. The nanochannel array of claim 1 wherein the at least a portion of the channels are pillar structures.

31. The nanochannel array of claim 1 wherein the channel period is less than about 200 nm.

32. The nanochannel array of claim 1 wherein the material of the surface is a film adjacently supported by a second substrate.

33. The nanochannel array of claim 1 wherein the substrate material is a conducting material.

34. The nanochannel array of claim 1 wherein the substrate material is a semi-conducting material.

35. The nanochannel array of claim 1 wherein the substrate material is a non-conducting material.

36. The nanochannel array of claim 1 further comprising at least one optically opaque layer adjacent to the sealing material.

37. The nanochannel array of claim 1 further comprising at least one near field slit above at least one channel.

38. The nanochannel array of claim 1 further comprising sealing material adjacent to a channel bottom.

39. The nanochannel array of claim 1 further comprising sealing material adjacent to a channel wall.

40. The nanochannel array of claim 1 wherein the dimensions of the sealing material are such that Δ

w′

is greater than or equal to Δ

w″

.

41. The nanochannel array of claim 1 wherein the length of the channels is greater than 1 millimeter.

42. The nanochannel array of claim 1 wherein the length of the channels is greater than 1 centimeter.

43. The nanochannel array of claim 1 wherein the length of the channels is greater than 5 centimeters.

44. The nanochannel array of claim 1 wherein the length of the channels is greater than 15 centimeters.

45. The nanochannel array of claim 1 wherein the length of the channels is greater than 25 centimeters.

46. The nanochannel array of claim 1 wherein the substrate is a flexible film material.

47. A method of preparing a nanochannel array, comprising the steps of:

providing a substrate having a surface;

forming a plurality of channels in the material of the surface; and

depositing a sealing material on the plurality of channels to surmount the plurality of channels to render such channels at least substantially closed, the substantially closed channels having a trench width of less than about 150 nanometers and a trench depth of less than 200 nanometers.

48. The method of claim 47 further comprising the step of providing at least one reservoir in fluid communication with at least one end of at least one of the channels.

49. The method of claim 48 wherein at least 2 reservoirs are provided in fluid communication with at least 2 separate channels.

50. The method of claim 48 wherein at least 10 reservoirs are provided in fluid communication with at least 10 separate channels.

51. The method of claim 48 wherein at least 96 reservoirs are provided in fluid communication with at least 96 separate channels.

52. The method of claim 48 wherein at least 500 reservoirs are provided in fluid communication with at least 500 separate channels.

53. The method of claim 47 wherein the plurality of channels is formed by:

nanoimprint lithography, spin coating, electron beam lithography, focused ion beam milling, photolithography, reactive ion-etching, wet-etching, plasma-enhanced chemical vapor deposition, electron beam evaporation, sputter deposition, and combinations thereof.

54. The method of claim 47 wherein the channels are linear channels formed by nanoimprint lithography, said linear channels having a trench width less than 100 nanometers and a trench depth less than 175 nanometers, wherein at least a portion of the sealing material is deposited using sputter deposition to provide sealing material adjacent to the channel wall material to narrow the trench width.

55. The method of claim 47 wherein the trench width is less than 100 nanometers.

56. The method of claim 47 wherein the trench width is less than 75 nanometers.

57. The method of claim 47 wherein the trench width is less than 50 nanometers.

58. The method of claim 47 wherein the trench width is less than 25 nanometers.

59. The method of claim 47 wherein the trench width is less than 15 nanometers.

60. The method of claim 47 wherein the trench width is about 10 nanometers.

61. The method of claim 47 wherein the trench width is greater than 5 nanometers.

62. The method of claim 47 wherein the trench width is greater than 2 nanometers.

63. The method of claim 47 wherein the trench depth is less than 175 nanometers.

64. The method of claim 47 wherein the trench depth is less than 125 nanometers.

65. The method of claim 47 wherein the trench depth is less than 100 nanometers.

66. The method of claim 47 wherein the trench depth is less than 75 nanometers.

67. The method of claim 47 wherein the trench depth is less than 50 nanometers.

68. The method of claim 47 wherein the trench depth is about 15 nanometers.

69. The method of claim 47 wherein the trench depth is greater than 5 nanometers.

70. The method of claim 47 wherein the trench depth is greater than 2 nanometers.

71. The method of claim 47 wherein the sealing material is deposited using electron beam evaporation or sputter deposition.

72. The method of claim 47 wherein the plurality of channels are formed substantially parallel.

73. The method of claim 47 wherein the plurality of channels are pillar structures.

74. The method of claim 47 wherein the channels in the substrate material have a periodicity of less than about 200 nm.

75. The method of claim 47 wherein the material of the surface is a film adjacently supported by a second substrate.

76. The method of claim 74 wherein the substrate material is conductive.

77. The method of claim 74 wherein the substrate material is semiconductive.

78. The method of claim 74 wherein the substrate material is non-conductive.

79. The method of claim 47 further comprising the step of depositing at least one optically opaque layer adjacent to the sealing material.

80. The method of claim 79 wherein the step of depositing at least one optically opaque layer occurs prior to depositing the sealing material.

81. The method of claim 79 wherein the step of depositing at least one optically opaque layer occurs subsequent to depositing the sealing material.

82. The method of claim 47 further comprising the step of providing at least one near field slit feature above at least one channel.

83. The method of claim 47 wherein a portion of the sealing material is deposited within the channels.

84. The method of claim 83 wherein a portion of the sealing material is deposited in the bottom of at least one channel.

85. The method of claim 83 wherein a portion of the sealing material is deposited on at least one wall of at least one channel.

86. The method of claim 47 wherein the dimensions of the deposited sealing material are such that Δ

w′

is greater than or equal to Δ

w″

.

87. The method of claim 47 further comprising the step of treating at least some of the channels with a surface-modifying agent to alter the surfaces interior to said channels.

88. The method of claim 87 wherein the surface-modifying agent reduces hydrophobicity of the surfaces interior to said channels.

89. The method of claim 87 wherein the surface-modifying agent increases hydrophobicity of the surfaces interior to said channels.

90. The method of claim 87 wherein the surface-modifying agent counteracts the electroosmosis effects inside the channels.

91. The method of claim 47 wherein the channels are substantially closed on the surface of the substrate and substantially open on the edges of the substrate.

92. The method of claim 47 wherein the sealing material surmounting the plurality of channels is at least the thickness of the atoms of the sealing material.

93. The method of claim 47 wherein the sealing material surmounting the plurality of channels is less than 500 nanometers thick.

94. The method of claim 47 wherein the sealing material surmounting the plurality of channels is less than 150 nanometers thick.

95. The method of claim 47 wherein the sealing material surmounting the plurality of channels is less than 100 nanometers thick.

96. The method of claim 47 wherein the sealing material surmounting the plurality of channels is less than 50 nanometers thick.

97. The method of claim 47 wherein the sealing material surmounting the plurality of channels is less than 25 nanometers thick.

98. The method of claim 47 wherein the sealing material surmounting the plurality of channels is less than 10 nanometers thick.

99. The method of claim 47 wherein the sealing material surmounting the plurality of channels is less than 5 nanometers thick.

100. The method of claim 47 wherein the sealing material surmounting the plurality of channels is at least 1 nanometer thick.

101. The method of claim 47 wherein the sealing material surmounting the plurality of channels is at least 2 nanometers thick.

102. The method of claim 47 further comprising the step of removing a portion of the sealing material to reduce the thickness of the sealing material above at least one channel.

103. A nanofluidic chip, comprising:

a) nanochannel array, comprising;

a substrate having a surface;

a plurality of parallel channels in the material of the surface, said channels having a trench width of less than about 150 nanometers and a trench depth of less than 200 nanometers;

at least some of the channels being surmounted by sealing material to render such channels at least substantially enclosed;

at least some of the channels are capable of admitting a fluid;

b) at least one sample reservoir in fluid communication with at least one of the channels, said sample reservoir capable of releasing a fluid; and

c) at least one waste reservoir in fluid communication with at least one of the channels, said waste reservoir capable of receiving a fluid.

104. The nanofluidic chip of claim 103 wherein the at least one sample reservoir is formed in the surface of the substrate.

105. The nanofluidic chip of claim 103 further comprising at least one waste reservoir in fluid communication with at least one of the channels.

106. The nanofluidic chip of claim 103 wherein the number of sample reservoirs is at least 10.

107. The nanofluidic chip of claim 103 wherein the number of sample reservoirs is at least 96.

108. The nanofluidic chip of claim 103 wherein the number of sample reservoirs is at least 1000.

109. The nanofluidic chip of claim 103 wherein at least a portion of the nanochannel array capable of being imaged with a two-dimensional detector.

110. The nanofluidic chip of claim 103 capable of transporting a plurality of elongated macromolecules from a sample reservoir and across the channels.

111. The nanofluidic chip of claim 103 further comprising at least one pair of electrodes capable of applying an electric field across at least some of the channels in at least one direction.

112. The nanofluidic chip of claim 111 wherein at least two pair of electrodes each provides an electric field in different directions.

113. A system, comprising:

A) a nanofluidic chip, comprising;

a) nanochannel array, comprising;

a substrate having a surface;

a plurality of parallel channels in the material of the surface, said channels having a trench width of less than about 150 nanometers and a trench depth of less than 200 nanometers;

at least one of the channels being surmounted by sealing material to render such channels at least substantially enclosed;

at least one of the channels capable of admitting a fluid; and

b) at least one sample reservoir in fluid communication with at least one of the channels, said sample reservoir capable of releasing a fluid; and

B) an apparatus for detecting at least one signal transmitted from the at least one fluid in the nanochannel array.

114. The system according to claim 113, further comprising a transporting apparatus to transport a fluid through at least one of the channels.

115. The system according to claim 113, further comprising a sample loading apparatus for loading at least one fluid to the at least one sample reservoir,

116. The system according to claim 113, further comprising a data processor.

118. The method according to claim 114 wherein the detected signal is correlated to at least one of the following properties:

length, conformation, and chemical composition.

119. The method according to claim 114 wherein the macromolecule is a synthetic polymer or biopolymer.

120. The method of claim 116 wherein the biopolymer is at least one of:

a protein, a polypeptide, and a nucleic acid.

122. The method of claim 114 wherein a plurality of reservoirs provide a plurality of macromolecules into a plurality of channels for determining the lengths of the macromolecules.

123. The method of claim 119 wherein more than one of the macromolecules enters a single channel.

124. The method of claim 119 wherein the macromolecules are biopolymers.

127. The method of claim 114 wherein the concentration of the macromolecules in the fluid is at least one attogram per milliliter.

128. The method of claim 114 wherein the concentration of the macromolecules in the fluid is at least one femtogram per milliliter.

129. The method of claim 114 wherein the concentration of the macromolecules in the fluid is at least one picogram per milliliter.

130. The method of claim 114 wherein the concentration of the macromolecules in the fluid is less than 5 micrograms per milliliter.

131. The method of claim 114 wherein the concentration of the macromolecules in the fluid is less than 0.5 micrograms per milliliter.

132. The method of claim 114 wherein the macromolecules have an elongated length in the channels of greater than 150 nanometers.

133. The method of claim 114 wherein the macromolecules have an elongated length in the channels of greater than 500 nanometers.

134. The method of claim 114 wherein the macromolecules have an elongated length in the channels of greater than 1 micron.

135. The method of claim 114 wherein the macromolecules have an elongated length in the channels of greater than 10 microns.

136. The method of claim 114 wherein the macromolecules are DNA having greater than 10 base pairs.

137. The method of claim 114 wherein the macromolecules are DNA having greater than 100 base pairs.

138. The method of claim 114 wherein the macromolecules are DNA having greater than 1,000 base pairs.

139. The method of claim 114 wherein the macromolecules are DNA having greater than 10,000 base pairs.

140. The method of claim 114 wherein the macromolecules are DNA having greater than 20,000 base pairs.

141. The method of claim 114 wherein the macromolecules are DNA having greater than 40,000 base pairs.

142. The method of claim 114 wherein the macromolecules are DNA having greater than 80,000 base pairs.

143. The method of claim 114 wherein the nanochannel array has at least 96 sample simultaneously reservoirs for analyzing at least 96 different macromolecular fluid samples.

144. The method of claim 114 wherein the at least one macromolecule is a restriction fragment length polymorphism.

145. The method of claim 114 wherein the at least one macromolecule is a chromosome.

146. The method of claim 142 wherein the at least one chromosome is analyzed to determine the presence of at least one single nucleotide polymorphism.

117. A method of analyzing at least one macromolecule, comprising the steps of:

providing a nanofluidic chip, comprising;

a) nanochannel array, comprising;

a surface having a plurality of parallel channels in the material of the surface, said channels having a trench width of less than about 150 nanometers and a trench depth of less than 200 nanometers;

at least one of the channels being surmounted by sealing material to render such channels at least substantially enclosed;

at least one of the channels capable of admitting a fluid;

b) at least one sample reservoir in fluid communication with at least one of the channels, said sample reservoir capable of releasing a fluid containing at least one macromolecule;

providing the at least one sample reservoir with at least one fluid, said fluid comprising at least one macromolecule;

transporting the at least one macromolecule into the at least one channel to elongate said at least one macromolecule;

detecting at least one signal transmitted from the at least one elongated macromolecule; and

correlating the detected signal to at least one property of the at least one macromolecule.

121. The method of claim 117 wherein the nucleic acid is DNA and the detected signals are correlated to the base pair sequence of said DNA.

125. The method of claim 121 wherein the biopolymers are proteins, polypeptides, DNA or RNA.

126. The method of claim 121 wherein the biopolymers are at least substantially unfolded in the channels.

147. A cartridge comprising at least one nanofluidic chip, said cartridge capable of being inserted and removed from a system for carrying out macromolecular analysis, said at least one nanofluidic chip comprising at least one nanonanochannel array, said nanonanochannel array comprising

a surface having a plurality of channels in the material of the surface, said channels having a trench width of less than about 150 nanometers and a trench depth of less than 200 nanometers;

at least some of the channels being surmounted by sealing material to render such channels at least substantially enclosed.