Molecular and atomic scale evaluation of biopolymers

• US 6,627,067 B1
• Filed: 06/22/2000
• Issued: 09/30/2003
• Est. Priority Date: 06/22/1999
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Branton, Daniel, Denison, Timothy J., Golovchenko, Jene A.
• Primary Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US09/602,650
• Time in Patent Office: 1,195 Days
• Field of Search: 204/403.06, 204/403.08, 204/403.07, 205/777.5, 205/778, 435/287.2, 435/287.1
• US Class Current: 205/778
• 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: B24B 37/013 Devices or means for detecting lapping completion
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/305 Denaturation or renaturation by physical action
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• 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: H01L 22/26 Acting in response to an ongoing measurement without interruption of processing, e.g. endpoint detection, in-situ thickness measurement endpoint detection arrangements in CMP apparatus B24B37/013, in discharge apparatus H01J37/32
• CPC Class Codes: H01L 2924/00 Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
• CPC Class Codes: H01L 2924/0002 Not covered by any one of groups H01L24/00, H01L24/00 and H01L2224/00

Abstract

A method for evaluating a polymer molecule including linearly connected monomer residues includes providing a polymer molecule in a liquid, contacting the liquid with an insulating solid-state membrane having a detector capable of detecting polymer molecule characteristics, and causing the polymer molecule to traverse a limited region of the solid-state membrane so that monomers of the polymer molecule traverse the limit region in sequential order, whereby the polymer molecule interacts linearly with the detector and data suitable to determine polymer molecule characteristics are obtained. The limited region may be defined by a nanometer-sized aperture in the membrane.

Claims

1. An apparatus for use in evaluating a linear polymer molecule, comprising:

a vessel for holding a liquid containing a candidate polymer molecule;

a solid-state membrane containing an aperture therein, said aperture comprising a constraining dimension, wherein the aperture includes an entry port and an exit port defining a channel there between, and wherein the membrane is positioned to be contactable with a liquid in the vessel;

means for causing a candidate polymer molecule to linearly traverse the aperture; and

a detector for detecting sensing time-dependent or monomer-dependent interactions of a candidate molecule with the aperture detector, said detector located in the aperture or on a face of the solid-state membrane.

2. The apparatus of claim 1, wherein the solid-state membrane comprises an electrically insulating material.

3. The apparatus of claim 1, wherein the channel is coated with an electrically insulating layer.

4. The apparatus of claim 1, wherein the channel is coated with a passivating layer.

5. The apparatus of claim 1, wherein the constraining dimension is located adjacent to, below, above or within the aperture.

6. The method of claim 5, wherein the detector comprises the constraining diameter of the aperture.

7. The apparatus of claim 1, wherein the constraining dimension is in the range of less than about 20 nm.

8. The apparatus of claim 1, wherein the constraining dimension is in the range of less than about 5 nm.

9. The apparatus of claim 1, wherein the constraining dimension is in the range of about less than 0.1-2 nm.

10. The apparatus of claim 1, wherein the constraining dimension comprises a feature integral with the aperture and tapering acutely to form a point of constriction in the aperture channel.

11. The apparatus of claim 10, wherein the taper is curvilinear.

12. The apparatus of claim 10, wherein the taper varies in acuteness along the length of the channel.

13. The apparatus of claim 10, wherein the feature is located at the exit or entry port of the aperture.

14. The apparatus of claim 10, wherein the feature is located within the channel of the aperture.

15. The apparatus of claim 1, wherein the constraining dimension extends through the channel for a length that is in the range of 0.1 to 10 nm.

16. The apparatus of claim 1, wherein the constraining dimension extends through the channel for a length that is in the range of 0.1 to 5 nm.

17. The apparatus of claim 1, wherein the solid-state membrane is selected from the group consisting of inorganic compounds, organic and inorganic polymers and glasses.

18. The apparatus of claim 1, wherein the solid-state membrane is selected from the group consisting of silicon nitrides, silica, alumina.

19. The apparatus of claim 1, wherein the solid-state membrane has a thickness in the range of about 10 nm to about 1 mm.

20. The apparatus of claim 1, wherein the solid-state membrane has a thickness in the range of about 50 nm to about 100 nm.

21. The apparatus of claim 1, wherein the solid-state membrane has a capacitance of less than about 0.1 ρ

F.

22. The apparatus of claim 1, wherein the detector comprises first and second electrodes on a face of the solid-state membrane adjacent to the aperture and in electrical communication with the channel.

23. The apparatus of claim 22, wherein the first and second electrodes are on the same side of the solid-state membrane.

24. The apparatus of claim 22, wherein the first and second electrodes are on opposing sides of the solid-state membrane.

25. The apparatus of claim 22, wherein said electrodes comprise a conductive metal layer deposited on the solid-state membrane.

26. The apparatus of claim 22, further comprising:

at least one insulating layer adjacent to the first and second electrodes.

27. The apparatus of claim 1, wherein the apparatus further comprises a monitoring means, and the monitoring means comprises an ammeter or an electrometer.

28. The apparatus of claim 1, wherein the means for causing a candidate polymer molecule to traverse the aperture is selected from the group consisting of voltage gradient means and biomotors.

29. The apparatus of claim 1 or 22, further comprising:

a substrate supporting the solid-state membrane.

30. The apparatus of claim 1, wherein a polymer replicating catalyst is in contact with the aperture.

31. The apparatus of claim 30, wherein the polymer replicating catalyst is located adjacent to, above, below, or within the membrane aperture.

32. The apparatus of claim 31, wherein the polymer replicating catalyst is the constraining dimension diameter feature.

33. The apparatus of claim 1, wherein the detector comprises an electrode pair located on opposing faces of the solid-state membrane, and the detector detects interactions of the electrode pair with a candidate molecule located between the electrode pair, and wherein the constraining dimension located at or between the electrode pair.

34. The apparatus of claim 33, wherein a detection signal arises from longitudinal electron tunneling along the length of the aperture channel.

35. The apparatus of claim 1, wherein the detector comprises an electrode pair located on a face of the solid-state membrane spaced apart from one another at opposing sides of the entry port or the exit port of the aperture.

36. The apparatus of claim 35, wherein the electrode pair comprises the constraining dimension.

37. The apparatus of claim 33 or 35, wherein the apparatus further comprises a dielectric layer disposed on each of the electrodes of the electrode pair.

38. The apparatus of claim 35, wherein a detection signal arises from transverse electron tunneling across the aperture.

39. An apparatus for use in evaluating a linear polymer molecule, comprising:

a first vessel having a first inlet therein;

a second vessel having a second inlet therein;

an elongated member having first and second ends, each end in sealing communication with the respective inlets of the first and second vessels;

a solid-state membrane containing an electrically insulating aperture therein disposed in the first end of the elongated member, wherein the aperture includes an entry port and an exit port defining a channel there between, and the membrane is positioned to be contactable with a liquid containing a candidate polymer molecule in the first vessel;

means for causing a candidate polymer molecule to linearly traverse the aperture; and

a detector for sensing time-dependent or monomer-dependent interactions of a candidate molecule with the detector, said detector located in the aperture or on a face of the solid-state membrane .

40. A method for evaluating a polymer molecule, the polymer molecule including linearly connected monomer residues, comprising:

contacting a liquid containing a polymer molecule with an insulating solid-state substrate having a detector capable of detecting polymer molecule characteristics;

causing the polymer molecule to traverse a limited volume on the solid-state substrate so that monomers of the polymer molecule traverse the limited volume in sequential order, whereby the polymer molecule interacts sequentially with the detector, whereby the detector comprises an electrode pair, and electron current is detected as the monomer traverses the limited volume and data suitable to determine polymer molecule characteristics are obtained.

41. The method of claim 40, wherein the limited volume of the solid-state substrate is a groove on the solid-state substrate surface, and the detector is located at the base of the groove, whereby the polymer molecule traverses the length of the groove.

42. The method of claim 40, wherein the polymer molecule is selected from the group consisting of polynucleic acids, DNA and RNA.

43. The method of claim 40, wherein the detector is located on the substrate surface, and further comprising:

a polymer replicating catalyst attached to the solid-state surface adjacent to the detector, whereby the polymer molecule is acted upon by the polymer replicating catalyst, so that the polymer molecule interacts sequentially with the detector as it advances through the polymer replicating catalyst.

44. The method of claim 43, wherein the liquid is a solution further including reagents necessary to replicate the polymer molecule.

45. The apparatus of claim 40, wherein the constraining dimension is located adjacent to, below, above or within the aperture.

64. The method of claim 40, 60 or 63, wherein the amplitude of duration of individual conductance measurements is indicative of sequential identity of monomers of the polymer molecule.

65. The method of claim 40, 60 or 63, wherein the number of changes in the conductance measurement is an indication of the number of monomers in the polymer.

66. The method of claim 40, 60 or 63, wherein the duration of the individual conductance measurement is an indication of the number of monomers in the polymer molecule.

67. The method of claim 40, 60 or 63, wherein multiple molecules of a heterogeneous mixture of individual polymer molecules are characterized to provide a size distribution of polymers in the mixture.

46. A method for evaluating a polymer molecule, the polymer molecule including linearly connected monomer residues, comprising:

contacting a liquid containing a polymer molecule with an insulating solid-state membrane having an aperture therein, wherein the aperture includes an entry port and an exit port defining a channel there between,

causing the polymer molecule to traverse the aperture of the membrane, whereby the polymer molecule interacts sequentially with a constraining dimension in, of or at the aperture and data arising from time-dependent or monomer-dependent interactions of a candidate molecule with a detector located in the aperture or on a face of the solid-state membrane are obtained.

47. The method of claim 46, wherein the solid-state membrane comprises an electrically insulating material.

48. The method of claim 46, wherein the aperture channel is coated with an electrically insulating layer, or a passivity layer.

49. The method of claim 46, wherein the constraining dimension is in the range of less than about 20 nm.

50. The method of claim 46, wherein the constraining dimension is in the range of less than about 5 nm.

51. The apparatus of claim 46, wherein the constraining dimension is in the range of about 1-2 nm.

52. The apparatus of claim 46, wherein the constraining dimension is located at the exit or entry port of the aperture.

53. The apparatus of claim 46, wherein the constraining dimension is located within the channel of the aperture.

54. The apparatus of claim 46, wherein the constraining dimension extends through the channel for a length that is in the range of 0.1 to 10 nm.

55. The apparatus of claim 46, wherein the constraining dimension extends through the channel for a length that is in the range of 0.1 to 5 nm.

56. The method of claim 46, wherein the solid-state substrate is selected from the group consisting of inorganic compounds, organic and inorganic polymers and glasses.

57. The method of claim 46, wherein the solid-state membrane has a thickness in the range of about 10 nm to about 1 mm.

58. The method of claim 46, wherein the solid-state membrane has a thickness in the range of about 50 nm to about 100 nm.

59. The method of claim 46, wherein the solid-state membrane has a capacitance of less than about 0.1 ρ

F.

60. The method of claim 46, wherein polymer molecule interactions with the detector are detected as electronic currents at first and second electrodes adjacent to the aperture and in electrical communication with said channel.

61. The method of claim 60, wherein translational current is detected.

62. The method of claim 60, wherein current along the length of the channel is detected.

63. The method of claim 46, wherein polymer molecule interactions with the detector are detected as ionic conductance in the channel.

68. The method of claim 46, wherein polymer molecule traverses the aperture by application of a voltage gradient or use of a biomotor.

69. The method of claim 46, wherein a polymer replicating catalyst is in contact with the aperture.

70. The method of claim 69, wherein polymer replicating catalyst is located adjacent to, below, above, or within the aperture.

71. The method of claim 46, wherein the constraining dimension comprises the detector.

72. A method for evaluating a polymer molecule, the polymer molecule including linearly connected monomer residues, comprising:

providing a candidate hybridized polynucleotide molecule in a liquid;

contacting a liquid containing a candidate hybridized polynucleotide molecule with an insulating solid-state membrane having an aperture therein, said aperture having a diameter insufficient to permit traversal of the hybridized molecule of the aperture;

causing the candidate polymer molecule to traverse the aperture of the membrane, whereby the hybridized polymer molecule is denatured and the single-stranded polymer interacts sequentially with the aperture and data suitable to determine polymer molecule characteristics are obtained.

73. The method of claim 72, whereby the hybridized polymer molecule oscillates between a first condition at which the polymer can not advance into the aperture and a second condition at which the hybridized molecule is denatured and a single strand of the polymer advances into the aperture.

74. The method of claim 73, wherein the rate of oscillation between the first and second conditions is selected to advance the polymer by about a single monomer with each oscillation.

75. The method of claim 74, wherein the condition varied is an applied potential gradient across the membrane.

76. The method of claim 72, wherein the rate of traversal of a single strand DNA is an order of magnitude slower when using hybridized polymer than when using a single strand polymer.