Capture, recapture, and trapping of molecules with a nanopore

• US 20090136958A1
• Filed: 10/02/2008
• Published: 05/28/2009
• Est. Priority Date: 10/02/2007
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Gershow, Marc H., Branton, Daniel, Golovchenko, Jene A.
• Granted Patent: US 8,273,532 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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

Abstract

In a molecular analysis system, there is provided a structure including a nanopore and first and second fluidic reservoirs. The two reservoirs are fluidically connected via the nanopore. A detector is connected to detect molecular species translocation of the nanopore, from one of the two fluidic reservoirs to the other of the two fluidic reservoirs. A controller is connected to generate a control signal to produce conditions at the nanopore to induce the molecular species to re-translocate the nanopore at least once after translocating the nanopore. This enables a method for molecular analysis in which a molecular species is translocated a plurality of times through a nanopore in a structure between two fluidic reservoirs separated by the structure.

Claims

1. A molecular analysis system comprising:

a structure including a nanopore;

first and second fluidic reservoirs, the two reservoirs being fluidically connected via the nanopore;

a detector connected to detect molecular species translocation of the nanopore, from one of the two fluidic reservoirs to the other of the two fluidic reservoirs; and

a controller connected to generate a control signal to produce conditions at the nanopore to induce the molecular species to re-translocate the nanopore at least once after translocating the nanopore.

2. The system of claim 1 wherein the controller is connected to the detector and is configured to generate the control signal in response to detection of the molecular species translocation of the nanopore.

3. The system of claim 2 wherein the controller includes a delay component configured to impose a time delay between detection of molecular species translocation of the nanopore and generation of the control signal.

4. The system of claim 1 wherein the controller is configured to generate the control signal after a prespecified delay period.

5. The system of claim 1 wherein the controller is configured to generate the control signal as a sequence of control signals to produce time-dependent conditions at the nanopore to induce the molecular species to re-translocate the nanopore a selected number of times.

6. The system of claim 1 wherein the controller is connected to the detector and is configured to generate the control signal in response to detection of molecular species translocation of the nanopore from the first fluidic reservoir to the second fluidic reservoir and to generate the control signal after a prespecified translocation duration corresponding to molecular species re-translocation of the nanopore from the second fluidic reservoir to the first fluidic reservoir.

7. The system of claim 1 wherein the controller is connected to the detector and is configured to generate the control signal in response to detection of molecular species translocation of the nanopore from the second fluidic reservoir to the first fluidic reservoir and to generate the control signal after a prespecified translocation duration corresponding to molecular species re-translocation of the nanopore from the first fluidic reservoir to the second fluidic reservoir.

8. The system of claim 1 wherein the detector is configured to detect a translocation duration during molecular species translocation of the nanopore and to detect a re-translocation duration during molecular species re-translocation of the nanopore.

9. The system of claim 1 further comprising a circuit connected to the controller to apply a voltage between the first and second fluidic reservoirs, the voltage being of a reversible polarity selected by the controller to induce molecular species translocation and re-translocation in selected directions through the nanopore.

10. The system of claim 1 wherein the detector comprises an amplifier connected to measure ionic current flow through the nanopore as an indicator of molecular species translocation of the nanopore.

11. The system of claim 10 wherein the controller comprises a high pass filter to filter measured current flow.

12. The system of claim 2 wherein the controller includes a comparator with a prespecified threshold to determine if a signal from the detector is indicative of molecular species translocation of the nanopore.

13. The system of claim 1 wherein the structure comprises a membrane.

14. The system of claim 1 wherein the nanopore has a diameter no greater than about 1 micron.

15. The system of claim 1 wherein the nanopore has a diameter no greater than about 100 nm.

16. The system of claim 1 wherein the nanopore has a diameter no greater than about 10 nm.

17. The system of claim 1 wherein the nanopore has a diameter no greater than about 5 nm.

18. The system of claim 1 wherein one of the fluidic reservoirs is configured to provide to the nanopore a molecular species comprising macromolecules.

19. The system of claim 1 wherein one of the fluidic reservoirs is configured to provide to the nanopore a molecular species comprising molecular components.

20. The system of claim 1 wherein one of the fluidic reservoirs is configured to provide to the nanopore a molecular species comprising polymer molecules.

21. The system of claim 1 wherein one of the fluidic reservoirs is configured to provide to the nanopore a molecular species comprising biomolecules.

22. The system of claim 1 wherein one of the fluidic reservoirs is configured to provide to the nanopore a molecular species comprising components of biomolecules.

23. The system of claim 1 wherein one of the fluidic reservoirs is configured to provide to the nanopore a molecular species comprising DNA molecules.

24. The system of claim 1 wherein one of the fluidic reservoirs is configured to provide to the nanopore a molecular species comprising oligonucleotides.

25. The system of claim 1 wherein one of the fluidic reservoirs is configured to provide to the nanopore a molecular species comprising nucleotides.

26. The system of claim 1 wherein one of the fluidic reservoirs is configured to provide to the nanopore a molecular species comprising at least one molecule including at least one attached species differing from the molecule in electronic charge.

27. The system of claim 1 wherein one of the fluidic reservoirs is configured to provide to the nanopore a molecular species comprising at least one molecule including at least one attached species differing from the molecule in diameter.

28. The system of claim 1 wherein one of the fluidic reservoirs is configured to include a species that is reactive with the molecular species.

29. The system of claim 28 wherein the reactive species comprises a protein.

30. The system of claim 28 wherein the reactive species comprises a complementary nucleic acid.

31. The system of claim 28 wherein the reactive species comprises an ionic species.

32. The system of claim 1 wherein one of the fluidic reservoirs is configured to include a condition that is reactive with the molecular species.

33. The system of claim 1 wherein the two fluidic reservoirs are configured with differing fluids.

34. The system of claim 33 wherein the two fluidic reservoirs are configured with fluids of differing pH.

35. The system of claim 33 wherein the two fluidic reservoirs are configured with fluids of differing concentrations of a solvent.

36. The system of claim 33 wherein the two fluidic reservoirs are configured with fluids of differing concentrations of a solute.

37. The system of claim 33 wherein the two fluidic reservoirs are configured with fluids of differing temperature.

38. A method for molecular analysis comprising translocating a molecular species a plurality of times through a nanopore in a structure between two fluidic reservoirs separated by the structure.

39. The method of claim 38 wherein translocating the molecular species comprises producing conditions at the nanopore to induce the molecular species to repeatedly translocate the nanopore into and out of the two fluidic reservoirs.

40. The method of claim 39 wherein repeated translocation of the nanopore by the molecular species comprises 2 translocations of the nanopore.

41. The method of claim 39 wherein repeated translocation of the nanopore by the molecular species comprises more than 2 translocations of the nanopore.

42. The method of claim 39 wherein repeated translocation of the nanopore by the molecular species comprises more than 10 translocations of the nanopore.

43. The method of claim 39 wherein repeated translocation of the nanopore by the molecular species comprises more than 50 translocations of the nanopore.

44. The method of claim 39 wherein producing conditions at the nanopore to induce the molecular species to repeatedly translocate the nanopore comprises alternating conditions at the nanopore to induce at least one molecular species translocation and re-translocation cycle.

45. The method of claim 38 wherein translocating a molecular species comprises detecting molecular species translocation of the nanopore and in response to the detection, producing conditions at the nanopore to induce the molecular species to re-translocate the nanopore at least once after the detected translocation.

46. The method of claim 38 wherein translocating a molecular species comprises carrying out at least one cycle comprising molecular species translocation of the nanopore a first time and production of conditions at the nanopore to induce the molecular species to re-translocate the nanopore.

47. The method of claim 46 wherein the conditions to induce the molecular species to re-translocate the nanopore are produced after a prespecified delay period.

48. The method of claim 38 further comprising detecting molecular species translocation of the nanopore during at least one in the plurality of translocations.

49. The method of claim 48 wherein detecting molecular species translocation of the nanopore comprises determining translocation duration.

50. The method of claim 48 wherein each of the two fluidic reservoirs includes an ionic solution, and wherein detecting molecular species translocation of the nanopore comprises detecting blockage of ionic solution flow through the nanopore by a molecular species.

51. The method of claim 38 wherein each of the two fluidic reservoirs between which the molecular species is translocated includes an ionic solution and wherein translocating a molecular species comprises applying a voltage between the first and second fluidic reservoirs in a sequence of voltage polarity reversals to induce repeated molecular species translocation of the nanopore between the fluidic reservoirs.

52. The method of claim 38 wherein the two fluidic reservoirs between which the molecular species is translocated comprise differing fluids.

53. The method of claim 38 wherein the molecular species comprises a macromolecule.

54. The method of claim 38 wherein the molecular species comprises a molecular component.

55. The method of claim 54 wherein the molecular component comprises a biomolecule component.

56. The method of claim 55 wherein the biomolecule component comprises a nucleotide fragment.

57. The method of claim 55 wherein the biomolecule component comprises an oligonucleotide.

58. The method of claim 38 wherein the molecular species comprises a polymer molecule.

59. The method of claim 55 wherein molecular species comprises a biomolecule.

60. The method of claim 38 wherein the molecular species comprises DNA.

61. The method of claim 38 wherein the molecular species includes at least one molecule including at least one attached species differing from the molecule in electronic charge.

62. The method of claim 38 wherein the molecular species includes at least one molecule including at least one attached species differing from the molecule in diameter.

63. A method for exposing a molecular species to a reactive environment comprising:

translocating a molecular species through a nanopore from a first fluidic reservoir to a second fluidic reservoir containing an environment that is reactive with the molecular species; and

producing conditions at the nanopore to induce molecular re-translocation of the nanopore from the second fluidic reservoir back to the first fluidic reservoir.

64. The method of claim 63 further comprising producing conditions at the nanopore in induce repeated molecular species translocation of the nanopore between the first and second fluidic reservoirs during reaction between the molecular species and the reactive environment.

65. The method of claim 64 further comprising detecting molecular species translocation during at least one of the repeated translocations between the first and second fluidic reservoirs.

66. The method of claim 64 wherein the environment of the second fluidic reservoir that is reactive with the molecular species comprises a reactive species in fluid.

67. The method of claim 64 wherein the environment of the second fluidic reservoir that is reactive with the molecular species comprises a protein.

68. The method of claim 64 wherein the environment of the second fluidic reservoir that is reactive with the molecular species comprises a biomolecule.

69. A method for spatially trapping a molecular species comprising:

translocating a molecular species a plurality of times through a nanopore in a structure between two fluidic reservoirs separated by the structure; and

applying trapping conditions at the nanopore to maintain the molecular species in one of the two reservoirs in a vicinity of the nanopore between each molecular species translation of the nanopore.

70. The method of claim 69 wherein each reservoir comprises an ionic solution, and wherein applying trapping conditions comprises applying a voltage bias between the two reservoirs that maintains the molecular species in the vicinity of the nanopore between each molecular species translation of the nanopore.

71. The method of claim 69 further comprising changing a characteristic of at least one of the fluidic reservoirs over time during the molecular species trapping.

72. The method of claim 71 wherein changing a characteristic of at least one of the fluidic reservoirs comprises changing a reservoir temperature.

73. The method of claim 71 wherein changing a characteristic of at least one of the fluidic reservoirs comprises changing a reservoir pH.

74. The method of claim 71 wherein changing a characteristic of at least one of the fluidic reservoirs comprises changing a reservoir ionic concentration.

75. The method of claim 71 wherein changing a characteristic of at least one of the fluidic reservoirs comprises providing a reactive species in one of the reservoirs.

76. A method for sequencing a genome comprising:

providing a plurality of nucleotide fragments from a genome in a first fluidic reservoir;

translocating each nucleotide fragment a selected number of times through a nanopore in a structure between the first fluidic reservoir and a second fluidic reservoir separated from the first reservoir by the structure; and

detecting each nucleotide fragment as that nucleotide fragment translocates the nanopore.

77. The method of claim 76 wherein each nucleotide fragment is translocated through the nanopore at least 5 times.

78. The method of claim 76 wherein each nucleotide fragment is translocated through the nanopore at least 10 times.

79. The method of claim 76 wherein each nucleotide fragment is translocated through the nanopore at least 20 times.

80. The method of claim 76 wherein detecting each nucleotide fragment comprises determining if a nucleotide fragment has previously been detected and translocated the selected number of times, and if so, discarding that nucleotide fragment into the second reservoir by discontinuing further translocations of that nucleotide fragment back to the first reservoir.

81. The method of claim 76 wherein detecting each nucleotide fragment comprises determining if a nucleotide fragment has previously been detected and translocated the selected number of times, and if not, translocating the nucleotide fragment through the nanopore at least one additional time.