Formation of Layers of Amphiphilic Molecules

• US 20090167288A1
• Filed: 12/19/2008
• Published: 07/02/2009
• Est. Priority Date: 12/19/2007
• Status: Abandoned Application

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Reid, Stuart William, Reid, Terence Alan, White, Steven Paul, Sanghera, Gurdial Singh, Clarke, James Anthony
• Application Number:
  US12/339,956
• Publication Number:
  US 20090167288A1
• Time in Patent Office: Days
• US Class Current: 324/72
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/161 Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0427 Electrowetting
• 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 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 27/453 Cells therefor
• 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

To form a layer separating two volumes of aqueous solution, there is used an apparatus comprising elements defining a chamber, the elements including a body of non-conductive material having formed therein at least one recess opening into the chamber, the recess containing an electrode. A pre-treatment coating of a hydrophobic fluid is applied to the body across the recess. Aqueous solution, having amphiphilic molecules added thereto, is flowed across the body to cover the recess so that aqueous solution is introduced into the recess from the chamber and a layer of the amphiphilic molecules forms across the recess separating a volume of aqueous solution introduced into the recess from the remaining volume of aqueous solution.

Claims

1. A method of forming a layer separating two volumes of aqueous solution, the method comprising:

(a) providing an apparatus comprising elements defining a chamber, the elements including a body of non-conductive material having formed therein at least one recess opening into the chamber, the recess containing an electrode;

(b) applying a pre-treatment coating of a hydrophobic fluid to the body across the recess;

(c) flowing aqueous solution, having amphiphilic molecules added thereto, across the body to cover the recess so that aqueous solution is introduced into the recess from the chamber and a layer of the amphiphilic molecules forms across the recess separating a volume of aqueous solution introduced into the recess from the remaining volume of aqueous solution.

2. A method according to claim 1, wherein step (c) comprises:

(c1) flowing aqueous solution across the body to cover the recess so that aqueous solution flows into the recess;

(c2) flowing the aqueous solution to uncover the recess, leaving some aqueous solution in the recess; and

(c3) flowing aqueous solution, having amphiphilic molecules added thereto, across the body and to re-cover the recess so that a layer of the amphiphilic molecules forms across the recess separating a volume of aqueous solution inside the recess from the remaining volume of aqueous solution.

3. A method according to claim 2, wherein

the apparatus is provided with a further electrode in the chamber outside said recess, in step (c1), the aqueous solution is flowed also to contact the further electrode, and step (c) further comprises, between steps (c1) and (c2):

(c4) applying a voltage across said electrode contained in the recess and said further electrode sufficient to reduce the amount of excess hydrophobic fluid covering said electrode contained in the recess.

4. A method according to claim 2, wherein the aqueous solution caused to flow in steps (c1) and (c2) is the same aqueous solution.

5. A method according to claim 1, wherein surfaces including one or both of (a) the outermost surface of the body around the recess, and (b) at least an outer part of the internal surface of the recess extending from the rim of the recess, are hydrophobic.

6. A method according to claim 5, wherein the body comprises an outermost layer formed of a hydrophobic material, the recess extending through the outermost layer and said outer part of the internal surface of the recess being a surface of the outermost layer.

7. A method according to claim 5, wherein an inner part of the internal surface of the recess inside the outer part is hydrophilic.

8. A method according to claim 7, wherein the body comprises an outermost layer formed of a hydrophobic material and an inner layer formed of a hydrophilic material, the recess extending through the outermost layer and inner layer, said outer part of the internal surface of the recess being a surface of the outermost layer, and said inner part of the internal surface of the recess being a surface of the inner layer.

9. A method according to claim 5, wherein said surfaces are modified by a fluorine species.

10. A method according to claim 9, wherein said surfaces are modified by a fluorine species by treatment with a fluorine plasma.

11. A method according to claim 1, wherein the electrode contained in the recess is provided on the base of the recess.

12. A method according to claim 1, wherein the body comprises a substrate and at least one further layer attached to the substrate, the recess extending through the at least one further layer.

13. A method according to claim 1, wherein the electrode has provided thereon a hydrophillic surface which repels the hydrophobic fluid applied in step (c) whilst allowing ionic conduction from the aqueous solution to the electrode.

14. A method according to claim 13, wherein the hydrophillic surface is the surface of protective material provided on the electrode.

15. A method according to claim 14, wherein the protective material is a covalently-attached hydrophillic species or a conductive polymer.

16. A method according to claim 1, wherein the electrode has a conductive polymer provided thereon.

17. A method according to claim 1, wherein the elements defining the chamber further include a cover extending over the body so that the chamber is a closed chamber.

18. A method according to claim 17, wherein the cover comprises at least one inlet and at least one outlet, the aqueous solution being introduced into the chamber through the inlet in step (c) and the outlet venting fluid displaced by the aqueous solution thus introduced.

19. A method according to claim 1, wherein the internal surface of the recess has no openings capable of fluid communication.

20. A method according to claim 1, wherein the at least one recess comprises plural recesses.

21. A method according to claim 1, wherein the layer of the amphiphilic molecules is a bilayer of the amphiphilic molecules.

22. A method according to claim 21, wherein the amphiphilic molecules are lipids.

23. A method according to claim 1, wherein the layer of the amphiphilic molecules has an electrical resistance of at least 1 GΩ

.

24. A method according to claim 1, further comprising, before step (c), depositing the amphiphilic molecules on an internal surface of the chamber or on an internal surface in the flow path of the aqueous solution into the chamber, the aqueous solution covering the internal surface during step (c) whereby the amphiphilic molecules are added to the aqueous solution.

25. A method according to claim 1, further comprising inserting a membrane protein into the layer of amphiphilic molecules.

26. A method according to claim 25, wherein the aqueous solution has a membrane protein added thereto, whereby the membrane protein is inserted spontaneously into the layer of amphiphilic molecules.

27. A method according to claim 25, further comprising, before step (c), depositing the membrane protein on an internal surface of the chamber, the aqueous solution covering the internal surface during step (c) whereby the membrane protein is added to the aqueous solution.

28. A method according to claim 1, wherein the at least one recess comprises plural recesses and the method comprises inserting different membrane protein into the layers of amphiphilic molecules formed in different recesses.

29. A method according to claim 25, wherein the apparatus is provided with a further electrode in the chamber outside the recess, and the method further comprises applying a potential across the electrode in the recess and the further electrode and monitoring an electrical signal developed between the electrode in the recess and the further electrode.

30. An apparatus for supporting a layer separating two volumes of aqueous solution, the apparatus comprising:

elements defining a chamber, the elements including a body of non-conductive material having formed therein at least one recess opening into the chamber; and

an electrode contained in the recess.

31. An apparatus according to claim 30, wherein surfaces including either or both of (a) the outermost surface of the body around the recess, and (b) at least an outer part of the internal surface of the recess extending from the rim of the recess, are hydrophobic.

32. An apparatus according to claim 31, wherein the body comprises an outermost layer formed of a hydrophobic material, the recess extending through the outermost layer and said outer part of the internal surface of the recess being a surface of the outermost layer.

33. An apparatus according to claim 31, wherein an inner part of the internal surface of the recess inside the outer part is hydrophilic.

34. An apparatus according to claim 33, wherein the body comprises an outermost layer formed of a hydrophobic material and an inner layer formed of a hydrophilic material, the recess extending through the outermost layer and inner layer, said outer part of the internal surface of the recess being a surface of the outermost layer, and said inner part of the internal surface of the recess being a surface of the inner layer.

35. An apparatus according to claim 31, wherein said surfaces are modified by a fluorine species.

36. An apparatus according to claim 35, wherein said surfaces are modified by a fluorine species by treatment with a fluorine plasma.

37. An apparatus according to claim 30, wherein the electrode contained in the recess is provided on the base of the recess.

38. An apparatus according to claim 30, wherein the body comprises a substrate and at least one further layer attached to the substrate, the recess extending through the at least one further layer.

39. An apparatus according to claim 38, wherein the at least one further layer is:

polycarbonate;

poly-vinyl chloride;

polyester;

a thermal laminating film;

a photoresist;

or an ink.

40. An apparatus according to claim 38, wherein the substrate comprises at least one of silicon, silicon oxide, silicon nitride or a polymer.

41. An apparatus according to claim 30, wherein the body has a conductive path extending from the electrode in the chamber to a contact allowing connection to an electrical circuit.

42. An apparatus according to claim 41, wherein the conductive path extends through the body to a contact disposed on the opposite side of the body from the recess.

43. An apparatus according to claim 41, wherein the conductive path extends across a surface of the substrate under the at least one further layer.

44. An apparatus according to claim 30, wherein the electrode has provided thereon a hydrophillic surface which repels the hydrophobic fluid applied in step (c) whilst allowing ionic conduction from the aqueous solution to the electrode.

45. An apparatus according to claim 44, wherein the hydrophillic surface is the surface of protective material provided on the electrode.

46. An apparatus according to claim 45, wherein the protective material is a covalently-attached hydrophillic species or a conductive polymer.

47. An apparatus according to claim 30, wherein the electrode has a conductive polymer provided thereon.

48. An apparatus according to claim 30, further comprising a further electrode in the chamber outside said recess.

49. An apparatus according to claim 30, wherein the elements defining the chamber further include a cover extending over the body so that the chamber is a closed chamber.

50. An apparatus according to claim 49, wherein the cover comprises at least one inlet and at least one outlet, the aqueous solution being introduced into the chamber through the inlet in step (c) and the outlet venting fluid displaced by the aqueous solution thus introduced.

51. An apparatus according to claim 30, wherein the internal surface of the recess has no openings capable of fluid communication.

52. An apparatus according to claim 30, wherein the recess has a width of at most 500 μ

m.

53. An apparatus according to claim 30, wherein the at least one recess is plural recesses.

54. An apparatus according to claim 30, further comprising amphiphilic molecules deposited on an internal surface of the chamber.

55. An apparatus according to claim 54, wherein the amphiphilic molecules are lipids.

56. An apparatus according to claim 30, further comprising a membrane protein deposited on the on an internal surface of the chamber.

57. An apparatus according to claim 30, further comprising a pre-treatment coating of a hydrophobic fluid applied to the body across the recess.

58. An apparatus according to claim 57, wherein the recess and the chamber contain aqueous solution.

59. An apparatus according to claim 58, further comprising a layer of amphiphilic molecules extending across the opening of the recess.

60. An apparatus according to claim 59, wherein the layer of the amphiphilic molecules has an electrical resistance of at least 1 GΩ

.

61. An apparatus according to claim 59, wherein the amphiphilic molecules are lipids.

62. An apparatus according to claim 59, the layer of amphiphilic molecules having a membrane protein inserted therein.

63. A method of using an apparatus according to claim 59, wherein the apparatus is provided with a further electrode in the chamber outside the recess, and the method comprises applying a potential across the electrode in the recess and the further electrode and monitoring an electrical signal developed between the electrode in the recess and the further electrode.

64. A method of improving the performance of an electrode in a recess in conducting electro-physiological measurements, the method comprising depositing a conductive polymer on the electrode.

65. A method according to claim 64, wherein the electrode is made of metal.

66. A method according to claim 65, wherein the electrode is made of silver, gold or platinum.

67. A method according to claim 64, wherein the conductive polymer is a polypyrrole.

68. An apparatus for conducting electro-physiological measurements, the apparatus comprising, a body having a recess in which an electrode is located, wherein a conductive polymer is deposited on the electrode.

69. A method according to claim 68, wherein the electrode is made of metal.

70. A method according to claim 69, wherein the electrode is made of silver, gold or platinum.

71. A method according to claim 68, wherein the conductive polymer is a polypyrrole.