Formation of Lipid Bilayers

• US 20100196203A1
• Filed: 02/18/2008
• Published: 08/05/2010
• Est. Priority Date: 02/20/2007
• Status: Abandoned Application

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Reid, Terence Alan, White, Steven Paul, Sanghera, Gurdial Singh
• Application Number:
  US12/527,687
• Publication Number:
  US 20100196203A1
• Time in Patent Office: Days
• US Class Current: 422/68.1
• CPC Class Codes: G01N 33/48728 Investigating individual cells, e.g. by patch clamp, voltage clamp investigating individual particles in general G01N15/10
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743

Abstract

A method for forming a lipid bilayer across an aperture, comprises: (a) providing a cell having a chamber adjacent to a septum comprising a membrane having an aperture capable of supporting a lipid bilayer; (b) depositing one or more lipids on an internal surface of the chamber; (c) introducing an aqueous solution into the chamber to cover the aperture and the internal surface and to form an interface between the solution and lipids; and (d) moving the interface past the aperture at least once to form a lipid bilayer across the aperture.

Claims

1. A method for forming a lipid bilayer across an aperture, comprising:

(a) providing a cell having a chamber adjacent to a septum comprising a membrane having an aperture capable of supporting a lipid bilayer;

(b) depositing one or more lipids on an internal surface of the chamber;

(c) introducing an aqueous solution into the chamber to cover the aperture and the internal surface and to form an interface between the solution and lipids; and

(d) moving the interface past the aperture at least once to form a lipid bilayer across the aperture.

2. A method according to claim 1, wherein:

(a) the lipids are dried;

(b) the lipids are dried and comprise less than 50 wt % solvent;

(c) the aqueous solution covers the internal surface before it covers the aperture;

(d) the internal surface on which the lipids are deposited is on the septum;

(e) the aperture has a diameter in at least one dimension which is 20 μ

m or less;

(f) the method further comprises pre-treating the membrane to increase its affinity to lipids;

(g) the method further comprises pre-treating the membrane with hexadecane;

(h) step (b) further comprises depositing one or more membrane proteins on the same or different internal surface, step (c) further comprises introducing the aqueous solution into the chamber to cover the membrane proteins and the method further comprises allowing the membrane proteins to insert into the lipid bilayer;

(i) the septum further comprises a support sheet on at least one side of the membrane;

(j) the membrane is made from polycarbonate (PC), polytetrafluoroethylene (PTFE), polyethylene, polypropylene, nylon and polyethylene naphthalate (PEN), polyvinylchloride (PVC), polyacrylonitrile (PAN), polyether sulphone (PES), polyimide, polystyrene, polyvinylfluoride (PVF), polyethylene telephthalate (PET), aluminized PET, nitrocellulose, polyetheretherketone (PEEK) or fluoroethylkene polymer (FEP); and

/or

(k) the lipids comprise diphantytanoyl-sn-glycero-3-phosphocholine.

9. A method according to claim 1, wherein the cell has two chambers.

10. A method according to claim 9, wherein the method comprises depositing the lipids on an internal surface of both chambers.

11. A method according to claim 9, wherein the aqueous solution is introduced into one chamber in step (c) and the other chamber comprises a gel, or a hydrogel.

13. A method according to claim 11, wherein the gel or hydrogel comprises one or more membrane proteins.

15. A method according to claim 2, wherein the internal surface on which the one or more membrane proteins are deposited is on the septum.

16. A method according to claim 2, wherein the membrane proteins are dried.

17. A method according to claim 16, wherein the membrane proteins comprise less than 20 wt % solvent.

18. A method according to any one of claims 2, wherein the one or more membrane proteins comprise α

-hemolysin or a variant thereof.

3-8. -8. (canceled)

12. (canceled)

14. (canceled)

19-21. -21. (canceled)

22. A device for forming a lipid bilayer comprising:

(a) a cell having a chamber adjacent to a septum comprising a membrane having an aperture capable of supporting a lipid bilayer; and

b) one or more lipids deposited on an internal surface of the chamber, wherein the cell comprises an inlet for introducing an aqueous solution into the chamber having lipid deposited therein.

23. A method according to claim 22, wherein:

(a) the lipids are dried;

(b) the lipids are dried and comprise less than 50 wt % solvent;

(c) the inlet, internal surface and aperture are arranged in such a manner that the aqueous solution covers the internal surface before it covers the aperture;

(d) the internal surface on which the lipids are deposited is on the septum;

(e) the aperture has a diameter in at least one dimension which is 20 μ

m or less;

(f) the membrane has a pre-treatment to increase its affinity to lipids;

(g) the membrane has a hexadecane pre-treatment;

(h) the cell has two chambers;

(i) the cell has two chambers and the inlet opens into one chamber and the other chamber comprises a gel or a hydrogel;

(j) the device further comprises one or more membrane proteins deposited on the same or different internal surface;

(k) the septum further comprises a support sheet material on at least one side of the membrane;

(l) the membrane is made from polycarbonate (PC), polytetrafluoroethylene (PTFE), polyethylene, polypropylene, nylon and polyethylene naphthalate (PEN), polyvinylchloride (PVC), polyacrylonitrile (PAN), polyether sulphone (PES), polyimide, polystyrene, polyvinylfluoride (PVF), polyethylene telephthalate (PET), aluminized PET, nitrocellulose, polyetheretherketone (PEEK) or fluoroethylkene polymer (FEP); and

/or

(m) the lipids comprise diphantytanoyl-sn-glycero-3-phosphocholine.

33. A device according to claim 23, wherein the gel or the hydrogel comprises one or more membrane proteins.

35. A device according to claim 23, wherein the internal surface on which the one or more membrane proteins are deposited is on the septum.

36. A device according to claim 23, wherein the one or more membrane proteins:

(a) are dried;

(b) are dried and comprise less than 20 wt % solvent;

or

(c) comprise α

-hemolysin or a variant thereof.

24-32. -32. (canceled)

34. (canceled)

37-41. -41. (canceled)