Delivery of molecules to a lipid bilayer

• US 7,939,270 B2
• Filed: 03/22/2006
• Issued: 05/10/2011
• Est. Priority Date: 03/23/2005
• Status: Active Grant

• Current Assignee: Oxford University Innovation Limited (University of Oxford)
• Original Assignee: Isis Innovation Limited
• Inventors: Holden, Matthew Alexander, Bayley, John Hagan Pryce
• Primary Examiner(s): CHEU, CHANGHWA J
• Application Number:
  US11/884,927
• Publication Number:
  US 20080153150A1
• Time in Patent Office: 1,875 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: C12M 35/00 Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q

Abstract

A method of delivering a molecule, such as a membrane protein, to a lipid bilayer uses a probe capable of holding the molecule on a carrier surface thereof. The molecule is deposited on the carrier surface and the probe is moved to engage the carrier surface against the lipid bilayer. The carrier surface may be the surface of a drop of hydrogel which adsorbs the molecule. The molecule may be a membrane protein which is thus inserted into the lipid bilayer. The method is fast and simple to perform thereby allowing high throughput experimentation.

Claims

1. A method of inserting a membrane protein into a planar lipid bilayer in an apparatus having an electrical circuit capable of measuring an electrical signal across the planar lipid bilayer, the method comprising:

providing a probe which is a macroscopic element arranged to be movable manually or by a mechanical system and having a carrier surface capable of holding the membrane protein;

depositing the membrane protein on the carrier surface of the probe so that the membrane protein is held on the carrier surface;

moving the probe manually or by a mechanical system to engage the carrier surface against the planar lipid bilayer wherein the carrier surface is capable of holding the membrane protein less strongly than the planar lipid bilayer and to cause the membrane protein to be inserted into the lipid bilayer, the insertion being detected on the basis of the electrical signal measured by the electrical circuit.

2. A method according to claim 1, wherein

the method further comprises, after said step of moving the probe to engage the carrier surface of the probe with the planar lipid bilayer, moving the probe to disengage the carrier surface of the probe against the planar lipid bilayer, thereby leaving the inserted membrane protein in the planar lipid bilayer.

3. A method according to claim 1, wherein the probe is capable of holding the membrane protein by adsorbing the membrane protein on said carrier surface.

4. A method according to claim 1, wherein the probe comprises a body having a drop of gel protruding from the body, the gel being capable of holding the membrane protein on a protruding surface thereof which protruding surface constitutes said carrier surface.

5. A method according to claim 4, wherein the gel is a hydrogel.

6. A method according to claim 5, wherein the gel is either agarose gel or a synthetic gel.

7. A method according to claim 6, wherein the gel is polyacrylamide.

8. A method according to claim 4, wherein the body has a recess and the drop of gel is in the recess.

9. A method according to claim 1, wherein the carrier surface is the surface of a material through which an aqueous solution may diffuse.

10. A method according to claim 1, wherein said carrier surface is a surface of a solid.

11. A method according to claim 10, wherein the carrier surface is treated to increase the affinity for the membrane protein.

12. A method according to claim 11, wherein the carrier surface is treated by a silane modification.

13. A method according to claim 10, wherein the carrier surface has a coating of material.

14. A method according to claim 10, wherein said solid is glass.

15. A method according to claim 1, wherein said carrier surface has an area which is greater than the area of the planar lipid bilayer.

16. A method according to claim 15, wherein said carrier surface has an area which is greater than the area of the planar lipid bilayer by at least an order of magnitude.

17. A method according to claim 1, wherein said carrier surface has an area which is at least 1 mm².

18. A method according to claim 1, wherein said carrier surface is smaller than the planar lipid bilayer.

19. A method according to claim 18, wherein said carrier surface has an area which is at most 70% of the area of the planar lipid bilayer.

20. A method according to claim 1, wherein said step of depositing the membrane protein on the carrier surface of the probe comprises depositing a solution containing the membrane protein on the carrier surface of the probe.

21. A method according to claim 1, wherein said step of depositing the membrane protein on the carrier surface of the probe comprises rubbing the carrier surface of the probe over a sample containing the membrane protein.

22. A method according to claim 1, wherein the membrane protein is a pore or a channel.

23. A method according to claim 22, wherein the membrane protein is a β

-barrel or an α

-helical bundle.

24. A method according to claim 1, wherein the planar lipid bilayer is formed in an apparatus comprising a chamber body defining a chamber, a wall of the chamber having an aperture in which the planar lipid bilayer is formed, the chamber containing an aqueous solution.

25. A method according to claim 24, wherein the molecule is a membrane protein which is a pore or a channel.

26. A method according to claim 24, wherein the probe is mounted on the chamber body to allow movement of the probe to engage the carrier surface against the planar lipid bilayer.

27. A method according to claim 24, wherein

the chamber has an opening in the upper surface of the chamber, the wall having an aperture being formed in a side surface of the chamber, and

the probe comprises:

a body having said carrier surface, and

an arm supporting the body in said chamber and extending out of the chamber through said opening to allow manipulation of the probe.

28. A method according to claim 27, wherein the probe further comprises a boom mounted on the chamber body and supporting the arm.

29. A method according to claim 26, wherein the probe is pivotally mounted on the chamber body.

30. A method according to claim 22, wherein the insertion of the membrane protein into the planar lipid bilayer is detected on the basis of a characteristic increase in the electrical current flowing across the planar lipid bilayer measured by the electrical circuit.