Formation of layers of amphiphilic molecules

• US 9,927,398 B2
• Filed: 06/30/2015
• Issued: 03/27/2018
• Est. Priority Date: 12/19/2007
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Reid, Stuart William, Reid, Terence Alan, Clarke, James Anthony, White, Steven Paul, Sanghera, Gurdial Singh
• Primary Examiner(s): Rufo, Louis J
• Application Number:
  US14/788,120
• Publication Number:
  US 20150300986A1
• Time in Patent Office: 1,001 Days
• 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 being capable of being filled by flowing aqueous solution across the body, and the recess containing an electrode, wherein the apparatus is provided with a further electrode in the chamber outside said recess;

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

(c1) flowing aqueous solution across the body to cover the recess so that aqueous solution is introduced into the recess wherein the aqueous solution is flowed also to contact the further electrode;

(c2) 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;

(c3) removing aqueous solution so that the recess is uncovered, leaving a volume of the aqueous solution in the recess; and

(c4) 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 having an electrical resistance of at least 1 GΩ

forms across the recess separating the volume of the aqueous solution in the recess from the aqueous solution that re-covers the recess.

2. A method according to claim 1, wherein the aqueous solution caused to flow in steps (c1) and (c4) is the same aqueous solution.

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

4. A method according to claim 3, 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.

5. A method according to claim 3, wherein an inner part of the internal surface of the recess inside the outer part is hydrophilic.

6. A method according to claim 5, 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.

7. A method according to claim 3, wherein said surfaces are modified by a fluorine species.

8. A method according to claim 7, wherein said surfaces are modified by a fluorine species by treatment with a fluorine plasma.

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

10. 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.

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

12. A method according to claim 11, wherein the hydrophilic surface is a surface of protective material provided on the electrode.

13. A method according to claim 12, wherein the protective material is a covalently-attached hydrophilic species or a conductive polymer.

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

15. A method according to claim 1, wherein the elements configured to define the chamber further comprise a cover extending over the body so that the chamber is a closed chamber.

16. A method according to claim 15, 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 steps (c1) and (c4) and the outlet venting fluid displaced by the aqueous solution thus introduced.

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

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

19. A method according to claim 18, wherein the amphiphilic molecules are lipids.

20. A method according to claim 1, further comprising, before step (c1), 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 (c1) whereby the amphiphilic molecules are added to the aqueous solution.

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

22. A method according to claim 21, wherein the aqueous solution has a membrane protein added thereto, whereby the membrane protein is inserted spontaneously into the layer of amphiphilic molecules.

23. A method according to claim 21, further comprising, before step (c1), depositing the membrane protein on an internal surface of the chamber, the aqueous solution covering the internal surface during step (c1) whereby the membrane protein is added to the aqueous solution.

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

25. A method according to claim 21, wherein step (c2) further comprises monitoring an electrical signal developed between the electrode in the recess and the further electrode.

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

27. A method according to claim 1, further comprising depositing the amphiphilic molecules on the body so that the amphiphilic molecules are added to the aqueous solution when the aqueous solution flows across the body.

28. A method according to claim 1, wherein the aqueous solution caused to flow in steps (c1) and (c4) are different aqueous solutions.

29. 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 being capable of being filled by flowing aqueous solution across the body, and the recess containing an electrode, wherein an inner part of an internal surface of the recess is hydrophilic, and wherein a further electrode is present in the chamber outside the recess;

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

(c1) flowing aqueous solution across the body both to cover the recess so that aqueous solution is introduced into the recess, and also to contact the further electrode;

(c2) removing aqueous solution so that the recess is uncovered, leaving a volume of the 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 having an electrical resistance of at least 1 GΩ

forms across the recess separating the volume of the aqueous solution in the recess from the aqueous solution that re-covers the recess,

wherein, at a time during which the volume of the aqueous solution is in the recess, a voltage is applied 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.