Methods for forming lipid bilayers on biochips

Patent metadata

Field	Value
Publication number	US-11725236-B2
Application number	US-202117242098-A
Country	US
Kind code	B2
Filing date	Apr 27, 2021
Priority date	Oct 23, 2013
Publication date	Aug 15, 2023
Grant date	Aug 15, 2023

Abstract

This disclosure provides a biochip comprising a plurality of wells. The biochip includes a membrane that is disposed in or adjacent to an individual well of the plurality of wells. The membrane comprises a nanopore, and the individual well comprises an electrode that detects a signal upon ionic flow through the pore in response to a species passing through or adjacent to the nanopore. The electrode can be a non-sacrificial electrode. A lipid bilayer can be formed over the plurality of wells using a bubble.

First claim

What is claimed is:

1. A method for forming a membrane for use in a nanopore sensing device,
comprising:
flowing a first aqueous buffer solution into a fluid flow path in fluid communication with a plurality of wells disposed on a chip, each well comprising an opening and an electrode, wherein the first aqueous buffer solution is ionic;
flowing a membrane material solution into the fluid flow path and over the plurality of wells to displace excess first aqueous buffer solution from the fluid flow path while leaving the first aqueous buffer within the plurality of wells;
flowing a second aqueous buffer solution into the fluid flow path to displace excess membrane material solution from the flow path, thereby leaving the openings of the plurality of wells covered with a layer of membrane material, wherein the second aqueous buffer solution is ionic;
applying a stimulus to the layer of membrane material covering the openings of the plurality of wells to thin the layer of membrane material into a membrane capable of receiving a nanopore, wherein the membrane comprises a polymeric material, wherein the stimulus is selected from the group consisting of a liquid flow over the plurality of wells, a sequential flow of one or more different liquids over the plurality of wells, a sequential flow of any combination of one or more different liquids and bubbles over the plurality of wells, an electrical pulse, a sonication pulse, a pressure pulse, or a sound pulse; and
inserting a nanopore into the membrane, wherein the first aqueous buffer solution and/or the second aqueous buffer solution comprises glutamate.

2. The method of claim 1 , wherein the stimulus comprises the sequential flow of one or more different liquids over the plurality of wells.

3. The method of claim 1 , wherein the stimulus comprises an electrical pulse.

4. The method of claim 1 , wherein the stimulus comprises the sequential flow of one or more different liquids over the plurality of wells and an electrical pulse.

5. The method of claim 1 , further comprising:
applying a voltage across the layer of membrane material; and
measuring an electrical signal in response to the applied voltage in order to determine whether the membrane comprising the polymeric material has been formed across the opening of the well.

6. The method of claim 5 , wherein the electrical signal is current.

7. The method of claim 5 , wherein the electrical signal is seal resistance.

8. A method for forming a membrane for use in a nanopore sensing device,
comprising:
flowing a first aqueous buffer solution into a fluid flow path in fluid communication with a plurality of wells disposed on a chip, each well comprising an opening and an electrode, wherein the first aqueous buffer solution is ionic;
flowing a lipid solution into the fluid flow path and over the plurality of wells to displace excess first aqueous buffer solution from the fluid flow path while leaving the first aqueous buffer within the plurality of wells;
flowing a second aqueous buffer solution into the fluid flow path to displace excess lipid solution from the flow path, thereby leaving the openings of the plurality of wells covered with a layer of lipid material, wherein the second aqueous buffer solution is ionic;
applying a stimulus to the layer of lipid material covering the openings of the plurality of wells to thin the layer of lipid material into a lipid bilayer capable of receiving a nanopore, wherein the stimulus is selected from the group consisting of a liquid flow over the plurality of wells, a sequential flow of one or more different liquids over the plurality of wells, a sequential flow of any combination of one or more different liquids and bubbles over the plurality of wells, an electrical pulse, a sonication pulse, a pressure pulse, or a sound pulse; and
inserting a nanopore into the lipid bilayer, wherein the first aqueous buffer solution and/or the second aqueous buffer solution comprises glutamate.

9. The method of claim 8 , wherein the lipid bilayer and/or the nanopore is stabilized by a sugar in the first aqueous buffer solution and/or the second aqueous buffer solution.

10. The method of claim 9 , wherein the sugar is trehalose.

11. The method of claim 8 , wherein the stimulus comprises a sequential flow of one or more different liquids over the plurality of wells.

12. The method of claim 8 , wherein the stimulus comprises an electrical pulse.

13. The method of claim 8 , wherein the stimulus comprises a sequential flow of one or more different liquids over the plurality of wells and an electrical pulse.

14. The method of claim 8 , further comprising:
applying a voltage across the layer of lipid material; and
measuring an electrical signal in response to the applied voltage in order to determine whether the lipid bilayer has been formed across the opening of the well.

15. The method of claim 4 , wherein the electrical signal is current.

16. The method of claim 4 , wherein the electrical signal is seal resistance.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Davis Randall W
• Liu Edward Shian
• Harada Eric Takeshi
• Aguirre Anne
• Trans Andrew
• Pollard James
• Cech Cynthia

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12M1/34

  Measuring or testing with condition measuring or sensing means, e.g. colony counters · CPC title

• G01N27/3277

  being a redox reaction, e.g. detection by cyclic voltammetry (voltammetry per se G01N27/42, G01N27/48) · CPC title

• G01N27/3278

  involving nanosized elements, e.g. nanogaps or nanoparticles (nanopores G01N33/48721; magnetic beads G01N27/745) · CPC title

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title