Methods for forming lipid bilayers on biochips

Patent metadata

Field	Value
Publication number	US-11021745-B2
Application number	US-201916272970-A
Country	US
Kind code	B2
Filing date	Feb 11, 2019
Priority date	Oct 23, 2013
Publication date	Jun 1, 2021
Grant date	Jun 1, 2021

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 and wherein the membrane is stabilized by a sugar in the first aqueous buffer solution and/or the second aqueous buffer solution, 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 nanopore is also stabilized by the sugar in the first aqueous buffer solution and/or the second aqueous buffer solution and 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 , wherein the sugar is trehalose.

6. 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.

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

8. The method of claim 6 , wherein the electrical signal is seal resistance.

9. A nanopore sensing system, the system comprising:
a chip comprising a fluid flow path in fluid communication with a plurality of wells, each well comprising an opening and a sensing electrode, wherein the sensing electrode is paired with a counter electrode; and
a control system, the control system programmed to:
flow a first ionic solution into the fluid flow path;
flow a membrane material solution into the fluid flow path;
flow a second ionic 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 first ionic solution and the second ionic solution comprises a sugar;
apply 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 and wherein the membrane is stabilized by the sugar in the first ionic solution and/or the second ionic solution, wherein the stimulus is selected from the group consisting of a liquid flow over the plurality of wells, the sequential flow of one or more different liquids over the plurality of wells, the sequential flow of any combination of one or more different liquids and bubbles over the plurality of wells, an electrical pulse, sonication pulse, pressure pulse, or sound pulse; and
insert a nanopore into the membrane, wherein the nanopore is also stabilized by the sugar in the first ionic solution and/or the second ionic solution and wherein the first aqueous buffer solution and/or the second aqueous buffer solution comprises glutamate.

10. The system of claim 9 , wherein the control system is external to the chip.

11. The system of claim 9 , wherein the control system comprises a computer processor.

12. The system of claim 9 , further comprising a fluid flow system operably coupled to the control system and the chip.

13. The system of claim 9 , wherein the sugar is trehalose.

14. The system of claim 9 , wherein the control system is further programmed to:
apply a voltage across the layer of membrane material; and
measure an electrical signal in response to the applied voltage in order to determine whether the membrane comprising the polymeric has been formed across the opening of the well.

15. The system of claim 14 , wherein the electrical signal is current.

16. The system of claim 14 , 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

• G01N33/48721

  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) · CPC title

• C12M1/34

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

• G01N27/44791

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

• G01N27/3278

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