Apparatus and methods for controlling insertion of a membrane channel into a membrane

Patent metadata

Field	Value
Publication number	US-12515215-B2
Application number	US-202418744948-A
Country	US
Kind code	B2
Filing date	Jun 17, 2024
Priority date	Nov 24, 2016
Publication date	Jan 6, 2026
Grant date	Jan 6, 2026

Abstract

Apparatus and methods for controlling the insertion of a membrane channel into a membrane are disclosed. The membrane separates first and second liquids. A driving unit applies a potential difference across the membrane via first and second electrodes to promote insertion of a membrane channel into the membrane. A membrane voltage reduction unit is connected in series with the membrane. The driving unit applies a driving voltage across the membrane voltage reduction unit and the membrane. The membrane voltage reduction unit is configured such that a reduction in resistance through the membrane caused by insertion of a membrane channel intrinsically increases a potential difference across the membrane voltage reduction unit thereby lowering the potential difference across the membrane, the lowering sufficient to prevent or reduce promotion of insertion of a further membrane channel.

First claim

The invention claimed is:

1 . An apparatus for controlling insertion of a membrane channel into a membrane, comprising:
a first electrode configured to contact a first liquid in contact with a first surface of the membrane; a second electrode configured to contact a second liquid in contact with a second surface of the membrane, wherein the membrane separates the first and second liquids; and a driving unit configured to apply a potential difference across the membrane via the first and second electrodes, to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid, and wherein the potential difference varies with time to provide a voltage waveform and is increased by (i) ramping up the potential difference over time, or (ii) stepping up the potential difference between values of potential at progressively higher values, and wherein the apparatus comprises a circuit comprising a membrane voltage reduction unit connected in series with the membrane, which redistributes the applied potential across the circuit such that the potential difference across the membrane is lowered following insertion of the membrane channel.

2 . The apparatus of claim 1 , wherein the driving unit is configured to apply a driving voltage across the membrane voltage reduction unit and the membrane, the driving voltage providing the potential difference across the membrane.

3 . The apparatus of claim 1 , wherein the initial potential value is between 0 mV and 150 mV and the final membrane potential is between 200 and 600 mV.

4 . The apparatus of claim 1 , wherein the potential difference is increased in 1-10 mV steps with 1 to 15 seconds per step.

5 . The apparatus of claim 1 , wherein the potential increased increase is linear or non-linear.

6 . The apparatus of claim 1 , wherein the potential assists the insertion by stretching and thinning the membrane.

7 . The apparatus of claim 1 , wherein the membrane channel is one of a naturally occurring pore, a mutated pore derived from a naturally occurring pore or a synthetic a pore.

8 . The apparatus of claim 1 , wherein the membrane voltage reduction unit is configured such that a reduction in resistance through the membrane caused by insertion of a membrane channel intrinsically increases a potential difference across the membrane voltage reduction unit thereby lowering the potential difference across the membrane, wherein the lowering of the potential difference across the membrane is sufficient to prevent or reduce promotion of insertion of a further membrane channel.

9 . The apparatus of claim 1 , further comprising: a first bath for holding the first liquid in contact with the first surface of the membrane; and a second bath for holding the second liquid in contact with the second surface of the membrane.

10 . The apparatus of claim 9 , wherein plural membrane voltage reduction units are provided to control insertion of channels in multiple second baths simultaneously while applying the same driving voltage to the multiple second baths.

11 . The apparatus of claim 1 , wherein:
the membrane voltage reduction unit comprises a current source configured to supply a constant current through resistances up to a maximum resistance; and the lowering of the potential difference across the membrane is caused by the resistance through the membrane being reduced from a resistance above said maximum resistance to a resistance below said maximum resistance.

12 . The apparatus of claim 1 , wherein:
the membrane voltage reduction unit comprises a resistive component in series with the membrane, wherein the resistance of the resistive component is selected to ensure that the lowering of the potential difference across the membrane is sufficient to prevent or reduce promotion of insertion of a further membrane channel, while also allowing the potential difference across the membrane to be sufficiently high prior to insertion to promote insertion of the membrane channel prior to insertion.

13 . The apparatus of claim 1 , wherein:
the membrane voltage reduction unit comprises a diode in series with the membrane, wherein the diode is configured such that the lowering of the potential difference across the membrane is sufficient to prevent or reduce promotion of insertion of a further membrane channel, while also allowing the potential difference across the membrane to be sufficiently high prior to insertion to promote insertion of the membrane channel prior to insertion.

14 . The apparatus of claim 13 , wherein:
a plurality of second baths are provided, each second bath being configured to support a different membrane; a plurality of the membrane voltage reduction units are provided, each membrane voltage reduction unit being connected in series with a different membrane or a different portion of the same membrane; and the driving unit is configured to apply the driving voltage in parallel across all of the pairs of membrane voltage reduction unit and different membrane or across all of the pairs of membrane voltage reduction unit and different portion of the same membrane.

15 . A method of controlling insertion of a membrane channel into a membrane, comprising:
providing a membrane in contact with and separating first and second liquids on respective first and second sides of the membrane; and using a driving unit to apply a potential difference across the membrane via first and second electrodes to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid; wherein the potential difference is varied with time to provide a voltage waveform and increased by (i) ramping the potential difference over time, or (ii) stepping between values of potential at progressively higher values, and wherein the apparatus comprises a circuit comprising a membrane voltage reduction unit connected in series with the membrane, which redistributes the applied potential across the circuit such that the potential difference across the membrane is lowered following insertion of the membrane channel.

16 . The method of claim 15 , wherein:
the membrane voltage reduction unit comprises a current source capable of supplying a predetermined constant current through resistances up to a maximum resistance; and the lowering of the potential difference across the membrane is caused by the resistance through the membrane being reduced from a resistance above said maximum resistance to a resistance below said maximum resistance.

17 . The method of claim 16 , wherein:
the membrane voltage reduction unit comprises a resistive component in series with the membrane, wherein the resistance of the resistive component is selected to ensure that the lowering of the potential difference across the membrane is sufficient to prevent or reduce promotion of insertion of a further membrane channel, while also allowing the potential difference across the membrane to be sufficiently high prior to insertion to promote insertion of the membrane channel prior to insertion.

18 . The method of claim 15 , wherein:
the membrane voltage reduction unit comprises a diode in series with the membrane, wherein the diode is configured such that the lowering of the potential difference across the membrane is sufficient to prevent or reduce promotion of insertion of a further membrane channel, while also allowing the potential difference across the membrane to be sufficiently high prior to insertion to promote insertion of the membrane channel prior to insertion.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Garalde Daniel Ryan
• Clarke James Anthony
• Jennison Mike
• Heron Andrew John

Classifications

• B81C1/00071

  Channels · CPC title

• B01L2400/0415

  electrical forces, e.g. electrokinetic · CPC title

• B01L2300/044

  pierceable, e.g. films, membranes · CPC title

• G01N33/48721 Primary

  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

• B01L3/502 Primary

  with fluid transport, e.g. in multi-compartment structures · CPC title