Coupling method

Patent metadata

Field	Value
Publication number	US-11946102-B2
Application number	US-202017064628-A
Country	US
Kind code	B2
Filing date	Oct 7, 2020
Priority date	May 27, 2011
Publication date	Apr 2, 2024
Grant date	Apr 2, 2024

Abstract

The invention relates to a new method of determining the presence, absence or characteristics of an analyte. The analyte is coupled to a membrane. The invention also relates to nucleic acid sequencing.

First claim

The invention claimed is:

1. A method for detecting analytes, comprising:
(a) providing a membrane in which is present a nanopore that provides a channel through the membrane;
(b) contacting the membrane, in an ionic solution, with one or more analytes, wherein following contact with the membrane the analytes are tethered to the membrane via a tethering group;
(c) applying a potential difference across the membrane and detecting a first analyte using the nanopore, from among the analytes tethered to the membrane; and
(d) detecting a second analyte, from among the analytes tethered to the membrane, using the same nanopore, wherein the second analyte is not the first analyte;
wherein the one or more analytes is an amino acid, peptide, polypeptide, or protein.

2. The method according to claim 1 , wherein the membrane is an amphiphilic layer or a solid state layer.

3. The method according to claim 1 , wherein the membrane is a lipid bilayer.

4. The method according to claim 1 , wherein the tethering group comprises a hydrophobic anchor.

5. The method according to claim 4 , wherein the hydrophobic anchor is capable of embedding in the membrane.

6. The method according to claim 4 , wherein the hydrophobic anchor is a lipid, fatty acid, sterol, carbon nanotube or amino acid.

7. The method according to claim 1 , wherein the tethering group comprises a linker.

8. The method according to claim 7 , wherein the linker comprises a polynucleotide, polyethylene glycol, polysaccharide, or polypeptide.

9. The method according to claim 1 , wherein the first analyte and the second analyte are detected based on ion flow through the nanopore that is measured via an electrical means.

10. The method according to claim 1 , wherein the nanopore is a protein nanopore.

11. The method according to claim 10 , wherein the protein nanopore is derived from Msp or α-hemolysin (α-HL).

12. The method according to claim 1 , wherein the nanopore comprises a molecular adaptor that mediates interaction of the analyte with the nanopore.

13. The method according to claim 1 , wherein the nanopore is coupled to a polynucleotide binding protein.

14. The method according to claim 13 , wherein the polynucleotide binding protein is an exonuclease or a polymerase.

15. The method according to claim 1 , wherein the first analyte and the second analyte are detected based on ion flow through the nanopore that is measured by measuring a current passing through the nanopore.

16. The method according to claim 1 , wherein the tethering group comprises a polypeptide.

17. The method according to claim 1 , wherein the analyte is present in the solution of (b) at a concentration of less than 100 pM.

18. The method according to claim 1 , wherein the analyte is present in the solution of (b) at a concentration of about 0.001 pM to about 1 nM.

19. The method according to claim 1 , wherein rate of interaction of the molecules of the analyte with the nanopore is increased as compared to rate of interaction of molecules of the analyte with the nanopore in the absence of the tethering group.

20. The method according to claim 19 , wherein the rate of interaction of the molecules of the analyte is increased by at least three orders of magnitude.

21. The method according to claim 1 , wherein the effective concentration of the analyte at the nanopore is increased as compared to the concentration of the analyte in the solution of (b).

22. The method according to claim 1 , wherein a single nanopore providing an ion channel through the membrane is present in the membrane.

23. The method according to claim 1 , wherein the analyte is functionalized with the tethering group.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Clarke James Anthony
• White James
• Milton John
• Brown Clive Gavin

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• B82Y15/00

  Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors · CPC title

• G01N27/44743

  Introducing samples · 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

• C12Q2565/631

  being a biochannel or pore · CPC title