Coupling method

Patent metadata

Field	Value
Publication number	US-2025297305-A1
Application number	US-202418966835-A
Country	US
Kind code	A1
Filing date	Dec 3, 2024
Priority date	May 27, 2011
Publication date	Sep 25, 2025
Grant date	—

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

1 .- 25 . (canceled)

26 . A method for detecting a peptide, polypeptide, or protein, 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 peptides, polypeptides, or proteins, wherein following contact with the membrane the peptides, polypeptides, or proteins are coupled to the membrane surface; and (c) applying a potential difference across the membrane and detecting a first peptide, polypeptide, or protein using the nanopore, from among the peptides, polypeptides, or proteins coupled to the membrane surface, wherein the first peptide, polypeptide, or protein is unfolded to form a chain before it is contacted with the pore.

27 . The method of claim 26 , wherein the peptides, polypeptides, or proteins are extracted or secreted from cells.

28 . The method of claim 26 , wherein the peptides, polypeptides, or proteins each comprises a modified amino acid.

29 . The method of claim 26 , wherein the peptides, polypeptides, or proteins are each coupled to the membrane surface via a protein fusion, peptide tag, or by a reactive amino acid.

30 . The method of claim 26 , wherein the nanopore is not a solid state nanopore.

31 . The method of claim 26 , further comprising: (d) detecting a second peptide, polypeptide, protein, from among the peptides, polypeptides, or proteins coupled to the membrane, using the same nanopore, wherein the second peptide, polypeptide, or protein is not the first peptide, polypeptide, or protein.

32 . The method of claim 26 , wherein the membrane is an amphiphilic layer.

33 . The method of claim 26 , wherein the membrane is a lipid bilayer.

34 . The method of claim 33 , wherein the lipid bilayer comprises a phosphatidylcholine (PC) or a phosphatidylethanolamine (PE) headgroup.

35 . The method of claim 26 , wherein the membrane is a block copolymer.

36 . The method of claim 35 , wherein the block copolymer is a triblock copolymer having outer hydrophilic layers and an inner hydrophobic core.

37 . The method of claim 26 , wherein the membrane comprises a mixture of lipids and copolymers.

38 . The method of claim 26 , wherein the nanopore is a protein nanopore.

39 . The method of claim 38 , wherein the protein nanopore is α-hemolysin (α-HL) nanopore or MspA pore.

40 . The method of claim 26 , wherein the peptides, polypeptides, or proteins each comprise a coupling group.

41 . The method of claim 40 , wherein the coupling group is hydrophobic.

42 . The method of claim 26 , wherein the peptides, polypeptides, or proteins are each tethered to the membrane transiently.

43 . The method of claim 26 , wherein the peptides, polypeptides, or proteins each comprises a leader sequence designed to preferentially thread into the nanopore.

44 . The method of claim 26 , wherein the polymers each further comprises polyethylene glycol (PEG).

45 . The method of claim 26 , wherein the detecting comprises measuring a current passing through the nanopore.

Assignees

• Oxford Nanopore Tech Plc

Inventors

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

Classifications

• 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

• B82Y15/00

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

• C12Q1/68

  involving nucleic acids · CPC title

• G01N33/487

  of liquid biological material · CPC title