# US 10,246,741 B2 · Coupling method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,741 B2 - Document ID: US10246741B2 - Status: Granted - Kind: B2 - Application number: 14/122,573 - Filed: 2012-05-25 - Published: 2014-09-18 - Granted: 2019-04-02 - Earliest claimed: 2011-05-27 - Assignees: Oxford Nanopore Technologies PLC - Applicants: James Clarke; James White; John Milton; Clive Brown - Inventors: James Clarke; James White; John Milton; Clive Brown - Web page: https://patentgrep.com/patent/US10246741B2 ## CPC - C12Q 1/6869: Methods for sequencing - B82Y 15/00: Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors - G01N 27/44743: {Introducing samples} - G01N 33/48721: {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)} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## 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. ## Claims ### Claim 1 A method for detecting polynucleotides, 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 polynucleotides, wherein following contact with the membrane the polynucleotides are tethered to the membrane via a tethering group; (c) applying a potential difference across the membrane and detecting a first polynucleotide using the nanopore, from among the polynucleotides tethered to the membrane; and (d) detecting a second polynucleotide, from among the polynucleotides tethered to the membrane, using the same nanopore, wherein the second polynucleotide is not the first polynucleotide. ### Claim 2 (depends on 1) The method according to claim 1, wherein the membrane is an amphiphilic layer or a solid state layer. ### Claim 3 (depends on 1) The method according to claim 1, wherein the membrane is a lipid bilayer. ### Claim 4 (depends on 1) The method according to claim 1, wherein the tethering group comprises a hydrophobic anchor. ### Claim 5 (depends on 4) The method according to claim 4, wherein the hydrophobic anchor is a lipid, fatty acid, sterol, carbon nanotube or amino acid. ### Claim 6 (depends on 4) The method according to claim 4, wherein the hydrophobic anchor is capable of embedding in the membrane. ### Claim 7 (depends on 1) The method according to claim 1, wherein the tethering group comprises a linker. ### Claim 8 (depends on 7) The method according to claim 7, wherein the linker comprises a polynucleotide, polyethylene glycol, polysaccharide, or polypeptide. ### Claim 9 (depends on 1) The method according to claim 1, wherein the polynucleotide is tethered transiently to the membrane. ### Claim 10 (depends on 1) The method according to claim 1, wherein the first polynucleotide and the second polynucleotide are detected based on ion flow through the nanopore that is measured via an electrical means. ### Claim 11 (depends on 1) The method according to claim 1, wherein the nanopore is a protein nanopore. ### Claim 12 (depends on 11) The method according to claim 11, wherein the protein nanopore is derived from Msp or α-hemolysin (α-HL). ### Claim 13 (depends on 1) The method according to claim 1, wherein the nanopore comprises a molecular adaptor that mediates interaction of the polynucleotide with the nanopore. ### Claim 14 (depends on 1) The method according to claim 1, wherein the nanopore is coupled to a polynucleotide binding protein, which is optionally an exonuclease or a polymerase. ### Claim 15 (depends on 1) The method according to claim 1, wherein the first polynucleotide and the second polynucleotide are detected based on ion flow through the nanopore that is measured by measuring a current passing through the nanopore. ### Claim 16 (depends on 1) The method according to claim 1, wherein the polynucleotide comprises a target polynucleotide. ### Claim 17 (depends on 16) The method according to claim 16, wherein the method comprises digesting a target polynucleotide to provide a fragment and the fragment sequence is determined. ### Claim 18 (depends on 1) The method according to claim 1, wherein the tethering group comprises a polypeptide. ### Claim 19 (depends on 1) The method according to claim 1, wherein the polynucleotide is present in the solution of (b) at a concentration of less than 0.01 pM. ### Claim 20 (depends on 1) The method according to claim 1, wherein the polynucleotide is present in the solution of (b) at a concentration of less than 0.1 pM. ### Claim 21 (depends on 1) The method according to claim 1, wherein the polynucleotide is present in the solution of (b) at a concentration of less than 1 pM. ### Claim 22 (depends on 1) The method according to claim 1, wherein the polynucleotide is present in the solution of (b) at a concentration of less than 10 pM. ### Claim 23 (depends on 1) The method according to claim 1, wherein the polynucleotide is present in the solution of (b) at a concentration of less than 100 pM. ### Claim 24 (depends on 1) The method according to claim 1, wherein the polynucleotide is present in the solution of (b) at a concentration of about 0.001 pM to about 1 nM. ### Claim 25 (depends on 1) The method according to claim 1, wherein rate of interaction of the molecules of the polynucleotide with the nanopore is increased as compared to rate of interaction of molecules of the polynucleotide with the nanopore in the absence of the tethering group. ### Claim 26 (depends on 25) The method according to claim 25, wherein the rate of interaction of the molecules of the polynucleotide is increased by at least three orders of magnitude. ### Claim 27 (depends on 1) The method according to claim 1, wherein the effective concentration of the polynucleotide at the nanopore is increased as compared to the concentration of the polynucleotide in the solution of (b). ### Claim 28 (depends on 1) The method according to claim 1, wherein a single nanopore providing an ion channel through the membrane is present in the membrane. ### Claim 29 (depends on 1) The method according to claim 1, wherein the polynucleotide is functionalized with the tethering group.