# US 11,994,525 B2 · Membrane protein analysis substrate, method of producing membrane protein analysis substrate, method of analyzing membrane protein and membrane protein analysis grid > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,994,525 B2 - Document ID: US11994525B2 - Status: Granted - Kind: B2 - Application number: 18/193,357 - Filed: 2023-03-30 - Published: 2023-08-17 - Granted: 2024-05-28 - Earliest claimed: 2020-10-28 - Assignees: Kyushu University, National University Corporation - Applicants: Kyushu University, National University Corporation - Inventors: Atsushi Shimada - Web page: https://patentgrep.com/patent/US11994525B2 ## CPC - G01N 33/92: involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)} - 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)} - G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)} - H01J 37/20: Means for supporting or positioning the object or the material; Means for adjusting diaphragms or lenses associated with the support - H01J 2237/2001: Maintaining constant desired temperature - H01J 2237/2004: Biological samples - 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) - H01J: ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS (spark-gaps H01T; electric arc lamps H05B31/00; particle accelerators H05H) ## Abstract A membrane protein analysis substrate including an electron microscope grid having a plurality of through-holes; a lipid bilayer membrane that is provided to cover at least one of the plurality of through-holes; and membrane proteins that are retained in a part planarly overlapping the through-holes of the lipid bilayer membrane, wherein the lipid bilayer membrane has a lipid monolayer, and wherein the lipid monolayer is larger than the through hole in a plan view, adheres to the grid, and constitutes a part of the lipid bilayer membrane. ## Claims ### Claim 1 A membrane protein analysis substrate, comprising: an electron microscope grid having a plurality of through-holes; a lipid bilayer membrane that is provided to cover at least one of the plurality of through-holes; and membrane proteins that are retained in a part planarly overlapping positions of the through-holes covered by the lipid bilayer membrane, wherein the lipid bilayer membrane has a lipid monolayer, and wherein the lipid monolayer is larger than the through-hole in a plan view, adheres to the electron microscope grid, and constitutes a part of the lipid bilayer membrane. ### Claim 2 (depends on 1) The membrane protein analysis substrate according to claim 1, wherein one lipid monolayer constituting the lipid bilayer membrane is formed of a first lipid, and wherein the other lipid monolayer constituting the lipid bilayer membrane is formed of a second lipid different from the first lipid. ### Claim 3 (depends on 2) The membrane protein analysis substrate according to claim 2, wherein the surface of the electron microscope grid is hydrophobic, wherein one lipid monolayer constituting the lipid bilayer membrane covers a first surface of the electron microscope grid, wherein the other lipid monolayer constituting the lipid bilayer membrane covers a second surface of the electron microscope grid, and wherein the one lipid monolayer and the other lipid monolayer are bonded inside the through-hole, form the lipid bilayer membrane, and cover the through-hole. ### Claim 4 (depends on 1) The membrane protein analysis substrate according to claim 1, wherein the two lipid monolayers constituting the lipid bilayer membrane are formed of the same lipid. ### Claim 5 (depends on 4) The membrane protein analysis substrate according to claim 4, wherein the surface of the electron microscope grid is hydrophobic, wherein one lipid monolayer constituting the lipid bilayer membrane covers one surface of the electron microscope grid, wherein the other lipid monolayer constituting the lipid bilayer membrane is discretely provided inside the plurality of through-holes, and wherein the one lipid monolayer and the other lipid monolayer are bonded inside the through-hole, form the lipid bilayer membrane, and cover the through-hole. ### Claim 6 (depends on 2, 4) The membrane protein analysis substrate according to claim 2 or claim 4, wherein the surface of the electron microscope grid is hydrophilic, and wherein the lipid bilayer membrane covers the through-hole from one surface of the electron microscope grid. ### Claim 7 (depends on 1) A method of analyzing membrane proteins, comprising a process of observing the membrane proteins retained by the membrane protein analysis substrate according to claim 1 under an electron microscope. ### Claim 8 (depends on 7) The method of analyzing membrane proteins according to claim 7, further comprising a process of staining the membrane proteins retained in the lipid bilayer membrane before the process of observing under an electron microscope. ### Claim 9 (depends on 7) The method of analyzing membrane proteins according to claim 7, wherein, in the observing process, the membrane proteins are observed by a low-temperature electron microscope method. ### Claim 10 A method of forming a lipid bilayer membrane of producing a membrane protein analysis substrate, comprising: a process of providing a first lipid monolayer on a first surface of an electron microscope grid having a plurality of through-holes; a process of providing a second lipid monolayer on a second surface of the electron microscope grid and forming a lipid bilayer membrane in which the first lipid monolayer and the second lipid monolayer are bonded; and a process of retaining membrane proteins in the lipid bilayer membrane; wherein, in the process of providing a first lipid monolayer, the plurality of through-holes are covered from the side of the first surface with the first lipid monolayer, wherein, in the process of forming a lipid bilayer membrane, the plurality of through-holes are covered from the side of the second surface with the second lipid monolayer, and wherein the first lipid monolayer and the second lipid monolayer are bonded inside the plurality of through-holes. ### Claim 11 (depends on 10) The method of producing a membrane protein analysis substrate according to claim 10, wherein the process of providing a first lipid monolayer includes bringing the first surface into contact with the first lipid monolayer formed on a liquid surface of a first solution. ### Claim 12 (depends on 10) The method of producing a membrane protein analysis substrate according to claim 10, wherein the process of providing a second lipid monolayer includes bringing the second surface into contact with the second lipid monolayer formed on a liquid surface of a second solution. ### Claim 13 (depends on 12) The method of producing a membrane protein analysis substrate according to claim 12, wherein, in the process of retaining membrane proteins, the membrane proteins are added to the second solution, and wherein the membrane proteins move from the second solution to the lipid bilayer membrane and are retained in the lipid bilayer membrane. ### Claim 14 (depends on 13) The method of producing a membrane protein analysis substrate according to claim 13, wherein, in the process of retaining membrane proteins, after the membrane proteins solubilized in a buffer solution with a surfactant are added to the second solution, the surfactant adsorbent is added to the second solution, and the surfactant is removed from the second solution. ### Claim 15 A method of producing a membrane protein analysis substrate, comprising: a process of bringing one surface of an electron microscope grid having a plurality of through-holes into contact with a lipid monolayer formed on a liquid surface of a solution and forming a lipid bilayer membrane inside the plurality of through-holes; and a process of adding membrane proteins to the solution and retaining the membrane proteins in the lipid bilayer membrane, wherein, in the process of forming a lipid bilayer membrane, the lipid monolayer is transferred to the one surface, and the plurality of through-holes are covered from the side of the one surface with the lipid monolayer, wherein the lipid monolayer forms a self-assembled lipid bilayer membrane inside the plurality of through-holes, and wherein, in the process of retaining membrane proteins, the membrane proteins move from the solution to the lipid bilayer membrane and are retained in the lipid bilayer membrane. ### Claim 16 (depends on 15) The method of producing a membrane protein analysis substrate according to claim 15, wherein, in the process of adding membrane proteins, after the membrane proteins solubilized in a buffer solution with a surfactant are added to the solution, the surfactant adsorbent is added to the solution, and the surfactant is removed from the solution. ### Claim 17 A method of producing a membrane protein analysis substrate, comprising: a process of forming a lipid bilayer membrane retaining membrane proteins; and a process of transferring the lipid bilayer membrane retaining the membrane protein to one surface of an electron microscope grid having a plurality of through-holes, wherein, in the process of transferring to one surface, the lipid bilayer membrane is brought into contact with the one surface in such a manner that it covers the entire through-holes. ### Claim 18 A membrane protein analysis grid, comprising: an electron microscope grid having a plurality of through-holes; and a lipid bilayer membrane that is provided to cover at least one of the plurality of through-holes, wherein one lipid monolayer constituting the lipid bilayer membrane covers a first surface of the electron microscope grid, wherein the other lipid monolayer constituting the lipid bilayer membrane covers a second surface of the electron microscope grid, and wherein the one lipid monolayer and the other lipid monolayer are bonded inside the through-hole, form the lipid bilayer membrane, and cover the through-hole. ### Claim 19 (depends on 18) The membrane protein analysis grid according to claim 18, wherein one lipid monolayer constituting the lipid bilayer membrane is formed of a first lipid, and wherein the other lipid monolayer constituting the lipid bilayer membrane is formed of a second lipid different from the first lipid. ### Claim 20 (depends on 18) The membrane protein analysis grid according to claim 18, wherein the two lipid monolayers constituting the lipid bilayer membrane are formed of the same lipid.