# US 10,481,120 B1 · Droplet based measurement of specific capacitance and interfacial tensions > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,481,120 B1 - Document ID: US10481120B1 - Status: Granted - Kind: B1 - Application number: 15/230,242 - Filed: 2016-08-05 - Granted: 2019-11-19 - Earliest claimed: 2015-08-06 - Assignees: University of Tennessee Research Foundation - Applicants: University of Tennessee Research Foundation - Inventors: Stephen Andrew Sarles; Graham Jeffrey Taylor - Web page: https://patentgrep.com/patent/US10481120B1 ## CPC - G01N 27/228: {Circuits therefor (measuring capacitance per se G01R27/26)} - C08J 5/00: Manufacture of articles or shaped materials containing macromolecular substances (manufacture of semi-permeable membranes B01D67/00 - B01D71/00) - G01N 21/27: using photo-electric detection (G01N21/31 takes precedence){; circuits for computing concentration (logarithmic circuits G06G7/24; photometric circuits in general G01J)} - G01N 27/226: {Construction of measuring vessels; Electrodes therefor} - G01N 33/4833: {of solid biological material, e.g. tissue samples, cell cultures (tissue in vivo A61B5/00; cell suspensions G01N33/48735)} - G01N 33/525: {Multi-layer analytical elements} - C08J 2383/12: containing polyether sequences - 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) - C08J: WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H (working, e.g. shaping, of plastics B29) ## Abstract Disclosed are methods and apparatus for determining the properties of artificial membranes (e.g., artificially prepared lipid bilayers and biomimetic membranes comprising amphiphilic copolymers). Multiple physical properties of the membranes can be determined via a combination of electrical and optical measurements. The optical and electrical measurements can be determined using a single apparatus, which can also be used to prepare the artificial membrane. Methods are also disclosed for determining the effects changes in the composition of the artificial membrane, such as the addition of a drug or a change in lipid content. ## Claims ### Claim 1 A method of determining a plurality of physical properties of an artificial cellular membrane, wherein the method comprises: (a) preparing an artificial cellular membrane comprising a bilayer of amphiphilic molecules at an interface between a first droplet and a second droplet, wherein the first and second droplets each comprise an aqueous solution, wherein the first droplet is attached to a first electrode and the second droplet is attached to second electrode, and wherein the first and second droplets are suspended in a hydrophobic liquid medium; (b) determining a specific capacitance of the artificial cellular membrane, wherein the determining comprises determining a nominal capacitance and an artificial cellular membrane area for the artificial cellular membrane at each of a plurality of artificial cellular membrane areas, wherein the area of the artificial cellular membrane is varied by changing the position of the first or second droplet relative to the other of the first or second droplet, and wherein the specific capacitance is calculated from the nominal capacitance and artificial cellular membrane area data, optionally from the slope of a linear least squares regression of nominal capacitance versus area data; and (c) determining an artificial cellular membrane tension, wherein the determining comprises fixing the positions of the first and the second droplets relative to each other, measuring a contact angle between the droplets at a plurality of applied bias voltages, and calculating monolayer tension and bilayer tension. ### Claim 2 (depends on 1) The method of claim 1, wherein the plurality of physical properties comprise specific capacitance, monolayer tension, and bilayer tension. ### Claim 3 (depends on 1) The method of claim 1, wherein the method further comprises determining one or more of membrane thickness, free energy of formation, and a parameter that describes electrowetting behavior, optionally alpha, beta, and/or B coefficients. ### Claim 4 (depends on 1) The method of claim 1, wherein the amphiphilic molecules are selected from the group consisting of a lipid, an amphiphilic polymer, a biological membrane extract, and a mixture thereof. ### Claim 5 (depends on 4) The method of claim 4, wherein the aqueous solution further comprises one or more of the group consisting of a small molecule, a peptide, a protein, a biomolecule, and a pharmaceutically active agent. ### Claim 6 (depends on 1) The method of claim 1, wherein the amphiphilic molecules comprise a lipid or lipid mixture selected from the group consisting of a fatty acyl, a glycerolipid, a glycerophospholipid, a shingolipid, a sterol lipid, a prenol lipid, a saccharolipid, a polyketide, a phospholipid, a fluorescent lipid, a glycolipid, cholesterol, a biological membrane extract, and a mixture thereof. ### Claim 7 (depends on 6) The method of claim 6, wherein the amphiphilic molecules comprise one or more of the group consisting of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), palmitoyl oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DoPhPC), dipalmitoyl phosphaditdylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), diphytanoyl phosphatidylglycerol (DPhPG), diphytanoyl phosphatidylserine (DPhPS), 1,2-dioleoyl-sn-glycero-3-phophoserine (DOPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), Rhodamine-DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NDB-DPPE), and cholesterol. ### Claim 8 (depends on 1) The method of claim 1, wherein the hydrophobic liquid medium comprises a natural oil, an alkane, an alkene, a fluorocarbon, a silicone oil, a hydrophobic polymer, an amphiphilic polymer, a wax, or a mixture thereof. ### Claim 9 (depends on 8) The method of claim 8, wherein the hydrophobic liquid medium is one or more of the group consisting of n-hexadecane, heptadecane, tetradecane, squalene, dodecane, silicone oil, decane and mixtures thereof. ### Claim 10 (depends on 1) The method of claim 1, wherein the hydrophobic liquid medium is contained in a transparent chamber, wherein a side of said chamber, optionally a top side of the chamber, is completely or partially open to receive the electrodes, further optionally wherein the transparent chamber comprises or consists of polydimethylsiloxane (PDMS), glass, acrylic or rubber. ### Claim 11 (depends on 1) The method of claim 1, wherein a microscope and/or camera is positioned to obtain optical images of the artificial cellular membrane. ### Claim 12 (depends on 1) The method of claim 1, wherein the first electrode and the second electrode are coated with one of the group consisting of a hydrogel, agarose, polyacrylamide, nitro-cellulose, cellulose acetate, glass, mesoporous silica, cross-linked polyethylene glycol, a chemically linked layer of biomolecules, proteins, or other species to provide a hydrophilic surface, and a patterned or textured surface created to display a desired wetting property. ### Claim 13 (depends on 1) The method of claim 1, wherein the first and second electrode each comprise silver/silver chloride. ### Claim 14 (depends on 1) The method of claim 1, wherein at least one of the first and the second electrodes is attached to a micromanipulator. ### Claim 15 (depends on 14) The method of claim 14, wherein a three-axis micromanipulator is attached to each of the first electrode and the second electrode, and the positioning of the first and/or second droplets is performed using one or more of the micromanipulators. ### Claim 16 (depends on 1) The method of claim 1, wherein the first and second droplets each have a volume of between about 10 nanoliters (nL) and about 1000 nL, optionally between about 200 nanoliters and about 500 nanoliters. ### Claim 17 (depends on 16) The method of claim 16, wherein the first and second droplets each have a volume of about 300 nL. ### Claim 18 (depends on 1) The method of claim 1, wherein the preparing of step (a) comprises: (i) preparing the aqueous solution, wherein the aqueous solution comprises an amphiphilic molecule or mixture of amphiphilic molecules; (ii) attaching the first droplet of the aqueous solution to a tip of the first electrode and attaching the second droplet of the aqueous solution to a tip of the second electrode; (iii) suspending the first and second droplets in the hydrophobic liquid medium; and (iv) moving the first and/or second electrode to position the first and second droplets relative to one another to form an interface between the first and second droplets. ### Claim 19 (depends on 18) The method of claim 18, wherein the amphiphilic molecule or mixture of amphiphilic molecules comprises an amphiphilic polymer, optionally an ABA block copolymer, and wherein step (iv) further comprises applying a voltage. ### Claim 20 (depends on 1) The method of claim 1, wherein the determining of a nominal capacitance and an artificial cellular membrane area at each of a plurality of artificial cellular membrane areas in step (b) comprises: (v) moving the first and/or second electrode to change the relative position of the first and second droplets and thus the artificial cellular membrane area; (vi) waiting for the nominal capacitance to stabilize, optionally wherein the waiting is for between about 30 seconds and about 60 seconds; (vii) recording the nominal capacitance and acquiring an optical image of the artificial cellular membrane; (viii) calculating the area of the artificial cellular membrane using data from the optical image; and (ix) repeating steps (v)-(viii) one or more times, optionally four times, prior to calculating the specific capacitance. ### Claim 21 (depends on 1) The method of claim 1, wherein the contact angle between droplets at each of the plurality of applied bias voltages is measured using an optical image of the droplets taken during the application of each of the applied bias voltages. ### Claim 22 (depends on 1) The method of claim 1, wherein calculating the monolayer tension comprises determining the slope of a linear least squares regression of a plot of the change in the cosine of the contact angle as a function of voltage squared and using the slope and the specific capacitance determined in step (b) to determine monolayer tension using the Young-Lippmann equation. ### Claim 23 (depends on 1) The method of claim 1, wherein calculating the bilayer tension comprises using the Young-Dupre equation. ### Claim 24 A method of determining the effect of a change in the chemical composition of an artificial cellular membrane, wherein the method comprises: (a) preparing an artificial cellular membrane at an interface between a first droplet and a second droplet of a first aqueous solution wherein the first aqueous solution comprises an amphiphilic molecule or mixture of amphiphilic molecules, and wherein the first droplet and the second droplet are each attached to separate electrodes and suspended in a hydrophobic liquid medium; (b) determining specific capacitance of the artificial cellular membrane, wherein the determining comprises determining a nominal capacitance and an artificial cellular membrane area at each of a plurality of artificial cellular membrane areas, wherein artificial cellular membrane area is varied by changing the position of the first or second droplet relative to the other of the first and second droplets, and optionally wherein the specific capacitance is calculated from the slope of a linear least squares regression of the nominal capacitance versus area data; (c) determining an artificial cellular membrane tension, wherein the determining comprises fixing the positions of the first and the second droplets relative to each other, measuring the contact angle of the droplets at a plurality of applied bias voltages, and calculating the monolayer and bilayer tension; (d) repeating steps (b)-(c) for an artificial membrane formed at the interface of a first and a second droplet of a second aqueous solution, wherein the second aqueous solution comprises an amphiphilic molecule or molecules, and wherein the chemical composition of the second aqueous solution differs from the chemical composition of first aqueous solution, and wherein the first and the second droplet of the second aqueous solution are each attached to separate electrodes and suspended in a hydrophobic liquid medium; and (e) comparing the data obtained in for the artificial cellular membrane formed with droplets of the first aqueous solution to that obtained for the artificial membrane formed with droplets of the second aqueous solution, thereby determining the effect of a change in chemical composition of the artificial cellular membrane. ### Claim 25 (depends on 24) The method of claim 24, wherein the second aqueous solution comprises a small molecule, peptide, protein, biomolecule, or pharmaceutically active agent that may or may not be present in the first aqueous solution, and wherein the method determines the effect of the small molecule, peptide, protein, biomolecule, or pharmaceutically active agent on the artificial cellular membrane. ### Claim 26 (depends on 25) The method of claim 25, wherein the second aqueous solution comprises a small molecule or pharmaceutically active agent, wherein the pharmaceutically active agent is an anesthetic or model anesthetic, optionally lidocaine or ethanol.