WO2011043008A1 - 人工脂質膜形成方法 - Google Patents
人工脂質膜形成方法 Download PDFInfo
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- WO2011043008A1 WO2011043008A1 PCT/JP2010/002348 JP2010002348W WO2011043008A1 WO 2011043008 A1 WO2011043008 A1 WO 2011043008A1 JP 2010002348 W JP2010002348 W JP 2010002348W WO 2011043008 A1 WO2011043008 A1 WO 2011043008A1
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- electrolytic solution
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- artificial lipid
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- electrolyte
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/5432—Liposomes or microcapsules
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
Definitions
- the present invention relates to a method for forming an artificial lipid membrane used for biosensing or membrane protein analysis.
- Patent Documents 1 to 3 disclose biosensors that use the excellent molecular recognition function of receptors.
- the biosensor includes an artificial lipid membrane having a receptor and an ion channel.
- Non-Patent Document 1 Examples of conventional methods for forming artificial lipid membranes are (1) bubble spraying method, (2) bonding method, and (3) ⁇ TAS (Micro Total Analysis System) (for example, Non-Patent Document 1).
- FIG. 20 shows a conventional method for forming an artificial lipid membrane by a bubble spraying method.
- the inside of the container 10 is partitioned by a flat plate 11 made of a hydrophobic resin such as Teflon (registered trademark) or polystyrene.
- the space partitioned by the flat plate 11 is filled with the electrolytic solution 12.
- a lipid solution 14 containing lipid molecules and an organic solvent is applied with a pipette 15 to the micropores 13 formed in the flat plate 11. Excess organic solvent contained in the lipid solution 14 applied to the micropores 13 is gradually transferred along the peripheral edge of the micropores 13 and removed.
- An artificial lipid film is formed in about 30 minutes to 3 hours after application.
- lipid is a phospholipid such as diphytanoyl phosphatidylcholine or glycerin monooleate.
- organic solvents are saturated hydrocarbons such as decane, hexadecane or hexane.
- FIG. 21A the inside of the container 20 is partitioned by a flat plate 21 having a hydrophobic surface.
- the flat plate 21 is made of a resin such as Teflon (registered trademark) or polystyrene.
- squalene is applied to the micro holes 22 formed in the flat plate 21 as a pretreatment.
- the electrolyte solution 23 is injected into the one chamber of the container 20 from the injection port 24 so that the height of the electrolyte solution 23 does not exceed the height of the lower end of the microhole 22.
- a lipid solution (mixed solution of lipid molecules 25 and an organic solvent) is dropped onto the electrolytic solution 23 from above the container 20 and left for several minutes.
- a lipid monomolecular film is formed at the gas-liquid interface of the electrolytic solution 23.
- the lipid molecule 25 has a hydrophilic portion and a hydrophobic portion, and the hydrophilic portion of the lipid molecule 25 is oriented so as to face toward the electrolytic solution 23.
- the electrolytic solution 23 is injected from the injection port 24 until the liquid level of the electrolytic solution 23 passes through the height of the upper end of the minute hole 22.
- the electrolytic solution 26 is injected from the injection port 27 so that the height of the liquid level does not exceed the height of the lower end of the microhole 22.
- a lipid solution is dropped onto the electrolyte solution 26 from above the container 20 and left for several minutes.
- a lipid monomolecular film is formed at the gas-liquid interface of the electrolyte solution 26.
- the electrolyte solution 26 is injected from the injection port 27 until the level of the electrolyte solution 26 passes through the height of the upper end of the minute hole 22.
- the other lipid monomolecular film is bonded to the lipid monomolecular film previously formed in the micropores 22.
- an artificial lipid film is formed in the micropores 22.
- Patent Documents 1 to 4 disclose a method of forming an artificial lipid membrane using ⁇ TAS technology in order to form a simpler artificial lipid membrane.
- FIG. 22 shows a small artificial lipid membrane forming apparatus using the ⁇ TAS technique described in Patent Document 1.
- the artificial lipid film forming apparatus shown in FIG. 22 includes a first chamber 31 and a second chamber 33 separated from the first chamber 31 by a partition wall 32.
- the partition wall 32 includes at least one small hole 34 that fluidly communicates the first chamber 31 and the second chamber 33.
- an artificial lipid film is formed as follows. First, the first chamber 31 is filled with the first aqueous solution, and then the second chamber 33 is filled with the lipid solution. The first aqueous solution contacts the lipid solution through the small holes 34. Further, the lipid solution filled in the second chamber 33 is replaced with the second aqueous solution, whereby an artificial lipid film 35 is formed in the small hole 34.
- Japanese Patent Laying-Open No. 2005-098718 page 15, FIG. 5
- Japanese Patent Laid-Open No. 5-007770 page 3, FIG. 1
- JP-A-8-152423 page 3, FIG. 1
- Japanese Patent Laid-Open No. 4-215052 5th page, FIG. 1
- the artificial lipid film forming apparatus disclosed in Patent Document 1 has a very excellent convenience because it is small and easy to carry.
- the artificial lipid membrane forming device is vibrated for carrying, or the artificial lipid membrane forming device is tilted or turned over to From the inlet / outlet opening may leak out of the chamber.
- the periphery of the artificial lipid film forming apparatus is contaminated by the electrolytic solution.
- a small artificial lipid membrane forming apparatus holds a very small amount of electrolyte, the electrolyte is rapidly evaporated and an artificial lipid membrane cannot be formed stably.
- the object of the present invention is to solve the above-mentioned conventional problems and provide a method for stably forming an artificial lipid membrane by preventing leakage of the electrolyte to the outside of the chamber and rapid evaporation of the electrolyte. That is.
- the present invention An artificial lipid film forming method comprising the following steps A to E: Step A for preparing the following artificial lipid film forming apparatus (100)
- the artificial lipid film forming apparatus (100) includes a first chamber (104), A second chamber (105); A partition wall (102) sandwiched between the first chamber (104) and the second chamber (105); An artificial lipid membrane forming part (103) comprising a through-hole provided in the partition wall (102),
- the first chamber (104) has a volume of 10 pl to 200 ⁇ l
- the second chamber (105) has a volume of 10 pl to 200 ⁇ l
- a second electrolytic solution (204) having a viscosity of
- At least one of the first electrolytic solution (201) or the second electrolytic solution (204) contains an organic compound having a hydroxyl group.
- the organic compound having a hydroxyl group is preferably an alcohol.
- the alcohol is preferably a lower alcohol.
- the alcohol is preferably glycerin.
- At least one of the first electrolytic solution (201) or the second electrolytic solution (204) contains a polymer.
- the polymer is preferably polyvinyl alcohol.
- the first electrolyte solution (201) is preferably injected into the first chamber (104) by an ink jet method.
- the second electrolytic solution (204) is preferably injected into the second chamber (105) by an ink jet method.
- the lipid solution (202) is preferably injected into the artificial lipid film forming part (103) by an ink jet method.
- the present invention preferably further comprises a step F of embedding at least one of a receptor or an ion channel in the artificial lipid membrane after the step E.
- the first chamber (104) is preferably filled with the first electrolyte (201).
- the second chamber (105) is preferably filled with the second electrolytic solution (204).
- the artificial lipid membrane forming method of the present invention it is possible to prevent the electrolyte from leaking out of the chamber by increasing the viscosity of the electrolyte while maintaining fluidity. As a result, contamination around the device due to the electrolyte can be prevented. Furthermore, since rapid evaporation of the electrolyte can be prevented, an artificial lipid membrane can be stably formed.
- FIG. 1 is an oblique projection of the artificial lipid film forming apparatus according to the first embodiment.
- FIG. 2 is a cross-sectional view of the artificial lipid film forming apparatus of the first embodiment.
- FIG. 3 shows a cross-section of a through hole that is an example of the artificial lipid film forming part of the first embodiment.
- FIG. 4 shows a first electrolyte solution injection step of the first embodiment.
- FIG. 5 shows processes from the lipid injection process to the artificial lipid film formation process of the first embodiment.
- FIG. 6 shows a state in which the artificial lipid film forming apparatus of Embodiment 1 is tilted.
- FIG. 7 shows a state where the artificial lipid film forming apparatus of Embodiment 1 is turned upside down.
- FIG. 1 is an oblique projection of the artificial lipid film forming apparatus according to the first embodiment.
- FIG. 2 is a cross-sectional view of the artificial lipid film forming apparatus of the first embodiment.
- FIG. 3 shows a
- FIG. 8 shows a state where the artificial lipid film forming apparatus of Embodiment 1 is carried in the hand of a handler.
- FIG. 9 shows a cross-sectional view of the artificial lipid film forming apparatus of the second embodiment.
- FIG. 10 is an exploded perspective view of the artificial lipid film forming apparatus according to the second embodiment.
- FIG. 11 shows processes from the first electrolyte injection process to the second electrolyte injection process of the second embodiment.
- FIG. 12 shows an artificial lipid film formation step of the second embodiment.
- FIG. 13 shows a state in which the artificial lipid film forming apparatus of Embodiment 2 is tilted.
- FIG. 14 shows a state where the artificial lipid film forming apparatus of Embodiment 2 is turned upside down.
- FIG. 15 shows a state where the artificial lipid film forming apparatus of Embodiment 2 is carried in the hand of a handler.
- FIG. 16 schematically shows a state in which a membrane protein is embedded in an artificial lipid membrane in the second embodiment.
- FIG. 17 shows the relationship between the glycerin concentration and the viscosity of the electrolytic solution.
- FIG. 18 shows the relationship between the PVA concentration and the viscosity of the electrolytic solution.
- FIG. 19 shows a photomicrograph of the electrolyte in the first chamber in the second embodiment.
- FIG. 20 shows a conventional artificial lipid film forming method (bubble spraying method).
- FIG. 21 shows a conventional artificial lipid film forming method (bonding method).
- FIG. 22 shows an artificial lipid film forming apparatus of Patent Document 1.
- Preparation process> 1 and 2 show an oblique projection and a cross-sectional view of the artificial lipid film forming apparatus 100 according to Embodiment 1 of the present invention, respectively.
- the artificial lipid film forming apparatus 100 includes a container 101.
- An example of the material of the container 101 is an organic material or an inorganic material. Organic materials are preferred.
- the organic material may be a thermoplastic resin or a thermosetting resin.
- the organic material may be general purpose plastic, engineering plastic or super engineering plastic. Examples of organic materials are phenol resin, melamine resin, epoxy resin, polyester resin, polyurethane resin, polyimide resin, polyethylene, polycarbonate, polyvinyl acetate, ABS (acrylonitrile butadiene styrene) resin, acrylic, polyethylene terephthalate, vinyl chloride, polypropylene, Polystyrene, polysulfone, PEEK (registered trademark), polyacetal, cyclic polyolefin, polyphenylene sulfide, polytetrafluoroethylene or polyamideimide.
- the organic material may be a composite resin.
- glass is preferable. Soda glass, quartz, borosilicate glass, low melting point glass, photosensitive glass can be used.
- silicon, germanium, indium phosphide, gallium arsenide, gallium nitride, aluminum oxide, silicon oxide, or silicon nitride can be used.
- the material of the container 101 may be a material combining a plurality of organic materials or inorganic materials.
- the material of the container 101 is preferably insulative regardless of whether it is an organic material or an inorganic material.
- At least a part of the outer peripheral surface of the container 101 has hydrophilicity.
- oxygen plasma treatment may be performed, or it may be coated with a hydrophilic material.
- the material of the container 101 is preferably transparent so that the artificial lipid film can be observed, but may be opaque.
- the capacity of the container 101 is preferably 2 pl or more and 2 ml or less from the viewpoint of operability.
- the volume of the container 101 is more preferably 1 nl or more and 400 ⁇ l or less.
- the container 101 is preferably a rectangular parallelepiped, but may be cylindrical or polygonal.
- the container 101 may be a flow path or a chamber.
- the container 101 is preferably molded by machining. As the machining, injection molding, extrusion molding, compression molding, hollow molding, cutting, molding, sand blasting, dry etching, wet etching, nanoimprinting, milling, photocuring, lithography or hot embossing is preferable.
- the container 101 is also preferably processed by a semiconductor process.
- the partition wall 102 is provided inside the container 101.
- the partition wall 102 is preferably provided so as to separate the container 101 into at least two chambers.
- the partition wall 102 is preferably provided at the center of the container 101, but may be provided at the end of the container 101.
- any material that can be used as the material of the container 101 can be used as the material of the partition wall 102.
- a part of the surface of the partition wall 102 may be covered with a thin film made of a material different from the material of the partition wall 102.
- the thickness of the thin film covering the surface of the partition wall 102 is preferably 10 nm or more and 100 ⁇ m or less.
- a part of the surface of the partition wall 102 may be covered with a thin film made of a self-assembled film (SAM film) or a water-repellent material.
- SAM film self-assembled film
- the material of the partition wall 102 is preferably insulative regardless of whether it is an organic material or an inorganic material.
- the electrical resistivity of the material of the partition wall 102 is preferably 10 10 ⁇ cm or more, and more preferably 10 12 ⁇ cm or more.
- the relative dielectric constant of the material of the partition wall 102 is preferably 2.0 or more and 50.0 or less, and more preferably 2.0 or more and 15.0 or less.
- the surface of the partition wall 102 is preferably water repellent.
- the contact angle of the surface of the partition wall 102 is preferably 90 ° or more, and more preferably 120 ° or more and 150 ° or less.
- the partition 102 is most preferably plate-shaped, but may be film-shaped.
- the thickness of the partition wall 102 is preferably 10 nm or more and 1 mm or less, and more preferably 30 ⁇ m or more and 500 ⁇ m or less.
- the thickness of the partition wall 102 may be the same or different over the entire surface.
- Area of the partition wall 102 is preferably 1 [mu] m 2 or more 100 cm 2 or less, more preferably 100 [mu] m 2 or more 1 cm 2 or less.
- the partition wall 102 is preferably molded by machining. As the machining, injection molding, extrusion molding, compression molding, hollow molding, cutting, solution casting, stretching, molding, sandblasting, dry etching, wet etching, nanoimprinting, milling, photocuring, lithography or hot embossing are preferable.
- the partition wall 102 is also preferably processed by a semiconductor process.
- partition wall 102 Only one partition wall 102 may be provided inside the container 101, or two or more partition walls 102 may be provided.
- the artificial lipid film forming part 103 is provided in the central part of the partition wall 102.
- the artificial lipid film forming unit 103 may be provided at the end of the partition wall 102.
- the artificial lipid membrane forming part 103 is most preferably a through-hole provided in the partition wall 102.
- the cross section of the through hole is preferably circular.
- FIG. 3 shows a cross-section of the through-hole as the artificial lipid membrane forming part 103 and viewed from the normal direction of the partition wall 102.
- FIG. 3A shows that the cross-section of the through hole as the artificial lipid membrane forming unit 103 is circular. Since the force applied to the artificial lipid membrane is evenly dispersed, the through hole preferably has a circular cross section.
- the cross section of the through hole may be oval, polygonal, trapezoidal or square as shown in FIGS.
- the through hole is more preferably tapered as shown in FIG.
- the diameter of the artificial lipid membrane forming portion 103 is preferably 10 nm or more and 1 mm or less, and more preferably 50 nm or more and 200 ⁇ m or less.
- the area of the artificial lipid membrane forming part 103 that is, the area of the graphic shown in FIGS. 3A to 3E is preferably 75 nm 2 or more and 0.75 mm 2 or less.
- the inner wall of the artificial lipid film forming unit 103 is preferably smooth, but may have a concavo-convex structure or a groove structure from the viewpoint of stabilizing the artificial lipid film.
- the artificial lipid film forming unit 103 can be molded in the same manner as the partition wall 102.
- One artificial lipid film forming unit 103 may be provided in the partition wall 102, or a plurality of artificial lipid film forming units 103 may be provided. It is preferable that the plurality of artificial lipid film forming units 103 are two-dimensionally arranged in an array.
- the plurality of artificial lipid film forming units 103 are preferably arranged in a square lattice, an orthorhombic lattice, a hexagonal lattice, a simple rectangular lattice, or a face-centered rectangular lattice.
- the shapes of the plurality of artificial lipid film forming units 103 may all be the same or different.
- the areas of the plurality of artificial lipid film forming units 103 may all be the same or different.
- the first chamber 104 is provided at one end of the container 101.
- the first chamber 104 is preferably provided between the inner wall of the container 101 and the partition wall 102, and is most preferably formed by the inner wall of the container 101 and the partition wall 102.
- the volume of the first chamber 104 is preferably 1 pl or more and 1 ml or less, more preferably 10 pl or more and 200 ⁇ l or less from the viewpoint of operability.
- the first chamber 104 preferably includes an inlet for injecting an electrolytic solution.
- the first chamber 104 preferably includes a discharge port for discharging the electrolytic solution.
- the first chamber 104 may be connected to the electrolyte reservoir via a flow path.
- the capacity of the electrolyte reservoir may or may not be included in the capacity of the first chamber 104.
- a lid or a stopper may be provided at the opening of the first chamber 104, or a film may be attached to the opening of the first chamber 104.
- the second chamber 105 is provided on the opposite side of the first chamber 104 through the partition wall 102.
- the second chamber 105 is preferably provided between the inner wall of the container 101 and the partition wall 102, and is most preferably formed by the inner wall of the container 101 and the partition wall 102.
- the volume of the second chamber 105 is preferably 1 pl or more and 1 ml or less, and more preferably 10 pL or more and 200 ⁇ l or less from the viewpoint of operability.
- the volume of the second chamber 105 may be the same as or different from the volume of the first chamber 104.
- the second chamber 105 preferably includes an inlet for injecting an electrolytic solution.
- the second chamber 105 preferably includes a discharge port for discharging the electrolytic solution.
- the second chamber 105 may be connected to the electrolyte reservoir through a flow path.
- the capacity of the electrolytic solution reservoir may or may not be included in the capacity of the second chamber 105.
- a lid or a stopper may be provided at the opening of the second chamber 105, or a film may be attached to the opening of the second chamber 105.
- FIGS. 1 and 2 show an artificial lipid film forming method according to Embodiment 1 of the present invention. 4 and 5, the same components as those in FIGS. 1 and 2 are denoted by the same reference numerals, and the description thereof is omitted.
- FIG. 4 shows a first electrolyte solution injection step.
- the first electrolyte solution 201 is injected into the first chamber 104 from the first opening 106 to fill the first chamber 104 with the first electrolyte solution 201. It is preferable that the first electrolytic solution 201 does not move from the first chamber 104 to the second chamber 105 through the artificial lipid film forming unit 103.
- the first electrolytic solution 201 preferably contains KCl, and more preferably isotonic KCl solution.
- the first electrolytic solution 201 is preferably the same as the physiological condition in the cell.
- the pH of the first electrolytic solution 201 is preferably around 7.
- the first electrolytic solution 201 may be a buffer solution such as HEPES, phosphate buffer (PBS), or phosphate buffered saline, or may be a general solution used in electrophysiological experiments.
- the Ca 2+ concentration of the first electrolytic solution 201 is preferably 10 to 100 nM. To adjust the Ca 2+ concentration, a Ca 2+ chelator such as EGTA may be used.
- the first electrolytic solution 201 preferably contains a Tyrode solution.
- the first electrolytic solution 201 preferably contains NaCl 137 mM, KCl 2.68 mM, CaCl 2 1.8 mM, NaH 2 PO 4 0.32 mM, Glucose 5.56 mM, and NaHCO 3 1.16 mM.
- the first electrolytic solution 201 may contain NaCl 140 mM, KCl 5.4 mM, CaCl 2 1.8 mM, MgCl 2 1 mM, NaHPO 4 0.3 mM, Glucose 5 mM, HEPES 5 mM (pH 7.4).
- the first electrolytic solution 201 may contain KCl 140 mM, MgCl 2 1 mM, CaCl 2 1 mM, EGTA 10 mM, Mg-ATP 2 mM, NaOH-HEPES 10 mM (pH 7.3).
- the Cl 2 ⁇ in the first electrolytic solution 201 is preferably replaced with SO 4 2 ⁇ , methanesulfonate, gluconate, glutamate, or aspartate which is a membrane-impermeable anion.
- the first electrolytic solution 201 is preferably stored frozen at ⁇ 20 ° C. so that microorganisms do not propagate. It is preferable that the cation in the first electrolytic solution 201 is replaced with a membrane-impermeable organic base. It is preferable that the cation in the first electrolytic solution 201 is replaced with tetraethylammonium or N-methyl-D-glucamine.
- EGTA contained in the first electrolytic solution 201 is preferably replaced with BAPTA.
- the first electrolytic solution 201 may contain ATP. In order to maintain the function of the receptor, the first electrolytic solution 201 may contain 0.1 to 0.3 mM GTP.
- the viscosity of the first electrolytic solution 201 is preferably 1.3 mPa ⁇ s or more and 200 mPa ⁇ s or less.
- the first electrolytic solution 201 preferably has fluidity from the viewpoint of reducing the voltage drop and increasing the ionic conductivity.
- the first electrolytic solution 201 is preferably liquid or semi-liquid.
- the viscosity of the first electrolytic solution 201 is adjusted with a water-soluble substance. It is preferable that the viscosity of the 1st electrolyte solution 201 is adjusted with a thickener.
- the viscosity of the first electrolyte 201 may be adjusted by an organic compound having a hydrophilic functional group such as a hydroxyl group, a carboxyl group, an amino group, or a sulfone group.
- an organic compound having 1 to 10 carbon atoms is preferable, and an organic compound having 1 to 5 carbon atoms is more preferable.
- the viscosity of the first electrolytic solution 201 is preferably adjusted with alcohol.
- the alcohol may be a monohydric alcohol or a polyhydric alcohol.
- the alcohol is preferably a lower alcohol such as glycerin.
- the viscosity of the first electrolyte 201 is adjusted with a sugar or sugar alcohol such as isopropyl alcohol, ethylene glycol, sorbitol, xylitol, dipropylene glycol, butylene glycol, polyethylene glycol, polyoxyethylene methyl glucoside, maltitol, mannitol or glucose. May be.
- a sugar or sugar alcohol such as isopropyl alcohol, ethylene glycol, sorbitol, xylitol, dipropylene glycol, butylene glycol, polyethylene glycol, polyoxyethylene methyl glucoside, maltitol, mannitol or glucose. May be.
- sugars monosaccharides, disaccharides, trisaccharides, tetrasaccharides or polysaccharides can be used.
- the first electrolytic solution 201 preferably has a viscosity higher than that of pure water at 20 ° C. (1.0 mPa ⁇ s).
- the viscosity of the first electrolytic solution 201 may be adjusted by a polymer.
- the viscosity of the first electrolytic solution 201 may be adjusted by a polymer having a hydrophilic functional group such as a hydroxyl group, a carboxyl group, an amino group, or a sulfone group.
- Polyvinyl alcohol is preferred as the polymer, but polyacrylamide or 2-hydroxyethyl methacrylate (HEMA) can also be used.
- the polymer may be either a homopolymer or a copolymer.
- the polymer is preferably a synthetic polymer, but may be a semi-synthetic polymer or a natural polymer.
- Polymers include gum arabic, carboxyvinyl polymer, sodium alginate, propylene glycol alginate, ethyl cellulose, sodium carboxymethyl cellulose, chitansan gum, synthetic sodium silicate, synthetic magnesium silicate, dimethyl distearyl ammonium hectorite, cyclodextrin, polyacrylic acid Sodium, gelatin, casein, collagen, hyaluronic acid, albumin, pectin, tamarind gum, guar gum, carrageenan or carob bean gum may be used.
- the substance that adjusts the viscosity of the first electrolytic solution 201 is preferably a substance that stabilizes a membrane protein such as an artificial lipid film.
- the concentration of the substance that adjusts the viscosity of the first electrolytic solution 201 is preferably 1% or more and 99% or less, and more preferably 1% or more and 50% or less from the viewpoint of ease of injection.
- the concentration of the substance that adjusts the viscosity of the first electrolytic solution 201 is preferably 0.087 w / w% or more and 20 w / w% or less from the viewpoint of ease of injection, and is 0.087 w / w% or more and 12 w / w. More preferably, it is w% or less.
- the glycerin concentration in the first electrolytic solution 201 is preferably 1% or more and 99% or less, and more preferably 1% or more and 50% or less.
- the PVA concentration in the first electrolytic solution 201 is preferably 0.087 w / w% or more and 12 w / w% or less. In the present specification, when simply expressed as%, it represents a volume concentration. When indicated as w / w%, it represents the weight concentration.
- the electrical resistivity of the first electrolytic solution 201 is preferably 1 ⁇ m or more and 100 k ⁇ m or less, and more preferably 1 m ⁇ m or more and 10 ⁇ m or less from the viewpoint of reducing the voltage drop.
- the first electrolytic solution 201 is preferably transparent from the viewpoint of observing the artificial lipid membrane, but may be opaque.
- the amount of the first electrolytic solution 201 injected into the first chamber 104 is preferably 10 pl to 200 ⁇ l, and more preferably 1 nl to 200 ⁇ l.
- the first electrolytic solution 201 is most preferably stationary, but may be flowing. When the first electrolytic solution 201 flows, it is preferable that the amount of the first electrolytic solution 201 that substantially participates in the formation of the artificial lipid membrane is within the above capacity range. After injecting the first electrolytic solution 201 into the first chamber 104, the amount of the first electrolytic solution 201 in the first chamber 104 may be adjusted by discharging the excess first electrolytic solution 201.
- the first electrolytic solution 201 is preferably injected into the first chamber 104 using a pipette, but may be injected into the first chamber 104 using a tube, a flow path, a dropper, or a syringe.
- the first electrolyte 201 may be continuously injected into the first chamber 104 or may be intermittently injected.
- the first electrolytic solution 201 may be injected into the first chamber 104 in the form of droplets.
- a method for injecting the droplet-shaped first electrolytic solution 201 an inkjet method, an electrostatic spray method, an ultrasonic method, a dot impact method, or a fine droplet coating method can be used.
- the ink jet method is a method in which a liquid is made into fine droplets and injected into a target position.
- the microdroplet coating method is a method of injecting a liquid filled in a capillary by filling the liquid in a capillary whose tip is narrowed and moving a needle inserted into the capillary tube.
- the inkjet method is most preferably a piezo method, but may be a thermal method.
- the microdroplet application method is a method in which a needle is provided inside a capillary having an opening at the tip, and a liquid filled in the capillary is applied to an object by moving the needle.
- the first electrolytic solution 201 is injected into the first chamber 104 manually, semi-manually or automatically.
- the injection time of the first electrolytic solution 201 is preferably 1 microsecond or more and 10 seconds or less, and more preferably 1 microsecond or more and 1 second or less.
- the injection rate of the first electrolytic solution 201 may be constant or may change in the first electrolytic solution injection step.
- the first electrolytic solution 201 is preferably maintained at room temperature in the first electrolytic solution injection step.
- the first electrolyte 201 is preferably maintained at 0 ° C. or higher and 40 ° C. or lower, and more preferably maintained at 10 ° C. or higher and 30 ° C. or lower.
- the relative humidity around the artificial lipid film forming apparatus 100 is preferably maintained at 50% or more and 100% or less.
- the first electrolyte solution 201 is preferably injected into the first chamber 104 by capillary force, gravity, surface tension, or centrifugal force.
- the first electrolyte solution injection step it may be detected that the first electrolyte solution 201 has been injected into the first chamber 104.
- the completion of injection may be detected by observation with an optical microscope, or may be detected by providing a plurality of electrodes in the first chamber 104 and measuring electrical conductivity.
- the first electrolyte solution injection step the first electrolyte solution 201 is preferably injected into the first chamber 104 until the upper end of the artificial lipid film forming unit 103 is exceeded.
- the first electrolytic solution 201 is most preferably a uniform electrolytic solution having a single viscosity.
- the first electrolytic solution 201 may be an electrolytic solution obtained by combining a plurality of electrolytic solutions having a viscosity of 1.3 to 200 mPa ⁇ s.
- the viscosity of the first electrolytic solution 201 may have a gradient.
- the viscosity gradient of the first electrolytic solution 201 may be continuous or discontinuous.
- the first electrolytic solution 201 is in a very small amount, if the viscosity of the first electrolytic solution 201 is insufficient, a lid or a stopper is provided at the opening of the first chamber 104 or the second chamber 105 or a film is applied.
- the first electrolyte 201 may evaporate. For this reason, attention is required for operation.
- FIG. 5 (a) shows a lipid solution injection step.
- the lipid solution 202 is injected into the artificial lipid film forming unit 103.
- the lipid solution 202 is preferably a solution in which the lipid 203 is dispersed in an organic solvent.
- the lipid 203 is preferably a complex lipid containing phosphoric acid or sugar in the molecule.
- Lipid 203 may contain simple lipids or derived lipids.
- the lipid 203 is most preferably a phospholipid, but may be a glycolipid, a lipolipid, a sulfolipid, a sphingophospholipid, a glycerophospholipid, an azolectin, or other naturally derived lipid, or a synthetic lipid. .
- Synthetic lipids are preferable to naturally derived lipids because they are easy to obtain highly pure and chemically stable reagents.
- lipid 203 diphytanoyl phosphatidylcholine, glycerin monooleate, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or dipalmitoylphosphatidylcholine may be used.
- the fatty acid portion of the lipid 203 is preferably a saturated or unsaturated fatty acid having 10 to 20 carbon atoms.
- one type of lipid may be used, or a lipid in which two or more types of lipids are mixed may be used.
- the organic solvent contained in the lipid solution 202 is preferably a saturated hydrocarbon such as decane, hexadecane, hexane, or chloroform.
- the concentration of lipid 203 relative to the organic solvent is preferably 1 to 50 mg / ml, more preferably 4 to 40 mg / ml.
- the lipid solution 202 may contain phosphatidylserine or phosphatidylinositol in order to give the artificial lipid membrane a net surface charge.
- the surface charge of the artificial lipid membrane is preferably negative.
- the phosphatidylserine or phosphatidylinositol may be mixed with the lipid solution 202 in advance before the lipid solution injection step, or may be mixed with the lipid solution 202 after the artificial lipid film formation step.
- the lipid solution 202 preferably contains a biological membrane protein or secreted protein such as a receptor, an ion channel or a G protein in addition to the lipid 203 and the organic solvent.
- the lipid solution 202 may contain a polypeptide such as gramicidin.
- One kind of biological membrane protein, secreted protein or polypeptide may be contained in the lipid solution 202, or a plurality of kinds of them may be contained in the lipid solution 202.
- the biological membrane protein, secreted protein or polypeptide may be introduced into the artificial lipid membrane by being mixed with the lipid solution 202 in advance before the lipid solution injecting step, and may be introduced into the artificial lipid membrane after the artificial lipid membrane forming step. May be introduced.
- a biological membrane protein, secreted protein or polypeptide is introduced into the artificial lipid membrane after the artificial lipid membrane formation step, the biological membrane protein, secreted protein or polypeptide is once incorporated into the vesicle and the vesicle is fused to the artificial lipid membrane.
- a known mixing technique may be used.
- a biological membrane protein, secreted protein or polypeptide is introduced into the artificial lipid membrane after the artificial lipid membrane formation step, a mechanism for mixing them may be provided in the artificial lipid membrane formation apparatus 100.
- the amount of the lipid solution 202 injected into the artificial lipid film forming unit 103 is preferably 1 pl or more and 10 ⁇ l or less, and more preferably 1 nl or more and 2 ⁇ l or less from the viewpoint of ease of producing the artificial lipid film.
- the lipid solution 202 is preferably injected by a pipette, but may be injected by a tube, a channel, a dropper or a syringe.
- the lipid solution 202 may be continuously injected or intermittently injected.
- the lipid solution 202 may be injected in the form of droplets.
- the lipid solution 202 may be injected into the artificial lipid film forming unit 103 by an inkjet method, a fine droplet coating method, a dot impact method, an electrostatic spray method, or an ultrasonic method.
- the inkjet method is most preferably a piezo method, but may be a thermal method.
- the lipid solution 202 is injected into the artificial lipid film forming unit 103 manually, semi-manually or automatically.
- the injection time of the lipid solution 202 is preferably 1 microsecond or more and 10 seconds or less, and more preferably 1 microsecond or more and 1 second or less.
- the injection speed of the lipid solution 202 may be constant or may change in the lipid solution injection process.
- the lipid solution 202 is preferably injected into the artificial lipid film forming unit 103 by capillary force, gravity, surface tension, or centrifugal force.
- the lipid solution injection step it may be detected that the lipid solution 202 has been injected into the artificial lipid film forming unit 103.
- the completion of injection may be detected by observation with an optical microscope, or may be detected by providing a plurality of electrodes on the partition wall 102 and measuring electrical conductivity.
- the first electrolytic solution 201 Since the first electrolytic solution 201 is in a very small amount, if the viscosity of the first electrolytic solution 201 is insufficient, the first electrolytic solution 201 evaporates while the lipid solution is being injected into the artificial lipid film forming unit 103 using a pipette. May end up. For this reason, attention is required for operation.
- FIG. 5B shows a second electrolyte solution injection step.
- the second electrolyte solution 204 is injected into the second chamber 105 through the second opening 107.
- the second electrolytic solution 204 preferably contains KCl, and more preferably isotonic KCl solution. It is preferable that the 2nd electrolyte solution 204 is the same as the physiological condition in a cell.
- the pH of the second electrolyte solution 204 is preferably around 7.
- the second electrolytic solution 204 may be a buffer solution such as HEPES, phosphate buffer (PBS), or phosphate buffered saline, or may be a general solution used in electrophysiological experiments.
- the Ca 2+ concentration of the second electrolytic solution 204 is preferably 10 to 100 nM. To adjust the Ca 2+ concentration, a Ca 2+ chelator such as EGTA may be used.
- the second electrolytic solution 204 preferably contains a Tyrode solution.
- the second electrolyte solution 204 preferably contains NaCl 137 mM, KCl 2.68 mM, CaCl 2 1.8 mM, NaH 2 PO 4 0.32 mM, Glucose 5.56 mM, and NaHCO 3 1.16 mM.
- the second electrolyte solution 204 may contain NaCl 140 mM, KCl 5.4 mM, CaCl 2 1.8 mM, MgCl 2 1 mM, NaHPO 4 0.3 mM, Glucose 5 mM, HEPES 5 mM (pH 7.4).
- the second electrolytic solution 204 may contain KCl 140 mM, MgCl 2 1 mM, CaCl 2 1 mM, EGTA 10 mM, Mg-ATP 2 mM, NaOH-HEPES 10 mM (pH 7.3).
- Cl 2 ⁇ in the second electrolytic solution 204 is preferably replaced with SO 4 2 ⁇ , methanesulfonate, gluconate, glutamate, or aspartate which is a membrane-impermeable anion.
- the second electrolyte solution 204 is preferably stored frozen at ⁇ 20 ° C. so that microorganisms do not propagate. It is preferable that the cation in the second electrolytic solution 204 is replaced with a membrane-impermeable organic base. It is preferable that the cation in the second electrolytic solution 204 is replaced with tetraethylammonium or N-methyl-D-glucamine.
- EGTA contained in the second electrolyte solution 204 is preferably replaced with BAPTA.
- the second electrolytic solution 204 may contain ATP. In order to maintain the function of the receptor, the second electrolyte solution 204 may contain 0.1 to 0.3 mM GTP.
- the viscosity of the second electrolytic solution 204 is preferably 1.3 mPa ⁇ s or more and 200 mPa ⁇ s or less.
- the viscosity of the second electrolytic solution 204 is preferably equal to the viscosity of the first electrolytic solution 201, but may be different from the viscosity of the first electrolytic solution 201.
- the second electrolytic solution 204 preferably has fluidity from the viewpoint of reducing the voltage drop and increasing the ionic conductivity.
- the second electrolytic solution 204 is preferably liquid or semi-liquid.
- the viscosity of the second electrolytic solution 204 is adjusted in the same manner as the first electrolytic solution 201.
- the second electrolytic solution 204 preferably has a viscosity higher than that of pure water at 20 ° C. (1.0 mPa ⁇ s).
- the electrical resistivity of the second electrolytic solution 204 is preferably 1 ⁇ m or more and 100 k ⁇ m or less, and more preferably 1 m ⁇ m or more and 10 ⁇ m or less from the viewpoint of reducing the voltage drop.
- the second electrolytic solution 204 is preferably transparent from the viewpoint of observing the artificial lipid membrane, but may be opaque.
- the amount of the second electrolytic solution 204 injected into the second chamber 105 is preferably 10 pl or more and 200 ⁇ l or less, and more preferably 1 nl or more and 200 ⁇ l or less.
- the second electrolytic solution 204 is most preferably stationary, but may be flowing. When the second electrolytic solution 204 flows, it is preferable that the amount of the second electrolytic solution 204 substantially involved in the formation of the artificial lipid membrane is within the above capacity range. After injecting the second electrolytic solution 204 into the second chamber 105, the amount of the second electrolytic solution 204 in the second chamber 105 may be adjusted by discharging the excess second electrolytic solution 204.
- the second electrolytic solution 204 is preferably injected into the second chamber 105 with a pipette, but may be injected into the second chamber 105 with a tube, a flow path, a dropper, or a syringe.
- the second electrolyte solution 204 may be continuously injected into the second chamber 105 or may be intermittently injected.
- the second electrolyte solution 204 may be injected into the second chamber 105 in the form of droplets.
- an inkjet method, a microdroplet coating method, a dot impact method, an electrostatic spraying method, an ultrasonic method, or a microdroplet coating method can be used as a method for injecting the droplet-like second electrolytic solution 204.
- the inkjet method is most preferably a piezo method, but may be a thermal method.
- the second electrolytic solution 204 is injected manually, semi-manually or automatically.
- the injection time of the second electrolyte solution 204 is preferably 1 microsecond or more and 10 seconds or less, and more preferably 1 microsecond or more and 1 second or less.
- the injection rate of the second electrolyte solution 204 may be constant or may change in the second electrolyte solution injection step.
- the second electrolytic solution 204 is preferably maintained at room temperature in the second electrolytic solution injection step.
- the second electrolytic solution 204 is preferably maintained at 0 ° C. or higher and 40 ° C. or lower, and more preferably maintained at 10 ° C. or higher and 30 ° C. or lower.
- the relative humidity around the artificial lipid film forming apparatus 100 is preferably maintained at 50% or more and 100% or less.
- the second electrolyte solution 204 is preferably injected into the second chamber 105 by capillary force, gravity, surface tension, or centrifugal force.
- the second electrolyte solution injection step it may be detected that the second electrolyte solution 204 has been injected into the second chamber 105.
- the end of injection may be detected by observation with an optical microscope, or may be detected by providing a plurality of electrodes in the second chamber 105 and measuring electrical conductivity.
- the second electrolyte solution injection step the second electrolyte solution 204 is preferably injected into the second chamber 105.
- the second electrolytic solution 204 is most preferably a uniform electrolytic solution having a single viscosity.
- the second electrolytic solution 204 may be an electrolytic solution in which a plurality of electrolytic solutions having a viscosity of 1.3 to 200 mPa ⁇ s are combined.
- the viscosity of the second electrolyte solution 204 may have a gradient.
- the viscosity gradient of the second electrolyte solution 204 may be continuous or discontinuous.
- the second electrolytic solution 204 is in a very small amount, if the viscosity of the second electrolytic solution 204 is insufficient, a lid or a stopper is provided at the opening of the first chamber 104 or the second chamber 105, or a film is applied.
- the second electrolytic solution 204 may evaporate. For this reason, attention is required for operation.
- FIG. 5 (c) shows an artificial lipid film formation step.
- the artificial lipid film 205 is formed in the artificial lipid film forming unit 103.
- the artificial lipid membrane 205 is most preferably a lipid bilayer membrane, but may include multiple membranes such as a monomolecular membrane, a quadruple membrane, or a hexalayer.
- Excess organic solvent and lipid 203 are preferably removed along the outer peripheral surface of partition wall 102.
- a structure for controlling a microfluid such as a groove structure or a concavo-convex structure is provided on at least one outer peripheral surface of the partition wall 102 so as to promote the removal of the organic solvent and the lipid 203 and prevent them from being removed more than necessary. Also good.
- the liquid level of the first electrolytic solution 201 and / or the second electrolytic solution 204 may be raised and lowered in order to remove excess organic solvent and lipid 203.
- a voltage may be applied to both surfaces of the artificial lipid membrane in order to remove excess organic solvent and lipid 203.
- the voltage applied to both surfaces of the artificial lipid membrane 205 is preferably 1 mV or more and 1 V or less, and more preferably 50 mV or more and 200 mV or less.
- the applied voltage may be a DC voltage or an AC voltage.
- the artificial lipid membrane forming step may include a step of detecting the formation of the artificial lipid membrane 205.
- the formation of the artificial lipid film 205 can be detected by observation using an optical microscope or by measuring the absorbance of the artificial lipid film 205.
- a plurality of electrodes 108 are provided in the first chamber 104 and the second chamber 105 to measure the membrane resistance, membrane capacitance, membrane current or other electrical characteristics of the artificial lipid membrane 205, thereby forming the artificial lipid membrane 205. It can also be detected.
- the first electrolytic solution 201 Since the first electrolytic solution 201 is in a very small amount, if the viscosity of the first electrolytic solution 201 is insufficient, the first electrolytic solution 201 may evaporate during the artificial lipid film forming step. For this reason, attention is required for operation. The same applies to the second electrolytic solution 204.
- the electrode 108 is preferably a non-polarizable electrode.
- the material of the electrode 108 is preferably an electrode material suitable for electrochemical measurement, and may be a single metal such as Au, Pt, or Ag.
- the electrode 108 is most preferably an Ag / AgCl electrode, but may be an electrode using an inorganic material such as a saturated calomel electrode, a hydrogen electrode carbon electrode, a graphite electrode, or a carbon nanotube electrode.
- the electrode 108 may be a field effect transistor (FET), and may be a gate electrode, a source electrode, or a drain electrode of the field effect transistor.
- the electrode 108 may be an ion sensitive field effect transistor (ISFET) or a gel electrode.
- ISFET ion sensitive field effect transistor
- the electrode 108 may be used to measure chemical substances such as ions, enzymes, reaction products, or substrates contained in the first electrolytic solution 201 or the second electrolytic solution 204.
- the electrode 108 is preferably in the form of a wire, but it may be in the form of a thin film, rod, plate, cylinder, quadrangular column, polygonal column, coil, or mesh. From the viewpoint of ease of handling, when the electrode 108 has a wire shape, the length of the electrode 108 is preferably 10 nm or more and 1 cm or less. When the electrode 108 is wire-shaped, the diameter of the electrode 108 is preferably 10 nm or more and 1 cm or less.
- the length, width, and thickness of the electrode 108 are each preferably 10 nm or more and 1 cm or less.
- the length and width of the electrode 108 are each preferably 10 nm or more and 1 cm or less.
- the thickness of the electrode 108 is preferably 10 nm or more and 100 ⁇ m or less, and more preferably 50 nm or more and 1 ⁇ m or less.
- the electrode 108 is preferably provided on the inner wall of the container 101, but may be provided on the side wall or bottom of the container 101.
- the electrode 108 may be provided at a position in the artificial lipid film forming apparatus 100 that does not contact the inner wall of the container 101.
- the electrode 108 may be one or plural. When a plurality of electrodes 108 are provided, all the electrodes 108 may be made of the same material or different materials. When a plurality of electrodes 108 are provided, all the electrodes 108 may have the same shape or different shapes. When a plurality of electrodes 108 are provided, all the electrodes 108 may have the same size or different sizes.
- the first electrolytic solution 201 and the second electrolytic solution 204 have high viscosities, so that the first electrolytic solution 201 and the second electrolytic solution 204 are formed in the openings of the inlet 24 and the outlet 304. From the first chamber 104 and the second chamber 105 can be suppressed. As a result, contamination by the electrolyte around the artificial lipid film forming apparatus 100 can be prevented. Furthermore, since a very small amount of the first electrolytic solution 201 and the second electrolytic solution 204 are suppressed from evaporating rapidly, the artificial lipid membrane 205 can be stably formed.
- the artificial lipid film forming apparatus 100 is preferably installed and operated on a horizontal plane, but can also be installed and operated on an inclined surface. This is because the first electrolytic solution 201 and the second electrolytic solution 204 have high viscosities, so that even if the artificial lipid film forming apparatus 100 is installed on the inclined surface, the first electrolytic solution 201 and the second electrolytic solution 204 remain in the first chamber. This is because leakage to the outside of 104 and the second chamber 105 is suppressed.
- vibration may be applied to the artificial lipid film forming apparatus 100, the artificial lipid film forming apparatus 100 may be tilted, or the artificial lipid film forming apparatus 100 may be turned over. .
- vibration may be applied to the artificial lipid film forming apparatus 100, the artificial lipid film forming apparatus 100 may be inclined, or the artificial lipid film forming apparatus 100 may be turned over.
- Such troubles are likely to occur particularly when the artificial lipid film forming apparatus 100 is small.
- Embodiment 1 since the viscosity of the first electrolytic solution 201 and the second electrolytic solution 204 is high, vibration is applied to the artificial lipid film forming apparatus 100, the artificial lipid film forming apparatus 100 is inclined, or the artificial lipid film is tilted. Even when the forming apparatus 100 is turned over, the first electrolytic solution 201 and the second electrolytic solution 204 are prevented from leaking out of the first chamber 104 and the second chamber 105.
- the artificial lipid film forming apparatus 100 can be operated in an inclined state. As shown in FIG. 7, the artificial lipid film forming apparatus 100 can also be operated in a state in which the state is reversed upside down from the state shown in FIG. 1.
- the artificial lipid film forming apparatus 100 may be stationary, moving, or vibrating.
- the artificial lipid film forming apparatus 100 may be operated while being carried by a handler. Since the viscosity of the first electrolyte solution 201 and the second electrolyte solution 204 is high, as shown in FIG. 8, even if the hand shake of the operator occurs, the leakage of the first electrolyte solution 201 and the second electrolyte solution 204 is suppressed. Because it can. As shown in FIG. 8, the artificial lipid film forming apparatus 100 may be incorporated in a part of the mobile terminal.
- a lipid solution injection step may be performed after the first electrolyte solution injection step and the second electrolyte solution injection step. That is, the conventional technique of spraying foam, pipetting, or brushing may be applied to this embodiment.
- the first electrolyte solution injection step and the lipid solution injection step may be performed simultaneously, and the second electrolyte solution injection step and the lipid solution injection step may be performed simultaneously. That is, a pasting method that is a conventional technique may be applied to this embodiment.
- the series of steps from the first electrolyte solution injection step to the artificial lipid membrane formation step is preferably performed at 20 ° C. or higher and 60 ° C. or lower, and more preferably performed at 25 ° C. or higher and 40 ° C. or lower. .
- the biosensor using the artificial lipid film forming method of the present embodiment is preferably used for detecting an organic compound.
- the organic compound is preferably a volatile organic compound, biomolecule, diagnostic marker, protein, peptide, base or metabolite. Since the effect of trapping or concentrating the substance to be detected by the biosensor can be expected, it is preferable to adjust the viscosity of the electrolytic solution.
- the biosensor using the artificial lipid film forming method of Embodiment 1 is preferably applied to an analyzer.
- the analytical apparatus include clinical laboratory analyzers, electrochemical analyzers, gas analyzers, taste analyzers, neurophysiological analyzers, ion channel analyzers, ion channel function analyzers, or drug screening devices.
- the method for forming an artificial lipid film of Embodiment 1 includes a chemical substance detection device, a biomolecule analysis device, an air pollutant analysis device, a water pollutant analysis device, a residual agricultural chemical analysis device, a food component analysis device, a drug analysis device, and a drinking determination device.
- a chemical substance detection device a biomolecule analysis device
- an air pollutant analysis device a water pollutant analysis device
- a residual agricultural chemical analysis device a food component analysis device
- a drug analysis device and a drinking determination device.
- Smoking determination device explosives search device, gas leak detector, fire alarm, unknown person search device, personal identification device, air purifier, lifestyle-related disease diagnosis device, urine analyzer, body fluid analyzer, breath analyzer, blood You may apply to an analyzer, a blood gas analyzer, or a stress measuring device.
- Preparation process> 9 and 10 show a cross-sectional view and an exploded oblique view of the artificial lipid film forming apparatus 100 according to Embodiment 2 of the present invention, respectively.
- the same components as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.
- the artificial lipid membrane 205 is formed by the ⁇ TAS technique in the second embodiment.
- the first chamber 104 and / or the second chamber 105 is a micro flow path or a micro hole. Since the first chamber 104 and the second chamber 105 are miniaturized by the ⁇ TAS technique, the ratio of the contact area S between the electrolyte and the chamber with respect to the amount V of the electrolyte, that is, the S / V value is increased. As a result, leakage of the first electrolytic solution 201 and the second electrolytic solution 204 from the first chamber 104 and the second chamber 105 can be further suppressed.
- any material that can be used as the material of the container 101 can be used as the material of the first substrate 301.
- the material of the first substrate 301 is an insulator.
- At least a part of the outer peripheral surface of the first substrate 301 has hydrophilicity.
- oxygen plasma treatment may be performed, or it may be coated with a hydrophilic material.
- the material of the first substrate 301 is preferably transparent so that the artificial lipid membrane 205 can be observed, but may be opaque.
- the first substrate 301 is most preferably a flat plate shape, but may be a disc shape, a trapezoidal shape, a polygonal shape, a cylindrical shape, or a prismatic shape.
- the first substrate 301 is preferably formed by machining.
- the machining is preferably injection molding, extrusion molding, compression molding, hollow molding, cutting, molding, sand blasting, dry etching, wet etching, nanoimprinting, milling, photocuring, lithography or hot embossing.
- the first substrate 301 is also preferably processed by a semiconductor process.
- the partition wall 102 is sandwiched between the first substrate 301 and the second substrate 302.
- the same material as the partition wall 102 of the first embodiment can be used for the partition wall 102 of the second embodiment.
- the artificial lipid membrane forming unit 103 preferably has a tapered shape as shown in FIG.
- the direction of the taper shape may be a direction narrowing toward the first chamber 104 or a direction narrowing toward the second chamber 105.
- the artificial lipid film forming unit 103 may be a through-hole having the same diameter.
- the first chamber 104 is provided on a part of the first substrate 301.
- the first chamber 104 is preferably provided between the first substrate 301 and the partition wall 102, and is most preferably formed by the first substrate 301 and the partition wall 102.
- the volume of the first chamber 104 is preferably 1 pl or more and 1 ml or less, more preferably 10 pl or more and 200 ⁇ l or less from the viewpoint of operability.
- the first chamber 104 preferably includes a first inlet 303 for injecting an electrolytic solution and an outlet 304.
- the first chamber 104 is preferably a flow path.
- the first chamber 104 may be a micropore, a capillary, a tube, or a reservoir.
- the first chamber 104 may be connected to the electrolyte reservoir via a flow path.
- the capacity of the electrolytic solution reservoir may or may not be included in the capacity of the first chamber 104.
- a lid or a stopper may be provided at the opening of the first chamber 104, or
- a holding structure is provided inside the first chamber 104 in order to suppress leakage of the electrolyte by increasing the surface area.
- the holding structure is preferably a pillar, porous, ball, bead, dot, sponge, fiber or foam.
- the holding structure may be nanopillars, micropillars, porous metals, porous ceramics, microbeads, nanobeads, nanofoams, porous silicon, porous silica or porous alumina.
- any material that can be used as the material of the container 101 can be used as the material of the holding structure.
- a part of the surface of the holding structure may be covered with a thin film made of a material different from the material of the holding structure.
- the thin film covering the surface of the holding structure is the same as the partition wall 102.
- the holding structure may be formed simultaneously with the formation of the first chamber 104, or may be formed after the first chamber 104 is formed.
- a holding structure may be formed in advance, and the holding structure may be filled into the first chamber 104 later.
- any material that can be used as the material of the container 101 can be used as in the holding structure.
- the material of the second substrate 302 is an insulator.
- At least a part of the outer peripheral surface of the second substrate 302 has hydrophilicity.
- oxygen plasma treatment may be performed, or it may be coated with a hydrophilic material.
- the material of the second substrate 302 is preferably transparent so that the artificial lipid film 205 can be observed, but may be opaque.
- the second substrate 302 is most preferably a flat plate shape, but may be a disk shape, a trapezoidal shape, a polygonal shape, a cylindrical shape, or a prismatic shape.
- the second chamber 105 is provided on the opposite side of the first chamber 104 through the partition wall 102.
- the second chamber 105 is preferably provided between the second substrate 302 and the partition wall 102, and is most preferably formed by the second substrate 302 and the partition wall 102.
- the volume of the second chamber 105 is the same as that of the first embodiment.
- the second chamber 105 preferably includes an inlet for injecting an electrolytic solution.
- the second chamber 105 is preferably a flow path, but may be a micro hole, a capillary, a tube, or a reservoir.
- the second chamber 105 may be connected to the electrolyte reservoir through a flow path.
- the capacity of the electrolytic solution reservoir may or may not be included in the capacity of the second chamber 105.
- a lid or a stopper may be provided at the opening of the second chamber 105, or a film may be attached to the opening of the second chamber 105.
- the second chamber 105 is provided with a holding structure similar to that of the first chamber 104 in order to suppress leakage of the electrolyte by increasing the surface area.
- the holding structure may be formed simultaneously with the formation of the second chamber 105, or may be formed after the formation of the second chamber 105.
- a holding structure may be formed in advance, and the holding structure may be filled into the second chamber 105 later.
- 11 and 12 show operation diagrams of the artificial lipid film forming apparatus 100 according to the second embodiment of the present invention. 11 and 12, the same components as those in FIGS. 9 and 10 are denoted by the same reference numerals, and the description thereof is omitted.
- FIG. 11A shows the first electrolyte injection process.
- the first electrolyte solution 201 is injected into the first chamber 104 from the first injection port 303, and the first chamber 104 is filled with the first electrolyte solution 201.
- Excess first electrolyte solution 201 may be discharged from the discharge port 304.
- the discharge port 304 may be used for letting bubbles in the first chamber 104 escape.
- the first electrolytic solution 201 can use the same electrolytic solution as in the first embodiment.
- the first electrolytic solution 201 has the same viscosity and electrical resistivity as in the first embodiment.
- the viscosity of the first electrolytic solution 201 can be adjusted in the same manner as in the first embodiment.
- the method of injecting the first electrolyte 201 into the first chamber 104 from the first inlet 303 is the same as that in the first embodiment.
- the temperature of the first electrolytic solution 201 and the relative humidity around the artificial lipid film forming apparatus 100 are the same as those in the first embodiment.
- the completion of the injection of the first electrolyte 201 into the first chamber 104 can be detected as in the first embodiment.
- FIG. 11B shows a lipid solution injection step.
- the lipid solution 202 is injected into the artificial lipid film forming unit 103.
- the same lipid solution as in Embodiment 1 can be used.
- the lipid solution 202 is injected into the artificial lipid film forming unit 103.
- a biological membrane protein, secreted protein or polypeptide can be introduced into an artificial lipid membrane.
- the completion of injecting the lipid solution 202 into the artificial lipid film forming unit 103 can be detected in the same manner as in the first embodiment.
- FIG. 11C shows the second electrolyte solution injection step.
- the second electrolyte solution 204 is injected into the second chamber 105.
- the second electrolytic solution 204 can use the same electrolytic solution as in the first embodiment.
- the second electrolytic solution 204 has the same viscosity and electrical resistivity as in the first embodiment.
- the viscosity of the second electrolytic solution 204 can be adjusted in the same manner as in the first embodiment.
- the method of injecting the second electrolytic solution 204 into the second chamber 105 is the same as that in the first embodiment.
- the temperature of the second electrolytic solution 204 and the relative humidity around the artificial lipid film forming apparatus 100 are the same as in the first embodiment.
- the completion of the injection of the second electrolytic solution 204 into the second chamber 105 can be detected as in the first embodiment.
- FIG. 12 shows an artificial lipid film formation step.
- the artificial lipid film 205 is formed in the artificial lipid film forming unit 103.
- the artificial lipid membrane 205 is the same as that in the first embodiment.
- the artificial lipid film formation step of Embodiment 2 is the same as that of Embodiment 1.
- the artificial lipid film forming apparatus 100 is installed and operated on a horizontal plane, but it can also be installed and operated on an inclined surface. This is because the first electrolytic solution 201 and the second electrolytic solution 204 have high viscosities, so that even if the artificial lipid film forming apparatus 100 is installed on the inclined surface, the first electrolytic solution 201 and the second electrolytic solution 204 remain in the first chamber. This is because leakage to the outside of 104 and the second chamber 105 is suppressed.
- the artificial lipid film forming apparatus 100 can be operated in an inclined state.
- the artificial lipid film forming apparatus 100 can be operated in a state reversed upside down from the state shown in FIG. 12.
- the artificial lipid film forming apparatus 100 may be stationary, moving, or vibrating.
- the artificial lipid film forming apparatus 100 may be operated while being carried by a handler.
- the artificial lipid film forming apparatus 100 may be incorporated in a part of the mobile terminal.
- a lipid solution injection step may be performed after the first electrolyte solution injection step and the second electrolyte solution injection step. That is, the conventional technique of spraying foam, pipetting, or brushing may be applied to this embodiment.
- the first electrolyte solution injection step and the lipid solution injection step may be performed simultaneously, and the second electrolyte solution injection step and the lipid solution injection step may be performed simultaneously. That is, a pasting method that is a conventional technique may be applied to this embodiment.
- a membrane protein may be embedded in the artificial lipid membrane 205.
- FIGS. 16A to 16C schematically show a state in which a membrane protein is embedded in the artificial lipid membrane 205. It is also preferable to embed the receptor type channel 305 in the artificial lipid membrane 205.
- FIG. 16A schematically shows a state in which the receptor type channel 305 is embedded in the artificial lipid film 205 formed in the artificial lipid film forming unit 103.
- Receptor-type channel 305 is triggered to open by direct ligand binding to the channel protein.
- FIG. 16A schematically shows how the ligand 306 binds to the receptor channel 305. Ions 307 pass through the open receptor channel 305.
- FIG. 16B schematically shows a state where the channel 308, the receptor protein 309, and the G protein 310 are embedded in the artificial lipid film 205 formed in the artificial lipid film forming unit 103.
- Channel 308 is triggered by an activated GTP binding protein (G protein 310) provided by ligand binding to an independent receptor protein 309.
- FIG. 16C schematically shows a state in which the channel 308, the receptor protein 309, the G protein 310, and the enzyme 311 are embedded in the artificial lipid film 205 formed in the artificial lipid film forming unit 103.
- Channel 308 is activated by a second messenger produced after G protein 310 activation.
- the membrane protein embedded in the artificial lipid membrane 205 may be an integral membrane protein or a superficial membrane protein.
- the membrane protein embedded in the artificial lipid membrane 205 may be a transmembrane protein or a single transmembrane protein.
- the membrane protein embedded in the artificial lipid membrane 205 may be a receptor, an ion channel or a G protein.
- the receptor embedded in the artificial lipid membrane 205 is preferably a transmembrane receptor or an intracellular receptor.
- the receptor embedded in the artificial lipid membrane 205 may be a metabotropic receptor or an ion channel type receptor.
- the receptor embedded in the artificial lipid membrane 205 is preferably a G protein-coupled receptor (GPCR).
- Receptors embedded in the artificial lipid membrane 205 are muscarinic acetylcholine receptor, adenosine receptor, adrenergic receptor, GABA receptor, angiotensin receptor, cannabinoid receptor, cholecystokinin receptor, dopamine receptor, glucagon receptor, Histamine receptor, olfactory receptor, opioid receptor, rhodopsin, secretin receptor, serotonin receptor, somatostatin receptor, gastrin receptor, P2Y receptor, tyrosine kinase receptor, erythropoietin receptor, insulin receptor, cell growth factor It may be a receptor, cytokine receptor, guanylate cyclase receptor, nicotinic acetylcholine receptor, glycine receptor, glutamate receptor, inositol triphosphate receptor, ryanodine receptor or P2X receptor.
- the G protein embedded in the artificial lipid membrane 205 is most preferably a heterotrimeric G protein related to a membrane receptor.
- the G protein embedded in the artificial lipid membrane 205 is preferably activated by GPCR.
- the ion channel embedded in the artificial lipid membrane 205 is preferably a potassium channel, but may be a calcium channel or a sodium channel.
- Embodiment 2 it is preferable to embed the receptor, ion channel, or G protein in the artificial lipid membrane 205 by an inkjet method, a microdroplet coating method, a dot impact method, an electrostatic spray method, an ultrasonic method, or an electroporation method.
- the membrane protein expressed on the cell membrane may be embedded by fusing the cell to the artificial lipid membrane 205.
- the cells may be fused to the artificial lipid membrane 205 by an inkjet method, a fine droplet coating method, a dot impact method, an electrostatic spray method, an ultrasonic method, or an electroporation method.
- the membrane protein arranged on the vesicle membrane may be embedded by fusing the vesicle to the artificial lipid membrane 205.
- the series of steps from the first electrolyte solution injection step to the artificial lipid membrane formation step is preferably performed at 20 ° C. or higher and 60 ° C. or lower, and more preferably performed at 25 ° C. or higher and 40 ° C. or lower. .
- biosensor using the artificial lipid film formation method of Embodiment 2 It is possible to produce a biosensor using the artificial lipid film formation method of Embodiment 2.
- the biosensor using the artificial lipid film forming method of the present embodiment can be applied to the same device as the biosensor using the artificial lipid film forming method of the first embodiment.
- the thickness of the first substrate 301 and the second substrate 302 was 1 mm.
- the thickness of the first substrate 301 and the second substrate 302 was 5 mm.
- the size of the first substrate 301 and the second substrate 302 was 20 mm ⁇ 20 mm.
- Container 101 was transparent.
- the diameters of the first chamber 104 and the second chamber 105 were set to 1 mm, 6 mm, and 10 mm according to the amounts of the first electrolytic solution 201 and the second electrolytic 204.
- the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 0.1 ⁇ l or 1 ⁇ l
- the diameters of the first chamber 104 and the second chamber 105 were 1 mm.
- the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 50 ⁇ l
- the diameters of the first chamber 104 and the second chamber 105 were 6 mm.
- the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 200 ⁇ l, 300 ⁇ l, or 400 ⁇ l
- the diameters of the first chamber 104 and the second chamber 105 were 10 mm.
- the partition wall 102 was a Teflon (registered trademark) film having a thickness of 50 ⁇ m and had an insulating property.
- the surface of the partition wall 102 showed water repellency.
- the area of the partition wall 102 was 4 cm 2 .
- the container 101 was divided into a first chamber 104 and a second chamber 105 by the partition wall 102.
- the artificial lipid film forming part 103 was a circular through hole having a diameter of 200 ⁇ m.
- the artificial lipid membrane forming part 103 was formed at one location in the center of the partition wall 102 using a drill.
- the artificial lipid film forming apparatus 100 was formed by sandwiching the partition wall 102 between the first substrate 301 and the second substrate 302.
- the above-described Compact chamber (Ionation GmbH) was used only for the artificial lipid film forming apparatus 100 in which the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 1 ml.
- the partition wall 102 was a Teflon (registered trademark) film having a thickness of 25 ⁇ m and had insulating properties.
- the surface of the partition wall 102 showed water repellency.
- the area of the partition wall 102 was 1 cm 2 .
- the surface where the partition wall 102 and the first electrolyte solution 201 contact each other was a circle having a diameter of 5 mm.
- the container 101 was divided into a first chamber 104 and a second chamber 105 by the partition wall 102.
- the artificial lipid film forming part 103 was a through hole having a diameter of 120 ⁇ m.
- the artificial lipid film forming part 103 was formed in the central part of the partition wall 102 using a laser.
- a Tyrode solution was used as the first electrolyte solution 201 and the second electrolyte solution 204.
- the composition of the Tyrode solution was NaCl (special grade Wako Pure Chemical) 137 mM, KCl (class Wako Pure Chemical) 2.68 mM, CaCl 2 (special grade Wako Pure Chemical) 1.8 mM, NaH 2 PO 4 (special grade Wako Pure Chemical) It was 32 mM, Glucose (SIGMA G-7021) 5.56 mM, NaHCO 3 (special grade Wako Pure Chemical Industries) 1.16 mM.
- glycerin special grade, Wako Pure Chemical Industries
- PVA polyvinyl alcohol
- PEG polyethylene glycol
- the viscosities of the electrolytic solution 201 and the second electrolytic solution 204 were adjusted.
- the viscosity of the first electrolytic solution 201 and the second electrolytic solution 204 was measured using a viscometer (TV-22) manufactured by Toki Sangyo.
- lipid solution 202 a mixed solution of phospholipid (1,2-diphytanoyl-sn-glycero-3-phosphocholine, “Avanti” Polar Lipids) and an organic solvent (chloroform) was used.
- the concentration of phospholipid was 1 mg / ml.
- the first electrolyte solution 201 was injected into the first chamber 104 by a pipette (Gilson).
- a pipette As the first electrolytic solution 201, a Tyrode solution whose viscosity was adjusted with glycerin, PVA, or PEG was used.
- the temperature of the 1st electrolyte solution 201 was 22 degreeC.
- Step C Lipid solution injection step> 1 ⁇ l of the lipid solution 202 was injected into the artificial lipid film forming unit 103 from the second chamber 105 side.
- a micro syringe Hemlton
- the lipid solution 202 was injected into the artificial lipid film forming unit 103, the lipid solution 202 reached the artificial lipid film forming unit 103 while spreading on the surface of the partition wall 102.
- Step D Second electrolyte injection step>
- the 2nd electrolyte solution 204 was inject
- a Tyrode solution whose viscosity was adjusted with glycerin, PVA or PEG was used as the second electrolytic solution 204.
- the temperature of the second electrolytic solution 204 was 22 ° C.
- the environment was 22 ° C. and relative humidity 50%.
- Table 1 shows the viscosities of the first electrolytic solution 201 and the second electrolytic solution 204 adjusted with glycerin.
- Table 2 shows the viscosities of the first electrolytic solution 201 and the second electrolytic solution 204 adjusted by PVA.
- FIG. 17 (a) and FIG. 17 (b) show the viscosities of the first electrolytic solution 201 and the second electrolytic solution 204 adjusted with glycerin.
- FIG. 17B shows an enlarged view of the low concentration region of FIG.
- FIG. 18 represents the viscosities of the first electrolytic solution 201 and the second electrolytic solution 204 adjusted by PVA.
- FIG. 18B shows an enlarged view of the low concentration region of FIG.
- the viscosity of the 1st electrolyte solution 201 and the 2nd electrolyte solution 204 increased with the increase in the density
- the ion concentration of the 1st electrolyte solution 201 and the 2nd electrolyte solution 204 was made constant.
- the viscosity of the first electrolytic solution 201 and the second electrolytic solution 204 the viscosity of the first electrolytic solution 201 and the second electrolytic solution 204 increased with the increase in the concentration of PEG.
- the ion concentrations of the first electrolytic solution 201 and the second electrolytic solution 204 were made constant.
- Tables 3 and 4 show the determination results of leakage of the first electrolytic solution 201 and the second electrolytic solution 204 whose viscosity is adjusted by glycerin.
- Table 5 shows the determination result of leakage of the first electrolytic solution 201 and the second electrolytic solution 204 whose viscosity is adjusted by PVA.
- the outer volume of the first chamber 104 and the second chamber 105 is less than 200 ⁇ l when the amount of the first electrolyte 201 and the second electrolyte 204 is 200 ⁇ l or less.
- the first electrolytic solution 201 and the second electrolytic solution 204 did not leak out.
- the amount of the first electrolytic solution 201 and the second electrolytic solution 204 is 300 ⁇ l or more, the first electrolytic solution 201 and the second electrolytic solution 204 may leak out of the first chamber 104 and the second chamber 105 chamber. It was.
- FIG. 19 shows a micrograph of droplets in the first chamber 104 immediately after 10 pl of the first electrolytic solution 201 is injected by the microdroplet coating method.
- the inside of a glass tube having an inner diameter of 300 ⁇ m is filled with the first electrolytic solution 201 or the second electrolytic solution 204, and the first electrolytic solution 201 or the second electrolytic solution is obtained by piston movement of a stainless steel needle having an outer diameter of 300 ⁇ m. 204 was applied in the first chamber 104 or the second chamber 105.
- FIG. 19A shows droplets when the viscosity of the first electrolytic solution 201 is 1.24 mPa ⁇ s.
- FIG. 19B is an enlarged view of FIG. FIG.
- FIG. 19C shows a droplet when the viscosity of the first electrolytic solution 201 is 2.71 mPa ⁇ s. The viscosity of the first electrolytic solution 201 was adjusted using glycerin.
- FIG. 19 (d) is an enlarged view of FIG. 19 (c).
- FIG. 19E shows droplets when the viscosity of the first electrolytic solution 201 is 2.85 mPa ⁇ s.
- FIG. 19 (f) is an enlarged view of FIG. 19 (e).
- the viscosity of the first electrolytic solution 201 was 2.71 mPa ⁇ s and 2.85 mPa ⁇ s
- the first electrolytic solution 201 did not leak out of the first chamber 104.
- the viscosity of the second electrolytic solution 204 was 2.71 mPa ⁇ s and 2.85 mPa ⁇ s
- the second electrolytic solution 204 did not leak out of the second chamber 105.
- the amount of the first electrolytic solution 201 and the second electrolytic solution 204 is less than 10 pl, the amount of the first electrolytic solution 201 and the second electrolytic solution 204 is too small, so that the first electrolytic solution 201 and the second electrolytic solution The liquid 204 could not be injected by the fine droplet coating method.
- the reagent of the Tyrode solution could not be dissolved in the first electrolytic solution 201.
- the first electrolyte 201 adhered to the inner wall of the first chamber 104 the artificial lipid membrane 205 could not be formed.
- the reagent of the Tyrode solution could not be dissolved in the second electrolytic solution 204.
- the artificial electrolyte membrane 205 could not be formed because the second electrolyte solution 204 adhered to the inner wall of the second chamber 105.
- the PVA concentration of the first electrolyte 201 is 20 w / w%, that is, when the viscosity of the first electrolyte 201 is 2000 mPa ⁇ s, the first electrolyte 201 adheres to the inner wall of the first chamber 104.
- the artificial lipid membrane 205 could not be formed.
- the PVA concentration of the second electrolytic solution 204 is 20 w / w%, that is, when the viscosity of the second electrolytic solution 204 is 2000 mPa ⁇ s, the second electrolytic solution 204 adheres to the inner wall of the second chamber 105.
- the artificial lipid membrane 205 could not be formed.
- the amount of the first electrolytic solution 201 and the second electrolytic solution 204 is 10 pl to 200 ⁇ l, and the viscosity of the first electrolytic solution 201 and the second electrolytic solution 204 is 1.3 mPa ⁇ s to 200 mPa ⁇ s.
- the first electrolyte solution 201 and the second electrolyte solution 204 did not leak out of the first chamber 104 and the second chamber 105 even when the artificial lipid film forming apparatus 100 was tilted or turned over.
- the state of evaporation of the first electrolytic solution 201 and the second electrolytic solution 204 was observed using a microscope (manufactured by KEYENCE, VH-6300). As a result, when the viscosity of the first electrolytic solution 201 and the second electrolytic solution 204 is 1.3 mPa ⁇ s or more and 200 mPa ⁇ s, the viscosity of the first electrolytic solution 201 and the second electrolytic solution 204 is 1.3 mPa ⁇ s. It was confirmed that evaporation of the 1st electrolyte solution 201 and the 2nd electrolyte solution 204 was suppressed compared with the case where it is less than.
- the size of the first substrate 301 and the second substrate 302 was 20 mm ⁇ 20 mm.
- Container 101 was transparent.
- the diameters of the first chamber 104 and the second chamber 105 were set to 1 mm, 6 mm, or 10 mm depending on the amount of the first electrolytic solution 201 and the second electrolytic solution 204.
- the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 0.1 ⁇ l or 1 ⁇ l
- the diameters of the first chamber 104 and the second chamber 105 were 1 mm.
- the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 50 ⁇ l
- the diameters of the first chamber 104 and the second chamber 105 were 6 mm.
- the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 200 ⁇ l, 300 ⁇ l, or 400 ⁇ l
- the diameters of the first chamber 104 and the second chamber 105 were 10 mm.
- the partition wall 102 was a Teflon (registered trademark) film having a thickness of 50 ⁇ m and had an insulator.
- the table of the partition wall 102 showed water repellency.
- the area of the partition wall 102 was 4 cm 2 .
- the container 101 was divided into a first chamber 104 and a second chamber 105 by the partition wall 102.
- the artificial lipid film forming part 103 was a circular through hole having a diameter of 200 ⁇ m.
- An artificial lipid film forming apparatus 100 in which an artificial lipid film forming unit 103 is formed at one central portion of the partition wall 102 using a drill was formed by sandwiching the partition wall 102 between the first substrate 301 and the second substrate 302.
- the Compact chamber (Ionation GmbH) described above was used only for the artificial lipid film forming apparatus 100 in which the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 1 ml.
- the partition wall 102 was a Teflon (registered trademark) film having a thickness of 25 ⁇ m and had insulating properties.
- the surface of the partition wall 102 showed water repellency.
- the area of the partition wall 102 was 1 cm 2 .
- the surface where the partition wall 102 and the first electrolyte solution 201 contact each other was a circle having a diameter of 5 mm.
- the container 101 was divided into a first chamber 104 and a second chamber 105 by the partition wall 102.
- the artificial lipid film forming part 103 was a through hole having a diameter of 120 ⁇ m.
- the artificial lipid film forming part 103 was formed in the central part of the partition wall 102 using a laser.
- the composition of the Tyrode solution was as follows: NaCl (special grade Wako Pure Chemical) 137 mM, KCl (special grade Wako Pure Chemical) 2.68 mM, CaCl 2 (special grade Wako Pure Chemical) 1.8 mM, NaH 2 PO 4 (special grade Wako Pure Chemical) It was 32 mM, Glucose (SIGMA G-7021) 5.56 mM, NaHCO 3 (special grade Wako Pure Chemical Industries) 1.16 mM.
- Viscosities of the first electrolytic solution 201 and the second electrolytic solution 204 were measured using a viscometer (Toki Sangyo, TV-22). The viscosity of the first electrolytic solution 201 and the second electrolytic solution 204 was 1.24 mPa ⁇ s.
- lipid solution 202 a mixed solution of phospholipid (1,2-diphytanoyl-sn-glycero-3-phosphocholine, “Avanti” Polar “Lipids”) and an organic solvent (chloroform) was used.
- the concentration of phospholipid was 1 mg / ml.
- the first electrolyte solution 201 was injected into the first chamber 104 by a pipette (Gilson).
- the temperature of the 1st electrolyte solution 201 was 22 degreeC.
- ⁇ Lipid solution injection process 1 ⁇ l of the lipid solution 202 was injected into the artificial lipid film forming unit 103 from the second chamber 105 side.
- a micro syringe Hamilton
- the lipid solution 202 was injected into the artificial lipid film forming unit 103, the lipid solution 202 reached the artificial lipid film forming unit 103 while spreading on the surface of the partition wall 102.
- the environment was 22 ° C. and relative humidity 50%.
- the first electrolytic solution 201 leaked out of the first chamber 104.
- the second electrolytic solution 204 leaked out of the second chamber 105.
- the first electrolytic solution 201 and the second electrolytic solution 204 had a viscosity of 1.24 mPa ⁇ s and a liquid volume of 10 pl. Since the first electrolytic solution 201 rapidly evaporated, the first electrolytic solution 201 could not be injected into the first chamber 104. Similarly, since the second electrolytic solution 204 rapidly evaporated, the second electrolytic solution 204 could not be injected into the second chamber 105.
- the first electrolytic solution 201 having a viscosity of 1.24 mPa ⁇ s evaporates higher than the first electrolytic solution 201 having a viscosity of 1.3 mPa ⁇ s or more and 200 mPa ⁇ s or less. Due to its speed, it was difficult to form the artificial lipid membrane 205.
- the second electrolytic solution 204 having a viscosity of 1.24 mPa ⁇ s evaporates higher than the second electrolytic solution 204 having a viscosity of 1.3 mPa ⁇ s or more and 200 mPa ⁇ s or less. Due to its speed, it was difficult to form the artificial lipid membrane 205.
- Comparative Example 2 As the first electrolytic solution 201 and the second electrolytic solution 204, an aqueous 0.1 KCl solution containing 0.1M glucose was used. In the same manner as in Comparative Example 1, leakage of the first electrolytic solution 201 and the second electrolytic solution 204 to the outside of the first chamber 104 or the second chamber 105 was determined.
- first electrolytic solution 201 and the second electrolytic solution 204 Glucose (SIGMA G-7021) 0.1M, KCl (special grade Wako Pure Chemical) 0.1M aqueous solution was used.
- the viscosities of the first electrolytic solution 201 and the second electrolytic solution 204 were measured with a viscometer (TV-22) manufactured by Toki Sangyo.
- the viscosity of the first electrolytic solution 201 and the second electrolytic solution 204 was 1.24 mPa ⁇ s.
- the lipid solution 202 was the same as Comparative Example 1.
- the first electrolyte solution 201 was injected into the first chamber 104 by a pipette (Gilson).
- the temperature of the 1st electrolyte solution 201 was 22 degreeC.
- ⁇ Lipid solution injection process 1 ⁇ l of the lipid solution 202 was injected into the artificial lipid film forming unit 103 from the second chamber 105 side.
- a micro syringe Hamilton
- the lipid solution 202 was injected into the artificial lipid film forming unit 103, the lipid solution 202 reached the artificial lipid film forming unit 103 while spreading on the surface of the partition wall 102.
- the environment was 22 ° C. and relative humidity 50%.
- Table 6 shows a determination result of leakage of the first electrolytic solution 201 and the second electrolytic solution 204 when a 0.1 M KCl aqueous solution containing 0.1 M glucose is used as the first electrolytic solution 201 and the second electrolytic solution 204. Represents.
- the first electrolytic solution 201 When the amount of the first electrolytic solution 201 was 200 ⁇ l or more, the first electrolytic solution 201 leaked out of the first chamber 104. When the amount of the second electrolytic solution 204 was 200 ⁇ l or more, the second electrolytic solution 204 leaked out of the second chamber 105.
- the first electrolytic solution 201 When the amount of the first electrolytic solution 201 was 10 pl, the first electrolytic solution 201 was rapidly evaporated, so that the first electrolytic solution 201 could not be injected into the first chamber 104.
- the second electrolytic solution 204 When the amount of the second electrolytic solution 204 was 10 pl, the second electrolytic solution 204 rapidly evaporated, and thus the second electrolytic solution 204 could not be injected into the second chamber 105.
- the first electrolytic solution 201 having a viscosity of 1.24 mPa ⁇ s evaporates higher than the first electrolytic solution 201 having a viscosity of 1.3 mPa ⁇ s or more and 200 mPa ⁇ s or less. Due to its speed, it was difficult to form an artificial lipid membrane.
- the second electrolytic solution 204 having a viscosity of 1.24 mPa ⁇ s has a higher evaporation rate than the second electrolytic solution 204 having a viscosity of 1.3 mPa ⁇ s or more and 200 mPa ⁇ s. Therefore, it was difficult to form an artificial lipid film.
- the artificial lipid membrane formation method of the present invention includes the environment, chemical industry, semiconductor, finance, food, housing, automobile, security, life, agriculture, forestry, fisheries, transportation, safety, nursing, welfare field, medical, pharmaceutical or healthcare Useful in the field.
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Abstract
Description
以下の工程A~Eを具備する、人工脂質膜形成方法:
以下の人工脂質膜形成装置(100)を準備する工程A
ここで、前記人工脂質膜形成装置(100)は
第1チャンバ(104)と、
第2チャンバ(105)と、
前記第1チャンバ(104)および前記第2チャンバ(105)の間に挟まれた隔壁(102)と、
前記隔壁(102)に設けられた貫通孔からなる人工脂質膜形成部(103)とを備え、
前記第1チャンバ(104)は、10pl以上200μl以下の容量を有し、
前記第2チャンバ(105)は、10pl以上200μl以下の容量を有し、
1.3 mPa・s以上200mPa・s以下の粘度を有する第1電解液(201)を、前記第1チャンバ(104)に注入する工程B、
脂質(203)および有機溶媒を含有する脂質溶液(202)を前記人工脂質膜形成部(103)に注入する工程C、
1.3mPa・s以上200mPa・s以下の粘度を有する第2電解液(204)を前記第2チャンバ(105)に注入し、前記第1電解液(201)および前記第2電解液(204)の間に前記脂質溶液(202)を挟む工程D、および
前記有機溶媒を除去して人工脂質膜を前記人工脂質膜形成部(103)に形成する工程E
に関する。
以下、本発明の実施形態1を、図面を参照しながら説明する。
図1および図2は、本発明の実施形態1における人工脂質膜形成装置100の斜投影図および断面図をそれぞれ示す。
図4は、第1電解液注入工程を表す。第1電解液注入工程では、第1電解液201を第1開口部106から第1チャンバ104内へ注入して、第1チャンバ104を第1電解液201により満たす。第1電解液201は、人工脂質膜形成部103を通過して、第1チャンバ104から第2チャンバ105へ移動しないことが好ましい。
図5(a)は、脂質溶液注入工程を表す。脂質溶液注入工程では、人工脂質膜形成部103へ脂質溶液202を注入する。脂質溶液注入工程では、第2チャンバ105側から脂質溶液202を注入することが好ましい。
図5(b)は、第2電解液注入工程を表す。第2電解液注入工程では、第2電解液204が第2開口部107を介して第2チャンバ105へ注入される。
図5(c)は、人工脂質膜形成工程を示す。人工脂質膜形成工程では、人工脂質膜形成部103に人工脂質膜205が形成される。人工脂質膜205は、脂質二重膜であることが最も好ましいが、単分子膜、四重膜または六重膜のような多重膜を含んでいても良い。人工脂質膜形成工程では、第1電解液201および第2電解液204の水圧により、または外部からの圧力により、脂質溶液202の薄膜から余剰な有機溶媒および脂質203を除去することが好ましい。余剰な有機溶媒および脂質203は、隔壁102の外周面に沿って、除去されることが好ましい。有機溶媒および脂質203の除去を促進し、かつ、これらが必要以上に除去されないように、隔壁102の少なくとも一方の外周面に、溝構造または凹凸構造のような微小流体を制御する構造を設けても良い。
以下本発明の実施形態2について、図面を参照しながら説明する。
図9および図10は、本発明の実施形態2における人工脂質膜形成装置100の断面図および分解斜投影図をそれぞれ示す。実施形態2については、実施形態1と同じ構成に同一符号を付し、その詳細な説明を省略する。
図11(a)は、第1電解液注入工程を表す。第1電解液注入工程では、第1電解液201を第1注入口303から第1チャンバ104へ注入して、第1チャンバ104を第1電解液201により満たす。余剰な第1電解液201は、排出口304から排出されても良い。排出口304は、第1チャンバ104の気泡を逃すために用いられても良い。
図11(b)は、脂質溶液注入工程を表す。脂質溶液注入工程では、人工脂質膜形成部103へ脂質溶液202が注入される。脂質溶液注入工程では、第2チャンバ105側から脂質溶液202を注入することが好ましい。
図11(c)は、第2電解液注入工程を表す。第2電解液注入工程では、第2チャンバ105へ第2電解液204が注入される。
図12は、人工脂質膜形成工程を表す。人工脂質膜形成工程では、人工脂質膜形成部103に人工脂質膜205が形成される。人工脂質膜205は、実施形態1と同様である。実施形態2の人工脂質膜形成工程は、実施形態1と同様である。
第1チャンバ104および第2チャンバ105の外への第1電解液201および第2電解液204の漏出を、以下の手順で判定した。第1電解液201および第2電解液204の蒸発の様子を、マイクロスコープ(KEYENCE社製、VH-6300)を用いて評価した。
図9に示す第1基板301および第2基板302として、アクリル板を用いた。第1基板301および第2基板302の厚みを、第1電解液201および第2電解液204の液量に応じて、0.5mm、1mmまたは5mmに調整した。第1電解液201および第2電解液204の液量が0.1μlまたは1μlの時、第1基板301および第2基板302の厚みは0.5mmであった。第1電解液201および第2電解液204の液量が50μlの時、第1基板301および第2基板302の厚みは1mmであった。第1電解液201および第2電解液204の液量が200μl、300μlまたは400μlの時、第1基板301および第2基板302の厚みは5mmであった。
第1電解液201をピペット(Gilson)により第1チャンバ104へ注入した。第1電解液201として、グリセリン、PVAまたはPEGにより粘度を調整されたTyrode液を用いた。第1電解液201の温度は22℃であった。
1μlの脂質溶液202を、第2チャンバ105側から人工脂質膜形成部103へ注入した。注入には、マイクロシリンジ(Hamilton)を用いた。人工脂質膜形成部103へ脂質溶液202を注入したとき、脂質溶液202は隔壁102の表面に広がりながら、人工脂質膜形成部103へ到達した。
第2電解液204をピペット(Gilson)により第2チャンバ105へ注入した。第2電解液204として、グリセリン、PVAまたはPEGにより粘度を調整されたTyrode液を用いた。第2電解液204の温度は22℃であった。
人工脂質膜形成装置100を静置した。
第1電解液201および第2電解液204として、Tyrode液を用いた。チャンバの外への第1電解液201および第2電解液204の漏出を、以下の手順で判定した。
図9に示す第1基板301および第2基板302として、アクリル板を用いた。第1基板301および第2基板302の厚みを、第1電解液201および第2電解液204の液量に応じて、0.5mm、1mmまたは5mmに調整した。第1電解液201および第2電解液204の液量が0.1μlまたは1μlの時、第1基板301および第2基板302の厚みは0.5mmであった。第1電解液201および第2電解液204の液量が50μlの時、第1基板301および第2基板302の厚みは1mmであった。第1電解液201および第2電解液204の液量が200μl、300μlまたは400μlの時、第1基板301および第2基板302の厚みは5mmであった。
第1電解液201をピペット(Gilson)により第1チャンバ104へ注入した。第1電解液201の温度は、22℃であった。
1μlの脂質溶液202を人工脂質膜形成部103へ第2チャンバ105側から注入した。注入には、マイクロシリンジ(Hamilton)を用いた。人工脂質膜形成部103へ脂質溶液202を注入したとき、脂質溶液202は隔壁102の表面に広がりながら、人工脂質膜形成部103へ到達した。
第2電解液204をピペット(Gilson)により第2チャンバ105へ注入した。第2電解液204の温度は22℃であった。
人工脂質膜形成装置100を静置した。
第1電解液201および第2電解液204として、0.1M Glucoseを含有する0.1 KCl水溶液を用いた。比較例1と同様にして、第1チャンバ104または第2チャンバ105の外への第1電解液201および第2電解液204の漏出を判定した。
第1電解液201をピペット(Gilson)により第1チャンバ104へ注入した。第1電解液201の温度は22℃であった。
1μlの脂質溶液202を、人工脂質膜形成部103へ第2チャンバ105側から注入した。注入には、マイクロシリンジ(Hamilton)を用いた。人工脂質膜形成部103へ脂質溶液202を注入したとき、脂質溶液202は、隔壁102の表面に広がりながら、人工脂質膜形成部103へ到達した。
第2電解液204をピペット(Gilson)により第2チャンバ105へ注入した。第2電解液204の温度は22℃であった。
人工脂質膜形成装置100を静置した。
11 平板
12 電解液
13 微小孔
14 脂質溶液
15 ピペット
20 容器
21 平板
22 微小孔
23 電解液
24 注入口
25 脂質分子
26 電解液
27 注入口
31 第1の室
32 隔壁
33 第2の室
34 小孔
35 人工脂質膜
100 人工脂質膜形成装置
101 容器
102 隔壁
103 人工脂質膜形成部
104 第1チャンバ
105 第2チャンバ
106 第1開口部
107 第2開口部
108 電極
201 第1電解液
202 脂質溶液
203 脂質
204 第2電解液
205 人工脂質膜
301 第1基板
302 第2基板
303 第1注入口
304 排出口
305 受容体型チャンネル
306 リガンド
307 イオン
308 チャンネル
309 受容体タンパク質
310 Gタンパク質
311 酵素
Claims (14)
- 以下の工程A~Eを具備する、人工脂質膜を形成する方法:
以下の人工脂質膜形成装置を準備する工程A
ここで前記人工脂質形成装置は、
第1チャンバと、
第2チャンバと、
前記第1チャンバおよび前記第2チャンバの間に挟まれた隔壁と、
前記隔壁に設けられた貫通孔からなる人工脂質膜形成部と
を備え、
前記第1チャンバは、10pl以上200μl以下の容量を有し、
前記第2チャンバは、10pl以上200μl以下の容量を有し、
1.3 mPa・s以上200mPa・s以下の粘度を有する第1電解液を、前記第1チャンバに注入する工程B、
脂質および有機溶媒を含有する脂質溶液を前記人工脂質膜形成部に注入する工程C、
1.3mPa・s以上200mPa・s以下の粘度を有する第2電解液を前記第2チャンバに注入し、前記第1電解液および前記第2電解液の間に前記脂質溶液を挟む工程D、および
前記有機溶媒を除去して人工脂質膜を前記人工脂質膜形成部に形成する工程E。 - 前記第1電解液または前記第2電解液の少なくとも一方は、水酸基を有する有機化合物を含有している、請求項1に記載の方法。
- 前記水酸基を有する有機化合物がアルコールである、請求項2に記載の方法。
- 前記アルコールが低級アルコールである、請求項3に記載の方法。
- 前記アルコールがグリセリンである、請求項3に記載の方法。
- 前記第1電解液または前記第2電解液の少なくとも一方は、高分子を含有している、請求項1に記載の方法。
- 前記第1電解液または前記第2電解液の少なくとも一方は、ポリビニルアルコールを含有している、請求項6に記載の方法。
- 前記工程Bにおいて、前記第1電解液はインクジェット法により前記第1チャンバに注入される、請求項1に記載の方法。
- 前記工程Dにおいて、前記第2電解液はインクジェット法により前記第2チャンバに注入される、請求項1に記載の方法。
- 前記工程Cにおいて、前記脂質溶液は、インクジェット法により前記人工脂質膜形成部(103)に注入される、請求項1に記載の方法。
- 前記工程Eの後に、さらに前記人工脂質膜に受容体またはイオンチャンネルの少なくとも一方を埋め込む工程Fを具備する、請求項1に記載の方法。
- 前記工程Bにおいて、前記第1チャンバが前記第1電解液で満たされる、請求項1に記載の方法。
- 前記工程Dにおいて、前記第2チャンバが前記第2電解液で満たされる、請求項1に記載の方法。
- 前記工程Dにおいて、前記第2チャンバが前記第2電解液で満たされる、請求項12に記載の方法。
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CN201080003225.2A CN102216773B (zh) | 2009-10-07 | 2010-03-31 | 人工脂质膜形成方法 |
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JP2015024936A (ja) * | 2013-07-26 | 2015-02-05 | 国立大学法人福井大学 | 脂質平面膜を形成するための貫通孔を有するガラス基板、およびその製造方法と用途 |
JP2015077559A (ja) * | 2013-10-17 | 2015-04-23 | 公益財団法人神奈川科学技術アカデミー | 脂質二重膜形成器具 |
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