WO2010023848A1 - 人工脂質膜形成方法および人工脂質膜形成装置 - Google Patents
人工脂質膜形成方法および人工脂質膜形成装置 Download PDFInfo
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- WO2010023848A1 WO2010023848A1 PCT/JP2009/003971 JP2009003971W WO2010023848A1 WO 2010023848 A1 WO2010023848 A1 WO 2010023848A1 JP 2009003971 W JP2009003971 W JP 2009003971W WO 2010023848 A1 WO2010023848 A1 WO 2010023848A1
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- lipid
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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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
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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
- B01L3/502769—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 multiphase flow arrangements
Definitions
- the present invention relates to a method for forming an artificial lipid membrane used for analysis of membrane proteins including ion channels.
- the present invention also relates to an artificial lipid membrane forming apparatus suitable for carrying out such a method.
- ⁇ / RTI> Material transport between the inside and outside of the cell is performed through a transmembrane protein.
- ion channels cause changes in membrane potential due to ion permeation, and are known to play an important role in information transmission by signal generation such as nerve action potentials, and research has been actively promoted in recent years. Yes.
- the patch clamp method An indispensable tool for conducting ion channel research is an experimental technique called the patch clamp method, which was developed in 1976 by Neher and Sakmann et al.
- the tip of a micro glass tube called a patch electrode is first brought into close contact with the cell membrane surface.
- the potential is fixed in a state in which the micromembrane region at the tip opening is electrically insulated from other regions, and the ion current passing through the ion channel included in the micromembrane region is measured.
- This method has helped to identify functional elements of channel protein molecules, elucidate their working mechanisms and structures, and brought great innovation in physiological research.
- the patch clamp method may not be applied even though it is a very effective method in physiological research as described above. For example, this is the case when it is difficult to access anatomically, that is, when analyzing a channel on an organelle or a channel on a microstructure such as a presynaptic membrane.
- the channel molecule in order to deepen the basic structure of the channel and detailed structure-function correlation research, it is not applicable to the case where it is necessary to conduct an experiment with a simple configuration.
- the channel molecule must be analyzed in a simple system, that is, a system composed of water, salts, phospholipids, and channels.
- the planar lipid membrane method has been developed as an effective means when the patch clamp method cannot be applied.
- the lipid planar membrane method is roughly classified into a bubble spraying method and a bonding method (for example, Non-Patent Document 1).
- FIG. 18 shows a conventional method for forming an artificial lipid membrane by a bubble spraying method.
- the container 10 is partitioned by a flat plate 11 made of a resin having a hydrophobic surface such as Teflon (registered trademark), polystyrene, and the space partitioned by the flat plate 11 is filled with the electrolytic solution 12, and the flat plate 11 is opened.
- a lipid solution 14, that is, a mixed solution of lipid molecules and an organic solvent is applied to the micropores 13 with a pipette 15. Excess organic solvent contained in the lipid solution 14 applied to the micropores 13 is gradually removed along the peripheral edge of the micropores 13. After waiting for about 30 minutes to 3 hours, an artificial lipid membrane is formed.
- a saturated hydrocarbon such as decane, hexadecane, or hexane is often used as the organic solvent.
- phospholipid is often used as the lipid.
- diphytanoyl phosphatidylcholine, glycerol monooleate, etc. are used.
- FIGS. 19 (a), (b), and (c) show a conventional method for forming an artificial lipid membrane by a bonding method.
- the container 20 is partitioned by a flat plate 21 made of a resin having a hydrophobic surface 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 added to the one chamber of the container 20 from the injection port 24 so that the level of the electrolyte solution 23 does not exceed the height of the lower end of the microhole 22.
- a lipid solution that is, a liquid mixture 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 is oriented so that the hydrophilic portion of the lipid molecule 25 faces toward the electrolyte solution 23.
- the electrolyte solution 23 is added from the injection port 24 until the height of the liquid level of the electrolyte solution 23 passes the height of the micropore 22 upper end.
- the same operation is performed in the other chamber of the container 20. That is, the electrolyte solution 26 is added from the injection port 27 so that the height of the liquid level does not exceed the height of the lower end of the minute hole 22.
- the lipid solution is added to 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 added 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. As a result, an artificial lipid film is formed in the micropores 22.
- FIG. 20 shows a conventional artificial lipid film forming apparatus described in Patent Document 1.
- a first chamber 31 and a second chamber 33 separated from the first chamber by a partition wall 32 are provided.
- the partition wall 32 includes a first chamber 31 and a second chamber 32.
- At least one small hole 34 is provided in fluid communication.
- An artificial lipid film is formed as follows using the artificial lipid film forming apparatus shown in FIG. 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 and the lipid solution are brought into contact with each other through the small holes 34. Furthermore, the artificial lipid membrane 35 can be formed in the small hole 34 by replacing the lipid solution in the second chamber 33 with the second aqueous solution.
- This artificial lipid film forming apparatus introduces a third introduction port for introducing a lipid solution into a fine channel, a first electrolytic solution containing a substance such as a biological substance, and a second electrolytic solution into a microchannel. A first inlet and a second inlet are provided. Then, a molecular film is formed on the boundary surface between the first electrolytic solution and the second electrolytic solution.
- This artificial lipid film forming apparatus forms an artificial lipid film covering micropores formed in a substrate.
- an artificial lipid membrane is formed by using a pore closing phenomenon with a solvent. That is, the lipid solution is supplied onto the substrate on which the micropores are formed, the substrate is swollen by the solvent, and the membrane is formed in a state where the micropores are closed. Thereafter, the micropores are opened by evaporation of the solvent, and the formed artificial lipid membrane is stretched.
- a micro flow operation is performed to move the mixed solution or the electrolyte solution at the interface.
- Japanese Patent Laying-Open No. 2005-098718 page 15, FIG. 5
- JP 2005-185972 A page 73, FIG. 1
- Japanese Patent Laying-Open No. 2005-245331 page 14, FIG. 2
- the conventional artificial lipid membrane device realized on a small chip is complicated and time-consuming.
- the conventional artificial lipid film forming apparatus is (1) a method of supplying an excess lipid solution once and discharging it, or (2) a method of combining a route for supplying a lipid solution and an electrolyte solution. Adopted. Therefore, as a method for discharging the surplus lipid solution, there are only (1) an external pump, a valve and a flow rate controller, or (2) waiting for the organic solvent in the lipid solution to vaporize and develop. .
- Patent Document 1 an electrolyte solution and a mixture of lipid molecules and an organic solvent are sequentially fed to a microchannel, so that a liquid feeding means such as a syringe pump, a diaphragm pump, or a peristatic pump provided outside the microchannel.
- a liquid feeding means such as a syringe pump, a diaphragm pump, or a peristatic pump provided outside the microchannel.
- Patent Document 2 supplies an electrolytic solution and a lipid solution using a pressurizing means, a flow rate adjusting means, and the like, similar to the artificial lipid film forming apparatus of Patent Document 1.
- Patent Document 3 is simple because the lipid solution and the electrolyte solution can be supplied by interfacial movement, but the discharge of the lipid solution must wait until the solvent evaporates. Took time.
- An object of the present invention is to solve the above-mentioned conventional problems and to provide a method and an apparatus for forming an artificial lipid membrane in a simple and short time.
- An artificial lipid film forming method using an artificial lipid film forming apparatus is A substrate, A first spacer provided at one end of the substrate; A first thin film provided on the substrate via the first spacer; A second spacer provided at one end of the first thin film; A second thin film provided on the first thin film via the second spacer; A cover provided at one end of the second thin film, A first chamber is provided between the substrate and the first thin film, The first thin film includes a first through hole penetrating both surfaces, A second chamber is provided between the first thin film and the second thin film; The second thin film includes a second through hole penetrating both surfaces, The cover is provided with an inlet connected to the second through hole, In plan view, the first through hole overlaps the second through hole, The first chamber is connected to the inlet through the first through hole and the second through hole, The method A first electrolyte injection step of injecting an electrolyte into the first chamber; A lipid solution injecting step of injecting a lipid
- the present invention also provides: An artificial lipid film forming device,
- the device is A substrate, A first spacer provided at one end of the substrate; A first thin film provided on the substrate via the first spacer; A second spacer provided at one end of the first thin film; A second thin film provided on the first thin film via the second spacer; A cover provided at one end of the second thin film,
- a first chamber is provided between the substrate and the first thin film,
- the first thin film includes a first through hole penetrating both surfaces,
- a second chamber is provided between the first thin film and the second thin film;
- the second thin film includes a second through hole penetrating both surfaces,
- the cover is provided with an inlet connected to the second through hole, In plan view, the first through hole overlaps the second through hole,
- the first chamber is an artificial lipid membrane forming apparatus connected to the inlet through the first through hole and the second through hole.
- the first thin film, the first spacer, and the second thin film are integrally formed.
- the first through hole has the same cross-sectional area as that of the second through hole.
- the inlet preferably overlaps the first chamber in plan view.
- the outer peripheral surface of the first chamber preferably has hydrophilicity.
- the outer peripheral surface of the second chamber is preferably hydrophobic.
- the outer peripheral surface of the inlet is preferably hydrophilic.
- At least one of the first chamber and the inlet has an electrode.
- At least one of the first chamber and the inlet has a sensor.
- the electrolyte solution in the first electrolyte solution injection step, is preferably injected into the first chamber by capillary action.
- the lipid solution injecting step in the lipid solution injecting step, is preferably injected into at least one of the first through hole or the second through hole by a capillary phenomenon.
- the artificial lipid film forming method and the artificial lipid film forming apparatus of the present invention since an appropriate amount of lipid solution can be introduced into the through-hole, there is no need to provide a discharge port for discharging excess lipid solution. There is no need to provide an external pump. Moreover, it is not necessary to wait for a long time until the artificial lipid membrane is formed. As a result, an artificial lipid membrane can be formed in a simpler and shorter time than a conventional artificial lipid membrane forming apparatus.
- FIG. 1 is a cross-sectional view of an artificial lipid film forming apparatus according to Embodiment 1 of the present invention.
- FIG. 2 is an exploded perspective view of the artificial lipid film forming apparatus according to Embodiment 1 of the present invention.
- FIG. 3 is an oblique projection of the artificial lipid film forming apparatus according to Embodiment 1 of the present invention.
- FIG. 4 is an enlarged view of the artificial lipid film forming apparatus according to Embodiment 1 of the present invention.
- FIG. 5 is an enlarged view of the first through hole and the second through hole in Embodiment 1 of the present invention.
- FIG. 6 is an operation diagram of the artificial lipid film forming apparatus in Embodiment 1 of the present invention.
- FIG. 1 is a cross-sectional view of an artificial lipid film forming apparatus according to Embodiment 1 of the present invention.
- FIG. 2 is an exploded perspective view of the artificial lipid film forming apparatus according to Embodiment 1 of the present invention.
- FIG. 7 is an explanatory diagram of a second electrolyte solution injection step in Embodiment 1 of the present invention.
- FIG. 8 is a cross-sectional view and an oblique projection of the artificial lipid film forming apparatus according to Embodiment 2 of the present invention.
- FIG. 9 is an exploded perspective view of the artificial lipid film forming apparatus according to Embodiment 2 of the present invention.
- FIG. 10 is a cross-sectional view and a perspective view of an artificial lipid film forming apparatus according to Embodiment 3 of the present invention.
- FIG. 11 is a cross-sectional view and a perspective view of an artificial lipid film forming apparatus according to Embodiment 4 of the present invention.
- FIG. 12 is a cross-sectional view and a perspective view of an artificial lipid film forming apparatus according to Embodiment 5 of the present invention.
- FIG. 13 is a photomicrograph of an artificial lipid membrane in an example of the present invention.
- FIG. 14 is a graph showing a transient response of the current flowing through the artificial lipid membrane in the example of the present invention.
- FIG. 15 is a diagram showing an artificial lipid film forming apparatus in a comparative example.
- FIG. 16 is a micrograph of the artificial lipid membrane in the comparative example.
- FIG. 17 is a comparison diagram of the formation rate of artificial lipid membranes.
- FIG. 18 is a schematic diagram of a conventional artificial lipid film forming apparatus (bubble blowing method).
- FIG. 19 is a schematic view of a conventional artificial lipid film forming apparatus (bonding method).
- FIG. 20 is a schematic diagram of a conventional artificial lipid film forming apparatus (Patent Document 1).
- FIG. 1 to 3 are a cross-sectional view, an exploded perspective view, and an oblique projection view of the artificial lipid film forming apparatus according to Embodiment 1 of the present invention.
- the artificial lipid film forming apparatus 100 includes a substrate 101.
- the material of the substrate 101 is most preferably glass.
- the glass may be soda glass, quartz, borosilicate glass, low-melting glass, photosensitive glass, or the like.
- the substrate 101 may be another inorganic material such as silicon or aluminum oxide, an organic material such as polyethylene, polypropylene, or vinyl chloride, or another organic material.
- the material of the substrate 101 may be a combination of a plurality of materials.
- At least a part of the outer peripheral surface of the substrate 101 preferably has a hydrophilic surface. In order to make at least a part of the outer peripheral surface of the substrate 101 hydrophilic, oxygen plasma treatment may be performed, or coating may be performed with a hydrophilic material. Moreover, you may perform the other hydrophilic process generally known.
- the substrate 101 is preferably transparent from the viewpoint of optical measurement. In the present invention, the shape of the substrate 101 is not limited.
- a first spacer 102 is provided on the substrate 101.
- the material of the first spacer 102 may be an organic material or an inorganic material.
- the organic material is most preferably Teflon (registered trademark), and may be an organic polymer such as polysulfone, polystyrene, polymethyl methacrylate, polyethylene, polyethylene terephthalate, vinyl chloride, and polydimethylsiloxane. Plastics such as polyethylene, polypropylene, and vinyl chloride may be used.
- a silicon-based, epoxy-based, or vinyl-based adhesive, a photoresist, a photosensitive organic material including polyimide, or the like may be used.
- the glass may be soda glass, quartz, borosilicate glass, low melting point glass, or the like.
- silicon, silicon oxide, aluminum oxide, silicon nitride, or the like may be used.
- the material of the first spacer 102 may be a combination of a plurality of materials.
- the outer peripheral surface of the first spacer 102 has a hydrophilic surface.
- the first spacer 102 is preferably subjected to a hydrophilic treatment.
- oxygen plasma treatment may be performed, or it may be covered with a hydrophilic material.
- the shape of the first spacer 102 is not limited. However, in general, the first spacer 102 is provided along the outer periphery of the substrate 101.
- the first thin film 103 is provided on the first spacer 102. That is, the first spacer 102 is provided between the substrate 101 and the first thin film 103.
- the material of the first thin film 103 is preferably an organic material.
- the organic material is most preferably Teflon (registered trademark), but organic polymers such as polysulfone, polystyrene, polymethyl methacrylate, polyethylene, polyethylene terephthalate, vinyl chloride, polydimethylsiloxane, and parylene may be used.
- organic materials silicon-based, epoxy-based, vinyl-based adhesives, photoresists, photosensitive organic materials including polyimide, and the like may be used.
- the first thin film 103 is made of an inorganic material such as glass, silicon oxide, and aluminum oxide, and the surface thereof is Teflon (registered trademark), polysulfone, polystyrene, polymethyl methacrylate, polyethylene, polyethylene terephthalate, vinyl chloride, polydimethyl. Cover with organic polymer such as siloxane, parylene, silicon-based, epoxy-based, vinyl-based adhesive, photoresist, photosensitive organic material including polyimide, and self-assembled film (SAM film) including hydrocarbon. Also good.
- the material of the first thin film 103 may be a combination of a plurality of materials.
- At least a part of the outer peripheral surface of the first thin film 103 has a hydrophobic surface. This is because the hydrophobic part of the lipid molecule is held by the hydrophobic surface of the first thin film 103, so that the artificial lipid film becomes stable.
- the thickness of the first thin film 103 is preferably 50 nm to 10 mm, and more preferably 1 ⁇ m to 1 mm.
- a second spacer 104 is provided on the first thin film 103. That is, the first thin film 103 is provided between the second spacer 104 and the first spacer 102.
- the material of the second spacer 104 may be an organic material or an inorganic material.
- the organic material is most preferably Teflon (registered trademark), and may be an organic polymer such as polysulfone, polystyrene, polymethyl methacrylate, polyethylene, polyethylene terephthalate, vinyl chloride, and polydimethylsiloxane. Plastics such as polyethylene, polypropylene, and vinyl chloride may be used.
- a silicon-based, epoxy-based, or vinyl-based adhesive a photosensitive organic material including a photoresist, polyimide, or the like may be used.
- the glass may be soda glass, quartz, borosilicate glass, low melting point glass, or the like.
- silicon, silicon oxide, aluminum oxide, silicon nitride, or the like may be used.
- the material of the second spacer 104 may be a combination of a plurality of materials. It is preferable that at least a part of the outer peripheral surface of the second spacer 104 has a hydrophilic surface.
- the second spacer 104 is subjected to a hydrophilic treatment.
- a hydrophilic treatment In order to make at least a part of the outer peripheral surface of the second spacer 104 hydrophilic, oxygen plasma treatment may be performed, or it may be coated with a hydrophilic material. Moreover, you may perform the other hydrophilic process generally known.
- the shape of the second spacer 104 is not limited. However, in general, the second spacer 104 is provided along the outer periphery of the substrate 101.
- the second thin film 105 is provided on the second spacer 104. That is, the second spacer 104 is provided between the second thin film 105 and the first thin film 103.
- the material of the second thin film 105 is preferably an organic material.
- the organic material is preferably plastic.
- the organic material is most preferably Teflon (registered trademark), and may be an organic polymer such as polysulfone, polystyrene, polymethyl methacrylate, polyethylene, polyethylene terephthalate, vinyl chloride, polydimethylsiloxane, and parylene.
- photosensitive organic materials including silicon-based, epoxy-based, and vinyl-based adhesives, photoresists, and polyimides may be used.
- the second thin film 105 is made of an inorganic material such as glass, silicon oxide, aluminum oxide, and the surface thereof is Teflon (registered trademark), polysulfone, polystyrene, polymethyl methacrylate, polyethylene, polyethylene terephthalate, vinyl chloride, polydimethylsiloxane. , Organic polymers such as parylene, silicon-based, epoxy-based, vinyl-based adhesives, photoresists, photosensitive organic materials including polyimide, and self-assembled films (SAM films) containing hydrocarbons good.
- the material of the second thin film 105 may be a combination of a plurality of materials.
- At least a part of the outer peripheral surface of the second thin film 105 has a hydrophobic surface. This is because the hydrophobic part of the lipid molecule is held by the hydrophobic surface of the second thin film 105, so that the artificial lipid film becomes stable.
- the thickness of the second thin film 105 is preferably 50 nm to 10 mm, and more preferably 1 ⁇ m to 1 mm.
- the cover 106 is provided on the second thin film 105. That is, the second thin film 105 is provided between the cover 106 and the second spacer 104.
- the material of the cover 106 is preferably an organic material.
- the organic material is preferably polydimethylsiloxane.
- the organic material may be an organic polymer such as polysulfone, polystyrene, polymethyl methacrylate, polyethylene, polyethylene terephthalate, vinyl chloride, polydimethylsiloxane, or parylene.
- a photosensitive organic material including a photoresist and polyimide may be used.
- the cover 106 may be made of an inorganic material such as glass, silicon oxide, or aluminum oxide. At least a part of the outer peripheral surface of the cover 106 preferably has a hydrophilic surface, but may have a hydrophobic surface.
- the material of the cover 106 may be a combination of a plurality of materials.
- the cover 106 is preferably transparent from the viewpoint of optical measurement. In the present invention, the shape of the cover 106 is not limited.
- the first chamber 107 is formed between the substrate 101 and the first thin film 103.
- the height 114 of the first chamber 107 is preferably 10 nm to 100 mm, and more preferably 10 nm to 1 mm.
- the width 120 of the first chamber 107 is preferably 10 nm or more and 100 mm or less, and more preferably 1 ⁇ m or more and 5 mm or less.
- the length 121 of the first chamber 121 is preferably 10 nm to 100 mm, and more preferably 1 ⁇ m to 5 mm.
- the height 114 of the first chamber 107 may be all the same or different in the first chamber 107.
- the width 120 of the first chamber 107 and the length 121 of the first chamber 107 may all be the same or different in the first chamber 107.
- the shape of the first chamber 107 is not limited.
- the shape of the first chamber 107 is most preferably a rectangular parallelepiped, but may be another shape such as a cylinder or a triangular prism.
- the first through hole 108 is formed so as to penetrate both surfaces of the first thin film 103.
- the first through hole 108 is most preferably circular.
- the first through hole 108 may have another shape such as an ellipse, a square, a rectangle, a rhombus, a hexagon, or a polygon.
- the diameter 122 of the first through hole 108 is preferably 10 nm or more and 1 mm or less, and more preferably 2 ⁇ m or more and 200 ⁇ m or less.
- the processing method of the first through hole 108 may be machining such as cutting or punching, lithography, etching, sandblasting, stereolithography, nanoimprinting, or the like.
- the inner wall of the first through hole 108 is preferably flat, but a groove structure or a concavo-convex structure may be provided.
- the number of the first through holes 108 is most preferably one, but may be two or more. When two or more first through holes 108 are provided, the shapes of the first through holes 108 may all be the same or different. Further, when two or more first through holes 108 are provided, the diameters 122 of the first through holes 108 may all be the same or different.
- the second chamber 109 is formed between the first thin film 103 and the second thin film 105.
- the second chamber 109 is connected to the first chamber 107 through the first through hole 108.
- the height 115 of the second chamber 109 is preferably 10 nm or more and 1 mm or less, and more preferably 10 nm or more and 10 ⁇ m or less.
- the width 123 of the second chamber 109 is preferably 10 nm to 100 mm, and more preferably 1 ⁇ m to 5 mm.
- the length 124 of the second chamber 109 is preferably 10 nm to 100 mm, and more preferably 1 ⁇ m to 5 mm.
- the height 115 of the second chamber 109 may be all the same or different in the second chamber 109.
- the lipid solution is preferably injected in one direction.
- the height decreases in the vicinity of the outer peripheral edges of the first through hole 108 and the second through hole 110, it is preferable because injection of an excessive lipid solution can be suppressed.
- the width 123 of the second chamber 109 and the length 124 of the second chamber 109 may all be the same or different in the second chamber 109.
- the shape of the second chamber 109 is not limited.
- the shape of the second chamber 109 is most preferably a rectangular parallelepiped, but may be another shape such as a cylinder or a triangular prism.
- a groove structure or an uneven structure may be provided on the inner wall of the second chamber 109.
- the second through hole 110 is formed so as to penetrate both surfaces of the second thin film 105.
- the shape of the second through hole 110 is most preferably circular.
- the shape of the second through hole 110 may be other shapes such as an ellipse, a square, a rectangle, a rhombus, a hexagon, and a polygon.
- the diameter 125 of the second through hole 110 is preferably 10 nm or more and 1 mm or less, and more preferably 2 ⁇ m or more and 200 ⁇ m or less.
- the diameter 125 of the second through hole 110 is preferably the same as the diameter 122 of the first through hole 108, but may be different.
- the processing method of the second through hole 110 may be machining such as cutting or punching, lithography, etching, sand blasting, stereolithography, nanoimprinting, or the like.
- the inner wall of the second through-hole 110 is preferably flat, but a groove structure or an uneven structure may be provided.
- the number of the second through holes 110 is most preferably one, but may be two or more. When two or more second through holes 110 are provided, the shapes of the second through holes 110 may all be the same or different. Further, when two or more second through holes 110 are provided, the diameters 125 of the second through holes 110 may all be the same or different.
- first through hole 108 and the second through hole 110 have the same shape.
- the shapes of the first through hole 108 and the second through hole 110 will be described in detail below.
- 4A to 4C are enlarged views of the periphery of the first through hole 108 and the second through hole 110 of the artificial lipid film forming apparatus according to Embodiment 1 of the present invention. 4A to 4C, only the first thin film 103 and the second thin film 105 are shown for ease of explanation.
- the first through hole 108 and the second through hole 110 are most preferably cylindrical.
- the first through hole 108 and the second through hole 110 are preferably the same size.
- the area of the cross section 108 b of the first through hole 108 is preferably substantially equal to the area of the cross section 110 a of the second through hole 110. This is because it is easy to inject a lipid solution.
- the diameter of the cross section 108b of the first through hole 108 is preferably 10 nm to 1 mm, and more preferably 2 ⁇ m to 200 ⁇ m.
- the diameter of the cross section 110a of the second through-hole 110 is preferably 10 nm or more and 1 mm or less, and more preferably 2 ⁇ m or more and 200 ⁇ m or less.
- At least one of the first through hole 108 and the second through hole 110 may be a trapezoidal column.
- the first through hole 108 and the second through hole 110 may be the same size.
- the area of the cross section 108 b of the first through hole 108 is preferably substantially equal to the area of the cross section 110 a of the second through hole 110.
- the area of the cross section 108b of the first through hole 108 is preferably smaller than the area of the cross section 108a of the first through hole 108.
- the area of the cross section 110 a of the second through hole 110 is preferably smaller than the area of the cross section 110 b of the second through hole 110.
- the first through hole 108 and the second through hole 110 may be trapezoidal columnar.
- the area of the cross section 108 b of the first through hole 108 is preferably substantially equal to the area of the cross section 110 a of the second through hole 110.
- the area of the cross section 108b of the first through hole 108 is preferably larger than the area of the cross section 108a of the first through hole 108.
- the area of the cross section 110 a of the second through hole 110 is preferably smaller than the area of the cross section 110 b of the second through hole 110.
- the shapes of the first through hole 108 and the second through hole 110 have been described only for the cylindrical type. However, the same applies to other shapes.
- first through hole 108 and the second through hole 110 are provided at overlapping positions.
- the positional relationship between the first through hole 108 and the second through hole 110 will be described in detail below.
- 5A to 5C show the first through hole 108 and the second through hole of the artificial lipid film forming apparatus according to Embodiment 1 of the present invention, as viewed from the normal direction of the first thin film 103 and the second thin film 105.
- FIG. 2 is an enlarged view of a hole 110.
- FIG. 5A to 5C only the first through hole 108 and the second through hole 110 are shown for ease of explanation.
- first through hole 108 and the second through hole 110 coincide.
- the shapes of the first through hole 108 and the second through hole 110 are cylindrical.
- the first through hole 108 and the second through hole 110 may partially overlap each other.
- FIG. 5B shows that the diameter of the first through hole 108 is smaller than the diameter of the second through hole 110. Note that the diameter of the first through hole 108 may be larger than the diameter of the second through hole 110.
- the center position of the first through hole 108 may be different from the center position of the second through hole 110.
- the diameter of the first through hole 108 may be different from the diameter of the second through hole 110.
- first through holes 108 and second through holes 110 their arrangement may be linear, circumferential, radial, square lattice, triangular lattice, or triangular lattice. It may be a shape.
- the first opening 111 is formed at one end of the substrate 101 and the first thin film 103. It is preferable that the first projecting portion 191 is formed by changing the position of the end portion of the substrate 101 and the end portion of the first thin film 103 to form the first opening 111.
- the length 130 of the first projecting portion 191 of the first opening 111 shown in FIG. 3 is preferably 1 mm or more and 10 mm or less.
- the width 131 of the first projecting portion 191 of the first opening 111 is preferably 1 mm or more and 20 mm or less.
- the first opening 111 may be flat as shown in FIG. 3 or may have a groove structure or a concavo-convex structure so that liquid can be easily injected.
- the second opening 112 is formed at one end of the first thin film 103 and the second thin film 105. As shown in FIG. 1, it is preferable that the second projecting portion 192 is formed by changing the position of the end portion of the first thin film 103 and the end portion of the second thin film 105 to form the second opening 112.
- the length 132 of the second overhanging portion 192 of the second opening 112 shown in FIG. 3 is preferably 1 mm or more and 10 mm or less.
- the width 133 of the second projecting portion 192 of the second opening 112 is preferably 1 mm or more and 20 mm or less.
- the second opening 112 may be flat as shown in FIG. 3, or may have a groove structure or a concavo-convex structure so that liquid can be easily injected.
- the inlet 113 is formed at one end of the cover 106.
- the inlet 113 is connected to the second chamber 109 through the second through hole 110. As shown in FIG. 1, it is preferably formed so as to penetrate the cover 106.
- the shape of the inlet 113 is preferably a cylindrical shape, but may be other shapes.
- the diameter 134 of the injection port 113 is preferably 0.5 mm or more and 2 mm or less.
- the substrate 101 and the first spacer 102 may be integrated.
- the first spacer 102 and the first thin film 103 may be integrated.
- the first thin film 103 and the second spacer 104 may be integrated.
- the first spacer 102, the first thin film 103, and the second spacer 104 may be integrated.
- the first thin film 103, the second spacer 104, and the second thin film 105 may be integrated.
- the second spacer 104 and the second thin film 105 may be integrated.
- the second thin film 105 and the cover 106 may be integrated.
- the substrate 101, the first spacer 102, the first thin film 103, the second spacer 104, the second thin film 105, and the cover 106 are preferably bonded after being laminated.
- Each layer may be adhered using an adhesive, or may be welded by applying heat.
- Each laminated layer may be sandwiched between two plates and fixed with bolts, or may be joined by other methods.
- FIG. 6 shows an operation diagram of the artificial lipid film forming apparatus in Embodiment 1 of the present invention.
- the same components as those in FIGS. 1 to 3 are denoted by the same reference numerals, and the description thereof is omitted.
- FIGS. 6A and 6B show the first electrolyte injection process.
- the first electrolyte 301 is injected into the first chamber 107 from the first opening 111.
- the first electrolytic solution 301 preferably contains KCl, and more preferably isotonic KCl solution. It is preferable that the 1st electrolyte solution 301 is a physiological condition in a cell.
- the pH is preferably around 7.
- a buffer solution such as HEPES may be used.
- a general solution used in electrophysiological experiments may be used.
- the Ca 2+ concentration is preferably 10 to 100 nM.
- a Ca 2+ chelator such as EGTA may be used.
- the amount of the first electrolytic solution 301 to be injected is most preferably about the same as the volume of the first chamber 107, but may be smaller or larger than the volume of the first chamber 107.
- the first electrolyte solution injection step it is most preferable to inject the first electrolyte solution 301 into the first chamber 107 by capillary action.
- the first electrolyte solution 301 may be injected by its own weight or by other methods. You may do it.
- 6A and 6B show a state in which the first electrolyte solution 301 is injected into the first chamber 107 by capillary action.
- the first electrolytic solution 301 is sequentially injected from the first opening 111 toward the first through hole 108 as shown in FIG. Then, as shown in FIG. 6B, the inside of the first chamber 107 is filled with the first electrolytic solution 301.
- the first electrolytic solution 301 When the first electrolytic solution 301 is injected into the first chamber 107 by capillary action, it is preferable that at least a part of the outer peripheral surface of the substrate 101 is subjected to a hydrophilic treatment. Of the outer peripheral surface of the substrate 101, it is preferable that a portion in contact with the first electrolytic solution 301 is subjected to a hydrophilic treatment. Of the outer peripheral surface of the substrate 101, the vicinity of the first through hole 108 is preferably subjected to a hydrophilic treatment.
- the first electrolyte solution injection step may include a step of detecting that the first electrolyte solution 301 has been injected into the first chamber 107.
- a step of detecting that the first electrolyte solution 301 has been injected into the first chamber 107 In order to detect that the first electrolytic solution 301 has been injected into the first chamber 107, observation with an optical microscope may be used. A plurality of electrodes may be provided in the first chamber 107, and the electrical conductivity may be measured and detected. You may use the method of detecting presence of other general electrolyte solution.
- FIG. 6C shows a lipid solution injection process.
- the lipid solution 302 is injected into the second chamber 109 from the second opening 112.
- the lipid solution 302 may be injected into the first through hole 108 and the second through hole 110 via the second chamber 109.
- the lipid solution 302 may be injected into the second through hole 110 via the second chamber 109.
- the lipid solution 302 is preferably a lipid dispersed in an organic solvent.
- the lipid is a phospholipid.
- the lipid may be a glycolipid, a lipolipid, or another lipid.
- the lipid may be azolectin, other naturally derived lipid, or synthetic lipid. Synthetic lipids are more preferred because they are easy to obtain highly pure and chemically stable. Specifically, it may be phospholipid diphytanoylphosphadylcholine, glycerol monooleate, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, dipalmitoylphosphatidylcholine, or other phospholipids.
- the fatty acid portion of the lipid molecule is preferably a saturated or unsaturated fatty acid having 10 to 20 carbon atoms. These lipids may be used alone or in combination of two or more.
- the lipid concentration relative to the organic solvent is preferably 3 to 50 mg / mL, more preferably 4 to 40 mg / mL.
- the lipid solution 302 may be mixed with a substance that gives the artificial lipid membrane a net surface charge.
- the surface charge of the artificial lipid membrane is preferably negative.
- phosphatidylserine, phosphatidylinositol or the like may be mixed.
- the substance for imparting electric charge to the artificial lipid membrane may be mixed in advance before the lipid solution injection step, or may be mixed after the artificial lipid membrane formation step. In the present invention, the amount of substance for imparting electric charge to the artificial lipid membrane is not limited.
- the lipid solution 302 may be mixed with biological membrane proteins such as receptors, ion channels, and G proteins, and secreted proteins.
- the lipid solution 303 may be mixed with a polypeptide such as gramicidin. Only one type of biological membrane protein, secreted protein, polypeptide or the like may be mixed, or a plurality of types may be mixed.
- Biological membrane proteins, secreted proteins, polypeptides and the like may be mixed in advance before the lipid solution injection step.
- Biological membrane proteins, secreted proteins, polypeptides and the like may be mixed after the artificial lipid membrane formation step.
- the biological membrane protein or secreted protein when mixing biological membrane protein or secreted protein after the artificial lipid membrane formation step, the biological membrane protein or secreted protein may be temporarily incorporated into the vesicle, and the vesicle may be fused to the artificial lipid membrane.
- the mixing technique may be used.
- the artificial lipid film forming apparatus 100 may be provided with a mechanism for mixing biological membrane proteins, secreted proteins, polypeptides and the like.
- the lipid solution injection step it is most preferable to inject the lipid solution 302 into the second chamber 109 by capillary action.
- the lipid solution 302 may be injected into the second chamber 109 by its own weight, or may be injected by other methods.
- the lipid solution injection step it is preferable to start injection of the lipid solution 302 into the second chamber 109 after the first chamber 107 is filled with the first electrolyte solution 301.
- the lipid solution injection step may include a step of detecting that the lipid solution 302 has been injected into the second chamber 109.
- a step of detecting that the lipid solution 302 has been injected into the second chamber 109 In order to detect the completion of the injection of the lipid solution 302 into the second chamber 109, observation with an optical microscope may be used. Other general methods for detecting the presence of organic solvents or lipid solutions may be used.
- FIG.6 (d) represents a 2nd electrolyte solution injection
- the second electrolyte solution injection step the second electrolyte solution 303 is injected into the injection port 113.
- the second electrolytic solution 303 preferably contains KCl, and more preferably isotonic KCl solution. It is preferable that the 2nd electrolyte solution 303 is a physiological condition in a cell.
- the pH is preferably around 7.
- a buffer solution such as HEPES may be used.
- the Ca 2+ concentration is preferably 10 to 100 nM.
- a Ca 2+ chelator such as EGTA may be used.
- the amount of the second electrolytic solution 303 to be injected is most preferably about the same as the volume of the inlet 113, but may be smaller or larger than the volume of the inlet 113.
- the second electrolytic solution 303 may be the same as or different from the first electrolytic solution 301.
- FIGS. 7A and 7B show how the second electrolytic solution 303 is injected into the injection port 113 in the second electrolytic solution injection step.
- the inner wall surface of the inlet 113 is disposed so as not to break the membrane of the lipid solution 302 formed in the first through-hole 108 when the second electrolytic solution 303 is dropped into the inlet 113. It is preferable to inject the second electrolytic solution 303 along 113a.
- the inner wall surface 113a of the inlet 113 is preferably inclined.
- the inner wall surface 110c of the second through hole 110 is preferably inclined.
- the inner wall surface 110c of the second through hole 110 may be inclined.
- the inclination angles of the inner wall surface 113a of the inlet 113 and the inner wall surface 110c of the second through hole 110 may be the same or different.
- the inner wall surface 113a of the inlet 113 is preferably subjected to a hydrophilic treatment.
- the inner wall surface 110c of the second through hole 110 is preferably subjected to a hydrophilic treatment.
- the inner wall surface 113a of the inlet 113 may be flat, or may have a groove structure or a concavo-convex structure so that the second electrolyte solution 303 can be easily injected.
- the injection of the second electrolyte solution 303 is preferably started after the first through hole 108 and the second through hole 110 are filled with the lipid solution 302.
- the injection of the second electrolyte solution 303 is preferably started after the second through hole 110 is filled with the lipid solution 302.
- the second electrolyte solution injection step may include a step of detecting that the second electrolyte solution 303 has been injected into the injection port 113.
- a step of detecting that the second electrolyte solution 303 has been injected into the injection port 113 In order to detect that the second electrolyte solution 303 has been injected into the injection port 113, observation with an optical microscope may be used. A plurality of electrodes may be provided at the inlet 113 and the electrical conductivity may be measured and detected. You may use the method of detecting presence of other general electrolyte solution.
- an artificial lipid membrane is formed in the first through hole 108.
- Artificial lipid membranes may be formed in the first through hole 108 and the second through hole 110.
- An artificial lipid film may be formed only in the second through hole 110.
- the artificial lipid membrane is a lipid bilayer membrane.
- the organic solvent is removed from the thin film of the lipid solution 302 by the weight of the second electrolytic solution 303. Excess organic solvent is preferably removed along the outer peripheral surface of at least one of the first thin film 103 and the second thin film 105. In order to promote the removal of the organic solvent, there is a groove structure, a concavo-convex structure, etc.
- a structure for controlling the microfluid may be provided on the outer peripheral surface of at least one of the first thin film 103 and the second thin film 105 and in the vicinity of the first through hole 108 and the second through hole 110 . Further, in order to prevent the removal of the organic solvent from proceeding more than necessary, at least one outer peripheral surface of the first thin film 103 and the second thin film 105 and in the vicinity of the first through hole 108 and the second through hole 110, A structure for controlling a microfluid such as a groove structure or an uneven structure may be provided.
- the artificial lipid film forming step may include a step of detecting the formation of the artificial lipid film. In order to detect the formation of the artificial lipid film, observation with an optical microscope may be used. The absorbance of the artificial lipid membrane may be measured. A plurality of electrodes may be provided in the first chamber 107 and the inlet 113, and the membrane resistance, membrane capacitance, membrane current, etc. of the artificial lipid membrane may be measured, or other electrical characteristics may be measured.
- the artificial lipid membrane device 100 may be installed and operated in the direction shown in FIG. 1, or may be operated in another direction.
- the artificial lipid film forming apparatus 100 shown in FIG. 1 may be installed and operated in a direction rotated 90 degrees counterclockwise in the paper.
- the series of steps from the first electrolyte injection step to the artificial lipid membrane formation step is preferably performed at 20 ° C. or more and 60 ° C. or less, and more preferably 25 ° C. or more and 40 ° C. or less.
- Analytical devices are used for clinical laboratory analyzers, electrochemical analyzers, gas analyzers, taste analyzers, neurophysiology analyzers, ion channel analyzers, ion channel functional analyzers, drug screening analyzers, biosensing devices, etc. May be.
- Embodiment 2 (Embodiment 2)
- the artificial lipid film forming method in Embodiment 2 of the present invention will be described with reference to the drawings.
- 8 (a) and 8 (b) are a cross-sectional view and an oblique projection view of the artificial lipid film forming apparatus according to the second embodiment.
- the same parts as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.
- the difference between the present embodiment and the first embodiment is the shape of the inlet 113. Moreover, the difference between this embodiment and Embodiment 1 is a 2nd electrolyte solution injection
- the artificial lipid film forming apparatus 100 includes a third spacer 201.
- a cover 106 is provided at one end of the third spacer 201.
- a third chamber 202 is formed between the second thin film 105 and the cover 106.
- the height 203 of the third chamber 202 is preferably 10 nm to 100 mm, and more preferably 10 nm to 1 mm.
- the height 203 of the third chamber 202 may be the same or different in the third chamber 202.
- FIG. 9 is an exploded perspective view of the artificial lipid film forming apparatus according to Embodiment 2 of the present invention.
- the width 126 of the third chamber 202 and the length 127 of the third chamber 202 may be the same or different in the third chamber 202.
- the shape of the third chamber 107 is not limited.
- the shape of the third chamber 107 is most preferably a rectangular parallelepiped, but may be another shape such as a cylinder or a triangular prism.
- the width 126 of the third chamber 202 is preferably 10 nm to 100 mm, and more preferably 1 ⁇ m to 5 mm.
- the length 127 of the third chamber 202 is preferably 10 nm to 100 mm, and more preferably 1 ⁇ m to 5 mm.
- the inlet 113 is formed at one end of the second thin film 105 and the cover 106. It is preferable that the third projecting portion 193 is formed by changing the positions of the end portions of the second thin film 105 and the cover 106 to serve as the inlet 113.
- the length 204 of the third projecting portion 193 of the inlet 113 shown in FIG. 8B is preferably 1 mm or more and 10 mm or less.
- the width 205 of the third projecting portion 193 of the inlet 113 is preferably 1 mm or more and 20 mm or less.
- the inlet 113 may be flat as shown in FIG. 8B, or may be provided with a groove structure or an uneven structure so that liquid can be easily injected.
- the second electrolyte solution injection step it is preferable to inject an appropriate amount of the second electrolyte solution 303 from the injection port 113 into the third chamber 202 by capillary action.
- the second electrolytic solution 303 is injected into the third chamber 202 by capillary action, it is preferable that at least a part of the outer peripheral surface of the cover 106 is subjected to a hydrophilic treatment.
- the portion in contact with the second electrolytic solution 303 is preferably subjected to a hydrophilic treatment.
- the vicinity of the second through hole 110 is preferably subjected to a hydrophilic treatment.
- oxygen plasma treatment may be performed, or a cover may be coated with a hydrophilic material.
- the artificial lipid membrane device 100 may be installed and operated in the direction shown in FIG. 8A, or may be operated in another direction.
- the artificial lipid film forming apparatus 100 shown in FIG. 8A may be installed and operated in a direction rotated 90 degrees counterclockwise within the paper surface.
- the openings are directed in the same direction, (2) when the second electrolyte solution 303 is injected into the third chamber 202, a capillary phenomenon can be used, so that it is easy. As a result, an artificial lipid membrane can be easily formed.
- Embodiment 3 (Embodiment 3)
- the artificial lipid film forming method in Embodiment 3 of the present invention will be described with reference to the drawings.
- 10 (a) and 10 (b) are a cross-sectional view and an oblique projection view of the artificial lipid film forming apparatus according to the third embodiment.
- the same parts as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.
- the difference between the present embodiment and the first embodiment is the shape of the first opening 111 and the second opening 112.
- the first opening 111 may be a through hole formed in the first thin film 103, the second thin film 105, and the cover 106.
- the second opening 112 may be a through-hole formed in the second thin film 105 and the cover 106.
- the openings are directed in the same direction, (2) the openings and the injection port can be made small, so the solution is difficult to evaporate, and as a result, an artificial lipid membrane is simply formed. it can.
- Embodiment 4 the artificial lipid film forming method in Embodiment 4 of the present invention will be described with reference to the drawings.
- FIG. 11 is a cross-sectional view and an oblique projection view of the artificial lipid film forming apparatus according to the fourth embodiment.
- the same parts as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.
- the difference between the present embodiment and the first embodiment is that an electrode 401 is provided in the first chamber 107 and the inlet 113.
- the electrode 401 may be one or plural.
- the electrode 401 is preferably an electrode suitable for electrochemical measurement.
- a non-polarizable electrode is preferred.
- the electrode 401 is most preferably an Ag / AgCl electrode, but may be a saturated calomel electrode, a hydrogen electrode, or the like.
- the electrode 401 may be a metal electrode such as an Ag electrode, a Pt electrode, or an Au electrode, or may be a carbon electrode, a graphite electrode, a carbon nanotube electrode, or the like.
- the conductance and electric capacity of the artificial lipid membrane may be measured using the electrode 401.
- the electrode 401 may be used to measure chemical substances such as ions, enzymes, reaction products, and substrates contained in the first electrolytic solution 301 or the second electrolytic solution 303.
- the electrode shape and size are not limited.
- the position of the electrode 401 is in the first chamber 107 and the inlet 113 and is provided in the vicinity of the first through hole 108 and the second through hole 110.
- the electrodes 401a and 401b may be the same electrode or different.
- a plurality of electrodes may be combined.
- the electrode 401 may be provided in advance before forming the artificial lipid membrane, or may be provided after forming the artificial lipid membrane.
- the electrode 401 may be fixed to the artificial lipid film forming apparatus 100 or may be removable.
- the electrode 401 a provided in the first chamber 107 is preferably formed on the outer peripheral surface of the substrate 101.
- the electrode 401 b provided at the inlet 113 is preferably formed on the outer peripheral surface of the second thin film 105 or the outer peripheral surface of the cover 106.
- the amplifier is most preferably a patch clamp amplifier, but an amplifier such as a field effect transistor, bipolar transistor, operational amplifier, or operational amplifier may be connected.
- the progress and end point of each process until the artificial lipid membrane fat is formed can be detected using the electrode 401.
- the first electrolyte solution injection step if two electrodes are provided in the first chamber 107 and the electrical conductivity between the two electrodes is measured, it is easily detected that the injection of the first electrolyte solution 301 has been completed. it can.
- the path for injecting the lipid solution and the path for injecting the electrolyte are provided separately, the electrode immersed in the electrolyte is not contaminated by the lipid solution. Therefore, since no troublesome steps such as protection of the electrode surface and cleaning of the electrode surface are required, an artificial lipid membrane can be easily formed.
- Embodiment 5 (Embodiment 5)
- the artificial lipid film forming method in Embodiment 5 of the present invention will be described with reference to the drawings.
- FIG. 12 is a cross-sectional view and an oblique projection of the artificial lipid film forming apparatus in the fifth embodiment.
- the same parts as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.
- a sensor 402 is provided in the first chamber 107.
- the sensor 402 may be provided in advance before forming the artificial lipid film, or may be provided after forming the artificial lipid film.
- the sensor 402 may be fixed to the artificial lipid film forming apparatus 100 or may be removable.
- the sensor 402 provided in the first chamber 107 is most preferably formed on the outer peripheral surface of the substrate 101.
- the senor 402 is most preferably a sensor suitable for electrochemical measurement.
- the sensor 402 is most preferably an ion electrode or an ion sensitive field effect transistor (ISFET).
- the ion electrode is preferably a potassium ion electrode, a sodium ion electrode, a calcium ion electrode, a chloride ion electrode or the like. It is preferable to detect potassium ions, sodium ions, calcium ions, chloride ions, etc. with an ion sensitive field effect transistor.
- the sensor 402 may be an optode, a QCM (Quartz crystal microbalance), a SAW (Surface acoustic wave) sensor, an SPR (Surface plasma resonance), an LSPR (Localized surface plasma microbalance), an organic electrochemical transistor, an enzyme sensor, or the like.
- a light source or detector for measuring optical properties such as absorbance and reflectance may be provided.
- the number, shape, and size of the sensor 402 are not limited.
- the position of the sensor 402 is more preferably provided in the first chamber 107 and in the vicinity of the first through hole 108.
- the sensor 402 may be provided at the inlet 113.
- the sensor 402 is preferably formed on the outer peripheral surface of the second thin film 105 or the outer peripheral surface of the cover 106.
- an electrode 403 may be provided at the inlet 113.
- the electrode 403 may be provided in advance before forming the artificial lipid membrane, or may be provided after forming the artificial lipid membrane.
- the electrode 403 may be fixed to the artificial lipid film forming apparatus 100 or may be removable.
- the electrode 403 is preferably formed on the outer peripheral surface of the second thin film 105 or the outer peripheral surface of the cover 106.
- the electrode 403 may be provided in the first chamber 107. When the electrode 403 is provided in the first chamber 107, it is most preferably formed on the outer peripheral surface of the substrate 101, and may be formed on the outer peripheral surface of the first thin film 103.
- the electrode 403 is preferably an electrode suitable for electrochemical measurement.
- a non-polarizable electrode is preferred.
- the electrode 403 is most preferably an Ag / AgCl electrode, but may be a saturated calomel electrode, a hydrogen electrode, or the like.
- the electrode 403 may be a metal electrode such as an Ag electrode, a Pt electrode, or an Au electrode, or may be a carbon electrode, a graphite electrode, a carbon nanotube electrode, or the like.
- the conductance and electric capacity of the artificial lipid membrane may be measured using the electrode 403.
- the electrode 403 may be used to measure chemical substances such as ions, enzymes, reaction products, and substrates contained in the first electrolytic solution 301 or the second electrolytic solution 303.
- the electrode shape and size are not limited.
- the position of the electrode 403 is more preferably provided in the vicinity of the second through-hole 110 in the inlet 113.
- the electrode 403 may be used as a reference electrode.
- the progress and end point of each process until the formation of artificial lipid membrane fat can be detected using the sensor 402.
- the sensor 402. For example, in the first electrolyte solution injection step, by providing an ion electrode as the sensor 402 in the first chamber 107, it can be easily detected that the injection of the first electrolyte solution 301 is completed.
- Borosilicate glass was used as the substrate 101.
- the borosilicate glass was 22 mm ⁇ 22 mm ⁇ 0.17 mm.
- borosilicate glass was ultrasonically washed with pure water, ethanol, and acetone for 10 minutes each.
- the outer peripheral surface of the borosilicate glass was hydrophilized with a UV ozone asher. The treatment time was 5 minutes.
- the first spacer 102, the first thin film 103, the second spacer 104, and the second thin film 105 were Teflon (registered trademark) films having a thickness of 100 ⁇ m.
- a single Teflon (registered trademark) film was used for the first spacer 102, the first thin film 103, the second spacer 104, and the second thin film 105.
- the size of the Teflon (registered trademark) film was 20 mm ⁇ 10 mm.
- One Teflon (registered trademark) film was folded at the center and molded.
- the cover 106 was made of polydimethylsiloxane (PDMS). Polydimethylsiloxane was formed into a film having a thickness of 0.5 mm, and a through hole having a diameter of 3 mm was formed at a position where the solution could be injected into the second through hole 110.
- PDMS polydimethylsiloxane
- the first through hole 108 was formed by a drill so as to penetrate both surfaces of the first thin film 103.
- the diameter of the first through hole 108 was 200 ⁇ m.
- the second through hole 110 was formed by a drill so as to penetrate both surfaces of the second thin film 105.
- the diameter of the second through hole 110 was 200 ⁇ m.
- the first through hole 108 and the second through hole 110 were simultaneously formed in a state where the first thin film 103 and the second thin film 105 were overlapped in order to suppress displacement.
- the first through hole 108 and the second through hole 110 are formed after the first through hole 108 and the second through hole 110 are separately formed in the first thin film 103 and the second thin film 105, respectively.
- the first thin film 103 and the second thin film 105 may be stacked.
- the first through hole 108 and the second through hole 110 were formed at a position 2 mm from one side of the first thin film 103 and the second thin film 105.
- the first through hole 108 and the second through hole 110 are preferably formed at a position of 0.5 mm to 3 mm from one side of the first thin film 103 and the second thin film 105.
- the second opening 112 was provided in the second thin film 105.
- the second opening 112 was a circular hole having a diameter of 1 mm that penetrated the second thin film 105.
- the second opening 112 was formed by a drill.
- the first opening 111 was provided in the first thin film 103 and the second thin film 105.
- the first opening 111 was a circular hole having a diameter of 1 mm that penetrates the first thin film 103 and the second thin film 105.
- the first opening 111 was formed by a drill simultaneously with the first thin film 103 and the second thin film 105 overlapped.
- the substrate 101, the first spacer 102, the first thin film 103, the second spacer 104, the second thin film 105, and the cover 106 were laminated.
- the substrate 101, the first spacer 102, the first thin film 103, the second spacer 104, the second thin film 105, and the periphery of the cover 106 laminated so as not to leak the solution from the first chamber 107 and the second chamber 109 are bonded by epoxy. Sealed with an agent.
- the cover 106 was spontaneously adhered to the second thin film 105.
- the laminated substrate 101, the first spacer 102, the first thin film 103, the second spacer 104, the second thin film 105, and the cover 106 are sandwiched between two polycarbonate plates (36 mm ⁇ 36 mm ⁇ 1 mm), and the polycarbonate plate 4 corners were fixed with bolts.
- the polycarbonate plate in contact with the cover 106 was provided with one circular hole having a diameter of 9 mm so that the solution could be injected into the first opening 111, the second opening 112, and the injection port 113.
- the procedure for forming an artificial lipid membrane will be described.
- the 1st electrolyte solution injection process was performed.
- a 1M KCl solution was used as the first electrolyte solution 301.
- the amount of the first electrolytic solution 301 was 1 ⁇ L.
- the first electrolytic solution 301 was dropped into the first opening 111.
- the dropped first electrolyte solution 301 was injected into the first chamber 107 by capillary action. Since the inner wall surface of the first chamber 107 was subjected to hydrophilic treatment, the first electrolyte solution 301 could be easily injected.
- the time required to inject the first electrolytic solution 301 was 1 second or less.
- the first chamber 107 was filled with the first electrolytic solution except for the first through hole 108 and the region surrounding the first through hole 108. Thereafter, the peripheral region of the first through hole 108 and the first through hole 108 were filled with the first electrolytic solution 301. The state in which the first electrolyte solution 301 was injected into the first chamber 107 was observed with an optical microscope.
- the lipid solution injection step was performed after the first electrolyte solution injection step.
- a mixed solution of phospholipid (1,2-diphytanoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids) and an organic solvent (chloroform) was used as the lipid solution 302 .
- the concentration of the lipid solution 302 was 25 mg / mL.
- the lipid solution 302 was dropped into the second opening 112.
- the liquid volume of the lipid solution 302 was 0.4 ⁇ L.
- the lipid solution 302 was injected into the first through hole 108 and the second through hole 110 via the second chamber 109.
- the lipid solution 302 was injected by capillary action.
- the lipid solution 302 could be easily injected.
- the time required to inject the lipid solution 302 was 1 second or less.
- the state in which the lipid solution 302 was injected into the first through hole 108 and the second through hole 110 was observed with an optical microscope.
- the second electrolyte solution injection step was performed after the lipid solution injection step.
- a 1M KCl solution was used as the second electrolytic solution 303.
- the amount of the second electrolytic solution 303 was 2 ⁇ L.
- the second electrolytic solution 303 was dropped into the injection port 113.
- the second electrolytic solution 303 was injected along the inner wall of the injection port 113.
- the time required to inject the second electrolyte solution 303 was 1 second or less.
- the state in which the second electrolyte solution 303 was injected into the injection port 113 was observed visually and with an optical microscope.
- FIGS. 13A and 13B are micrographs of artificial lipid membranes.
- FIG. 13B shows the boundary line between the first through-hole 108 and the artificial lipid membrane region 501 in FIG. 13A by a white dotted line.
- the region 501 appeared darker than the surrounding area. With the passage of time, the area of the region 501 expanded, and it was possible to observe how the artificial lipid membrane was made thinner.
- an electrode 401a and an electrode 401b were provided in the first opening 111 and the injection port 113, respectively.
- a patch clamp amplifier (EPC-10, HEKA) was used to measure the electrical characteristics.
- the electrode 401a was connected to the ground line, and the electrode 401b was connected to the signal line.
- a pulse voltage of 5 mV and 10 msec was applied to the electrode 401b with respect to the electrode 401a.
- the transient response of the current flowing through the artificial lipid membrane was recorded.
- FIG. 14 shows the transient response of the current flowing through the artificial lipid membrane. The transient response was measured by the capacitive membrane current found in artificial lipid membranes.
- the electrode 401a and the electrode 401b provided in the first opening 111 and the injection port 113 keep the electrode surface clean without touching the lipid solution 302.
- An artificial lipid membrane could be formed.
- the electrical properties of the artificial lipid membrane were successfully measured.
- FIG. 15 shows an artificial lipid film forming apparatus of a comparative example.
- the first chamber 601 was prepared by opening a circular through hole having a diameter of 1.5 mm in the thin film 602.
- the first chamber 601 was produced by punching.
- Polydimethylsiloxane was used as the thin film 602.
- the size of the thin film 602 was 10 mm ⁇ 10 mm ⁇ 0.5 mm.
- the through-hole 603 forming the artificial lipid membrane was prepared by opening a circular through-hole having a diameter of 200 ⁇ m in a Teflon (registered trademark) film 604 having a thickness of 100 ⁇ m.
- the through hole 603 was formed by a drill.
- the injection port 605 was prepared by opening a circular through hole having a diameter of 1.5 mm in the thin film 606.
- the inlet 605 was produced by punching.
- Polydimethylsiloxane was used as the thin film 606.
- the size of the thin film 606 was 10 mm ⁇ 10 mm ⁇ 0.5 mm.
- a Teflon (registered trademark) film 604, a thin film 602, and a thin film 606 were laminated on a glass substrate 607.
- Borosilicate glass was used as the glass substrate 607.
- the size of the glass substrate 607 was 22 mm ⁇ 22 mm ⁇ 0.17 mm.
- an electrolytic solution was injected into the first chamber 601 using a micropipette.
- 1M KCl was used as the electrolyte.
- the amount of electrolyte injected was 1 ⁇ L.
- a Teflon (registered trademark) film 604 was laminated on the thin film 602. Excess electrolyte was removed.
- a thin film 606 was laminated on the Teflon (registered trademark) film 604.
- the electrolyte solution was inject
- 1M KCl was used as the electrolyte. The amount of electrolyte injected was 1 ⁇ L.
- the lipid solution was sprayed to the through-hole 603 using a micropipette.
- a mixed solution of phospholipid (1,2-diphytanoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids) and an organic solvent (chloroform, Wako Pure Chemical Industries) was used as the lipid solution.
- the concentration of the lipid solution was 25 mg / mL.
- FIGS. 16A and 16B are optical micrographs of the artificial lipid membrane in the comparative example.
- FIG. 16B clearly shows the boundary line between the through hole 603 and the artificial lipid membrane region 608 in FIG. 16A by a white dotted line.
- the area 608 appeared darker than the surrounding area. It was observed that the area of the region 608 increased with the passage of time, and the artificial lipid membrane became thinner.
- FIG. 17 is a diagram comparing the formation rates of artificial lipid membranes in the examples of the present invention and comparative examples.
- the formation rate mentioned here is a numerical value obtained by dividing the number of times that an artificial lipid membrane has been formed by the number of trials and multiplying by 100. Specifically, an attempt was made to form an artificial lipid membrane 10 times each in the examples and comparative examples. As a result, an artificial lipid membrane could be formed at a formation rate of 60% in the examples and 10% in the comparative example. This result shows that an artificial lipid membrane can be formed at a high rate by a simple operation as compared with the conventional example.
- the artificial lipid membrane can be formed in a shorter time and more simply than the conventional artificial lipid membrane forming method and artificial lipid forming apparatus.
- artificial lipid membranes incorporating membrane proteins such as ion channels and receptors can be applied to basic structural analysis, functional elucidation of membrane proteins, and membrane protein-membrane protein correlation studies.
- the present invention not only directly contributes to the above-described research development, but also has potential for industrial application in the medical and pharmaceutical fields such as diagnosis of diseases caused by ion channels and screening for new drug development.
- it can be applied to biosensors and the like by utilizing the specific molecular recognizability of membrane proteins.
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Abstract
Description
人工脂質膜形成装置を用いた人工脂質膜形成方法であって、
前記装置は、
基板と、
前記基板の一端に設けられた第1スペーサと、
前記第1スペーサを介して前記基板に設けられた第1薄膜と、
前記第1薄膜の一端に設けられた第2スペーサと、
前記第2スペーサを介して前記第1薄膜に設けられた第2薄膜と、
第2薄膜の一端に設けられたカバーと
を備え、
前記基板と前記第1薄膜との間には第1チャンバが備えられ、
前記第1薄膜は両面を貫通する第1貫通孔を備え、
前記第1薄膜と前記第2薄膜との間には第2チャンバが備えられ、
前記第2薄膜は両面を貫通する第2貫通孔を備え、
前記カバーには前記第2貫通孔へ接続する注入口が備えられ、
平面視において前記第1貫通孔は前記第2貫通孔と重なり、
前記第1チャンバは、前記第1貫通孔と前記第2貫通孔を介して前記注入口に接続され、
前記方法は、
前記第1チャンバへ電解液を注入する第1電解液注入工程、
前記第2チャンバを経て、前記第1貫通孔または前記第2貫通孔の少なくとも一方へ、脂質溶液を注入する脂質溶液注入工程、および
前記注入口へ電解液を注入して、前記脂質溶液が注入された貫通孔の内部に人工脂質膜を形成する第2電解液注入工程、
を順に包含する人工脂質膜形成方法である。
人工脂質膜形成装置であって、
前記装置は、
基板と、
前記基板の一端に設けられた第1スペーサと、
前記第1スペーサを介して前記基板に設けられた第1薄膜と、
前記第1薄膜の一端に設けられた第2スペーサと、
前記第2スペーサを介して前記第1薄膜に設けられた第2薄膜と、
第2薄膜の一端に設けられたカバーと
を備え、
前記基板と前記第1薄膜との間には第1チャンバが備えられ、
前記第1薄膜は両面を貫通する第1貫通孔を備え、
前記第1薄膜と前記第2薄膜との間には第2チャンバが備えられ、
前記第2薄膜は両面を貫通する第2貫通孔を備え、
前記カバーには前記第2貫通孔へ接続する注入口が備えられ、
平面視において前記第1貫通孔は前記第2貫通孔と重なり、
前記第1チャンバは、前記第1貫通孔と前記第2貫通孔を介して前記注入口に接続されている、人工脂質膜形成装置である。
以下、本発明の実施形態について、図面を参照しながら説明する。
以下、本発明の実施形態2における人工脂質膜形成方法について、図面を参照しながら説明する。
以下、本発明の実施形態3における人工脂質膜形成方法について、図面を参照しながら説明する。
以下、本発明の実施形態4における人工脂質膜形成方法について、図面を参照しながら説明する。
以下、本発明の実施形態5における人工脂質膜形成方法について、図面を参照しながら説明する。
まず、人工脂質膜形成装置の作製方法を説明する。基板101としてホウケイ酸ガラスを用いた。ホウケイ酸ガラスは22mm×22mm×0.17mmであった。まず、ホウケイ酸ガラスを純水、エタノール、アセトンで10分ずつ超音波洗浄した。次に、UVオゾンアッシャーによりホウケイ酸ガラスの外周面を親水化処理した。処理時間は5分間とした。
従来の人工脂質膜形成方法の1つである、泡吹き付け法を用いて、人工脂質膜を形成した。
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 基板
102 第1スペーサ
103 第1薄膜
104 第2スペーサ
105 第2薄膜
106 カバー
107 第1チャンバ
108 第1貫通孔
108a、108b 断面
109 第2チャンバ
110 第2貫通孔
110a、110b 断面
110c 内壁面
111 第1開口
112 第2開口
113 注入口
113a 内壁面
114 第1チャンバの高さ
115 第2チャンバの高さ
120 第1チャンバの幅
121 第1チャンバの長さ
122 第1貫通孔の直径
123 第2チャンバの幅
124 第2チャンバの長さ
125 第2貫通孔の直径
126 第3チャンバの幅
127 第3チャンバの長さ
130 第1開口の張り出し部の長さ
131 第1開口の張り出し部の幅
132 第2開口の張り出し部の長さ
133 第2開口の張り出し部の幅
134 注入口の直径
191 第1張り出し部
192 第2張り出し部
193 第3張り出し部
201 第3スペーサ
202 第3チャンバ
203 第3チャンバの高さ
204 注入口の張り出し部の長さ
205 注入口の張り出し部の幅
301 第1電解液
302 脂質溶液
303 第2電解液
401、401a、401b 電極
402 センサ
403 電極
501 領域
601 第1チャンバ
602 薄膜
603 貫通孔
604 テフロン(登録商標)フィルム
605 注入口
606 薄膜
607 ガラス基板
608 領域
Claims (20)
- 人工脂質膜形成装置を用いた人工脂質膜形成方法であって、
前記装置は、
基板と、
前記基板の一端に設けられた第1スペーサと、
前記第1スペーサを介して前記基板に設けられた第1薄膜と、
前記第1薄膜の一端に設けられた第2スペーサと、
前記第2スペーサを介して前記第1薄膜に設けられた第2薄膜と、
第2薄膜の一端に設けられたカバーと
を備え、
前記基板と前記第1薄膜との間には第1チャンバが備えられ、
前記第1薄膜は両面を貫通する第1貫通孔を備え、
前記第1薄膜と前記第2薄膜との間には第2チャンバが備えられ、
前記第2薄膜は両面を貫通する第2貫通孔を備え、
前記カバーには前記第2貫通孔へ接続する注入口が備えられ、
平面視において前記第1貫通孔は前記第2貫通孔と重なり、
前記第1チャンバは、前記第1貫通孔と前記第2貫通孔を介して前記注入口に接続され、
前記方法は、
前記第1チャンバへ電解液を注入する第1電解液注入工程、
前記第2チャンバを経て、前記第1貫通孔または前記第2貫通孔の少なくとも一方へ、脂質溶液を注入する脂質溶液注入工程、および
前記注入口へ電解液を注入して、前記脂質溶液が注入された貫通孔の内部に人工脂質膜を形成する第2電解液注入工程、
を順に包含する人工脂質膜形成方法。 - 前記第1薄膜、第1スペーサ、および前記第2薄膜は、一体的に形成されている、請求項1に記載の人工脂質膜形成方法。
- 前記第1貫通孔は、前記第2貫通孔の断面積と同じ断面積を有する、請求項1に記載の人工脂質膜形成方法。
- 前記注入口は、平面視において前記第1チャンバと重なり合う、請求項1に記載の人工脂質膜形成方法。
- 前記第1チャンバの外周面は、親水性を有する、請求項1に記載の人工脂質膜形成方法。
- 前記第2チャンバの外周面は、疎水性を有する、請求項1に記載の人工脂質膜形成方法。
- 前記注入口の外周面は、親水性を有する、請求項1に記載の人工脂質膜形成方法。
- 前記第1チャンバおよび前記注入口のうち少なくとも一方は、電極を備えている、請求項1に記載の人工脂質膜形成方法。
- 前記第1チャンバおよび前記注入口のうち少なくとも一方は、センサを備えている、請求項1に記載の人工脂質膜形成方法。
- 前記第1電解液注入工程では、毛細管現象により前記第1チャンバへ前記電解液が注入される、請求項1に記載の人工脂質膜形成方法。
- 前記脂質溶液注入工程では、毛細管現象により前記第1貫通孔または前記第2貫通孔の少なくとも一方へ、前記脂質溶液が注入される、請求項1に記載の人工脂質膜形成方法。
- 人工脂質膜形成装置であって、
前記装置は、
基板と、
前記基板の一端に設けられた第1スペーサと、
前記第1スペーサを介して前記基板に設けられた第1薄膜と、
前記第1薄膜の一端に設けられた第2スペーサと、
前記第2スペーサを介して前記第1薄膜に設けられた第2薄膜と、
第2薄膜の一端に設けられたカバーと
を備え、
前記基板と前記第1薄膜との間には第1チャンバが備えられ、
前記第1薄膜は両面を貫通する第1貫通孔を備え、
前記第1薄膜と前記第2薄膜との間には第2チャンバが備えられ、
前記第2薄膜は両面を貫通する第2貫通孔を備え、
前記カバーには前記第2貫通孔へ接続する注入口が備えられ、
平面視において前記第1貫通孔は前記第2貫通孔と重なり、
前記第1チャンバは、前記第1貫通孔と前記第2貫通孔を介して前記注入口に接続されている、人工脂質膜形成装置。 - 前記第1薄膜、第1スペーサ、および前記第2薄膜は、一体的に形成されている、請求項12に記載の人工脂質膜形成装置。
- 前記第1貫通孔は、前記第2貫通孔の断面積と同じ断面積を有する、請求項12に記載の人工脂質膜形成装置。
- 前記注入口は、平面視において前記第1チャンバと重なり合う、請求項12に記載の人工脂質膜形成装置。
- 前記第1チャンバの外周面は、親水性を有する、請求項12に記載の人工脂質膜形成装置。
- 前記第2チャンバの外周面は、疎水性を有する、請求項12に記載の人工脂質膜形成装置。
- 前記注入口の外周面は、親水性を有する、請求項12に記載の人工脂質膜形成装置。
- 前記第1チャンバと前記注入口のうち少なくとも一方は、電極を備えている、請求項12に記載の人工脂質膜形成装置。
- 前記第1チャンバと前記注入口のうち少なくとも一方は、センサを備えている、請求項12に記載の人工脂質膜形成装置。
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JP2011149868A (ja) * | 2010-01-22 | 2011-08-04 | Kanagawa Acad Of Sci & Technol | 脂質二重膜、それを形成するために用いられる自己支持性フィルム及びそれを具備するマイクロ流路デバイス |
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WO2017170075A1 (ja) * | 2016-03-30 | 2017-10-05 | シャープ ライフ サイエンス (イーユー)リミテッド | 微小流体装置 |
US11040345B2 (en) | 2016-03-30 | 2021-06-22 | Sharp Life Science (Eu) Limited | Microfluidic device |
JP2018140478A (ja) * | 2017-02-28 | 2018-09-13 | 国立大学法人東北大学 | シリコンチップ及びその製造方法 |
WO2019142784A1 (ja) * | 2018-01-19 | 2019-07-25 | 地方独立行政法人神奈川県立産業技術総合研究所 | 脂質二重膜形成用隔壁及びその製造方法 |
US11607871B2 (en) | 2018-01-19 | 2023-03-21 | Kanagawa Institute Of Industrial Science And Technology | Partition wall for formation of lipid bilayer membrane, and method for producing same |
Also Published As
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JPWO2010023848A1 (ja) | 2012-01-26 |
US7828947B2 (en) | 2010-11-09 |
CN101971013B (zh) | 2013-06-19 |
US20100213070A1 (en) | 2010-08-26 |
CN101971013A (zh) | 2011-02-09 |
JP4469024B2 (ja) | 2010-05-26 |
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