JP4717961B2 - Artificial lipid membrane formation method - Google Patents

Description

  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.

  Examples of conventional methods for forming artificial lipid membranes are (1) a bubble spraying method, (2) a bonding method, and (3) μTAS (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. In FIG. 20, 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.

  Examples of such lipids are phospholipids such as diphytanoyl phosphatidylcholine or glycerol monooleate. Examples of such organic solvents are saturated hydrocarbons such as decane, hexadecane or hexane.

  21A to 21C show a conventional method for forming an artificial lipid membrane by a bonding method. In 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.

  First, 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. Next, 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. As shown in FIG. 21A, 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.

  Then, as shown in FIG. 21B, the electrolytic solution 23 is injected from the injection port 24 until the level of the electrolytic solution 23 passes the height of the upper end of the minute hole 22.

  The same operation is performed in the other chamber of the container 20. That is, as shown in FIG. 21C, 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. Next, 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. In this manner, 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.

  Using the above-mentioned two methods to form a highly reproducible and stable artificial lipid membrane requires skill.

  Patent Documents 1 to 4 disclose a method of forming an artificial lipid film using the μTAS technique in order to form a simpler artificial lipid film.

  FIG. 22 shows a small artificial lipid film forming apparatus using the μTAS technology 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. Using the artificial lipid film forming apparatus, 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)

Okada Yasunobu "Patch clamp experiment technique" Yoshioka Shoten September 25, 1996 (p.133-139)

  Since the artificial lipid film forming apparatus disclosed in Patent Document 1 is small and easy to carry, it has very excellent convenience. However, during or after the formation of the artificial lipid membrane, 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. As a result, the periphery of the artificial lipid film forming apparatus is contaminated by the electrolytic solution. Furthermore, since 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 of preparing the following artificial lipid film forming apparatus (100)
Here, 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 μl,
The second chamber (105) has a volume of 10 pl to 200 μl,
A step B of injecting a first electrolytic solution (201) having a viscosity of 1.3 mPa · s to 200 mPa · s into the first chamber (104);
A step C of injecting a lipid solution (202) containing a lipid (203) and an organic solvent into the artificial lipid film forming unit (103);
A second electrolytic solution (204) having a viscosity of 1.3 mPa · s to 200 mPa · s is injected into the second chamber (105), and the first electrolytic solution (201) and the second electrolytic solution (204). Step D for sandwiching the lipid solution (202) between the steps, and Step E for removing the organic solvent to form an artificial lipid membrane in the artificial lipid membrane forming part (103)
About.

  At least one of the first electrolytic solution (201) or the second electrolytic solution (204) preferably 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 electrolyte solution (201) and the second electrolyte solution (204) preferably contains a polymer.

  The polymer is preferably polyvinyl alcohol.

  In the step B, it is preferable that the first electrolyte solution (201) is injected into the first chamber (104) by an inkjet method.

  In the step D, the second electrolyte solution (204) is preferably injected into the second chamber (105) by an ink jet method.

  In the step C, 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.

  In the step B, the first chamber (104) is preferably filled with the first electrolyte (201).

  In the step D, the second chamber (105) is preferably filled with the second electrolyte (204).

  The above object, other objects, features and advantages of the present invention will become apparent from the following detailed description of preferred embodiments with reference to the accompanying drawings.

  According to 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. 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.

(Embodiment 1)
Embodiment 1 of the present invention will be described below with reference to the drawings.

<Process A: 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.

  In Embodiment 1, 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 as the inorganic material. Soda glass, quartz, borosilicate glass, low melting point glass, photosensitive glass can be used. As an inorganic material other than glass, 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 is preferably hydrophilic. In order to make at least a part of the outer peripheral surface of the container 101 hydrophilic, 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 μl 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 μm 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.

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 Ωcm or more, and more preferably 10 ¹² Ωcm 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 μm or more and 500 μm or less. The thickness of the partition wall 102 may be the same over the entire surface, or may be different. 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.

  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 unit 103 is preferably provided at the center 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.

When the artificial lipid membrane forming portion 103 is a through-hole having a circular cross section, 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 μm or less. The area of the artificial lipid membrane forming part 103, that is, the area of the figure 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 μl 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 via 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 μl 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.

  Next, a procedure for forming an artificial lipid membrane will be described. 4 and 5 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.

<Process B: 1st electrolyte solution injection process>
FIG. 4 shows a first electrolyte solution injection step. In the 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).

Cl ⁻ 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 preferably 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. As the organic compound, 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. As sugars, monosaccharides, disaccharides, trisaccharides, tetrasaccharides or polysaccharides can be used.
It is preferable that the 1st electrolyte solution 201 has a viscosity higher than the viscosity (1.0 mPa * s) of the pure water in 20 degreeC.

  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 μΩm or more and 100 kΩm or less, and more preferably 1 mΩm or more and 10Ωm 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 electrolyte 201 injected into the first chamber 104 is preferably 10 pl to 200 μl, and more preferably 1 nl to 200 μl. 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. As 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 electrolyte 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.

  From the viewpoint of suppressing the drying of the first electrolytic solution 201, 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. In the first electrolyte solution injection step, the relative humidity around the artificial lipid film forming apparatus 100 is preferably maintained at 50% or more and 100% or less.

  It is preferable to remove dust or congestion contained in the first electrolyte 201 through the membrane filter.

  In the first electrolyte solution injection step, the first electrolyte solution 201 is preferably injected into the first chamber 104 by capillary force, gravity, surface tension, or centrifugal force.

  In 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. In 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 electrolyte 201 is most preferably a uniform electrolyte 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 mPa · s 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.

  Since 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.

<Step C: Lipid solution injection step>
FIG. 5 (a) shows a lipid solution injection step. In the lipid solution injection step, the lipid solution 202 is injected into the artificial lipid film forming unit 103. In the lipid solution injection step, it is preferable to inject the lipid solution 202 from the second chamber 105 side.

  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. As the 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. As the lipid 203, 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 the lipid 203 with respect to the organic solvent is preferably 1 to 50 mg / ml, and more preferably 4 to 40 mg / ml.

  In addition to the lipid 203 and the organic solvent, 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.

  When 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. Alternatively, a known mixing technique may be used. When 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 to be injected into the artificial lipid film forming unit 103 is preferably 1 pl or more and 10 μl or less, and more preferably 1 nl or more and 2 μl or less from the viewpoint of ease of creating 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.

  In the lipid solution injection step, the lipid solution 202 is preferably injected into the artificial lipid film forming unit 103 by capillary force, gravity, surface tension, or centrifugal force.

  In 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.

  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 injecting the lipid solution into the artificial lipid film forming unit 103 using a pipette. May end up. For this reason, attention is required for operation.

<Step D: Second electrolyte injection step>
FIG. 5B shows a second electrolyte solution injection step. In the 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 ⁻ 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 substituted 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 electrolytic 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.

  It is preferable that the 2nd electrolyte solution 204 has a viscosity higher than the viscosity (1.0 mPa * s) of the pure water in 20 degreeC.

  The electrical resistivity of the second electrolytic solution 204 is preferably 1 μΩm or more and 100 kΩm or less, and more preferably 1 mΩm or more and 10Ωm 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 to 200 μl, and more preferably 1 nl to 200 μl. 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 by a pipette, but may be injected into the second chamber 105 by 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. As a method for injecting the droplet-like second electrolytic solution 204, 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. 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.

  From the viewpoint of suppressing the drying of the second electrolyte solution 204, the second electrolyte solution 204 is preferably maintained at room temperature in the second electrolyte 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.

  It is preferable to remove dust or congestion contained in the second electrolytic solution 204 through the membrane filter.

  In the second electrolyte solution injection step, the second electrolyte solution 204 is preferably injected into the second chamber 105 by capillary force, gravity, surface tension, or centrifugal force.

  In 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. In the second electrolyte solution injection step, it is preferable to inject the second electrolyte solution 204 into the second chamber 105 until the upper end of the artificial lipid film forming unit 103 is exceeded. In 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 obtained by combining a plurality of electrolytic solutions having a viscosity of 1.3 mPa · s to 200 mPa · s. 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.

  Since 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.

<Process E: Artificial lipid film formation process>
FIG. 5 (c) shows an artificial lipid film formation step. In the artificial lipid film forming 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. In the artificial lipid film forming step, it is preferable to remove excess organic solvent and lipid 203 from the thin film of the lipid solution 202 by the hydraulic pressure of the first electrolytic solution 201 and the second electrolytic solution 204 or by external pressure. 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.

  In the artificial lipid film forming step, 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.

  In the artificial lipid film forming step, a voltage may be applied to both surfaces of the artificial lipid film 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 film forming step may include a step of detecting the formation of the artificial lipid film 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.

  Since the first electrolytic solution 201 is in a 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.

  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 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.

  When the electrode 108 is flat, the length, width, and thickness of the electrode 108 are each preferably 10 nm or more and 1 cm or less. When the electrode 108 is a thin film, the length and width of the electrode 108 are each preferably 10 nm or more and 1 cm or less. When the electrode 108 is a thin film, the thickness of the electrode 108 is preferably 10 nm or more and 100 μm or less, and more preferably 50 nm or more and 1 μm 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.

  There may be one electrode 108 or a plurality of electrodes 108. 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.

  According to this configuration and operation procedure, 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.

  In the first embodiment, as shown in FIG. 1, it is preferable that 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.

  During the process of forming the artificial lipid film 205, 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. . After the artificial lipid film 205 is formed, 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.

  However, in 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.

  As shown in FIG. 6, 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.

  As shown in FIG. 8, 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.

  In Embodiment 1, 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. In the present 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.

  In Embodiment 1, 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. .

  A biosensor can be manufactured using the artificial lipid film formation method of Embodiment 1. 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.

  Compared with the conventional solid gel, the substance to be detected diffuses quickly in the fluid electrolyte. Therefore, a biosensor using an electrolytic solution is expected to be sensed more rapidly than a biosensor using a solid gel. The biosensor using the artificial lipid film forming method of Embodiment 1 is preferably applied to an analyzer. Examples of 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. , 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.

(Embodiment 2)
Embodiment 2 of the present invention will be described below with reference to the drawings.

<Process A: 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. In the second embodiment, the same components as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  The biggest difference between the second embodiment and the first embodiment is that in the second embodiment, the artificial lipid membrane 205 is formed by the μTAS technique. Specifically, 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 μTAS 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. Most preferably, the material of the first substrate 301 is an insulator.

  It is preferable that at least a part of the outer peripheral surface of the first substrate 301 has hydrophilicity. In order to make at least a part of the outer peripheral surface of the first substrate 301 hydrophilic, 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 film 205 can be observed, but may be opaque.

  The first substrate 301 is most preferably a flat plate, but may be a disc, trapezoid, polygon, column, or prism.

  The first substrate 301 is preferably molded 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.

  As in Embodiment 1, the artificial lipid film 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. As shown in FIGS. 9 and 10, 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 μl 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 film may be attached to the opening of the first chamber 104.

  The first chamber 104 is preferably provided with a holding structure in order to suppress leakage of the electrolytic solution 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.

  As the material of the holding structure, any material that can be used as the material of the container 101 can be used. 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 formation of the first chamber 104. 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 the material of the second substrate 302, as in the holding structure. Most preferably, the material of the second substrate 302 is an insulator.

  It is preferable that at least a part of the outer peripheral surface of the second substrate 302 has hydrophilicity. In order to make at least a part of the outer peripheral surface of the second substrate 302 hydrophilic, 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 flat, but may be disc-shaped, trapezoidal, polygonal, cylindrical, or prismatic.

  The second chamber 105 is provided on the opposite side of the first chamber 104 via 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 preferably provided with a holding structure similar to 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.

  Next, a procedure for forming an artificial lipid membrane will be described. 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.

<Process B: 1st electrolyte solution injection process>
FIG. 11A shows the first electrolyte injection process. In the first electrolyte solution injection step, 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 for injecting the first electrolyte 201 into the first chamber 104 from the first inlet 303 is the same as 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.

<Step C: Lipid solution injection step>
FIG. 11B shows a lipid solution injection step. In the lipid solution injection step, the lipid solution 202 is injected into the artificial lipid film forming unit 103. In the lipid solution injection step, it is preferable to inject the lipid solution 202 from the second chamber 105 side.

  The lipid solution 202 may use the same lipid solution as in the first embodiment. As in the first embodiment, the lipid solution 202 is injected into the artificial lipid film forming unit 103. In the same manner as in Embodiment 1, 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.

<Step D: Second electrolyte injection step>
FIG. 11C shows the second electrolyte solution injection step. In the second electrolyte solution injection step, the second electrolyte solution 204 is injected into the second chamber 105.

  As the second electrolytic solution 204, the same electrolytic solution as in the first embodiment can be used. 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 electrolyte 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.

<Process E: Artificial lipid film formation process>
FIG. 12 shows an artificial lipid film formation step. In the artificial lipid film forming 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.

  In the second embodiment, as shown in FIG. 12, 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.

  Even when vibration is applied to the artificial lipid film forming apparatus 100, the artificial lipid film forming apparatus 100 is tilted, or the artificial lipid film forming apparatus 100 is turned over, the first electrolytic solution 201 and the second electrolytic solution 204 are Leaking out of the first chamber 104 and the second chamber 105 can be suppressed. For example, as shown in FIG. 13, the artificial lipid film forming apparatus 100 can be operated in an inclined state. As shown in FIG. 14, 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. As shown in FIG. 15, the artificial lipid film forming apparatus 100 may be operated while being carried by a handler. Since the viscosity of the first electrolytic solution 201 and the second electrolytic solution 204 is high, leakage of the first electrolytic solution 201 and the second electrolytic solution 204 can be suppressed even if hand movement as shown in FIG. 15 occurs. is there. As shown in FIG. 15, the artificial lipid film forming apparatus 100 may be incorporated in a part of the mobile terminal.

  In Embodiment 2, 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. In the present 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.

  In Embodiment 2, a membrane protein may be embedded in the artificial lipid membrane 205. FIGS. 16A to 16C schematically show a state where 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.

  It is also preferable to embed the G protein 310 in the artificial lipid membrane 205. 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.

  It is preferable to embed a second messenger-controlled channel in the artificial lipid membrane 205. 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.

  In Embodiment 2, the G protein embedded in the artificial lipid membrane 205 is most preferably a heterotrimeric G protein associated with a membrane receptor. The G protein embedded in the artificial lipid membrane 205 is preferably activated by GPCR.

  In the second embodiment, 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.

  In 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. . In the present embodiment, 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. In this embodiment, the membrane protein arranged on the vesicle membrane may be embedded by fusing the vesicle to the artificial lipid membrane 205. In the present 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. .

  A biosensor can be manufactured using the artificial lipid film forming method of the second embodiment. 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.

[Example]
Leakage of the first electrolytic solution 201 and the second electrolytic solution 204 to the outside of the first chamber 104 and the second chamber 105 was determined by the following procedure. The state of evaporation of the first electrolytic solution 201 and the second electrolytic solution 204 was evaluated using a microscope (manufactured by KEYENCE, VH-6300).

<Process A: Preparation process>
Acrylic plates were used as the first substrate 301 and the second substrate 302 shown in FIG. The thickness of the 1st board | substrate 301 and the 2nd board | substrate 302 was adjusted to 0.5 mm, 1 mm, or 5 mm according to the liquid volume of the 1st electrolyte solution 201 and the 2nd electrolyte solution 204. FIG. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 0.1 μl or 1 μl, the thickness of the first substrate 301 and the second substrate 302 was 0.5 mm. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 50 μl, the thickness of the first substrate 301 and the second substrate 302 was 1 mm. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 200 μl, 300 μl, or 400 μl, 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. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 0.1 μl or 1 μl, the diameters of the first chamber 104 and the second chamber 105 were 1 mm. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 50 μl, the diameters of the first chamber 104 and the second chamber 105 were 6 mm. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 200 μl, 300 μl, or 400 μl, 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 μm 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 μm. 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 μm 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 μm. 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. First, by glycerin (special grade, Wako Pure Chemical Industries) or polyvinyl alcohol (PVA) (primary, Wako Pure Chemical Industries, degree of polymerization 3100-3900), polyethylene glycol (PEG) (first grade, Wako Pure Chemical Industries, average molecular weight 7300-9300). The viscosities of the electrolytic solution 201 and the second electrolytic solution 204 were adjusted. The viscosity of the 1st electrolyte solution 201 and the 2nd electrolyte solution 204 was measured using the viscometer (TV-22) of Toki Sangyo.

  As the lipid solution 202, 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. The concentration of phospholipid was 1 mg / ml.

<Process B: 1st electrolyte solution injection process>
The first electrolyte solution 201 was injected into the first chamber 104 by a pipette (Gilson). 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 μl of the lipid solution 202 was injected into the artificial lipid film forming unit 103 from the second chamber 105 side. For injection, a micro syringe (Hamilton) was used. When 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 | poured into the 2nd chamber 105 with the pipette (Gilson). As the second electrolytic solution 204, a Tyrode solution whose viscosity was adjusted with glycerin, PVA or PEG was used. The temperature of the second electrolytic solution 204 was 22 ° C.

<Process E: Artificial lipid film formation process>
The artificial lipid film forming apparatus 100 was allowed to stand.

  Existence of leakage of the first electrolyte solution 201 and the second electrolyte solution 204 when the artificial lipid film forming apparatus 100 is installed at angles of 45 °, 90 °, and 180 ° with respect to the horizontal plane after the artificial lipid film forming step. Was judged. To install at 90 ° to the horizontal plane means to install the opening of the second chamber 105 in the horizontal direction. The installation at 180 ° with respect to the horizontal plane means that the opening of the second chamber 105 is installed directly below. By this determination, it was confirmed whether or not the first electrolytic solution 201 and the second electrolytic solution 204 leaked out when the artificial lipid film forming apparatus 100 was tilted or turned over.

  From the first electrolyte solution injection process to the artificial lipid film formation process, the environment was a room temperature of 22 ° C. and a relative humidity of 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 viscosity of the 1st electrolyte solution 201 and the 2nd electrolyte solution 204 adjusted by PVA.

  FIGS. 17A and 17B 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 | concentration of glycerol or PVA. When adjusting the viscosity of the 1st electrolyte solution 201 and the 2nd electrolyte solution 204 with glycerol or PVA, the ion concentration of the 1st electrolyte solution 201 and the 2nd electrolyte solution 204 was made constant. When adjusting 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. When adjusting the viscosities of the first electrolytic solution 201 and the second electrolytic solution 204 by 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.

  “+” In the table means that the first electrolytic solution 201 and the second electrolytic solution 204 did not leak out of the first chamber 104 and the second chamber 105 from the inlet and outlet openings. To do. “-” In the table means that the first electrolytic solution 201 and the second electrolytic solution 204 leaked out of the first chamber 104 and the second chamber 105 from the inlet and outlet openings. To do.

  As shown in Tables 3 and 4, when the viscosity is adjusted with glycerin, the outer volume of the first chamber 104 and the second chamber 105 is less than 200 μl when the amount of the first electrolyte 201 and the second electrolyte 204 is 200 μl or less. The first electrolytic solution 201 and the second electrolytic solution 204 did not leak out. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 is 300 μl 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.

  As shown in Table 4, when the viscosity is adjusted by glycerin, when the amount of the first electrolytic solution 201 and the second electrolytic solution 204 is 10 pl, the first electrolytic solution 201 and the second electrolytic solution 204 are out of the chamber. Did not leak.

  FIG. 19 shows a micrograph of droplets in the first chamber 104 immediately after the injection of 10 pl of the first electrolytic solution 201 by the microdroplet coating method. In this embodiment, the inside of a glass tube having an inner diameter of 300 μm 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 μm. 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. 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).

  When 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. When 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.

  When 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.

  As shown in Table 5, when the viscosity is adjusted by PVA, when the amount of the first electrolytic solution 201 and the second electrolytic solution 204 is 200 μl or less, the first electrolytic solution 201 and the second electrolytic solution are moved out of the chamber. The electrolytic solution 204 did not leak out. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 300 μl or more, the first electrolytic solution 201 and the second electrolytic solution 204 leaked out of the chamber.

  When the viscosity was adjusted by PEG, when the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 200 μl or less, the first electrolytic solution 201 and the second electrolytic solution 204 did not leak out of the chamber. . When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 300 μl or more, the first electrolytic solution 201 and the second electrolytic solution 204 leaked out of the chamber.

  When the glycerin concentration of the first electrolytic solution 201 was 99%, that is, when the viscosity of the first electrolytic solution 201 was 1500 mPa · s, the reagent of the Tyrode solution could not be dissolved in the first electrolytic solution 201. In this case, since the first electrolyte 201 adhered to the inner wall of the first chamber 104, the artificial lipid membrane 205 could not be formed.

  When the glycerin concentration of the second electrolytic solution 204 was 99%, that is, when the viscosity of the second electrolytic solution 204 was 1500 mPa · s, the reagent of the Tyrode solution could not be dissolved in the second electrolytic solution 204. In this case, 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.

  When 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. When 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.

  From the above, the amount of the first electrolytic solution 201 and the second electrolytic solution 204 is 10 pl to 200 μl, and the viscosity of the first electrolytic solution 201 and the second electrolytic solution 204 is 1.3 mPa · s to 200 mPa · s. In some cases, 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.

[Comparative Example 1]
A Tyrode solution was used as the first electrolyte solution 201 and the second electrolyte solution 204. The leakage of the first electrolytic solution 201 and the second electrolytic solution 204 to the outside of the chamber was determined by the following procedure.

<Preparation process>
Acrylic plates were used as the first substrate 301 and the second substrate 302 shown in FIG. The thickness of the 1st board | substrate 301 and the 2nd board | substrate 302 was adjusted to 0.5 mm, 1 mm, or 5 mm according to the liquid volume of the 1st electrolyte solution 201 and the 2nd electrolyte solution 204. FIG. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 0.1 μl or 1 μl, the thickness of the first substrate 301 and the second substrate 302 was 0.5 mm. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 50 μl, the thickness of the first substrate 301 and the second substrate 302 was 1 mm. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 200 μl, 300 μl, or 400 μl, 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, or 10 mm depending on the amount of the first electrolytic solution 201 and the second electrolytic solution 204. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 0.1 μl or 1 μl, the diameters of the first chamber 104 and the second chamber 105 were 1 mm. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 50 μl, the diameters of the first chamber 104 and the second chamber 105 were 6 mm. When the amount of the first electrolytic solution 201 and the second electrolytic solution 204 was 200 μl, 300 μl, or 400 μl, 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 μm 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 μm. 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 μm 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 μm. 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.

  As the lipid solution 202, 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. The concentration of phospholipid was 1 mg / ml.

<First electrolyte injection process>
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 μl of the lipid solution 202 was injected into the artificial lipid film forming unit 103 from the second chamber 105 side. For injection, a micro syringe (Hamilton) was used. When 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.

<Second electrolyte injection process>
The 2nd electrolyte solution 204 was inject | poured into the 2nd chamber 105 with the pipette (Gilson). The temperature of the second electrolytic solution 204 was 22 ° C.

<Artificial lipid membrane formation process>
The artificial lipid film forming apparatus 100 was allowed to stand.

  Existence of leakage of the first electrolyte solution 201 and the second electrolyte solution 204 when the artificial lipid film forming apparatus 100 is installed at angles of 45 °, 90 °, and 180 ° with respect to the horizontal plane after the artificial lipid film forming step. Was judged. By this determination, it was confirmed whether or not the first electrolytic solution 201 and the second electrolytic solution 204 leaked out when the artificial lipid film forming apparatus 100 was tilted or turned over.

  From the first electrolyte solution injection process to the artificial lipid film formation process, the environment was a room temperature of 22 ° C. and a relative humidity of 50%.

  When the amount of the first electrolytic solution 201 was 300 μl or more, the first electrolytic solution 201 leaked out of the first chamber 104. When the amount of the second electrolytic solution 204 was 300 μl or more, 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.

  When the liquid volume is 10 pl or more and 200 μl, 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. When the liquid volume is 10 pl or more and 200 μl, 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.

  As the first electrolytic solution 201 and the second electrolytic solution 204, aqueous solutions of Glucose (SIGMA G-7021) 0.1M and KCl (special grade Wako Pure Chemical) 0.1M were 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 in Comparative Example 1.

<First electrolyte injection process>
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 μl of the lipid solution 202 was injected into the artificial lipid film forming unit 103 from the second chamber 105 side. For injection, a micro syringe (Hamilton) was used. When 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.

<Second electrolyte injection process>
The 2nd electrolyte solution 204 was inject | poured into the 2nd chamber 105 with the pipette (Gilson). The temperature of the second electrolytic solution 204 was 22 ° C.

<Artificial lipid membrane formation process>
The artificial lipid film forming apparatus 100 was allowed to stand.

  Existence of leakage of the first electrolyte solution 201 and the second electrolyte solution 204 when the artificial lipid film forming apparatus 100 is installed at angles of 45 °, 90 °, and 180 ° with respect to the horizontal plane after the artificial lipid film forming step. Was judged. By this determination, it was confirmed whether or not the first electrolytic solution 201 and the second electrolytic solution 204 leaked out when the artificial lipid film forming apparatus 100 was tilted or turned over.

  From the first electrolyte solution injection process to the artificial lipid film formation process, the environment was a room temperature of 22 ° C. and a relative humidity of 50%.

  Table 6 shows the 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.

  When the amount of the first electrolytic solution 201 was 200 μl 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 μl or more, the second electrolytic solution 204 leaked out of the second chamber 105.

  When the amount of the first electrolytic solution 201 was 10 pl, the first electrolytic solution 201 rapidly evaporated, and thus the first electrolytic solution 201 could not be injected into the first chamber 104. 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.

  When the liquid volume is 10 pl or more and 200 μl, 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. When the liquid volume is 10 pl or more and 200 μl, 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.

  From the foregoing description, many modifications and other embodiments of the present invention are apparent to persons skilled in the art. Accordingly, the foregoing description should be construed as illustrative only and is provided for the purpose of teaching those skilled in the art the best mode of carrying out the invention. The details of the structure and / or function may be substantially changed without departing from the spirit of the invention.

  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.

DESCRIPTION OF SYMBOLS 10 Container 11 Flat plate 12 Electrolyte 13 Micropore 14 Lipid solution 15 Pipette 20 Container 21 Flat plate 22 Micropore 23 Electrolyte 24 Inlet 25 Lipid molecule 26 Electrolyte 27 Inlet 31 First chamber 32 Partition 33 Second chamber 34 Small pore 35 Artificial lipid membrane 100 Artificial lipid membrane forming device 101 Container 102 Partition wall 103 Artificial lipid membrane forming portion 104 First chamber 105 Second chamber 106 First opening 107 Second opening 108 Electrode 201 First electrolyte 202 Lipid solution 203 Lipid 204 Second Electrolyte 205 Artificial Lipid Membrane 301 First Substrate 302 Second Substrate 303 First Inlet 304 Ejector 305 Receptor Channel 306 Ligand 307 Ion 308 Channel 309 Receptor Protein 310 G Protein 311 Enzyme

Claims (14)

A method for forming an artificial lipid membrane comprising the following steps A to E:
Step A for preparing the following artificial lipid film forming apparatus
Here, the artificial lipid forming device is:
A first chamber;
A second chamber;
A partition wall sandwiched between the first chamber and the second chamber;
An artificial lipid membrane forming part comprising a through-hole provided in the partition wall,
The first chamber has a volume of 10 pl to 200 μl;
The second chamber has a volume of 10 pl to 200 μl;
A step B of injecting a first electrolytic solution having a viscosity of 1.3 mPa · s to 200 mPa · s into the first chamber;
A step C of injecting a lipid solution containing a lipid and an organic solvent into the artificial lipid film forming part,
Injecting a second electrolyte having a viscosity of 1.3 mPa · s to 200 mPa · s into the second chamber, and sandwiching the lipid solution between the first electrolyte and the second electrolyte; and Step E of removing the organic solvent to form an artificial lipid film in the artificial lipid film forming part.   The method according to claim 1, wherein at least one of the first electrolytic solution or the second electrolytic solution contains an organic compound having a hydroxyl group.   The method according to claim 2, wherein the organic compound having a hydroxyl group is an alcohol.   The method of claim 3, wherein the alcohol is a lower alcohol.   4. The method of claim 3, wherein the alcohol is glycerin.   The method according to claim 1, wherein at least one of the first electrolytic solution and the second electrolytic solution contains a polymer.   The method according to claim 6, wherein at least one of the first electrolytic solution and the second electrolytic solution contains polyvinyl alcohol.   The method according to claim 1, wherein in the step B, the first electrolytic solution is injected into the first chamber by an inkjet method.   The method according to claim 1, wherein in the step D, the second electrolytic solution is injected into the second chamber by an inkjet method. The method according to claim 1, wherein in step C, the lipid solution is injected into the artificial lipid film forming unit by an inkjet method.   The method according to claim 1, further comprising a step F of embedding at least one of a receptor and an ion channel in the artificial lipid membrane after the step E.   The method of claim 1, wherein in step B, the first chamber is filled with the first electrolyte.   The method of claim 1, wherein in step D, the second chamber is filled with the second electrolyte.   The method of claim 12, wherein in step D, the second chamber is filled with the second electrolyte.