JP2019022872A - Method for forming lipid bilayer membrane and instrument for the same - Google Patents

Abstract

To provide a method for forming a lipid bilayer membrane, the method allowing for direct contact of aqueous medium with a gas composition in atmosphere with a lipid bilayer membrane formed, and achieving electrical insulation of both sections partitioned by the lipid bilayer membrane while the direct contact is allowed, and to provide an instrument for the method for forming a lipid bilayer membrane.SOLUTION: The method for forming a lipid bilayer membrane includes: a step of adding lipid solution in which lipid membrane-forming lipid is dissolved into organic solvent to a through-hole of a partition wall having the through-hole, thereby forming a membrane of lipid solution covering the through-hole; and a step of immersing an obtained partition wall into aqueous medium, thereby forming a lipid bilayer membrane in the lipid solution covering the through-hole. The aqueous medium is housed in a chamber, the chamber is separated into a first section and a second section in an insulated manner by immersing the partition wall into the aqueous medium, and the aqueous medium housed in at least either one of the first section and the second section directly contacts outside air.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for forming a lipid bilayer membrane and a device therefor.

  Cells that make up living organisms, various organelles such as mitochondria, Golgi bodies, and endoplasmic reticulum, cell nuclei, etc. are covered with a biological membrane on the outside. This biological membrane is basically a lipid bilayer membrane. It is composed of Various proteins having physiological activity, that is, receptors, enzymes, and the like, are retained on the lipid bilayer membrane in a form that penetrates the lipid bilayer membrane. These transmembrane proteins play an important role in vivo. In particular, it has been found that various receptors present on cell membranes trigger various physiological reactions by binding to ligands present in the living body. For this reason, various ligands that enhance the function of the receptor, inhibitors that inhibit the function of the receptor, and the like are used as pharmaceuticals, and natural or artificial ligands and inhibitors that can be used as new pharmaceuticals have been studied. ing. Research is also underway to develop odor sensors by holding olfactory receptors on lipid bilayers.

  In order to develop these transmembrane proteins and their ligands, inhibitors, odor sensors, etc., various measurements must be performed in the same state as in the living body, that is, with the transmembrane protein held on the biological membrane. desired. Conventionally, a lipid bilayer membrane formation method itself is well known, and a droplet contact method, a brush coating method, a spraying method, and a bonding method are known. Among these, the droplet contact method is widely used because the lipid bilayer membrane formation operation is simple and the measurement operation after the lipid bilayer membrane formation can be easily performed. In the droplet contact method, for example, a double well chamber in which two shallow cylindrical recesses (wells) are arranged adjacent to each other and a boundary portion where the two wells are in contact with each other is formed as a gap (about several mm on a side). The method used is known. In this method, each well of the double well chamber is filled with a lipid solution, and then an aqueous buffer solution is added to each well to form droplets of the buffer solution in the lipid solution. This utilizes the fact that an interface between a solution and a buffer solution is formed, and a lipid bilayer is formed in this portion.

  In the droplet contact method, water droplets are placed in an organic solvent in which lipids are dispersed, and a lipid bilayer membrane is obtained at the interface between the water droplets. Therefore, the aqueous phase is surrounded by an oil phase. When a chemical receptor, a membrane protein, is reconstituted in a lipid bilayer membrane and used for chemical substance detection, it is difficult to add chemical substances from the outside into droplets that have submerged in oil. The problem is that it is difficult to reach because of the phase. As a solution, there is a known technique (Non-Patent Document 1) in which the surface of the droplet is partially exposed. This system drops aqueous droplets on a substrate impregnated with an oil phase, but it has not been demonstrated that gas components have dissolved in the aqueous droplets. The measurement operation is complicated. Furthermore, when measuring an ionic current through a membrane protein reconstituted into a lipid bilayer membrane, the two regions separated by the membrane must be electrically insulated in advance. In the conventional droplet contact method, stable insulation is obtained by covering the droplets with an oil phase, but the price is compensated by the low melting efficiency of the atmospheric gas.

JB Boreykoa, G. Polizos, PG Datskos, SA Sarles, and CP Collier: “Air-stable droplet interface bilayers on oil-infused surfaces”, Proc. Nat. Acad. Sci. USA, Vol. 111, pp. 7588-7593 (2014).

  Accordingly, an object of the present invention is to allow the gas component in the atmosphere to be in direct contact with the aqueous medium in a state where the lipid bilayer membrane is formed, and in this state, both compartments separated by the lipid bilayer membrane. It is intended to provide a method for forming a lipid bilayer membrane and a device therefor in which electrical insulation is achieved.

  As a result of earnest research, the inventors of the present application added a lipid solution in which a lipid film-forming lipid is dissolved in an organic solvent to a through-hole of a partition wall having a through-hole to form a lipid solution membrane covering the through-hole, In this state, the partition wall is immersed in an aqueous medium to form a lipid bilayer in the lipid solution covering the through-hole. At this time, the aqueous medium is accommodated in the chamber, and the partition wall is stored in the aqueous medium. The chamber is insulated into a first compartment and a second compartment by dipping in, and the aqueous medium accommodated in at least one of the first compartment and the second compartment is In the state in which the lipid bilayer membrane is formed, the gas component in the atmosphere can be brought into direct contact with the aqueous medium, and in this state, it is separated by the lipid bilayer membrane. Found that electrical insulation of both compartments was achieved The present invention has been completed.

That is, the present invention includes a step of adding a lipid solution in which a lipid film-forming lipid is dissolved in an organic solvent to the through-hole of the partition wall having a through-hole to form a membrane of the lipid solution covering the through-hole,
Immersing the obtained partition wall in an aqueous medium to form a lipid bilayer in the lipid solution covering the through-hole,
The aqueous medium is accommodated in a chamber, and the chamber is insulated into a first compartment and a second compartment by immersing the partition wall in the aqueous medium, and the first compartment And a method for forming a lipid bilayer, wherein the aqueous medium accommodated in at least one of the second compartments is in direct contact with outside air.

  In the present invention, the lipid bilayer membrane formed by the above-described method of the present invention is reconstituted with a desired transmembrane bioactive protein, and the test substance is used as the aqueous medium with the outside air as the test substance-containing atmosphere. A method for investigating a test substance including supplying is provided.

Furthermore, the present invention is a lipid bilayer membrane forming device for forming a lipid bilayer membrane by the method of the present invention,
A bottom plate and a chamber having a sidewall and containing an aqueous medium therein;
A partition having a through-hole, wherein a lipid solution in which a lipid film-forming lipid is dissolved in an organic solvent is added to the through-hole to form a membrane of the lipid solution that covers the through-hole. A partition wall that is insulatively separated into a first compartment and a second compartment by immersing in the chamber containing
Comprising
One or a plurality of through holes are provided in the side wall of the chamber, and at least one of the aqueous medium accommodated in the first compartment and the second compartment is outside air through the through holes. Direct contact,
A device for forming a lipid bilayer membrane is provided.

  According to the present invention, the gas component in the atmosphere can be directly brought into contact with the aqueous medium in a state where the lipid bilayer membrane is formed, and in this state, the electrical insulation of both compartments separated by the lipid bilayer membrane is achieved. For the first time, a method for forming a lipid bilayer membrane and a device therefor have been provided. According to the method of the present invention, the gas component in the atmosphere can be sufficiently dissolved in the aqueous medium, the interaction between the membrane protein and the like carried on the lipid bilayer membrane and the gas component, and the odor sensor It is thought to contribute greatly to development.

It is a schematic diagram for demonstrating an example of the method of this invention. It is a schematic diagram for demonstrating other one Embodiment of this invention. It is a schematic diagram for demonstrating the chamber device produced in the Example of this invention. It is a schematic diagram for demonstrating the method of coat | covering the through-hole of a partition with a lipid solution performed in the Example of this invention. It is a schematic diagram for demonstrating the electrical measurement performed in the Example of this invention. It is a schematic diagram for demonstrating the formation method of a lipid bilayer membrane performed in the Example of this invention. It is a figure which shows the time-dependent change of the electric current at the time of reconstituting (alpha) -hemolysin to the lipid bilayer membrane performed in the Example of this invention. It is a schematic diagram for demonstrating the method performed in the Example of this invention which investigates the uptake | capture of the gas in atmosphere. It is a figure which shows the result of the uptake | capture test of octenol performed in the Example of this invention.

  First, the principle of the method for forming a lipid bilayer membrane of the present invention will be described with reference to FIG.

  In the method of the present invention, a chamber (recess) 10 for containing an aqueous medium is used. The chamber shown in FIG. 1 is in the form of a double well chamber (a gourd-shaped chamber in which two cylindrical wells are fused) used in the droplet contact method, but is not limited to this and will be described later. It may be cylindrical or other shapes as used in the embodiments. As the aqueous medium, water or a buffer solution containing water as a solvent is usually used. As the buffer solution, a known buffer solution known to be biocompatible, such as a phosphate buffer solution, can be used. Since the chamber 10 is a recess, it necessarily has a side wall and a bottom. In the example shown in FIG. 1, a groove 12 is formed in a portion where two cylindrical wells are fused (a central portion of a gourd). A partition wall described later is inserted into the groove 12. The groove 12 may penetrate or may not penetrate if the depth of the chamber 12 is sufficient. A through hole 14 is formed in the side wall of the chamber 10. When the aqueous medium is accommodated in the chamber 10, the aqueous medium comes into direct contact with the outside air through the through hole 14. In addition, although the number of the through holes 14 may be one, it is also possible to provide a plurality of aqueous media so that the aqueous media in both compartments separated after the partition wall is inserted are in direct contact with the outside air.

  In the method of the present invention, a partition wall 16 is used that is immersed in an aqueous medium accommodated in the chamber and divides the chamber into a first compartment and a second compartment. Although the diameter of the through-hole provided in the partition 16 is not specifically limited, Usually, it is about 300 micrometers-1.2 mm. A lipid solution 18 in which a lipid film-forming lipid is dissolved in an organic solvent is added to the through-hole to form a membrane of the lipid solution 18 that covers the through-hole. As lipid membrane-forming lipids, lipid bilayer membranes, that is, lipid molecules having a hydrophilic region and a hydrophobic region in one molecule are arranged in two layers with the hydrophobic region on the inside and the hydrophilic region on the outside. It is not particularly limited as long as it is a lipid capable of forming a membrane, but in order to simulate a reaction in a biological membrane, the same or similar to the biological membrane is preferable, and phospholipids that have been widely used in this field, for example, Diphytanoyl phosphatidylcholine (diphytanoyl phosphatidylcholine, DPhPC), dipalmytoyl phosphatidylcholine, dipalmytoyl phosphatidylcholine, 1-Palmitoyl 2-Oleoyl phosphatidylcholine, POPC A preferred example is dioleoyl phosphatidylcholine (DOPC). Since many of these are commercially available, commercially available products can be preferably used. As the organic solvent for dissolving the lipid, an aliphatic hydrocarbon solvent such as n-decane is preferable. The lipid concentration in the lipid solution is not particularly limited as long as the lipid bilayer can be formed, but is usually about 1 g / L to 50 g / L, preferably about 5 g / L to 25 g / L.

  The chamber and partition used in the present invention are preferably subjected to water repellent treatment as a whole. By this water repellent treatment, as will be described later, when the partition wall is inserted into the chamber, insulation between the first partition and the second partition separated by the partition wall is easily achieved. The through-hole 14 is usually about 3 mm in diameter, but the aqueous medium does not leak out even if a through-hole having such a size opens on the side wall of the chamber. Similarly, the width of the groove 12 is usually about 1 mm to 2 mm, but the aqueous medium does not leak even if the groove having this width penetrates. The water-repellent treatment can be preferably performed by immersing the partition walls or the entire chamber in a fluorine coating agent, and a fluorine resin solution such as polytetrafluoroethylene can be preferably used as the fluorine coating agent. Since such a fluorine coating agent is commercially available, a commercially available product can be preferably used. If the water-repellent treatment is performed up to the periphery of the through hole of the partition wall, the lipid solution becomes difficult to adhere to the partition wall, and it becomes difficult to cover the through hole with the lipid solution. It is preferable to immerse in a fluorine coating agent in a coated state.

  When forming the lipid bilayer membrane, the partition wall 16 in which the through hole is covered with the lipid solution 18 as described above is inserted into the groove 12, and the lipid solution 18 covering the through hole is immersed in the aqueous medium. Then, the partition 10 separates the chamber 10 into the first compartment 20 and the second compartment 22. The lipid molecules in the lipid solution are aligned with the hydrophilic portion facing outward (aqueous medium side) and the hydrophobic portion facing inward to form a lipid bilayer membrane. In this state, if the dimensions of the partition wall 16 and the groove 12 are appropriately designed so that almost no gap is generated, the first partition and the second partition 22 are insulated by the water repellent treatment described above. Here, “insulation” means that the first compartment and the second compartment are electrically insulated, and an aqueous medium is accommodated in the first compartment and the second compartment. This means that there is no flow of ions moving between the first compartment and the second compartment in the aqueous medium. Whether or not the first compartment and the second compartment are insulated is determined by carrying a channel protein such as α-hemolysin in the lipid bilayer (reconstitution), as specifically described in the following examples. It can be confirmed that the current flows between both compartments only after the ion channel is opened, and the current increases stepwise as the number of reconstituted channel proteins increases.

  Further, in the state in which the lipid bilayer membrane is formed as described above, the aqueous medium in the first compartment 20 is in direct contact with the outside air through the through-hole 14 that opens to the side wall of the chamber. In the embodiment shown in FIG. 1, the upper part of the chamber is also open. However, by inserting the partition wall 16 covered with the through hole with the lipid solution into the aqueous medium, a part of the lipid solution is detached from the partition wall and the aqueous system is removed. There is a possibility that a thin layer of lipid solution is formed on the top of the medium (even if it is a layer that cannot be observed visually), because the specific gravity of lipid solution is smaller than that of water. However, it is not always ensured that the aqueous medium is in direct contact with the outside air. On the other hand, when the through hole 14 is provided on the side wall of the chamber, the lipid solution forms a layer above the aqueous medium even if a part of the lipid solution is detached from the partition wall. It is always ensured that at least a part of the exposed aqueous medium is in direct contact with the outside air.

  Next, a second embodiment in which the chamber configuration is different from that of FIG. 1 will be described with reference to FIG. FIG. 2A is a plan view of the partition wall. FIG. 2B shows the structure of the chamber device, which includes an upper plate 23 and a lower plate 24. The state where the upper plate 23 and the lower plate 24 are overlapped and heat-sealed is shown in FIG. 2 (C), and the end view of the ab line cut portion and the end view of the cd line cut portion in FIG. 2 (C) are shown. 2 (D). The upper plate 23 is provided with a cylindrical through hole 26 in the horizontal direction, and this through hole constitutes a chamber. That is, the through-hole 26 constitutes a cylindrical chamber, and it can be considered that the side walls at both ends of the cylindrical chamber extending in the horizontal direction are fully opened and the chamber communicates with the outside. In this aspect, the aqueous medium accommodated in both the first compartment and the second compartment separated by the partition walls is in direct contact with the outside air. As described above, the water repellent treatment prevents the liquid from leaking from the opening having a diameter of about 3 mm. The upper plate 23 is provided with a through slit 28 orthogonal to the through hole 26, and a partition wall is inserted into the through slit 28. Further, the upper plate 23 is provided with through holes 30a and 30b on both sides of the extension line of the through slit 28, respectively, and fluorocarbon to be described later is injected from either of these through holes. Furthermore, through holes 31a and 31b for inserting electrodes to be described later are formed at the bottom of the through hole 26 (see FIG. 2D).

  A groove 32 is formed in the lower plate 24 at a portion that is directly below the through slit 28 when the upper plate is overlaid. This groove accommodates fluorocarbon to be described later. Further, the lower plate 24 is provided with two through holes 34a and 34b in a portion that is directly below the through hole 26 when the upper plate is overlapped, and an electrode is inserted therein, and the top of the electrode is Through the through holes 34a and 34b and the through holes 31a and 31b provided in the upper plate 23, the through holes 26, that is, the chambers are reached.

  As in the embodiment shown in FIG. 1, in the embodiment shown in FIG. 2, the whole of the partition wall (excluding the periphery of the through hole) and the upper plate 23 and the lower plate 24 which are heat-sealed with a fluorine coating agent are used. It is preferable to perform a water repellent treatment.

  In the case of forming a lipid bilayer membrane, an aqueous medium is injected from the through hole 26 and the aqueous medium is accommodated in the through hole. Further, an insulating liquid, such as fluorocarbon, having a specific gravity greater than that of water and not mixed with water is injected from the through hole 30a or 30b and accommodated in the groove 32. On the other hand, the through-hole 17 is covered with a lipid solution and inserted into the through-slit 28 in the same manner as in the embodiment of FIG. If the partition 16 has a T-shape as shown in FIG. 2A and the portion of the T-shaped horizontal bar is longer than the through-slit 28, the portion of the T-shaped horizontal bar becomes the upper side of the through-slit 28. It only has to be inserted until it stops, and the operation is simple. The through-hole 17 of the partition wall 16 has a cross-section of the through-hole 26 (a circular cross-section in a direction perpendicular to the longitudinal direction) when inserted to a position where the T-shaped horizontal bar is on the upper side and stops at the through-slit 28. Design the dimensions so that they are near the center. When the partition wall 16 is inserted into the through slit 28, the through hole 17 of the partition wall 16 covered with the lipid solution is positioned in the aqueous medium in the through hole 26, and a lipid bilayer membrane is formed in the through hole 17 portion. It is formed. At this time, the size is designed so that the lower end portion of the partition wall 16 is inserted into the groove 32 and immersed in the insulating liquid accommodated in the groove 32. By doing so, since the lower end part of the partition 16 is immersed in an insulating liquid and the bottom boundary of the 1st division and the 2nd division separated by the partition 16 is reliably insulated, it is preferable. As described above, if an electrode is inserted, the current flowing between the first compartment and the second compartment can be measured.

  By the method described above, with the lipid bilayer membrane formed in the through-hole part of the partition wall, the gas component is effectively acted on the lipid bilayer membrane by bringing the aqueous medium directly into contact with the aqueous medium. Can be made. A desired membrane-spanning physiologically active protein is reconstituted in the lipid bilayer membrane, and the test substance is supplied to the aqueous medium using the outside air as the test substance-containing atmosphere, and the first compartment and the second compartment By measuring the current flowing between them, the test substance can be investigated. That is, the action of the test substance on the desired transmembrane bioactive protein held in the lipid bilayer membrane can be examined, and if the olfactory receptor is reconstituted in the lipid bilayer membrane, the test substance It is possible to detect odors.

  Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.

Example 1
1. Preparation of organic solvent sample with dissolved lipid
300 μL of 25 mg / mL 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) / chloroform solution and 25 mg / mL 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ) / Chloroform solution (100 μL) was mixed and placed in a transparent glass vial, and the vial was loosened and degassed in a low vacuum desiccator for 2 hours or more at room temperature to volatilize chloroform. To the above dry mixed lipid sample, 500 μL of n-decane was added and stirred well by vortexing or the like to obtain a 20 mg / mL lipid decane solution (weight ratio DOPC: DOPE = 3: 1). The lipid decane solution was stored at room temperature and used for an expiration date of about one week.

2. Fabrication and surface treatment of perforated partition walls and chamber devices A 75 μm thick acrylic film was cut with a milling device to produce a T-shaped perforated partition wall with the dimensions shown on the left in FIG. 2 is the same figure, but FIG. 3 shows the dimensions of the instrument actually produced in the example). Similarly, a 4 mm thick acrylic plate is cut with a milling machine to produce two parts with the dimensions shown in the center of FIG. 3, and then joined with a thermocompression bonding machine to obtain a chamber device with an overview shown on the right of FIG. It was.

The produced perforated partition wall was subjected to surface treatment and the like by the following procedure (FIG. 4).
● A silicon rubber piece 3mm in diameter (no matter the thickness) punched out using a biopsy trepan is manually placed from both sides of the partition wall so as to cover the through-hole using a mask as a mask, and then a fluorine coating agent (AGC Asahi Glass) 2 μL each of SFE-DP02H (manufactured by Sangyo Co., Ltd.) was dropped on both sides of the partition wall, and left to stand at room temperature for 20 minutes to dry.
After removing the silicone rubber, 0.5 μL of the above-described lipid decane solution was added dropwise around the through-holes on both sides of the partition wall, allowed to stand at room temperature for 30 minutes or more, and dried to deposit a lipid film.
-Immediately before inserting the perforated partition into the chamber device, 0.5 μL of the aforementioned lipid decane solution was again dropped around the through hole on one side of the partition.

The manufactured chamber device after thermocompression bonding was subjected to surface treatment and electrode installation according to the following procedure.
● The entire chamber device was immersed in SFE-DP02H (manufactured by AGC Asahi Glass Co., Ltd.) for several seconds and then taken out and dried at room temperature for 20 minutes.
● Two silver wires with a diameter of 1 mm (regardless of length) were inserted into the chamber device, the gap was filled with silver / silver chloride paste, and electrode was fixed on the exposed portion of the electrode by applying dotite.
● The lower end of the above two electrodes was joined to the wiring on the BNC socket side using dotite for the BNC socket to connect to the amplifier (Fig. 5).

3. Formation of lipid membrane A fluorocarbon solution (Fluorinert (registered trademark) FC-40, manufactured by Sigma-Aldrich) was previously stored in the chamber device, and 50 μL of a buffer solution was injected into the side hole. A lipid decane solution of 0.5 μL dropped on one side of the perforated partition wall is manually inserted into the chamber device as shown in FIG. 6 to divide the buffer solution into two, and the interface between the two regions A lipid membrane was formed.

Example 2
Α-hemolysin was mixed with the buffer solution in the above lipid membrane formation so that the final concentration was 2 μM. The electrode was connected to an amplifier, and the change in current value was measured while applying a voltage of +100 mV. The results are shown in FIG.

  As shown in FIG. 7, a change in current value due to nanopores formed by reconstituting α-hemolysin into a lipid bilayer was observed. It has been shown that the current increases stepwise with time, and that the current increases as the number of α-hemolysin molecules reconstituted in the lipid bilayer increases. From this, it can be seen that the first section and the second section separated by the partition are insulated by the above method.

Example 3
1 mL of octenol stock solution, which is a volatile chemical substance, was soaked in filter paper and allowed to stand in a sealed container at room temperature. The device in which the lipid bilayer membrane shown in FIG. 6 was formed in the same container was placed next to the filter paper, and left at room temperature for 30 minutes (b in FIG. 8). Thereafter, the buffer solution was recovered and analyzed by chromatography. On the other hand, a system in which n-decane was added to a buffer solution was also tested in order to simulate the incorporation of octenol into an aqueous medium in the conventional droplet contact method (FIG. 8a).

  The results are shown in FIG. As shown in FIG. 9, octenol peaks were observed for both a and b, but a much larger peak was observed for b according to the present invention. From this, it became clear that according to the method of the present invention, a large amount of gas components can be taken into the aqueous medium as compared with the droplet contact method that has been widely used conventionally.

DESCRIPTION OF SYMBOLS 10 Chamber 12 Groove 14 Through-hole 16 Partition 17 Through-hole 18 Lipid solution 20 1st division 22 2nd division 23 Upper plate 24 Lower plate 26 Through-hole 28 Through slit 30a Through-hole 30b Through-hole 31a Through-hole 31b Through-hole 32 Groove 34a Through hole 34b Through hole

Claims (15)

Adding a lipid solution in which a lipid film-forming lipid is dissolved in an organic solvent to the through-hole of the partition wall having a through-hole to form a membrane of the lipid solution covering the through-hole,
Immersing the obtained partition wall in an aqueous medium to form a lipid bilayer in the lipid solution covering the through-hole,
The aqueous medium is accommodated in a chamber, and the chamber is insulated into a first compartment and a second compartment by immersing the partition wall in the aqueous medium, and the first compartment And a method of forming a lipid bilayer in which the aqueous medium housed in at least one of the second compartments is in direct contact with outside air.   The method according to claim 1, wherein the aqueous medium accommodated in the first compartment and / or the second compartment is in direct contact with outside air through a through hole provided in a side wall of the chamber.   The method according to claim 1, wherein the partition wall and the chamber are subjected to water repellent treatment.   The method according to claim 3, wherein the water repellent treatment is performed by applying a fluorine coating agent.   The method according to claim 3 or 4, wherein the chamber is in the form of a cylindrical through hole extending in the horizontal direction.   Under the aqueous medium, containing an insulating liquid having a specific gravity greater than that of the aqueous medium and not mixed with water, and immersing the lower end of the partition wall in the insulating liquid, the first compartment and the 6. A method according to any one of the preceding claims, wherein the bottom boundary of the second compartment is insulated.   The method of claim 6, wherein the insulating liquid is a fluorocarbon.   A lipid transmembrane formed by the method according to any one of claims 1 to 7, wherein a desired transmembrane bioactive protein is reconstituted, and the test substance is used as the test substance-containing atmosphere. A method for investigating a test substance, including supplying to an aqueous medium.   9. The method of claim 8, comprising measuring a current flowing between the first compartment and the second compartment. A lipid bilayer membrane forming device for forming a lipid bilayer membrane according to the method of claim 1,
A chamber having a bottom and side walls and containing an aqueous medium therein;
A partition having a through-hole, wherein a lipid solution in which a lipid film-forming lipid is dissolved in an organic solvent is added to the through-hole to form a membrane of the lipid solution that covers the through-hole. A partition wall that is insulatively separated into a first compartment and a second compartment by immersing in the chamber containing
Comprising
One or a plurality of through holes are provided in the side wall of the chamber, and at least one of the aqueous medium accommodated in the first compartment and the second compartment is outside air through the through holes. Direct contact,
Lipid bilayer forming device.   The instrument according to claim 10, wherein the partition wall and the chamber are subjected to water repellent treatment.   The instrument according to claim 11, wherein the water repellent treatment is performed by applying a fluorine coating agent.   13. A device according to claim 11 or 12, wherein the chamber is in the form of a cylindrical through-hole extending in the horizontal direction.   A groove into which the lower end of the partition wall is inserted is formed at the bottom of the chamber. The groove contains an insulating liquid having a specific gravity greater than that of the aqueous medium, and the lower end of the partition wall is 14. A device according to any one of claims 10 to 13, wherein the bottom boundary of the first compartment and the second compartment is insulated by immersion in an insulating liquid.   The instrument according to any one of claims 10 to 14, wherein the bottom plate is provided with a through-hole for inserting an electrode, which communicates with the first compartment and the second compartment, respectively.