Method for producing lipid membrane

Abstract

To provide a production method of a lipid membrane, capable of forming a homogeneous lipid membrane having a large area.SOLUTION: A production method of a lipid membrane includes a step of storing in a tank, a first liquid comprising a lipid dissolving liquid and a second liquid immiscible with the lipid dissolving liquid, forming a lipid membrane on a boundary surface between the first liquid and the second liquid, and moving a base material from one of the first liquid and the second liquid to the other so as to carry the lipid membrane on the surface of the base material. In the production method of the lipid membrane, an amount of lipid in the lipid dissolving liquid is 4 times or more and less than 40 times the minimum amount necessary for forming the lipid membrane on the whole boundary surface between the first liquid and the second liquid in the tank.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a lipid membrane. More specifically, the present invention relates to a method for producing a lipid membrane by utilizing a method for forming a lipid membrane at the interface between a lipid solution and a liquid that is immiscible with the lipid solution. [Background technology]

[0002] Patent document 1 describes a method for producing a lipid membrane in which a lipid solution and a liquid that is immiscible with the lipid solution are placed in a tank, a lipid membrane is formed at the interface between the two liquids, and this lipid membrane is attached to the tip of a probe to form the lipid membrane on the probe surface.

[0003] That is, the method of Patent Document 1 is "a method for producing an artificial biomembrane, which comprises layering a lipid solution in which lipids are dissolved in a solvent on the liquid surface of an aqueous solution to form a first lipid layer in which the lipids are arranged side by side along the interface between the aqueous solution and the lipid solution, covering the surface of the tip portion with a hydrophilic membrane, and immersing the tip portion of the metal probe having a membrane transport protein bound to the hydrophilic membrane in the lipid solution to form a second lipid layer in which the lipids are arranged side by side along the hydrophilic membrane, binding the second lipid layer to the first lipid layer to form a lipid bilayer, and simultaneously incorporating the membrane transport protein into the lipid bilayer to form an artificial biomembrane." [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2017-29090 A Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method for producing a lipid membrane, which is capable of forming a homogeneous lipid membrane with a large area. [Means for solving the problem]

[0006] The method for producing a lipid membrane of the present invention comprises the steps of placing a first liquid consisting of a lipid solution and a second liquid immiscible with the lipid solution in a tank, forming a lipid membrane at the interface between the first and second liquids, and supporting the lipid membrane on the surface of a substrate by moving the substrate from one of the first and second liquids to the other, wherein the amount of lipid in the lipid solution is 4 to 40 times the minimum amount required to form a lipid membrane over the entire interface between the first and second liquids in the tank.

[0007] In one embodiment of the present invention, the solvent of the lipid dissolving liquid is hexane, chloroform or perfluorohexane, and the second liquid is water.

[0008] In one embodiment of the present invention, the amount of lipid in the first liquid is 4 to 30 times the minimum required amount.

[0009] In one aspect of the invention, the lipid is a phospholipid.

[0010] In one embodiment of the present invention, a protein is contained in the lipid membrane formed at the interface between the first liquid and the second liquid.

[0011] In one aspect of the present invention, the substrate is transferred from the one liquid to the other liquid, and then transferred from the other liquid to the one liquid.

[0012] In one embodiment of the present invention, the minimum required amount is a value calculated by dividing the area of ​​the interface between the first liquid and the second liquid by the area occupied by one lipid molecule. Effect of the Invention

[0013] In the method for producing a lipid membrane of the present invention, the amount of lipid in the first liquid (lipid solution) is set to be 4 to 40 times the minimum amount required to form a lipid membrane over the entire interface between the first and second liquids, making it possible to form a homogeneous lipid membrane with a large area.

[0014] The lipid membrane supported on the substrate is formed at the interface between the first and second liquids, and because lipids are supplied from a hydrophobic solvent, it is possible to produce a large, defect-free lipid membrane.

[0015] According to the present invention, it is possible to produce lipid membranes necessary for constructing reaction fields and separation fields utilizing biological functions, and the lipid membranes can be applied to the production of sensors, reactors, selectively permeable membranes, etc. [Brief description of the drawings]

[0016] [Figure 1] 13 is a graph showing experimental results. [Diagram 2] 13 is a graph showing experimental results. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention will now be described in further detail.

[0018] The method for producing a lipid membrane of the present invention comprises the steps of placing a first liquid consisting of a lipid solution and a second liquid immiscible with the lipid solution in a tank, forming a lipid membrane at the interface between the first and second liquids, and supporting the lipid membrane on the surface of a substrate by moving the substrate from one of the first and second liquids to the other, wherein the amount of lipid in the lipid solution is 4 to 40 times the minimum amount required to form a lipid membrane over the entire interface between the first and second liquids in the tank.

[0019] The minimum required amount of lipid in the first liquid can be obtained by dividing the interface area between the first liquid and the second liquid by the area occupied by one lipid molecule. For example, the minimum required amount (molar amount) of lipid in the first liquid can be obtained by dividing the interface area between the first liquid and the second liquid by the area occupied by one lipid molecule, and then dividing this by Avogadro's number.

[0020] Examples of the hydrophobic solvent used in the first liquid include hexane, chloroform, perfluorohexane, etc. Examples of the solvent in the second liquid include hydrophilic solvents, specifically water.

[0021] In one embodiment of the present invention, a hydrophilic solvent as the second liquid and a first liquid in which lipids are dissolved in a hydrophobic solvent are placed in one tank to form two phases, and a lipid membrane is formed at the interface. Then, a substrate on which the lipid membrane is to be supported is moved from one phase to the other phase, thereby supporting the lipid membrane on the substrate surface.

[0022] In the present invention, since the lipid membrane is formed at the interface in advance, defects are reduced, and since the lipid is replenished from a hydrophobic solvent, a large lipid membrane can be prepared.

[0023] In the present invention, the amount of lipid dissolved in the hydrophobic solvent is less than 40 times the amount (minimum required amount) required to form a lipid membrane over the entire interface. If the amount is more than this, a multi-layer lipid membrane will be formed.

[0024] The amount of lipid in the first liquid is preferably 4 to 40 times, and particularly preferably 10 to 30 times, the minimum required amount.

[0025] Examples of lipids include phospholipids, glycolipids, glycerol, ceramide, fatty acids, and steroids.

[0026] The phospholipid may be either a neutral phospholipid or an anionic phospholipid.

[0027] Examples of the neutral phospholipid include 1-palmitoyl-2-oleoylphosphatidylcholine, 1,2-dioleoylphosphatidylcholine, 1,2-dipalmitoylphosphatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylethanolamine.

[0028] Examples of cationic phospholipids include 1,2-dioleoyl-3-trimethylammonium propane, 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, and the like.

[0029] Examples of the anionic phospholipid include 1-palmitoyl-2-oleoylphosphatidylglycerol, 1-palmitoyl-2-oleoylphosphatidic acid, and 1-palmitoyl-2-oleoylphosphatidylserine.

[0030] Examples of steroids include cholesterol, ergosterol, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride, and the like.

[0031] The shape of the substrate on which the lipid membrane is supported may be any of a plate, a membrane, a cube, a rectangular parallelepiped, a rod, a sphere, etc. Materials for the substrate include glass, plastic, metal, ceramic, etc., and functional materials such as selectively permeable membranes may also be used. In order to facilitate the spreading of lipids on the surface, hydrophilic groups such as cationic groups or anionic groups, or hydrophobic groups such as octyl groups or octadecyl groups may be introduced on the surface.

[0032] Examples of biofunctional materials include channel substances, sensing substances, biocatalyst substances, etc. As channel substances, aquaporin, gramicidin, amphotericin B, or derivatives thereof can be used.

[0033] In the method for producing a lipid membrane of the present invention, the substrate is moved from one of the first liquid and the second liquid to the other to support the lipid membrane on the substrate surface, and then the substrate may be moved from the other liquid to the first liquid, or this process may be repeated.

[0034] The moving speed of the substrate is preferably about 0.1 to 5.0 mm / min, particularly about 0.5 to 2.0 mm / min, but is not limited thereto. EXAMPLES

[0035] [Film forming material] Container for holding first and second liquid: 50 mL beaker (diameter 4 cm) Second liquid (hydrophilic solvent): Water Hydrophobic solvent for the first liquid: Hexane (Fujifilm Wako Pure Chemical) Lipid: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (weight average molecular weight = 760.1: POPC, NOF) 1,2-Dimethylammonium Propane Chloride (weight average molecular weight = 698.5: DOTAP, NOF) Substrate: Cover glass (C218181, Matsunami Glass Industry Co., Ltd. 18mm x 18mm x 0.13~0.17mm, cut to 10mm x 5mm x 0.13~0.17mm)

[0036] [Calculation method of minimum required amount and lipid concentration] When 15 mL of lipid (POPC) solution and water are placed in a 50 mL beaker, the amount of lipid (minimum required amount) required for lipid molecules to exist in a monolayer state across the entire interface can be calculated as follows.

[0037] Occupied area per POPC molecule (a): approx. 7×10 -1 nm 2 Interfacial area of ​​lipid solution (b): π(4 / 2) 2 cm 2 = approx. 12×10 14 nm 2 The molar amount of lipid required to form a monolayer at the interface is (b) / ((a)×6×10 23 ) = approx. 3×10 -9 The result is mol.

[0038] The minimum required molar amount (3×10 -9 The lipid concentration of a solution in which 100 mol of lipid was dissolved in 15 mL of hexane (hereinafter referred to as the minimum concentration) was 3 × 10 -9 mol / 15mL ≒ 2 × 10 -4 mmol / L.

[0039] <Experiment 1: Measurement of interfacial tension and calculation of the amount of lipid required for monolayer formation based on the measurement> When lipid molecules cover the entire interface between the lipid solution (in hexane) and water in a monolayer state, the interfacial tension is thought to decrease.-4 , 6×10 -4 , 7×10 -4 , 8×10 -4 , 1×10 -3 , 4×10 -3 The interfacial tension between hexane solutions with each lipid concentration (mmol / L) and water was measured by the Wilhelmy method using an interfacial tensiometer (DY-300, Kyowa Interface Science Co., LTD, Saitama, Japan). The results are shown in Figure 1.

[0040] As shown in Figure 1, 8 x 10 -4 The interfacial tension drops sharply at concentrations around mmol / L, and it takes 8×10 -4 mmol / L or more, i.e., the minimum concentration obtained from the above calculation (2 × 10 -4 It was found that the minimum concentration required for the DOTAP to be dissolved in water was four times that of POPC (1 mmol / L). Since the weight-average molecular weight of DOTAP is almost the same as that of POPC, the minimum concentration for DOTAP is considered to be the same as that for POPC.

[0041] <Example 1> [Preparation of lipid solution] DOTAP was used as the lipid, and 6 × 10 -3 A lipid solution was prepared at a concentration of mmol / L (30 times the minimum concentration required for forming a lipid monolayer at the interface).

[0042] [Lipid membrane formation on substrate surface] The substrate made of the above cover glass was subjected to plasma treatment for 10 minutes at 150 W using a small plasma device (PR200; Yamato Scientific Co., Ltd., Tokyo, Japan), and was immersed in pure water until immediately before film formation. Next, 30 mL of pure water (second liquid) was added to a 50 mL beaker to form an aqueous phase, and the substrate was immersed in the water in a horizontal position. Then, 30 mL of the above lipid solution was gently poured onto the aqueous phase and left to stand for 5 minutes to form a lipid membrane at the interface.

[0043] The substrate was then moved vertically upward at a speed of 1 mm / min into the lipid solution and allowed to stand in the lipid solution for 5 minutes. The substrate was then moved downward at a speed of 1 mm / min into the water. The lipid solution was then removed by sucking it out using a Pasteur pipette, and the substrate was recovered from the aqueous phase. A dip coater (ND-0407, SDI, Kyoto, Japan) was used to move the substrate.

[0044] [Structural Evaluation] The structure of lipid membranes was evaluated by FRAP. FRAP is a method to observe the fluidity of lipid molecules on a planar substrate, and can distinguish whether the lipid molecules on the substrate form a planar bilayer membrane or are adsorbed as liposomes. First, lipids are adsorbed onto a planar substrate using lipid molecules labeled with a fluorescent substance (3,3-dioctadecyloxacarbo-cyanine perchlorate; DiO, MW=882.0, Biotium, Fremont, USA). Then, the fluorescence of the lipid molecules is locally quenched with a laser, and the change in fluorescence intensity over time in the quenched region is measured. When lipids form a planar bilayer membrane on the planar substrate, the fluorescence intensity recovers because fluorescent molecules are supplied from the non-quenched region, but when lipids are adsorbed as liposomes, the movement of lipid molecules does not occur and the fluorescence intensity does not recover.

[0045] Using a confocal laser scanning microscope (FV1000-D, Olympus Co., Ltd, Tokyo, Japan), a circular spot of about 20 μm in diameter was irradiated with a laser to partially quench the fluorescence of the sample, and the recovery of the fluorescence in that area was observed. The fluorescence intensities at an excitation wavelength of 484 nm and a fluorescence wavelength of 501 nm were Ib and Ir, respectively, and the relative fluorescence intensity RI(t)=Ib / Ir was calculated after t minutes of quenching, and the fluorescence recovery rate FR(t) was calculated by the following formula.

[0046] FR(t)=(RI(t)-RI(0)) / (1-RI(0)) The recovery rate was 91.1%.

[0047] [Example 2] The lipid dissolving concentration is 4 x 10 -3 A lipid membrane was prepared in the same manner as in Example 1, except that the concentration was set to mmol / L (20 times the minimum concentration at which a lipid monolayer is formed at the interface). When the fluidity of the lipid membrane was evaluated by the above-described FRAP, the recovery rate was 53.4%.

[0048] [Example 3] The lipid dissolving concentration is 2 x 10 -3 A lipid membrane was prepared in the same manner as in Example 1, except that the concentration was set to mmol / L (10 times the minimum concentration at which a lipid monolayer is formed at the interface). When the fluidity of the lipid membrane was evaluated by the above-described FRAP, the recovery rate was 46.3%.

[0049] [Example 4] A lipid membrane was prepared in the same manner as in Example 1, except that a lipid mixture consisting of DOTAP and POPC in a molar ratio of 95:5 was used. The recovery rate by FRAP was 97.4%.

[0050] [Example 5] A lipid membrane was prepared in the same manner as in Example 1, except that a cation exchange membrane (CMB, Astom Corporation) was used instead of the substrate made of plasma-treated cover glass. The recovery rate by FRAP was 66.3%.

[0051] [Comparative Example 1] Lipid dissolving concentration 8 x 10 -3 A lipid membrane was prepared in the same manner as in Example 1, except that the concentration was set to mmol / L (40 times the minimum concentration at which a lipid monolayer is formed at the interface). The recovery rate by FRAP was 0.6%.

[0052] [Comparative Example 2] Lipid dissolution concentration 1 x 10 -2 A lipid membrane was prepared in the same manner as in Example 1, except that the concentration was set to mmol / L (50 times the minimum concentration at which a lipid monolayer is formed at the interface). The recovery rate by FRAP was 2.7%.

[0053] [Comparative Example 3] The lipid dissolving concentration is 4 x 10 -4 Except for using a concentration of 0.5 mmol / L (twice the minimum concentration at which a lipid monolayer is formed at the interface), a lipid membrane was prepared in the same manner as in Example 1. The recovery rate by FRAP was 14.6%.

[0054] <Consideration> In Examples 1, 2, 3, 4, and 5, the FRAP recovery rates were higher than those in Comparative Examples 1, 2, and 3, and it was confirmed that the lipid membrane was developed without defects and without multilayering.

Claims

1. A first liquid comprising a lipid solution and a second liquid immiscible with the lipid solution are placed in a tank, and a lipid membrane is formed at the interface between the first liquid and the second liquid; The substrate is moved from one of the first liquid and the second liquid to the other, thereby supporting the lipid membrane on the surface of the substrate. A method for producing a lipid membrane comprising the steps of: The amount of lipid in the lipid solution is 4 times or more and less than 40 times the minimum amount required to form a lipid membrane over the entire interface between the first liquid and the second liquid in the tank, The minimum required amount is a value obtained by dividing the area of ​​the interface between the first liquid and the second liquid by the area occupied by one lipid molecule, The method for producing a lipid membrane, wherein the lipid is a phospholipid.

2. A first liquid comprising a lipid solution and a second liquid immiscible with the lipid solution are placed in a tank, and a lipid membrane is formed at the interface between the first liquid and the second liquid; The substrate is moved from one of the first liquid and the second liquid to the other, thereby supporting the lipid membrane on the surface of the substrate. A method for producing a lipid membrane comprising the steps of: The amount of lipid in the lipid solution is 4 times or more and less than 40 times the minimum amount required to form a lipid membrane over the entire interface between the first liquid and the second liquid in the tank, The minimum required amount is a value obtained by dividing the area of ​​the interface between the first liquid and the second liquid by the area occupied by one lipid molecule, A method for producing a lipid membrane, comprising the step of incorporating a protein into the lipid membrane formed at the interface between the first liquid and the second liquid.

3. 3. The method for producing a lipid membrane according to claim 1, wherein the solvent of the lipid dissolving solution is hexane, chloroform or perfluorohexane, and the second liquid is water.

4. 4. The method for producing a lipid membrane according to claim 1, wherein the amount of lipid in the first liquid is 4 to 30 times the minimum required amount.

5. 5. The method for producing a lipid membrane according to claim 1, wherein the substrate is transferred from the one liquid to the other liquid, and then transferred from the other liquid to the one liquid.

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