WO2005029056A1 - 人工脂質二重膜の形成装置および人工脂質二重膜の形成方法、並びにその利用 - Google Patents
人工脂質二重膜の形成装置および人工脂質二重膜の形成方法、並びにその利用 Download PDFInfo
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- WO2005029056A1 WO2005029056A1 PCT/JP2004/013680 JP2004013680W WO2005029056A1 WO 2005029056 A1 WO2005029056 A1 WO 2005029056A1 JP 2004013680 W JP2004013680 W JP 2004013680W WO 2005029056 A1 WO2005029056 A1 WO 2005029056A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
Definitions
- Apparatus for forming artificial lipid bilayer membrane method for forming artificial lipid bilayer membrane, and use thereof
- the present invention relates to an apparatus for forming an artificial lipid bilayer and a method for forming an artificial lipid bilayer, and to its use, for example, to detect microcurrents via membrane proteins, peptides, etc.
- the present invention relates to an apparatus and a method for forming an artificial lipid bilayer membrane suitably used for a planar lipid membrane method to be performed, and an example of typical use thereof.
- Lipid bilayers are the basic structure of biological membranes and are found in all living biological membranes. Most biological membranes are in a state in which various membrane proteins are embedded in the lipid bilayer, and these membrane proteins transport and signal substances such as various ions, sugars, amino acids, and nucleotides. Alternatively, it is involved in lipid synthesis and the like.
- a biological membrane is a place where a physiological function is exhibited, and plays various important roles such as recognition of external force information and transmission into the membrane, conversion and transport of substances, and the like. Also, if the functions of biological membranes are applied, the possibility of application to various industrial uses is found. Therefore, it is extremely important to create an artificial membrane as a biological membrane model.
- An artificial lipid bilayer is a thin film in which amphipathic molecules such as phospholipids are arranged with the alkyl chains of the hydrophobic part facing inward.For example, it is used to measure ion current in ion channels. It is applied to etc.
- a lipid planar membrane method As a method of forming an artificial lipid bilayer membrane, a lipid planar membrane method is known. Patch clamp method is used as a method to measure the ion current of a single ion channel.In order to deepen the correlation study on the structural function of the force channel, it is necessary to use a simple reconstruction system when conducting experiments. There are cases.
- the method of forming an artificial lipid bilayer used in such a case is the lipid planar membrane method.
- This planar lipid membrane method uses ion, water, artificial lipid bilayer membrane, and ion channels to minimize the basic structure and detailed structure-function correlation of ion channels using a minimally simple system. If you look at it, you'll find that it's a bug (see Non-Patent Document 1).
- an ion channel 112 is incorporated into an artificial lipid bilayer membrane 111, and a current flowing through the ion channel 112 is measured.
- the artificial lipid bilayer membrane 111 is formed in a small hole 115 formed in a partition plate 114 such as a plastic plate that partitions the aqueous solution tank 113.
- An electrode 116 is charged into one of the two partitioned aqueous solution tanks 113, and a current measuring device 117 is provided via the electrodes 116.
- An electrode 118 is put into the other tank, and an earth 119 is made to the aqueous solution tank 113 via the electrode 118!
- the lipid solution 110 is applied to a small hole 115 formed in a support such as a partition plate 114 for partitioning the liquid (not shown in FIG. 14) with a thin glass tube or the like. In this state, the lipid solution 110 is raised so as to protrude from both the surface side of the partition plate 114 while closing the small holes 115.
- This lipid solution 110 is a solution obtained by dissolving lipid in an organic solvent such as decane.
- the lipid solution 110 moves on the surface of the partition plate 114, whereby a naturally thinned artificial lipid bilayer membrane 111 is obtained.
- the term “thinning” refers to a process in which an organic solvent or the like moves in the central part of the applied lipid solution 110 and a lipid bilayer is formed in the central part.
- a lipid monomolecular film 121 is developed at a gas-liquid interface in an aqueous solution tank 113 (not shown in FIG. 15). I do.
- the gas-liquid interface is set at the same position as the lower end of the small hole 115 formed in the partition plate 114.
- the liquid surface (gas-liquid interface) in one of the two aqueous solution tanks 113 (right side in the figure) is raised to separate the monomolecular film 121.
- the surface of the plate 114 is opened. Thereby, one opening of the small hole 115 is formed by the monomolecular film 121. It will be in a blocked state.
- the liquid surface (gas-liquid interface) in the other tank (left side in the figure) of the aqueous solution tank 113 divided into two is raised, whereby the monomolecular film 121 is formed. It is spread on the surface of the partition 114. As a result, the other opening of the small hole 115 is also in a state where the monomolecular film 121 is closed. As a result, the monomolecular film 121 is attached to both the openings of the small holes 115, and finally the artificial lipid bilayer membrane 111 is formed.
- the aqueous solution tank 113 shown in FIG. 16 (a) the small holes 115 formed in the partition plate 114 are closed with the lipid solution 110, and the lipid solution 110 waits until the lipid solution 110 is naturally thinned to become the artificial lipid bilayer 111.
- the artificial lipid bilayer membrane 111 is formed by increasing the water pressure in the tank above the small holes 115 to expand and thin the lipid solution 110 downward.
- the artificial lipid bilayer membrane 111 is used. Formation cannot be artificially controlled at all. Therefore, thinning may take several hours or more.
- the resulting human lipid bilayer 111 is referred to as “lipid bilayer”. This results in a thinner part and a thicker part surrounding it, called an annular Balta layer. Therefore, the artificial lipid bilayer 111 obtained by this method is based on the physical equilibrium of each part described above. The lamina 111 tears quickly . Since it is difficult to accurately control the pressure difference between the upper and lower tanks in the aqueous solution tank 113, the resulting artificial lipid bilayer membrane 111 is more likely to be unstable.
- the present inventors have proposed a technique for improving the above-mentioned conventional problems in an artificial lipid bilayer membrane used for a current measuring device (for example, see Non-Patent Document 2).
- a current measuring device obtained by this technology, it is possible to simultaneously measure the structure and function of ion channel molecules using an artificial lipid bilayer membrane.
- the current measuring device includes two solution tanks, an upper solution tank 101 and a lower solution tank 102.
- a film 103 having a small hole 105 at the center is attached to the bottom surface of the upper solution tank 101.
- the lower solution tank 102 has an opening 104 on the bottom surface, and a cover glass 106 is fixed with an adhesive.
- An agarose gel layer (not shown) is formed on the cover glass 106.
- a thick film of the lipid solution is formed in the small holes 105 by moving the lower part of the upper solution tank 101 in the lipid solution.
- the upper solution tank 101 is put into the lower solution tank 102, and the upper solution tank 101 is lowered until the thick film formed in the small hole 105 contacts the agarose gel layer formed on the cover glass 106.
- the pressure (water pressure) of the upper solution tank 101 excess lipid solution is pushed out from between the agarose gel layers, and the thick membrane is thinned to form an artificial lipid bilayer.
- the conventional technology for forming an artificial lipid bilayer membrane described above may have difficulty in stably forming an artificial lipid bilayer membrane, and thus has a problem that a more stable formation technology is required. are doing.
- the present invention has been made in view of the above-mentioned conventional problems, and has an object to quickly form a stable artificial lipid bilayer membrane, for example, to simultaneously measure the structure and function of a single channel.
- An object of the present invention is to provide an apparatus and a method for forming an artificial lipid bilayer membrane which can be used for an electric current measuring device to be measured, and an example of typical use thereof.
- the apparatus for forming an artificial lipid membrane according to the present invention has a membrane-forming opening formed in a flat plate-shaped portion, and is capable of accumulating a membrane support and an aqueous solution.
- a membrane-forming solution tank, and a support layer for supporting the artificial lipid bilayer membrane is provided in the membrane-forming solution tank, and the artificial lipid formed at the membrane-forming opening of the membrane support is provided.
- a lipid solution discharging means for discharging at least one surface of the membrane-forming opening.
- the membrane support is charged into the membrane-forming solution tank so as to be in contact with the solution, and then a lipid solution is applied to the membrane-forming opening, and the lipid solution is sandwiched between the aqueous solution and the support layer.
- the method is characterized in that a thin artificial lipid bilayer membrane is formed at the membrane-forming opening by contacting the support layer and discharging excess lipid solution by the lipid solution removing means in this state.
- the lipid solution discharging means since the lipid solution discharging means is provided, it is possible to discharge the excess lipid solution from the lipid solution applied to the membrane-forming opening without changing the water pressure. Therefore, since no pressure is applied from above the film, the film does not break or become unstable when thinning. Therefore, a stable and highly durable artificial lipid bilayer membrane can be rapidly formed.
- the method for forming an artificial lipid membrane according to the present invention uses a membrane support in which a membrane-forming opening is formed in a flat plate-like portion, and at least one surface of the membrane-forming opening. Is placed in contact with an aqueous solution, and a lipid solution application step of applying a lipid solution to the membrane-forming opening, and applying the lipid solution applied to the membrane-forming opening to the aqueous solution with a hydrophilic surface.
- the lipid solution applied to the membrane-forming opening can also discharge the excess lipid solution without changing the water pressure. Therefore, since no pressure is applied from above the membrane, the membrane is not broken or becomes unstable in the lipid membrane thinning step. Therefore, a stable and highly durable artificial lipid bilayer membrane can be rapidly formed.
- the current measuring device according to the present invention is characterized by being formed using the artificial lipid bilayer forming device according to the present invention.
- a stable artificial lipid bilayer membrane can be used for current measurement through the membrane, so that current measurement can be performed accurately.
- FIG. 1 is a cross-sectional view illustrating a configuration of an apparatus for forming an artificial lipid bilayer membrane according to one embodiment of the present invention.
- FIG. 2 shows a portion of the apparatus for forming an artificial lipid bilayer membrane shown in FIG. 1 in which a lipid solution is applied to a small hole of an upper solution tank and excess lipid solution is sucked by a suction unit. It is a sectional view, (b) is a partial sectional view showing a state where an artificial lipid bilayer membrane was formed by sucking an excess lipid solution.
- FIG. 3 is a cross-sectional view illustrating an example of a configuration of a suction unit in the apparatus for forming an artificial lipid bilayer shown in FIG. 1.
- FIG. 4 (a)-(c) are cross-sectional views showing steps of a method for forming an artificial lipid bilayer membrane according to one embodiment of the present invention.
- FIG. 5 is a cross-sectional view illustrating a current measuring device according to one embodiment of the present invention.
- FIG. 6 is a partial cross-sectional view of a small hole and a support layer in an artificial lipid bilayer membrane forming apparatus according to another embodiment of the present invention.
- (B) shows a state in which the lipid solution and the support layer are in contact with each other, and
- (c) shows a state in which an artificial lipid bilayer is formed.
- FIG. 7 (a) Partial cross-section showing the appearance of the artificial lipid bilayer formed by pressing the membrane support coated with the lipid solution against the polymer gel and the polymer gel penetrating into the pores.
- FIG. 3B is a partial cross-sectional view showing a state in which an artificial lipid bilayer is formed on a polymer gel in which a portion in contact with a small hole is previously formed higher than other portions.
- FIG. 8 (a)-(c) is a cross-sectional view showing a step of a method for forming an artificial lipid bilayer membrane according to another embodiment of the present invention.
- FIG. 9 is a photograph showing a state where the thinning of the artificial lipid bilayer membrane is completed in [Example 1].
- FIGS. 10 (a) and (b) are diagrams showing current traces obtained in [Example 1].
- FIG. 11 is a drawing showing potential-current characteristics obtained in [Example 1].
- FIG. 12 (a) is a drawing showing a fluorescence image of fluorescently labeled aramethicin observed in [Example 2], and (b) is a drawing showing a current trace measured in [Example 2]. It is.
- FIG. 13 is a schematic diagram showing a conventional lipid planar membrane method.
- FIG. 14 is a drawing showing a conventional vertical painting method.
- FIG. 15 is a drawing showing a conventional vertical bonding method.
- FIG. 16 (a) and (b) are drawings showing a conventional method for forming a horizontal artificial lipid bilayer membrane.
- FIG. 17 is a drawing showing a conventional current measuring device.
- FIG. 18 is a drawing showing a conventional artificial lipid bilayer formed on a polymer gel layer.
- an apparatus for forming an artificial lipid bilayer membrane according to the present invention includes at least an upper solution tank 1 and a lower solution tank (membrane forming solution tank). 2, a support layer 3, and a suction unit (suction unit) not shown.
- the suction section functions as a lipid solution discharging means for discharging the excess lipid solution from the lipid solution applied to the film forming opening without changing the water pressure.
- the bottom surface of the upper solution tank 1 is a membrane support 5, and an artificial lipid bilayer membrane is formed in the small holes (openings for membrane formation) 11 formed in the membrane support 5.
- the specific configuration of the membrane support 5 is not particularly limited as long as the configuration is such that small holes 11 serving as membrane-forming openings are formed in a flat plate-like portion.
- the membrane support 5 is a bottom portion of the upper solution tank 1 capable of storing a solution.
- the membrane support 5 is not particularly limited as long as it has a plate shape or a film shape.
- a small hole 11 is formed in the bottom surface of the tank 1 itself, so that the film having the small hole 11 formed in the opening is provided in the bottom of the upper solution tank 1. It may have a bonded configuration.
- the material of the membrane support 5 is not particularly limited, but specifically, for example, plastics such as polypropylene, polychlorinated vinyl, and polystyrene, and fluorine resin are preferably used. . Further, the thickness of the membrane support 5 is preferably 0.1 mm or more and 0.3 mm or less. By making only the periphery of the small hole 11 thinner than the other regions, a stable artificial lipid bilayer membrane can be quickly formed.
- the pores 11 preferably have a diameter of lO / zm or more and 500 ⁇ m or less, more preferably 50 ⁇ m or more and 200 ⁇ m or less. Thereby, an artificial lipid bilayer membrane can be favorably formed.
- the small holes 11 can be formed by, for example, a conventionally known method as described below. First, a stainless steel rod sharply cut in a conical shape to the very tip is heated with a gas burner or the like. Next, this is strongly pressed against the surface on which the small holes 11 are formed, and the pressing is continued until a small bulge is formed on the side opposite to the pressed surface. A small hole 11 is formed by cutting out the bulge with a razor. In addition, the small holes 11 are cleaned with a form-form methanol methanol to remove impurities and the like.
- the method for forming the small holes 11 is not limited to this, and any known method can be used.
- the upper solution tank 1 is provided above the lower solution tank 2 and is capable of storing an aqueous solution, and is not particularly limited as long as the membrane support 5 is configured to serve as the bottom surface of the upper solution tank 1. Not a thing.
- the shape of the upper solution tank 1 is not particularly limited, and examples thereof include a cylindrical shape.
- the size of the upper solution tank 1 is not particularly limited as long as it is larger than the small holes 11, but, for example, in the case of a cylindrical shape, the inner diameter is preferably 0.5 mm or more and 20 mm or less. Mass 1. More preferably Omm or more and 10 mm or less. Further, the size of the upper solution tank 1 can be preferably reduced to a size of several tens / zm. Further, the volume of the upper solution tank 1 is not particularly limited, but is preferably not less than 0.1 Olcm 3 and not more than 1.0 Ocm 3 . Thereby, the artificial lipid double according to the present invention A film forming apparatus can be formed on a small chip, and a smaller sensor can be manufactured.
- the material of the upper solution tank 1 other than the portion where the small holes 11 are formed is not particularly limited.
- glass, plastic, or the like is used. Are listed.
- the upper solution tank 1 is capable of storing an aqueous solution.
- Each side of the artificial lipid bilayer formed in the small hole 11 is in contact with the aqueous solution filled in the upper solution tank 1 and the lower solution tank 2.
- the aqueous solution is not particularly limited as long as it does not contain a surfactant, an organic solvent and the like.
- Preferred examples of the aqueous solution include aqueous solutions of potassium salt, sodium salt, calcium chloride and the like.
- the upper solution tank 1 be capable of moving vertically.
- the vertical movement of the upper solution tank 1 may be performed manually, or a device for movement may be used.
- a device for movement a micro-multiplier or the like can be given.
- the lower solution tank 2 may be disposed below the upper solution tank 1 so that the aqueous solution can be accumulated therein, and may be a film forming solution tank for forming the membrane support 5.
- a support layer 3 for supporting the artificial lipid bilayer membrane is provided on the bottom surface of the lower solution tank 2. Then, the artificial lipid bilayer formed in the small holes 11 of the upper solution tank 1 is supported by being brought into contact with the support layer 3.
- the volume of the lower solution tank 2 is not particularly limited as long as the bottom of the upper solution tank 1 can be put on the support layer 3 formed thereon.
- the material of the lower solution tank 2 is not particularly limited, but examples thereof include glass and plastic such as polystyrene.
- the aqueous solution contains a surfactant, an organic solvent, and the like, like the aqueous solution in the upper solution tank 1. It is not particularly limited unless it is inevitable.
- Preferable examples of the aqueous solution include aqueous solutions of potassium salt sodium, sodium salt sodium, calcium salt sodium and the like.
- the support layer 3 provided on the bottom surface of the lower solution tank 2 may have any configuration as long as it is in contact with and supports the artificial lipid bilayer membrane formed by the small holes 11 of the membrane support 5.
- the support layer 3 is not particularly limited as long as it can penetrate an aqueous solution and can support the artificial lipid bilayer membrane 2.
- Specific examples of the support layer 3 include a porous membrane such as a polymer gel and a cellulose membrane. Above all, it is more preferable that the support layer 3 also has a polymer gel force.
- the polymer gel is not specifically limited, but polysaccharides such as agarose and hydrophilic resins such as polyacrylamide can be preferably used. By using these, the support layer 3 can be easily formed using an inexpensive and highly reliable material.
- the thickness of the support layer 3 is not particularly limited, but is preferably 100 nm or more and 2 mm or less. When the thickness of the support layer 3 is within this range, the formed artificial lipid bilayer membrane is vertically stabilized.
- the method of forming the support layer 3 is not particularly limited, and a conventionally known method may be used.
- a dispersion of agarose is prepared, and heated to dissolve the agarose.
- a method in which the solution is applied to the bottom of the lower solution tank 2 and dried at room temperature may be used.
- the artificial lipid bilayer membrane is formed in the small hole 11 formed in the bottom surface 5 of the upper solution tank 1 as described above. As will be described later, the artificial lipid bilayer is formed by applying a lipid solution to the small holes 11 and then bringing the lipid solution into contact with the support layer 3 of the lower solution tank 2 and sucking excess lipid solution by the suction unit. You.
- the lipid is not particularly limited as long as it forms an artificial lipid bilayer membrane, but phospholipids are preferably used. Specifically, for example, phosphatidylcholine, difita Nylphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and the like.
- the two hydrocarbon chains in these phospholipids may be saturated hydrocarbons, or may be unsaturated hydrocarbons. These lipids may be used pure or may be a mixture of at least two kinds of lipids.
- the lipid solution refers to a solution in which the lipid is dispersed in an organic solvent.
- the organic solvent used is not particularly limited as long as it is a non-polar organic solvent.
- a saturated hydrocarbon such as decane, hexadecane, and hexane is suitably used.
- the lipid concentration is more preferably 5 to 40 mg / mL, more preferably 15 to 20 mg / mL.
- the suction unit may be configured so that the lipid solution applied to the small hole 11 can be discharged by sucking the excess lipid solution.
- the lipid solution 13 is applied to the small holes 11 of the membrane support 5. This lipid solution 13 is brought into contact with the support layer by sandwiching it between the aqueous solution and the support layer as described later. In this state, the excess lipid solution 13 is discharged using the suction unit 12, and a thin artificial lipid bilayer membrane 14 is formed in the small holes 11.
- the suction unit 12 is a tubular member that can suck an excess lipid solution 13 other than an amount necessary for forming a lipid bilayer from the lipid solution 13 to which the peripheral force is also applied. Anything is fine.
- a glass tube connected to a syringe, a silicon dropper and the like can be mentioned.
- the suction may be performed using a syringe 17 to which a glass tube 15 is stretched by heating and thinly drawn, and then attached to a lipid solution 13 and a silicon tube 16 is connected thereto.
- a syringe 17 to which a glass tube 15 is stretched by heating and thinly drawn, and then attached to a lipid solution 13 and a silicon tube 16 is connected thereto.
- sucking the lipid solution 13 in order to form the membrane without breaking, it is preferable to suck from the periphery of the opening, and it is particularly preferable to suck from the Balta portion.
- the method of suction is not limited to the above.
- an artificial lipid bilayer membrane which is useful in the present invention
- the artificial lipid described in (1) above is used.
- the case where a double-film forming apparatus is used will be described with reference to FIGS.
- lipid solution application step will be described. As shown in FIG. 4 (a), a support layer 3 is previously formed at the bottom of the lower solution tank 2, and the upper solution tank 1 and the lower solution tank 2 are filled with the above aqueous solution. Then, the lipid solution 13 is applied to the small holes 11 formed in the membrane support 5 serving as the bottom surface of the upper solution tank 1.
- the support layer contacting step will be described.
- the bottom surface of the upper solution tank 1 is put into the lower solution tank 2 and brought into contact with the support layer 3, and the lipid solution 13 coated with the small holes 11 in the above-mentioned lipid solution coating step is lifted up. It is sandwiched between the aqueous solution in the solution tank 1 and the support layer 3. Thereby, the applied lipid solution 13 can be brought into contact with the support layer 3.
- lipid membrane thinning step As shown in FIG. 4 (c), a suction unit 12 having a tubular member is attached to the lipid solution 13 that has been brought into contact with the support layer 3, and as described above, the excess lipid solution 13 is sucked and discharged, and the pores are removed. An artificial lipid bilayer membrane 14 thinned to 11 is formed.
- the excess lipid solution 13 is discharged from the lipid solution 13 applied to the small holes 11 without changing the water pressure, that is, without increasing the water pressure applied to the lipid solution 13 as in the related art.
- the artificial lipid bilayer membrane can be formed in the small holes 11 in a state of being in contact with the support layer 3.
- the upper solution tank 1 is an open system
- the bottom of the lower solution tank 2 is closed and not an open system. Therefore, the artificial lipid bilayer is stabilized in a direction parallel to the bottom surface, which was conventionally unstable, so that the durability of the artificial lipid bilayer can be further improved.
- the apparatus for forming an artificial lipid bilayer membrane according to the present invention can form a stable and highly durable artificial lipid bilayer membrane in a short time. Further, the present invention also includes an artificial lipid bilayer formed using the apparatus for forming an artificial lipid bilayer according to the present invention.
- any change in the artificial lipid bilayer membrane for example, change in membrane potential, electric capacity, ion permeability, luminescence, heat generation, endotherm, endotherm, etc.
- a dissolved substance in a sample liquid And its concentration can be detected.
- a lipid bilayer membrane include an artificial lipid bilayer membrane substantially composed of only lipids, and an artificial lipid bilayer membrane containing various molecules such as proteins and sugars without adhering thereto. be able to.
- Examples of the membrane protein include an ion channel.
- the method for embedding the ion channel in the artificial lipid bilayer membrane can be a conventionally known method, and is not particularly limited. Specifically, for example, there is a method in which a membrane fraction containing an ion channel is solubilized with a surfactant, reconstituted into a membrane vesicle, and fused with an artificial lipid bilayer membrane.
- a current measuring device can be formed using the device for forming an artificial lipid bilayer membrane, and this current measuring device is also included in the present invention.
- This current measuring device is not particularly limited as long as it can measure the current flowing through the artificial lipid duplex.
- the current measuring device according to the present embodiment has a configuration in which the current electrically connected to the upper solution tank 1 is added to the configuration of the artificial lipid bilayer forming device shown in FIG.
- a measuring instrument (current measuring means) 18 and an earth (earth means) 19 electrically connected to the lower solution tank 2 are provided.
- the configuration of the current measuring device is not limited to the above.
- An electrode 20 is supplied to the upper aqueous solution tank 1, and a current measuring device 18 is provided via the electrode 20.
- An electrode 21 is supplied to the lower aqueous solution tank, and an earth 19 is provided to the lower aqueous solution tank 2 via the electrode 21.
- the bottom surface of the lower solution tank 2 is made of a light-transmitting material, and below the bottom surface, optical observation is performed so that the artificial lipid bilayer membrane on the support layer 3 can be observed.
- Means 18 may be provided.
- Examples of the optical observation means include an optical microscope. Observation with an optical microscope includes, for example, changes in the fluorescence intensity of a fluorescently labeled ion channel due to opening and closing of the gate, movement of the ion channel, and spectrum transfer due to energy transfer between two fluorescent dyes. Observation of changes and the like. In addition, it is possible to confirm with an optical microscope that an artificial lipid bilayer is formed. Furthermore, the movement of lipid molecules can also be observed using an artificial lipid bilayer using fluorescently labeled lipids. Of course, the optical measurement is not limited to these, and any conventionally known method can be applied.
- the apparatus for forming an artificial lipid bilayer membrane of the present invention can further stabilize the formed artificial lipid bilayer membrane. Therefore, even if the membrane protein is incorporated in the artificial lipid bilayer membrane, it can be sufficiently obtained.
- the structure and function of a membrane protein can be simultaneously measured in a stable state.
- Ion channel proteins are distributed in almost all cells of many types. Therefore, these are proteins that are likely to cause disease, and it is said that 30-40% of drug discovery targets are ion channel proteins.
- the effect is measured by administering a reagent to an experimental animal, but if a stable artificial lipid bilayer membrane 2 is formed, it is possible to directly investigate the effect on the target ion channel protein in drug discovery. Screening can be performed.
- drugs that act on the nervous system such as psychotropic drugs
- the current measuring device of the present invention can be used for visualization analysis of protein-protein (drug) interaction on the artificial lipid bilayer membrane. Furthermore, by changing the type of molecule incorporated into the artificial lipid bilayer membrane, it can be applied to the detection of various substances.
- the artificial lipid bilayer forming apparatus has the same configuration as the artificial lipid bilayer forming apparatus shown in FIG.
- the excess lipid solution is drained from the lipid solution applied to the opening without changing the water pressure by the membrane support having a movable part.
- the movable membrane support functions as a lipid solution discharging unit.
- the support layers are different as described below.
- the movable membrane support is not particularly limited as long as it can move in the direction of pressing the support layer provided in the lower solution tank 2.
- the movable membrane support is a bottom portion of the upper solution tank 1 in which a solution can be stored.
- the lipid solution 13 applied to the small holes 11 of the membrane support 50 is pressed against the support layer 30 by using the movable membrane support 50 as a lipid solution discharging means, and the lipid solution 13 By pushing out, the excess lipid solution is discharged from the lipid solution 13 applied to the small holes 11 without changing the water pressure.
- the lipid solution 13 is extruded through the gap between the movable membrane support 50 and the support layer 30, and the artificial lipid bilayer membrane is thinned.
- the movable membrane support 50 be capable of moving up and down.
- the membrane support 50 serves as the bottom surface of the upper solution tank 1, it can move together with the upper solution tank 1.
- the movement of the upper solution tank 1 may be performed manually, or a device for movement may be used.
- a micromanipulator or the like can be given.
- the support layer 30 is provided on the bottom surface of the lower solution tank 2 as in the first embodiment, and is configured to contact and support the artificial lipid bilayer formed by the small holes 11 of the membrane support 50. Good.
- the support layer 30 is preferably resilient because it is pressed through the movable membrane support 50.
- a polymer gel layer may be used as the support layer 30 in the same manner as in Embodiment 1.
- the thickness of the polymer gel layer is preferably 0.5 mm or more and 2.Omm or less. With this thickness, when the support layer 30 and the lipid solution 13 applied to the small holes 11 of the membrane support 50 are pressed, excess lipid solution 13 can be preferably extruded.
- the support layer 30 When the movable membrane support 50 coated with the lipid solution is pressed against the support layer 30, when the support layer 30 is, for example, a polymer gel layer, the shape of the support layer 30 is deformed. As shown in FIG. 7 (a), it may enter the small hole 11. Therefore, as shown in FIG. 7 (b), the support layer 30 may be previously set to be 50 m or more and 200 m or less higher in the area in contact with the small hole 11 than in the other area. In this way, the support layer 30 can be pushed into the small holes 11 by being raised. Therefore, the support layer 30 can be in good contact with the lipid bilayer, and a stable artificial lipid bilayer can be formed.
- FIG. 8 (a) — (c) Explain based on
- lipid solution application step will be described. As shown in FIG. 8 (a), a support layer 30 is previously formed at the bottom of the lower solution tank 2, and the upper solution tank 1 and the lower solution tank 2 are filled with the above aqueous solution. Then, the lipid solution 13 is applied to the small holes 11 formed in the membrane support 50 serving as the bottom surface of the upper solution tank 1.
- the support layer contacting step will be described. As shown in FIG. 8 (b), the bottom surface of the upper solution tank 1 was put into the lower solution tank 2 and was brought into contact with the support layer 30. It is sandwiched between the aqueous solution in the tank 1 and the support layer 30. Thereby, the applied lipid solution 13 can be brought into contact with the support layer 30.
- the lipid membrane thinning step will be described.
- the lipid solution 13 is pressed against the support layer 30 by pressing the movable membrane support 50 against the support layer 30, and the lipid solution 13 is discharged.
- the lipid solution 13 is extruded through the gap between the membrane support 50 and the support layer 30, and the artificial lipid bilayer membrane is thinned.
- the excess lipid solution 13 is discharged from the lipid solution 13 applied to the small holes 11 without changing the water pressure, and the artificial lipid bilayer membrane is brought into contact with the support layer 30 in the small holes 11. Can be formed.
- the upper solution tank 1 is an open system
- the bottom of the lower solution tank 2 is closed and not an open system. Therefore, the artificial lipid bilayer is stabilized in a direction parallel to the bottom surface, which has been unstable in the past, so that the durability of the artificial lipid bilayer can be further improved.
- the present invention will be described in more detail with reference to Examples and FIGS. 9 to 13, but the present invention is not limited thereto.
- the upper solution tank 1 is a polypropylene having a volume of 0.1 cm 3 , a bottom surface of 0.2 mm—0.3 mm, and a small hole 11 having a diameter of 0.15 mm.
- An agarose gel layer having a thickness of 150 nm was formed on the bottom of the lower tub 2 and used as a support layer 3.
- an Ag—AgCl electrode obtained by plating an Ag foil was used.
- a glass tube connected to a silicon tube was used as a suction unit for thinning lipids.
- the current was measured using a notch clamp amplifier (CEZ-2400, manufactured by Nihon Kohden) and recorded on a DAT tape using a DAT recorder.
- the upper solution chamber 1 and the lower solution chamber 2 lOOmM KC1, ImM CaCl (or 10- 9 MC
- the current was measured in the same manner as described above.
- FIG. 10 shows a trace of the obtained current
- FIG. 11 shows a membrane potential-current characteristic.
- 10 (a) is, if the aqueous solution is LOOmM KC1, including 10- 9 M CaCl
- FIG. 10 (b) an aqueous solution of 100
- the ratio of ion channel opening increased.
- the obtained current trace pattern was the same as the pattern that was clearly apparent from the conventional experimental results.
- the current measuring device using the artificial lipid bilayer forming device of the present invention a stable artificial lipid bilayer membrane can be formed quickly and easily, and reliable current measurement can be performed. It turns out that a result is obtained. From Fig. 11, the magnitude of the single-channel current (single-channel inductance) agreed with the value clarified by the conventional method. This indicates that the current measurement device of the present invention can measure a current without the agarose gel layer directly below the artificial lipid bilayer affecting the properties of the channel.
- Cy3 a methanol solution of Aramechishin fluorescently-labeled with (Amanshamu 'manufactured by Pharmacia) (manufactured by Sigma), final concentration urchin upper solution chamber 1 by which is about 10- 8 M Added to the aqueous solution.
- the fluorescent labeling of aramethicin was performed by adding glycine to the C-terminus of aramethicin, fixing Cy3 to the amino group of the glycine using a Cy3 monoflual dye kit (manufactured by Amansham Pharmacia), and binding. .
- Aramethicin is an amphipathic peptide that naturally migrates from the liquid phase to an artificial lipid bilayer to form an ion channel.
- an aqueous solution to be filled in the upper and lower solution tanks an aqueous solution of 100 mM KC1 and 10 mM Hepes (pH 7.4) was used.
- the fluorescence image and the ion current due to aramethicin were simultaneously observed and measured. The fluorescence image was measured using an objective lens type total internal reflection fluorescence microscope (TIRFM).
- FIG. 12 (a) shows the obtained fluorescent image of fluorescent alamethicin and the locus of Brownian motion in the film
- FIG. 12 (b) shows the current trace.
- an opening for forming a membrane is formed in a flat plate-shaped portion, and a membrane support and an aqueous solution can be accumulated.
- a membrane-forming solution tank, and a support layer for supporting the artificial lipid bilayer membrane is provided in the membrane-forming solution tank, and the artificial lipid formed at the membrane-forming opening of the membrane support is provided.
- the apparatus for forming an artificial lipid bilayer which supports the bilayer by contacting the bilayer on the support layer, further comprising an excess lipid solution from the lipid solution applied to the membrane-forming opening without changing the water pressure.
- the membrane support is put into a membrane-forming solution tank so that at least one surface of the membrane-forming opening is in contact with the aqueous solution.
- a lipid solution is applied to the opening for formation, and the lipid solution is sandwiched between the aqueous solution and the support layer.
- the lipid-removing means is used to discharge the excess lipid solution, thereby forming a thin artificial lipid bilayer membrane at the membrane-forming opening. It is.
- the apparatus for forming an artificial lipid membrane according to the present invention is capable of accumulating an aqueous solution with a membrane support in which a membrane-forming opening is formed in a flat plate-like portion. And a lipid solution discharging means for discharging the lipid solution applied to the film forming opening by sucking the excess lipid solution. Configuration.
- the artificial lipid membrane forming apparatus has a configuration in which the membrane forming solution tank is provided with a support layer that supports an artificial lipid bilayer formed at the membrane-forming opening of the membrane support. Yo Is more preferable.
- the membrane support is charged into the membrane-forming solution tank so that at least one surface of the membrane-forming opening is brought into contact with the aqueous solution, and then the lipid solution is poured into the membrane-forming opening.
- the lipid solution is sandwiched between the aqueous solution and the support layer and brought into contact with the support layer, and in this state, the excess lipid solution is discharged by the lipid solution removing means, thereby forming the membrane-forming opening.
- a thin artificial lipid bilayer membrane can be formed in the part.
- the lipid solution discharging means since the lipid solution discharging means is provided, the excess lipid solution can be discharged from the lipid solution applied to the membrane-forming opening without changing the water pressure. Therefore, since no pressure is applied from above the film, the film does not break or become unstable when thinning. Therefore, a stable and highly durable artificial lipid bilayer membrane can be rapidly formed.
- the apparatus for forming an artificial lipid membrane according to the present invention is capable of storing an aqueous solution provided above the lower solution tank when the membrane forming solution tank is a lower solution tank.
- a liquid tank may be provided, and the support layer may be provided on a bottom surface of the lower solution tank, and the membrane support may be a bottom surface of the upper solution tank.
- the bottom surface of the upper solution tank to which the lipid solution has been applied can be easily brought into contact with the support layer provided on the bottom surface of the lower solution. Therefore, a stable artificial lipid bilayer membrane can be quickly and easily formed with a simple configuration.
- the lipid solution discharging means may be a suction means for suctioning the lipid solution applied to the opening for forming a membrane by a tubular member.
- the excess lipid solution is sucked from the lipid solution applied to the forming opening by using the suction means. Therefore, the excess lipid solution can be easily discharged in a short time, and the artificial lipid bilayer can be thinned.
- the membrane support is movable in a direction of pressing a support layer provided in the membrane forming solution tank, and is capable of moving.
- the membrane support as the lipid solution discharging means, the lipid solution applied to the membrane-forming opening is pressed against the support layer and the lipid solution is extruded to remove excess lipid solution. May be issued.
- the membrane support is movable in a direction of pressing a support layer provided in the membrane forming solution tank, and the movable body is movable.
- the membrane support as the lipid solution discharging means and pressing the membrane support applied to the membrane-forming opening against the support layer, the lipid solution is applied to the membrane-forming opening and other regions. It is more preferable to separate them by ⁇ .
- the membrane support is movable, an excess lipid solution can be discharged by pressing the lipid solution onto the support layer and pushing out the lipid solution. Therefore, since there is no need to prepare a member for discharging the excess lipid solution, the artificial lipid bilayer can be easily and inexpensively thinned.
- the support layer is preferably made of a polymer gel.
- the thickness of the polymer gel layer is arbitrary and does not vary.However, when the lipid solution discharging means is a suction member, it is not less than 100 nm and not more than 2.0 mm, and is a movable membrane support. Is preferably 0.5 mm or more and 2 mm or less. Further, it is preferable that the polymer gel is higher than other regions in a region in contact with the film forming opening, and is higher than 50 ⁇ m to 200 ⁇ m. Is more preferred.
- the artificial lipid double membrane is vertically supported by the polymer gel layer when the thin film is formed.
- the diameter of the membrane-forming opening is preferably 10 ⁇ m or more and 500 ⁇ m or less! / ⁇ .
- an artificial lipid bilayer membrane can be formed favorably.
- the method for forming an artificial lipid membrane according to the present invention uses a membrane support in which a membrane-forming opening is formed in a plate-like portion, and at least one surface of the membrane-forming opening is treated with an aqueous solution.
- a lipid solution application step of applying a lipid solution to the membrane-forming opening after contacting the lipid solution, and supporting the lipid solution applied to the membrane-forming opening with the aqueous solution and a hydrophilic surface. Support layer contact between the support layer and the applied lipid solution sandwiched between the layers. Discharging the excess lipid solution from the lipid solution applied to the membrane-forming opening without changing the water pressure, thereby forming the thinned artificial lipid bilayer in the membrane-forming opening. And forming a lipid membrane thinning step.
- the lipid solution applied to the membrane-forming opening can also discharge the excess lipid solution without changing the water pressure. Therefore, since no pressure is applied from above the membrane, the membrane is not broken or becomes unstable in the lipid membrane thinning step. Therefore, a stable and highly durable artificial lipid bilayer membrane can be rapidly formed.
- an excess lipid solution may be discharged by sucking the lipid solution by a tubular member.
- the excess lipid solution can be easily discharged in a short time, and the artificial lipid bilayer can be thinned.
- the lipid solution applied to the membrane-forming opening is pressed against the support layer to extrude the lipid solution.
- the excess lipid solution may be discharged.
- the artificial lipid bilayer membrane can be easily and inexpensively thinned.
- the artificial lipid bilayer membrane of the present invention is characterized by being formed using the above-described method for forming an artificial lipid bilayer membrane.
- the artificial lipid bilayer membrane is formed using the above-described method.
- the artificial lipid bilayer is stable and durable.
- the artificial lipid bilayer membrane of the present invention has a membrane protein incorporated into the above-described artificial lipid bilayer membrane! I do not work.
- the current measuring device according to the present invention is characterized by being formed using the artificial lipid bilayer forming device according to the present invention.
- a stable artificial lipid bilayer membrane can be used when measuring current through a membrane, so that current measurement can be performed accurately.
- the current measuring device is provided with a support layer for the film forming solution tank.
- a support layer for the film forming solution tank In addition to the fact that the side surface also has a light-transmitting material, an optical observation means for observing the artificial lipid bilayer membrane on the support layer is provided outside the side surface! / ⁇ .
- the artificial lipid bilayer membrane can be optically observed through the membrane forming solution tank and the support layer.
- the current measuring device includes current measuring means electrically connected to the upper solution tank, and ground means electrically connected to the lower solution tank. Is preferred.
- the current measuring device is capable of controlling the artificial lipid bilayer membrane even if the membrane protein force ion channel may be a membrane protein.
- lipid bilayer forming apparatus of the present invention As described above, by using the lipid bilayer forming apparatus of the present invention, a stable and highly durable artificial lipid bilayer membrane can be formed in a short time. Moreover, the lipid bilayer membrane of the present invention formed using the above-described artificial lipid forming apparatus is stable and has high durability.
- the use of the current measuring device of the present invention enables the detection of various substances depending on the molecules incorporated in the stable artificial lipid bilayer membrane. For example, when an ion channel is incorporated, the state of ion transmission can be confirmed. Therefore, the above-described current measuring device can be used for functional analysis of the ion channel.
- Ion channels are distributed in almost all types of cells and play an important role for the survival of living organisms, and thus are likely to cause diseases. For this reason, many drugs targeting the ion channel are needed. Since the present invention has a possibility that it can be used for screening pharmacology tests in drug development targeting ionic channels, it can be said that its usefulness is high.
- lipid bilayer forming apparatus of the present invention As described above, by using the lipid bilayer forming apparatus of the present invention, a stable and highly durable artificial lipid bilayer membrane can be formed in a short time.
- the above-described artificial lipid forming apparatus The lipid bilayer membrane of the present invention formed using a device is stable and has high durability.
- the current measuring device of the present invention it can be used for detection of various substances depending on molecules incorporated in a stable artificial lipid bilayer membrane.
- the present invention can be used in a wide range of fields such as bioscience, biomedical, pharmaceutical, and other life science using a biomembrane model, and the field of electronics using a biomembrane model as a device.
- fields such as bioscience, biomedical, pharmaceutical, and other life science using a biomembrane model
- the field of electronics using a biomembrane model as a device For example, it can be used in the development of various sensors using biological membrane models, screening and pharmacological tests in the development of drugs targeting membrane proteins.
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Abstract
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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EP04773299A EP1677102A4 (en) | 2003-09-19 | 2004-09-17 | DOUBLE MEMBRANE DEVICE AND METHOD FOR FORMING ARTIFICIAL LIPIDS, AND METHOD OF USE |
US10/572,205 US7858030B2 (en) | 2003-09-19 | 2004-09-17 | Artificial lipid bilayer membrane formation device, artificial lipid bilayer membrane formation method, and usage thereof |
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JP2003328651A JP4394916B2 (ja) | 2003-09-19 | 2003-09-19 | 人工脂質二重膜の形成装置および人工脂質二重膜の形成方法、並びにその利用 |
JP2003-328651 | 2003-09-19 |
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US (1) | US7858030B2 (ja) |
EP (1) | EP1677102A4 (ja) |
JP (1) | JP4394916B2 (ja) |
WO (1) | WO2005029056A1 (ja) |
Cited By (1)
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EP1710578A1 (en) * | 2005-04-08 | 2006-10-11 | Charite-Universitätsmedizin Berlin | Method and apparatus for forming a lipid bilayer membrane |
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CN102145259A (zh) * | 2005-05-20 | 2011-08-10 | 水通道蛋白有限公司 | 用于过滤水的膜 |
JP5114702B2 (ja) | 2005-07-29 | 2013-01-09 | 国立大学法人 東京大学 | 両親媒性単分子膜の接触による二分子膜の形成方法およびその装置 |
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GB0716264D0 (en) * | 2007-08-21 | 2007-09-26 | Isis Innovation | Bilayers |
GB0724736D0 (en) | 2007-12-19 | 2008-01-30 | Oxford Nanolabs Ltd | Formation of layers of amphiphilic molecules |
CN101971013B (zh) * | 2008-08-26 | 2013-06-19 | 松下电器产业株式会社 | 人工脂质膜形成方法和人工脂质膜形成装置 |
JP4717961B2 (ja) * | 2009-10-07 | 2011-07-06 | パナソニック株式会社 | 人工脂質膜形成方法 |
JP5544186B2 (ja) * | 2010-02-17 | 2014-07-09 | 独立行政法人科学技術振興機構 | 平面脂質二重膜の形成方法 |
JP5614642B2 (ja) * | 2010-10-10 | 2014-10-29 | 公益財団法人神奈川科学技術アカデミー | 脂質二重膜の形成方法及びそのための器具 |
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JP6211273B2 (ja) * | 2013-02-27 | 2017-10-11 | 国立大学法人 東京大学 | 脂質二重膜デバイス、脂質二重膜デバイスアレイ、脂質二重膜デバイス製造装置及び脂質二重膜デバイスの製造方法 |
US10576456B2 (en) * | 2014-06-30 | 2020-03-03 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Systems and methods of preparing stabilized lipid assemblies |
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EP1710578A1 (en) * | 2005-04-08 | 2006-10-11 | Charite-Universitätsmedizin Berlin | Method and apparatus for forming a lipid bilayer membrane |
Also Published As
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EP1677102A4 (en) | 2010-09-01 |
US20060251709A1 (en) | 2006-11-09 |
JP4394916B2 (ja) | 2010-01-06 |
US7858030B2 (en) | 2010-12-28 |
EP1677102A1 (en) | 2006-07-05 |
JP2005091305A (ja) | 2005-04-07 |
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