WO2005029054A1 - 人工脂質二重膜を有する電流測定装置 - Google Patents
人工脂質二重膜を有する電流測定装置 Download PDFInfo
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- WO2005029054A1 WO2005029054A1 PCT/JP2004/013671 JP2004013671W WO2005029054A1 WO 2005029054 A1 WO2005029054 A1 WO 2005029054A1 JP 2004013671 W JP2004013671 W JP 2004013671W WO 2005029054 A1 WO2005029054 A1 WO 2005029054A1
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- solution tank
- lipid bilayer
- artificial lipid
- current measuring
- membrane
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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
- the present invention relates to a current measuring device having an artificial lipid bilayer membrane, and more particularly to an artificial lipid bilayer membrane that only measures channel current in a single ion channel using a human lipid bilayer membrane.
- the present invention relates to a current measuring device capable of optically observing a multilayer film.
- An ion channel is a protein that penetrates a biological membrane based on a lipid bilayer membrane as a basic structure, regulates the flow of ions in response to a stimulus, and generates an electric signal or a calcium signal in a cell. That is, ion channels are important protein molecules that convert stimuli into intracellular signals.
- Such an ion channel is composed of a pore as a path for ions and a gate for controlling the opening and closing of the channel, and the gate is opened and closed by sensing a membrane potential or a physiologically active substance.
- This opening and closing function can be confirmed by measuring the ionic current when passing through the ionic force on-channel.
- the patch clamp method is used as a method of measuring the ion current of a single ion channel, but the ion current is also measured by a lipid planar membrane method.
- lipid planar membrane method In order to deepen the correlation study of the structure and function of ion channels, it is necessary to conduct experiments with a simple reconstructed system. In this case, a lipid planar membrane method is used. This lipid planar membrane method provides a minimal simple system of ion, water, artificial lipid bilayer, and ion channels. Further, development of a sensor using a system of the lipid planar membrane method is also actively carried out at present (for example, Non-Patent Document 1 and the like).
- an ion channel 112 is incorporated in 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 supplied to one of the tanks 113, and a current measuring device 117 is provided via the electrode 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!
- methods for forming the artificial lipid bilayer membrane 111 in the small holes 115 include the following (A) a vertical painting method, (B) a vertical bonding method, and (C) a horizontal method. Either of the methods can be mentioned.
- the lipid solution 110 is applied to the small holes 115 with a thin glass tube or the like. In this state, the lipid solution 110 is raised so as to protrude from both surfaces of the partition plate 114 in a state of closing the small holes 115.
- This lipid solution 110 is a solution obtained by dissolving lipid in an organic solvent such as decane. After the application, the lipid solution 110 moves on the surface of the partition plate 114 as shown on the right side of FIG.
- the term “thinning” refers to a process in which an organic solvent or the like at the center of the painted lipid solution 110 moves and a lipid double membrane is formed at the center.
- a lipid monomolecular film 121 is developed at a gas-liquid interface in an aqueous solution tank 113 (not shown in FIG. 9). 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 is raised to separate the monomolecular film 121 from the partition plate.
- Spread on the surface of 114 thus, one opening of the small hole 115 is closed by the monomolecular film 121.
- 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. It will be cut off.
- the small holes 115 formed in the partition plate 114 are closed with the lipid solution 110, and the lipid solution 110 is naturally thinned to form the artificial lipid bilayer 111.
- the waiting force as shown in FIG. 10 (b), raises the water pressure in the tank above the small holes 115, thereby causing the lipid solution 110 to swell downward and thinner, thereby forming the artificial lipid bilayer membrane 111.
- the artificial lipid bilayer membrane 111 is used. Formation cannot be artificially controlled at all. Therefore, thinning may take several hours or more.
- the thin portion that becomes the “lipid bilayer membrane” and the ring surrounding the perimeter are formed. A thick part called the Balta phase occurs. Therefore, the artificial lipid bilayer 111 obtained by this method is based on the physical equilibrium of each of the above-mentioned parts. The bilayer 111 tears quickly. Also, 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.
- This current measuring device includes two solution tanks, an upper solution tank 101 and a lower solution tank 102, as shown in FIG.
- a film 103 having a small hole 105 in the center is attached to the bottom 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.
- the upper solution tank 101 is provided with an electrode 116 in the same manner as in the system of the lipid planar membrane method, and a current measuring device 117 is provided via the electrode 116. Also, an electrode 118 is put into the lower solution tank 102, and an earth 119 is made to the lower solution tank 102 via the electrode 118!
- 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. Thereafter, 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 thick membrane is thinned to form an artificial lipid bilayer membrane.
- Non-patent document 2 Ide'T., Takeuchi'U., Yanagida'T. Development of an Experimental Apparatus for Simultaneous Observation of Optical and Electrical Signals from Single Ion Cannels, Single Mol. 3 (2002) l, 33-42
- the conventional current measuring devices described above still have insufficient points in terms of stability and miniaturization of the artificial lipid bilayer membrane, and current measuring devices having higher performance are demanded.
- the artificial lipid bilayer membrane 111 formed in the small holes 105 of the film 103 is attached to the agarose on the cover glass 106. Being supported by the sgel layer 108, it is stable in the vertical direction.
- both the upper solution tank 101 and the lower solution tank 102 are open systems, when the pressure of the upper solution tank 101 is increased, the direction parallel to the bottom surface of the upper solution tank 101 (not shown in FIG. 12) ( In the direction of arrow H), the artificial lipid bilayer membrane 111 becomes unstable due to vibration caused by the flow of the aqueous solution.
- the present invention has been made in view of the above-mentioned conventional problems, and has as its object to quickly form a stable artificial lipid bilayer membrane, to reduce the size,
- An object of the present invention is to provide a current measuring device having an artificial lipid double membrane, which can be suitably used for simultaneously measuring the structure and function of a channel.
- the current measurement device that works in the present invention is a current measurement device that can measure a current flowing through an artificial lipid bilayer membrane, and includes an upper solution tank that can store an aqueous solution, An upper solution tank is provided below the upper solution tank. An opening is formed, and a support layer for supporting the artificial lipid bilayer membrane is provided on the bottom surface of the lower solution tank. The artificial lipid bilayer formed in the membrane formation opening of the upper solution tank is provided.
- the current measurement device further includes a bottom plate on which the support layer is mounted on a surface, and a spacing member for keeping a predetermined spacing between the upper solution tank and the bottom plate.
- the lower solution tank is formed below the upper solution tank by being surrounded by the bottom plate and the spacing member, and is provided below the artificial lipid bilayer formed at the film forming opening of the upper solution tank.
- the artificial lipid bilayer membrane is brought into contact with a support layer in a thinned state by being expanded toward the solution tank side, and is supported on the support layer.
- FIG. 1 is a sectional view showing a schematic configuration of a current measuring device according to the present invention.
- FIG. 2 (a) is a partial cross-sectional view showing a state where a lipid solution is applied to small holes of an upper solution tank in the current measuring device shown in FIG. 1, and (b) is an artificial lipid bilayer membrane.
- FIG. 4 is a partial cross-sectional view showing a state in which a thin film is in contact with a support layer.
- FIG. 3 is a partial exploded view showing each member constituting a lower solution tank in the current measuring device shown in FIG. 1.
- FIG. 4 is a cross-sectional view showing another schematic configuration of the current measuring device according to the present invention.
- FIG. 5 is a partial cross-sectional view showing a more specific configuration of a negative pressure suction unit in the current measuring device shown in FIG. 4.
- FIG. 6 (a) is a partial cross-sectional view showing a state in which a lipid solution is applied to small holes of an upper solution tank in the current measuring device shown in FIG. 1, and (b) is an artificial lipid bilayer membrane.
- FIG. 4 is a partial cross-sectional view showing a state in which a thin film is in contact with a support layer.
- FIG. 7 is a schematic diagram showing a conventional lipid planar membrane method.
- FIG. 8 is a drawing showing a conventional vertical painting method.
- FIG. 9 is a drawing showing a conventional vertical bonding method.
- FIG. 10 is a drawing showing a conventional method for forming an artificial lipid bilayer membrane by a horizontal method.
- FIG. 11 is a drawing showing a conventional current measuring device having an artificial lipid bilayer membrane.
- FIG. 12 is a drawing showing a conventional artificial lipid bilayer formed on a polymer gel layer.
- FIG. 13 is a photograph showing a state in which thinning of the artificial lipid bilayer membrane is completed in [Example 1].
- FIG. 14 is a drawing showing a current trace measured in [Example 1].
- FIG. 15 is a drawing showing the measured membrane potential and current characteristics in [Example 1].
- FIG. 16 (a) is a drawing showing a fluorescence image of fluorescently labeled aramethicin observed in [Example 2]
- FIG. 16 (b) is a drawing showing a current trace measured in [Example 2]. is there
- FIG. 17 (a) is a drawing showing current traces measured or observed in [Example 3] before ryanodine addition, and (b) is a diagram measured or observed in [Example 3].
- FIG. 2 is a drawing showing a fluorescent image of a ryanodine receptor channel
- (c) is a drawing showing a fluorescent image of ryanodine measured or observed in [Example 3]
- (d) is a drawing showing a fluorescent image of the ryanodine receptor channel.
- 3E is a drawing showing the observed current trace after the addition of ryanodine, and (e) is a drawing showing a fluorescent image of the ryanodine receptor channel measured or observed in [Example 3]; 3 is a drawing showing a fluorescent image of ryanodine measured or observed in [Example 3].
- FIG. 1 is a sectional view showing a schematic configuration of a current measuring device according to the present invention.
- Figures 2 (a) and (b) show how an artificial lipid bilayer is formed in the current measurement device shown in Figure 1.
- FIG. FIG. 3 is an exploded view of the current measuring device shown in FIG. In FIG. 3, the upper solution tank is shown not as a whole but as a bottom.
- the current measuring device 1 includes an artificial lipid bilayer membrane 2, an upper solution tank 3, a support layer 5, a bottom plate 6, a spacing member 7a, A current measuring unit 13 and a grounding unit 14 are provided.
- the artificial lipid bilayer membrane 2 is formed in a small hole (opening for membrane formation) 4 provided on the bottom surface 9 of the upper solution tank 3.
- the lower solution tank 8 is formed below the upper solution tank 3 by being surrounded by the bottom plate 6 and the spacing member 7a.
- the current measuring device 1 thins the artificial lipid bilayer 2 by expanding the artificial lipid bilayer 2 formed in the small holes 4 of the upper solution tank 3 toward the lower solution tank 8. In this state, the support is brought into contact with the support layer 5 and is supported on the support layer 5.
- an optical microscope (optical observation means) 17 is provided below the bottom plate 6.
- the lower solution tank 8 is not an open system, vibration caused by the flow of the aqueous solution is suppressed, and the artificial lipid bilayer membrane 2 formed in the small holes 4 is It is possible to maintain stability even in a direction parallel to the bottom surface.
- the artificial lipid bilayer membrane 2 is formed in the small hole 4 opened in the bottom surface 9 of the upper solution tank 3 as described above. As described later, the artificial lipid bilayer membrane 2 is formed by applying a lipid solution to the pores 4 and then lowering the pressure inside the lower solution tank 8 so that the upper part of the artificial lipid bilayer membrane 2 The aqueous solution in the tank 3 is caused to flow into the lower solution tank 8, and the artificial lipid bilayer membrane 2 is swollen downward and is formed by contact with the support layer 5.
- the artificial lipid bilayer membrane 2 simulates a biological membrane including an ion channel through which ions are opened and closed by opening and closing the gate. It is possible to provide a model channel that has been ridden. The details of the ion channel will be described later.
- the lipid is not particularly limited as long as it forms the artificial lipid bilayer 2, but phospholipids are preferably used. Specifically, for example, phosphatidylcholine, diphthalylphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and the like can be mentioned. [0033]
- 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. Further, for example, cholesterol or the like may be added as needed to maintain the activity of the ion channel.
- the lipid solution is a solution obtained by dispersing the lipid 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 preferably 5 mgZmL or more and 40 mgZmL or less, more preferably 15 mgZmL or more and 20 mgZmL or less.
- the configuration of the upper solution tank 3 is not particularly limited as long as it is a container having a small hole 4 formed in the bottom surface 9 and capable of storing an aqueous solution. With such a configuration, the artificial lipid bilayer membrane 2 can be formed in the small hole 4 as described later.
- the small hole 4 may be formed in the bottom surface 9 of the upper solution tank 3, but is more preferably formed in the center of the bottom surface. Thereby, optical observation from below can be easily performed. Further, the small holes 4 may be formed directly on the bottom surface 9, or an opening may be provided on the bottom surface 9 of the upper solution tank 3, and a film having the small holes 4 may be bonded to the opening. You may.
- the pores 4 preferably have a diameter of 10 / zm or more and 500 m or less, more preferably 50 / zm or more and 200 m or less. Thereby, an artificial lipid bilayer membrane can be favorably formed.
- the material of the bottom surface 9 or the film for forming the small holes 4 is not particularly limited, but specifically, for example, a plastic such as polypropylene, polyvinyl chloride, polystyrene, or the like, Fluororesin such as polyethylene is preferably used. Further, it is preferable that the thickness of the bottom surface 9 or the film forming the small holes 4 is 0.1 mm or more and 0.3 mm or less. Further, it is more preferable to process the thickness of the bottom surface 9 or the film only around the opening of the small hole 4 smaller than the thickness of the bottom surface 9 or other portions of the film. As a result, a stable artificial lipid bilayer membrane can be rapidly formed.
- the small holes 4 can be formed, for example, by a conventionally known method as shown below. it can. 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 4 are formed, and the pressing is continued until a small bulge is formed on the side opposite to the pressed surface. A small hole 4 is formed by cutting the bulge with a razor. In addition, the small holes 4 are cleaned with a form-Z form methanol to remove impurities and the like.
- the method for forming the small holes 4 is not limited to this, and any known method can be used.
- the shape of the upper solution tank 3 is not particularly limited, but may be, for example, a cylindrical shape.
- the size of the upper solution tank 3 is not particularly limited, 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 1.Omm or more, More preferably, it is 10 mm or less. Further, the size of the upper solution tank 3 can be reduced to a diameter of preferably 0.5 mm, more preferably 10 m. Further, the volume of the upper solution tank 3 is not particularly limited, but is preferably not less than 0.1 Olcm 3 and not more than 1.0 Ocm 3 . Further, the volume of the upper solution tank 3 can be reduced to 0.001 cm 3 . Accordingly, the current measuring device 1 according to the present invention can be formed on a small chip, and a smaller sensor can be manufactured.
- the material of the upper solution tank 3 other than the portion forming the small holes 4 is not particularly limited, and examples thereof include glass and plastic.
- the upper solution tank 3 is capable of storing an aqueous solution.
- Each side of the artificial lipid bilayer 2 formed in the small hole 4 is in contact with the aqueous solution filled in the upper solution tank 3 and the lower solution tank.
- 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 above aqueous solution include aqueous solutions of potassium salt, sodium salt, potassium salt and the like.
- the upper solution tank 3 be capable of moving vertically.
- the vertical movement of the upper solution tank 3 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 8 is disposed below the upper solution tank 3 and may be formed by being surrounded by the bottom plate 6 and the spacing member 7a.
- a support layer 5 for supporting the artificial lipid bilayer membrane 2 is provided on the bottom surface of the lower solution tank 8, that is, the surface of the bottom plate 6, and the artificial lipid bilayer membrane 2 formed by the small holes 4 of the upper solution tank 3 is provided. The support is made to come into contact with the support layer 5.
- the artificial lipid bilayer membrane 2 is formed in the small holes 4 on the bottom surface 9 of the upper solution tank 3, and the formed artificial lipid bilayer membrane 2 is stably supported by the support layer 5.
- the configuration of the lower solution tank 8 is not specifically limited in the current measuring device 1 according to the present invention.
- the bottom plate 6 and the space It is preferably formed by being surrounded by the holding member 7a.
- [0045] are not limited to particular volume of the lower solution chamber 8 but, 0. 1 mm 3 or more, 10 mm 3 preferably be less that instrument 0. 5 mm 3 or more, it 3. is 5 mm 3 or less preferable.
- the current measuring device 1 according to the present invention can be formed on a small chip, and a smaller sensor can be manufactured.
- the bottom plate 6 is disposed below and substantially parallel to the bottom surface of the upper solution tank 3, and a spacing member 7 a is disposed between the bottom plate 6 and the upper solution tank 3.
- the lower solution tank 8 is formed below the upper solution tank 3 by being surrounded by the bottom plate 6 and the spacing member 7a.
- the configuration of the bottom plate 6 is not particularly limited as long as the support layer 5 can be placed on the surface. Therefore, the shape, size, surface condition, etc. of the bottom plate 6 are not particularly limited, either.The appropriate shape depends on the configuration of the finally obtained current measuring device 1 and the support layer 6 to be formed. , Size, surface condition, etc. may be selected.
- the material and the like of the bottom plate 6 are not particularly limited.
- a material having a light-transmitting property is preferable. Rustic and the like. Thus, the downward force can be observed by the optical microscope 17.
- the thickness of the bottom plate 6 is not particularly limited, but is preferably 0.1 mm or more and 1.0 mm or less. Thereby, good mechanical strength is obtained.
- the thickness of the bottom plate 6 is not particularly limited as long as an appropriate thickness is selected according to the conditions of the optical observation (for example, the working distance of the objective lens). Although not required, it is more preferably 0.1 mm or more and 0.17 mm or less. This makes it possible to favorably perform optical observation using an objective lens having a high numerical aperture.
- the support layer 5 for supporting the artificial lipid bilayer membrane 2 is formed on the surface of the bottom plate 6 on the side facing the upper solution tank 3, that is, on the bottom surface of the lower solution tank 8. Have been.
- the support layer 5 is not particularly limited as long as it can permeate an aqueous solution and can support the artificial lipid bilayer membrane 2.
- Specific examples of the support layer 5 include a polymer gel and a porous film such as a cellulose film. Above all, it is more preferable that the support layer 5 also has a polymer gel force.
- the high molecular gel is not specifically limited, polysaccharides such as agarose and hydrophilic resins such as polyacrylamide can be preferably used. By using these, the support layer 5 can be easily formed using an inexpensive and highly reliable material.
- the thickness of the support layer 5 is not particularly limited, but is preferably 50 nm or more and 2 mm or less, more preferably 100 nm or more and 1 mm or less. This makes it possible to manufacture a current measuring device having a suitable size.
- the thickness of the support layer 5 is not particularly limited as long as it is smaller than the working distance of the objective lens, but may be 50 nm or more and 20 m or less. More preferably, it is more than 10 Onm and less than 20 m. Thereby, it becomes possible to favorably perform optical observation using an objective lens having a high numerical aperture.
- the distance between the artificial lipid bilayer membrane and the objective lens is preferable to reduce the distance between the artificial lipid bilayer membrane and the objective lens.
- the artificial lipid bilayer membrane 2 is placed on the support layer 5 side of the bottom plate 6. It is preferably located at a position of 20 / zm or less from the surface. This allows the objective lens to work effectively.
- the thickness of the support layer 5 is more preferably 100 nm or more and 200 nm or less. This makes it possible to observe the fluorescent substance in the solution well.
- the artificial lipid bilayer membrane 2 be located at a position of 100 nm or more and 200 nm or less from the surface of the bottom plate 6 on the support layer 5 side. Thereby, optical observation using near-field light can be favorably performed.
- the method for forming the support layer 5 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 of applying it to the bottom plate 6 and drying it at room temperature can be used.
- the artificial lipid bilayer membrane 2 formed in the small hole 4 of the upper solution tank 3 is expanded toward the lower solution tank 8, so that the artificial lipid bilayer membrane 2 is thinned onto the support layer 5. It can be brought into contact and supported on the support layer 5. Thereby, even when there is a pressure difference between the upper solution tank 3 and the lower solution tank, the artificial lipid bilayer membrane 2 is supported by the support layer 5 and stabilized vertically.
- the spacing member 7a is arranged between the upper solution tank 3 and the bottom plate 6, as shown in FIGS. 1 to 3, and holds a predetermined distance between the upper solution tank 3 and the bottom plate 6.
- the lower solution tank 8 is formed below the upper solution tank 3 by being surrounded by the bottom plate 6 and the spacing member 7a, as described above.
- the spacing member 7a is not particularly limited as long as it has a shape capable of holding a predetermined spacing between the upper solution tank 3 and the bottom plate 6 and forming the lower solution tank 8,
- the lower solution tank 8 is hermetically sealed by the spacing member 7a, the upper solution tank 3, the bottom plate 6, and the artificial lipid bilayer 2.
- the lower solution tank 8 is a closed space, even when there is a pressure difference between the upper solution tank 3 and the lower solution tank 8, the artificial solution is stable in a direction parallel to the bottom plate 6. Lipid bilayer 2 can be formed. This is because in the closed lower solution tank 8, the aqueous solution does not flow when both the upper and lower solution tanks are release systems (see FIG. 12).
- the spacing member 7a used in the present embodiment has an open upper and lower part, and has a hollow cylindrical or prismatic shape inside.
- the gap held by the gap holding member 7a that is, the height of the cylinder or prism, is such that the artificial lipid bilayer membrane 2 formed on the bottom surface 9 of the upper solution tank 3 swells toward the lower solution tank side, and the bottom plate 6 Any height may be used as long as it can contact the support layer 5 formed at the bottom.
- the spacing member 7a more preferably has a cylindrical shape, and the inner diameter thereof is preferably not less than 0.25 mm and not more than 2.5 mm. It is more preferable that it is 1.5 mm or less.
- the volume of the lower solution tank 8 can be set within the above range.
- the difference between the inner diameter and the outer diameter, that is, the thickness of the side surface of the lower solution tank 8 is preferably 0.05 mm or more and 0.5 mm or less. More preferably, it is 0.1 mm or more and 0.3 mm or less. Thereby, the lower solution tank 17 can be sealed more sufficiently.
- the spacing member 7a used in the present embodiment is capable of changing the distance between the upper solution tank 3 and the bottom plate 6, and the change in the distance causes the small holes 4 of the upper solution tank 3 to change.
- the formed artificial lipid bilayer 2 can be expanded toward the lower solution tank 8.
- the lipid solution 12 is applied to the small holes 4 of the upper solution tank 3 with the interval held by the interval holding member 7a reduced. Thereafter, as shown in FIG. 2 (b), when the above interval is increased, the aqueous solution in the upper solution tank 3 flows into the lower solution tank 8 from above the artificial lipid bilayer 2, and the artificial lipid bilayer 2 moves downward. And swells into contact with the support layer 5. In this state, the artificial lipid bilayer membrane 2 is supported by the support layer 5. As described above, the artificial lipid bilayer membrane 2 can be quickly formed by changing the interval held by the interval holding member 7a.
- the interval holding member 7a capable of changing the interval is not particularly limited as long as the interval to be held can be changed. It may have such a configuration, or may be capable of such a change depending on the properties of the material of the spacing member 7a.
- a preferred example is the example
- the elastic body for example, various elastomers can be preferably used, and among them, silicone rubber is more preferable in terms of durability and stability.
- the method for forming lower solution tank 8 using spacing member 7a and bottom plate 6 and sealing lower solution tank 8 with bottom surface 9 of upper solution tank 3 is not particularly limited.
- a method in which the above three members are closely attached to each other and screwed thereto, or a method in which the above three members are pressure-bonded using a fixing means such as a clip can be used.
- the current measuring section (current measuring means) 13 is electrically connected to the upper solution tank 3 and can measure a current flowing through an ion channel incorporated in the artificial lipid bilayer membrane 2. If there is, it is not particularly limited.
- an electrode 15 put into the upper solution tank 3 an amplifier 18 connected to this electrode 15, A configuration including an ammeter (not shown) electrically connected to 18 can be given.
- the electrode 15 include an Ag—AgCl electrode, but are not particularly limited.
- the amplifier 18 and the ammeter are not particularly limited, and known devices can be used.
- the earth portion (earth means) 14 used at this time is not particularly limited, and may be any means that is electrically connected to the lower solution tank 8.
- the electrode 16 can be the same as the electrode 15 described above.
- the specific method of measuring the current by the current measuring unit 13 is not particularly limited, but includes, for example, a patch clamp method, a method of measuring the current embedded in a lipid bilayer membrane, and the like. be able to. This makes it possible to observe the function of the ion channel, and to identify and quantify the analyte from the shape, magnitude and frequency of the current change.
- an optical microscope (optical observation means) 17 is provided on the side opposite to the side on which the layer 5 is formed. This makes it possible to optically observe the ion channel simultaneously with the measurement of the current flowing through the ion channel.
- any means other than the optical microscope 17 can be suitably used as long as the means can optically observe the ion channel (optical observation means).
- a near-field light excitation fluorescence microscope and the like are mentioned, but there is no particular limitation.
- the observation by the optical microscope 17 includes, for example, a change in the fluorescence intensity of the fluorescently labeled ion channel due to the opening and closing of the gate, a movement of the ion channel, a change in the spectrum due to an energy transition between two fluorescent dyes, and the like. Observation. Further, the formation of the artificial lipid bilayer membrane 2 can be confirmed by the optical microscope 17. Furthermore, the movement of lipid molecules can also be observed using an artificial lipid bilayer membrane 2 using fluorescently labeled lipids. Of course, the optical measurement is not limited to these, and any conventionally known method can be applied.
- the current measuring device of the present invention can be suitably used for the purpose of simultaneously measuring the structure and function of the ion channel. That is, using an artificial lipid bilayer membrane 2 incorporating an ion channel, the current flowing through the ion channel is measured, and the current is inhibited or activated by binding to a physiologically active substance or a sample as a stimulus S ion channel. This makes it possible to observe dangling and to measure the concentration of a physiologically active substance or a specimen.
- the ion channel incorporated into the artificial lipid bilayer membrane 2 may be isolated and purified from a biological membrane, or may be prepared by a genetic engineering technique or the like. There is no particular limitation as long as it is artificially synthesized. Specific examples include a Na + channel, a K + channel, a Ca 2+ channel, an aramethicin channel, a ryanodine receptor channel, and a hemolysin channel.
- the method of embedding the ion channel in the artificial lipid bilayer membrane can be a conventionally known method, and is not particularly limited. Specifically, for example, a membrane fraction containing an ion channel is solubilized with a surfactant, reconstituted into a membrane vesicle, and melted into an artificial lipid bilayer membrane. And a method of combining them.
- the spacing member 7a has a configuration in which the spacing between the upper solution tank 3 and the bottom plate 6 can be changed. Therefore, when an elastic body is employed as the space holding member 7a, a procedure for changing the above-mentioned space by utilizing the expansion and contraction of the elastic body in the vertical direction and thereby rapidly forming the artificial lipid bilayer 2 will be described. I do.
- the upper solution tank 3 and the lower solution tank 8 are filled with the above aqueous solution.
- the lipid solution 12 is applied to the small holes 4 in a state where the spacing member 7a is contracted.
- the upper solution tank 3 is gradually pulled up, so that the aqueous solution of the upper solution tank 3 enters the lower solution tank 8 of the artificial lipid bilayer membrane 2 and the artificial lipids.
- the double membrane 2 swells downward. In this way, the artificial lipid bilayer 2 comes into contact with the support layer 5 and becomes thinner.
- the artificial fat double membrane 2 can be formed in a few seconds by expanding and contracting the spacing member 7a to change the spacing. Further, it is possible to form the artificial lipid bilayer membrane 2 more stable than in the conventional case where the pressure of the upper solution tank 3 is increased and the artificial lipid bilayer membrane 2 is expanded toward the lower solution tank side.
- the curvature of the artificial lipid bilayer membrane 2 is not constant, particularly in the release system.
- the lipid on the bottom surface 9 around the annular Balta phase and the subsequent pores 4 diffuses laterally, and the artificial lipid bilayer membrane 2 bulges excessively laterally, and the curvature increases with time. This leads to destabilization of the artificial lipid bilayer 2.
- the method of expanding and contracting the spacing member 7a since the pressure of the lower solution tank 8 is reduced, the anxiety due to the lateral diffusion of lipid does not occur.
- a large pressure is required to determine the curvature of the artificial lipid bilayer membrane 2 having a small area. It is. That is, it is necessary to store more water solution in the upper solution tank in order to increase the water pressure in the upper solution tank. For example, to determine the curvature of an artificial lipid bilayer membrane with a diameter of 500 m, an aqueous solution depth of about 3-5 mm is required. In this case, the depth of the aqueous solution must be 20mm or more. Therefore, there is a problem that the apparatus becomes large in order to form the artificial lipid bilayer membrane 2 having a small area.
- the artificial lipid bilayer membrane 2 having a small area without increasing the size of the upper solution tank 3 can be formed because the above problem does not occur. Further, since the artificial lipid bilayer 2 can be made smaller, electric noise can be reduced.
- the lower solution tank 8 is sealed by the artificial lipid bilayer membrane 2, the bottom surface 9 of the upper solution tank 3, the spacing member 7a, and the bottom plate 6. System. Therefore, the artificial lipid bilayer membrane 2 is stabilized in a direction parallel to the bottom plate 6, which was conventionally unstable, so that the durability of the artificial lipid bilayer membrane 2 can be further improved.
- the present invention also includes a method of forming the artificial lipid bilayer membrane 2 by the above procedure.
- an opening (small hole 4) for forming a membrane is formed on the bottom surface 9 of the upper solution tank 3 and the lower solution tank 8.
- the lower solution tank 8 is disposed below the upper solution tank 3 and has a bottom plate 6 on which a support layer 5 supporting the artificial lipid bilayer membrane 2 is placed on the surface, and a predetermined distance between the upper solution tank 3 and the bottom plate 6.
- the spacing member 7a can change the spacing between the upper solution tank 3 and the bottom plate 6.
- the surfaces of the upper solution tank 3 side and the lower solution tank 8 side of the above-mentioned membrane forming opening (small hole 4) are brought into contact with an aqueous solution, A lipid solution application step of applying the lipid solution 12 with the above-mentioned interval reduced, and a lipid that is brought into contact with the support layer 5 with the artificial lipid bilayer membrane 2 thinned by increasing the above-mentioned interval. Film thinning step.
- the formed artificial lipid bilayer membrane 2 can be more stable, and therefore, even when the ion channel is incorporated in the artificial lipid bilayer membrane 2.
- the structure and function of the ion channel can be simultaneously measured in a sufficiently stable state.
- Ion channel proteins are distributed in almost all cells of various 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 2. Furthermore, by changing the type of ion channel, it can be applied to the detection of various substances.
- FIGS. 4 and 6 Another embodiment of the present invention will be described with reference to FIGS. 4 and 6.
- FIG. 4 is a cross-sectional view showing a schematic configuration of another current measuring device according to the present invention.
- FIG. 5 is a sectional view showing a more specific configuration of the negative pressure forming section shown in FIG. 6 (a) and 6 (b) are cross-sectional views showing how an artificial lipid bilayer membrane is formed in the current measuring device shown in FIG.
- the current measuring device basically has the same configuration as that of the current measuring device described in the first embodiment, but has a spacing member. 7b Force The pressure is not configured to be variable, and a negative pressure forming unit (negative pressure forming means) 10 for forming a negative pressure is provided in the lower solution tank 8.
- the basic configuration of the spacing member 7b used in the present embodiment is the same as that of the spacing member 7a in the first embodiment, but has a configuration that cannot be changed in the vertical direction. . Therefore, the material is not particularly limited as long as it is a material that can be in close contact with the upper solution tank 3 and the bottom plate 6 and can seal the lower solution tank 8. Specifically, silicone rubber such as polydimethylsiloxane (PDMS), epoxy resin, latex rubber and the like can be preferably used.
- PDMS polydimethylsiloxane
- epoxy resin epoxy resin
- latex rubber latex rubber
- the negative pressure forming section 10 is not particularly limited as long as the pressure inside the lower solution tank 8 formed by being surrounded by the bottom plate 6 and the spacing member 7b can be made lower than the pressure of the upper solution tank 3.
- the more specific configuration of the negative pressure forming section 10 is not particularly limited.
- a suction port 21 connected to the outside of the measuring device from the lower solution tank 8 is provided in the spacing member 7b, and further connected to the suction port 21 and the current measuring device is connected to the lower solution tank 8 from the lower solution tank 8.
- a configuration in which a drawn-out tube 22 is provided outside and a suction unit (suction means, not shown) is connected via the tube 22 can be given. Thereby, the aqueous solution is sucked from the lower solution tank 8 through the suction port 21 and the tube 22 by the suction unit, and the pressure inside the lower solution tank 8 can be reduced.
- the specific configuration of the suction port 21 is not particularly limited.
- the diameter of the suction port 21 is such that the aqueous solution inside the lower solution tank 8 can be sucked out so that the pressure inside the lower solution tank 8 can be favorably reduced. What is necessary is just a structure which has.
- the method of forming the suction port 21 is not particularly limited, either, and an appropriate method may be selected according to the material and size of the spacing member 7b.
- the specific configuration of the tube 22 is not particularly limited either, and the tube 22 may have a strength, a diameter, and a length enough to sufficiently suck out the aqueous solution inside the lower solution tank 8 by suction of the suction unit.
- the material of the tube 22 is not particularly limited, but specific examples thereof include fluorine resin such as polytetrafluoroethylene and silicone. Etc.
- the specific configuration of the suction unit is not particularly limited as long as it can suck the aqueous solution in the lower solution tank 8 through the suction port 21 and the tube 22.
- Specific examples include, for example, pipettes, spoids, syringes, and the like.
- the material of the suction unit is not particularly limited, and an appropriate material may be selected according to the specific configuration of the suction unit.
- a silicone rubber spoid can be particularly preferably used.
- the specific configuration of the negative pressure forming section is not limited to the above configuration, and any known means can be suitably used as long as the inside of the lower solution tank 8 can be depressurized. it can.
- the upper solution tank 3 and the lower solution tank 8 are filled with the above aqueous solution.
- the lipid solution 12 is applied to the small holes 4.
- the aqueous solution is transferred from the lower solution tank 8 through a suction port 21 connected to the outside from the lower solution tank 8 using a tube 22 (not shown) connected to the suction port 21 and a suction unit. Aspirate.
- the aqueous solution in the upper solution tank 3 enters the lower solution tank 8 from the upper force of the artificial lipid bilayer membrane 2, and the artificial lipid bilayer membrane 2 swells downward. In this way, the artificial lipid bilayer 2 comes into contact with the support layer 5 and becomes thinner.
- the artificial lipid bilayer membrane 2 can be formed in a few seconds as compared with the case where there is no pressure difference. Also, in comparison with the case where the pressure of the upper solution tank 3 is increased to provide a pressure difference, the method of the present embodiment makes it possible to easily provide the pressure difference. Further, as described in the above [Embodiment 1] (2), compared to the case where the pressure of the upper solution tank 3 is increased to inflate the artificial lipid bilayer 2 toward the lower solution tank, The morphological method can form a stable artificial lipid bilayer membrane 2 and increase the size of the device to form an artificial lipid bilayer membrane 2 with a small area. There is no problem.
- the lower solution tank 8 is a system closed by the artificial lipid bilayer membrane 2, the bottom surface 9 of the upper solution tank 3, the spacing member 7a, and the bottom plate 6. Has become. Therefore, the artificial lipid bilayer membrane 2 is stabilized in a direction parallel to the bottom plate 6 which was conventionally unstable, and the durability of the artificial lipid bilayer membrane 2 can be further improved.
- the present invention also includes a method for forming the artificial lipid bilayer membrane 2 by the above procedure.
- an opening (small hole 4) for forming a membrane is formed on the bottom surface 9 of the upper solution tank 3 and the lower solution tank 8.
- the lower solution tank 8 is arranged below the upper solution tank 3 and has a predetermined distance between the bottom plate 6 on which the support layer 5 for supporting the artificial lipid bilayer membrane 2 is placed and the upper solution tank 3 and the bottom plate 6. It has a spacing member 7b for holding, and is formed by being surrounded by the bottom plate 6 and the spacing member 7b.
- the lower solution tank 8 is provided with a negative pressure forming means (negative pressure forming section 10) for reducing the pressure inside the lower solution tank 8.
- the method of forming an artificial lipid bilayer membrane according to the present invention is performed by bringing the surfaces of the upper solution tank 3 side and the lower solution tank 8 side of the membrane formation opening (small hole 4) into contact with the aqueous solution.
- the current measuring device according to the present embodiment can also be used for the same applications as in the first embodiment.
- the spacing member 7b in the present embodiment may be provided with only the negative pressure forming part 10, but as in the first embodiment, the upper solution tank 3 and the bottom plate are provided. 6 may be combined with a variable configuration. That is, the spacing member 7b may be an elastic body, and the pressure in the lower solution tank 8 may be reduced and the spacing may be variable.
- the current measuring device shown in Fig. 5 and working for the present invention was used.
- the upper solution tank 3 was made of polypropylene having a volume of 0.1 cm 3 and a bottom surface 9 having a thickness of 0.2 mm-0.3 mm.
- a small hole 4 having a diameter of 0.15 mm was formed in the bottom surface 9.
- the bottom plate 6 a square glass plate having a thickness of 0.17 mm and a side of 18 mm is used, and an agarose gel layer having a thickness of 100 nm is formed as the support layer 5 on the side of the bottom plate 6 facing the solution tank 3. did.
- the agarose gel layer was formed by preparing a dispersion of agarose (manufactured by Sigma), heating it to dissolve the agarose, applying it to the bottom plate 6, and drying it at room temperature.
- a member having an opening at an upper portion and a lower portion and having a hollow cylindrical shape inside was used as the spacing member 7b.
- the inner diameter of the gap holding member 7b was 1. Omm, and the height was 0.2 mm.
- the spacing member 7b used also has a silicone rubber force.
- the spacing member 7b was provided with a suction port 21 and a polytetrafluoroethylene tube 22 connected to the suction port 21 and having a diameter of 50 ⁇ m or less.
- a silicone rubber spoid was connected to the polytetrafluoroethylene tube 22 as a suction part.
- the electrode 16 an Ag-AgCl electrode made of Ag foil was used in each of the upper and lower solution tanks, and the Ag-AgCl electrode of the lower solution tank 8 used was one embedded in silicone rubber during silicone rubber molding.
- the channel current was measured using a patch clamp amplifier (CEZ-2400, manufactured by Nihon Kohden) and recorded on a DAT tape using a DAT recorder.
- the upper solution chamber 3 and the lower solution chamber 8 lOOmM KC1, 10- 9 M CaCl, lOmM He
- the pes (pH 7.4) aqueous solution was filled.
- the current measuring device of the present invention was fabricated by incorporating the film into the film 2. The current was measured over time using the prepared current measuring device.
- a current measuring device was prepared in the same manner as above, and the current was measured.
- FIG. 13 shows a state where the formed artificial lipid bilayer membrane 2 has been thinned. The completion of the thin film was completed by the visual recognition of the boundary between the artificial lipid bilayer 2 (indicated as “double membrane” in the figure) and the circular Balta phase surrounding it (indicated as "balta phase” in the figure). Can be confirmed.
- Fig. 14 shows the obtained current trace
- Fig. 15 shows the membrane potential-current characteristics.
- FIG. 14 (a) if the aqueous solution is LOOmM KC1, including 10- 9 M CaCl
- FIG. 14 (b) the aqueous solution is 100m
- Cy3 Aramechishin fluorescently labeled (Amersham full Alma made shear) (manufactured Sigma) in methanol at a final concentration of the upper solution chamber 3 so that the order of 10- 8 M Added to the aqueous solution.
- Aramethicin adds glycine to its C-terminus, kit, manufactured by Pharmacia) to bind to Cy3 for fluorescent labeling.
- Arameticisin is an amphipathic peptide, and migrates spontaneously to the artificial lipid bilayer 2 from the liquid phase 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 a self-made objective lens type total reflection fluorescence microscope.
- FIG. 16 (a) shows the obtained fluorescent image of fluorescent alamethicin and the locus of Brownian motion in the film
- FIG. 16 (b) shows the current trace.
- the upper solution tank 3 and the lower solution tank 8 were filled with 500 mM Na-methanesulfonic acid, 40 mM Hepes (pH 7.4), 0.01-0 .: LM Ca 2+ aqueous solution.
- a phosphatidylcholine 20mgZml decane solution was applied to the small holes 4 on the bottom surface 9 of the upper solution tank 3, and the aqueous solution inside the lower solution tank 8 was aspirated using the above-mentioned spoid, and the artificial lipid double membrane was formed. 2 was expanded toward the lower solution tank 8 to form an artificial lipid bilayer membrane 2.
- a cell membrane vesicle prepared from porcine myocardium was fused with this artificial lipid bilayer, and the ryanodine receptor channel (Ca-channel) on the vesicle membrane was incorporated into the artificial lipid bilayer 2.
- the ryanodine receptor channel on the vesicle membrane was labeled with a fluorescent dye Cy5 (manufactured by Amarsham Pharmacia) in advance, and then the cell membrane vesicle was fused to the artificial lipid bilayer 2.
- the fluorescent labeling of the ryanodine receptor channel was performed by labeling a specific monoclonal antibody thereto with Cy5, and binding the labeled antibody to the ryanodine receptor channel.
- FIG. 17 shows a current trace (a) before addition of ryanodine (shown as “current” in the figure and the same in (d) described later), and a fluorescence image of the ryanodine receptor channel (b) (in the figure, Shows "channel", The same applies to (e) described later) and the fluorescence image of ryanodine (c) (indicated as “ligand” in the figure, the same applies to (f) described later), and the current trace after ryanodine addition (d), A fluorescent image of a ryanodine receptor channel (e) and a fluorescent image of ryanodine (f) are shown.
- the current measuring device is a current measuring device capable of measuring a current flowing through an artificial lipid bilayer membrane, and is capable of storing an aqueous solution!
- An upper solution tank and a lower solution tank disposed below the upper solution tank are provided.
- An opening for film formation is formed on the bottom surface of the upper solution tank, and a bottom surface of the lower solution tank is formed on the bottom surface of the lower solution tank.
- a bottom plate for mounting the support layer on the surface, and a spacing member for maintaining a predetermined spacing between the upper solution tank and the bottom plate are provided, and the lower solution tank is provided on the bottom plate and the spacing member. By being surrounded, it is formed below the upper solution tank, and the film forming opening of the upper solution tank is formed.
- the artificial lipid bilayer membrane formed in step 2 is expanded toward the lower solution tank so that the artificial lipid bilayer membrane is brought into contact with the support layer in a thinned state and is supported on the support layer. .
- the current measuring device is a current measuring device capable of measuring a current flowing through an artificial lipid bilayer membrane, capable of accumulating an aqueous solution, and has a bottom surface for forming a membrane.
- An upper solution tank having an opening formed therein; and a lower solution tank disposed below the upper solution tank and capable of storing the aqueous solution, wherein the lower solution tank includes a bottom plate, an upper solution tank, and a bottom.
- the bottom surface of the upper solution tank and the bottom plate are in close contact with each other so as to maintain a predetermined distance between the plates, and the bottom plate is constituted by a space holding member, and the bottom plate supports the artificial lipid bilayer membrane. May be provided.
- the artificial lipid bilayer formed in the membrane-forming opening of the upper solution tank is expanded toward the lower solution tank so that the artificial lipid bilayer is thinned.
- the current measuring device includes negative pressure forming means for reducing the pressure inside the lower solution tank formed by being surrounded by the bottom plate and the spacing member.
- the negative pressure forming means includes a suction port formed on the spacing member and connected to an external force of the lower solution tank, and a suction means connected to the suction port for suctioning an aqueous solution inside the lower solution tank. Is preferred.
- the interval holding member can change the interval between the upper solution tank and the bottom plate, and the change in the interval causes the opening for film formation in the upper solution tank.
- the artificial lipid bilayer formed in the section may be expanded toward the lower solution tank.
- the spacing member also has elasticity and is capable of extending and contracting up and down.
- the polymer gel in which the support layer preferably has a polymer gel force. Further, the thickness of the polymer gel layer is preferably 50 nm or more and 2 mm or less.
- the artificial lipid bilayer membrane is supported by the polymer gel layer and is vertically stabilized.
- the diameter of the opening for forming a film is 10?
- the bottom plate is made of a light-transmitting material, and the artificial lipid bilayer membrane on the support layer can be observed below the bottom plate. It is preferable that observation means be provided.
- the current measuring device may further include a current measuring means electrically connected to the upper solution tank and a ground means electrically connected to the lower solution tank.
- the artificial lipid bilayer preferably includes an ion channel.
- the use of the current measuring device according to the present invention makes it possible to obtain an artificial lipid bilayer membrane that is stable and durable even in a direction parallel to the bottom surface 9 of the upper solution tank 3. 2 can be easily formed in a short time.
- the ion channel can be optically observed simultaneously with the measurement of the channel current.
- miniaturization is possible, and by manufacturing on a small chip, a smaller sensor can be manufactured.
- the current measuring device of the present invention can be used, for example, for screening in drug discovery using ion channel proteins involved in diseases.
- Ion channel proteins are distributed in almost all types of cells. 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 that is effective in the present invention is a protein measuring device on the artificial lipid bilayer membrane 2. It can be used for visualization analysis of protein (drug) interaction. Furthermore, by changing the type of ion channel, it can be applied to the detection of various substances.
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Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/572,569 US7408359B2 (en) | 2003-09-19 | 2004-09-17 | Current measuring device having artificial lipid bilayer membrane |
EP04773290.4A EP1669746A4 (en) | 2003-09-19 | 2004-09-17 | ELECTRIC VOLTAGE MEASURING INSTRUMENT HAVING DOUBLE ARTIFICIAL LIPID MEMBRANE |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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JP2003328696A JP4394917B2 (ja) | 2003-09-19 | 2003-09-19 | 人工脂質二重膜を有する電流測定装置 |
JP2003-328696 | 2003-09-19 |
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Publication Number | Publication Date |
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WO2005029054A1 true WO2005029054A1 (ja) | 2005-03-31 |
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PCT/JP2004/013671 WO2005029054A1 (ja) | 2003-09-19 | 2004-09-17 | 人工脂質二重膜を有する電流測定装置 |
Country Status (4)
Country | Link |
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US (1) | US7408359B2 (ja) |
EP (1) | EP1669746A4 (ja) |
JP (1) | JP4394917B2 (ja) |
WO (1) | WO2005029054A1 (ja) |
Families Citing this family (18)
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WO2006030523A1 (ja) * | 2004-09-17 | 2006-03-23 | Japan Science And Technology Agency | 人工脂質二重膜における脂質置換方法、その脂質置換方法を用いて得られる人工脂質二重膜、その人工脂質二重膜を製造する装置、および、イオン透過測定装置 |
JP4953044B2 (ja) * | 2005-05-09 | 2012-06-13 | 財団法人生産技術研究奨励会 | 脂質二重膜の形成方法およびその装置 |
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 |
JP4734542B2 (ja) * | 2006-01-13 | 2011-07-27 | 財団法人生産技術研究奨励会 | 人工二分子膜を用いた膜輸送分子の膜輸送機能測定方法およびその測定装置 |
GB2447043A (en) * | 2007-02-20 | 2008-09-03 | Oxford Nanolabs Ltd | Lipid bilayer sensor system |
WO2008102120A1 (en) * | 2007-02-20 | 2008-08-28 | Oxford Nanopore Technologies Limited | Lipid bilayer sensor system |
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 |
US8062489B2 (en) | 2009-10-07 | 2011-11-22 | Panasonic Corporation | Method for forming artificial lipid membrane |
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 |
JP5770615B2 (ja) * | 2011-12-08 | 2015-08-26 | 日本電信電話株式会社 | 脂質二分子膜基板の製造方法 |
GB201202519D0 (en) | 2012-02-13 | 2012-03-28 | Oxford Nanopore Tech Ltd | Apparatus for supporting an array of layers of amphiphilic molecules and method of forming an array of layers of amphiphilic molecules |
GB201313121D0 (en) | 2013-07-23 | 2013-09-04 | Oxford Nanopore Tech Ltd | Array of volumes of polar medium |
GB201418512D0 (en) | 2014-10-17 | 2014-12-03 | Oxford Nanopore Tech Ltd | Electrical device with detachable components |
GB201611770D0 (en) | 2016-07-06 | 2016-08-17 | Oxford Nanopore Tech | Microfluidic device |
JP6734210B2 (ja) * | 2017-02-20 | 2020-08-05 | 日本電信電話株式会社 | 脂質二分子膜基板、及びその製造方法 |
CN111936846B (zh) * | 2018-06-14 | 2022-12-20 | Nok株式会社 | 银-氯化银电极的制造方法 |
AU2020239385A1 (en) | 2019-03-12 | 2021-08-26 | Oxford Nanopore Technologies Plc | Nanopore sensing device and methods of operation and of forming it |
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US20020144905A1 (en) * | 1997-12-17 | 2002-10-10 | Christian Schmidt | Sample positioning and analysis system |
JP3486171B2 (ja) * | 1997-12-17 | 2004-01-13 | エコル・ポリテクニック・フェデラル・ドゥ・ロザンヌ(エ・ペー・エフ・エル) | ミクロ構造キャリア上における細胞単体および再構成膜系のポジショニングおよび電気生理学的特性決定 |
US6300108B1 (en) * | 1999-07-21 | 2001-10-09 | The Regents Of The University Of California | Controlled electroporation and mass transfer across cell membranes |
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2004
- 2004-09-17 WO PCT/JP2004/013671 patent/WO2005029054A1/ja active Application Filing
- 2004-09-17 EP EP04773290.4A patent/EP1669746A4/en not_active Withdrawn
- 2004-09-17 US US10/572,569 patent/US7408359B2/en not_active Expired - Fee Related
Non-Patent Citations (3)
Title |
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MACDONALD A.G.: "Combined Spectroscopic and Electrical Recording Techniques in Membrane Research:Prospects for Single Channel Studies", PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY, vol. 63, no. 1, 1995, pages 1 - 29, XP002982427 * |
See also references of EP1669746A4 * |
TORU IDE: "An Artificial Lipid Bilayer Formed on an Agarose-Coated Glass for Simultaneous Electrical and Optical Measurement of Single Ion Channels", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 265, no. 2, 1999, pages 595 - 599, XP002982426 * |
Also Published As
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JP2005091308A (ja) | 2005-04-07 |
US20070035308A1 (en) | 2007-02-15 |
US7408359B2 (en) | 2008-08-05 |
EP1669746A1 (en) | 2006-06-14 |
EP1669746A4 (en) | 2013-09-25 |
JP4394917B2 (ja) | 2010-01-06 |
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