JP2008194573A - Lipid double film forming method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method not requiring the connection with an external pump at each time when a lipid double film is formed in an apparatus for forming the lipid double film. <P>SOLUTION: A lipid double film forming apparatus has a first chamber 209 and a second chamber 205 so that both chambers 209 and 205 are connected through a lipid double film forming part 207 and the cross-sectional area of the lipid double film forming part 207 is smaller than each of the cross-sectional areas of both first and second chambers 209 and 205. The lipid double film can be simply formed by successively performing a first electrolyte filling process for filling the second chamber 205 with an electrolyte, a raw material filling process for arranging a mixed liquid of a raw material of the lipid double film and an organic solvent to the lipid double film forming part 207, a second electrolyte filling process for filling the first chamber 209 with the electrolyte and a centrifugal process for forming the lipid double film to the lipid double film forming part 207 by centrifuging the lipid double film forming apparatus. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for forming a lipid bilayer membrane used for analysis of membrane proteins including ion channels.

  Mass transport between intracellular and extracellular cells is performed through transmembrane proteins. Among them, ion channels cause changes in membrane potential due to ion permeation, and are known to play an important role in information transmission by signal generation such as nerve action potentials, and research has been actively promoted in recent years. Yes.

  An indispensable tool for conducting ion channel research is an experimental technique called the patch clamp method, which was developed in 1976 by Neher and Sakmann. In the patch clamp method, the tip of a micro glass tube called a patch electrode is first brought into close contact with the cell membrane surface. The potential is fixed in a state in which the micromembrane region at the tip opening is electrically insulated from other regions, and the ion current passing through the ion channel included in the micromembrane region is measured. The development of this method has helped to identify functional elements of channel protein molecules, elucidate their working mechanisms and structures, and brought great innovation in physiological research.

  However, the patch clamp method may not be applied although it is a very effective method in physiological research as described above. For example, this is the case when it is difficult to access anatomically, that is, when analyzing a channel on an organelle or a channel on a microstructure such as a presynaptic membrane. In addition, in order to deepen the basic structure of the channel and detailed structure-function correlation research, it is not applicable to the case where it is necessary to conduct an experiment with a simple configuration. At this time, the channel molecule must be analyzed in a simple system, that is, a system composed of water, salts, phospholipids, and channels.

  As described above, the planar lipid membrane method has been developed as an effective means when the patch clamp method cannot be applied. The lipid planar membrane method is roughly classified into a painting method and a bonding method (for example, Non-Patent Document 1).

  FIG. 11 shows a conventional method for forming a lipid bilayer membrane by a painting method. In FIG. 11, a container 10 is partitioned by a flat plate 11 made of a resin having a hydrophobic surface such as Teflon (registered trademark), polystyrene, etc., and a space partitioned by the flat plate 11 is filled with an electrolytic solution 12, and a minute opening opened on the flat plate 11. A mixed solution 14 of lipid molecules and an organic solvent is applied to the holes 13 with a brush 15. Since the excess organic solvent contained in the mixed solution 14 applied to the micropores 13 is gradually removed along the peripheral edge of the micropores 13, a lipid bilayer is formed after waiting for about 30 minutes to 3 hours.

  On the other hand, FIG. 12 (a), (b), (c) shows the formation method of the conventional lipid bilayer membrane by the bonding method. In FIG. 12A, the container 20 is partitioned by a flat plate 21 made of a resin having a hydrophobic surface such as Teflon (registered trademark) or polystyrene. First, squalene is applied to the micro holes 22 formed in the flat plate 21 as a pretreatment. The electrolyte solution 23 is added to the one chamber of the container 20 from the injection port 24 so that the level of the electrolyte solution 23 does not exceed the height of the lower end of the microhole 22. Next, when a mixed liquid of lipid molecules 25 and an organic solvent is dropped into the electrolytic solution 23 and left for several minutes, a lipid monomolecular film is formed at the gas-liquid interface of the electrolytic solution 23 as shown in FIG. It is formed. The lipid molecule 25 has a hydrophilic portion and a hydrophobic portion, and is oriented so that the hydrophilic portion of the lipid molecule 25 faces toward the electrolyte solution 23.

  Then, as shown in FIG. 12B, the electrolytic solution 23 is added until the level of the electrolytic solution 23 passes the height of the upper end of the microhole 22.

  Next, in FIG. 12C, the same operation is performed in the other chamber of the container 20. That is, the electrolytic solution 26 is added so that the height of the liquid level does not exceed the height of the lower end of the microhole 22. Next, when a mixed liquid of lipid molecules and an organic solvent is added to the electrolytic solution 26 and left for several minutes, a lipid monomolecular film is formed at the gas-liquid interface of the electrolytic solution 26. Then, the electrolytic solution 26 is added until the level of the electrolytic solution 26 passes the height of the upper end of the micropore 22. By performing the above operation, the other lipid monomolecular film is bonded to the lipid monomolecular film previously formed in the micropore 22, and as a result, a lipid bilayer membrane is formed.

  Forming a stable and reproducible lipid bilayer membrane by the two methods described above requires considerable skill. As a simpler method for forming a lipid bilayer membrane, there has been a method of forming a lipid bilayer membrane on a small chip using micro / nano processing technology (see, for example, Patent Document 1).

FIG. 13 shows a conventional lipid bilayer membrane forming apparatus described in Patent Document 1. In FIG. 13, microchambers 32 and microchannels 33 are provided on the front and back of the substrate 31, and micropores 34 that penetrate the microchambers 32 and microchannels 33 are provided. An electrolytic solution is injected into the microchamber 32, and an electrolytic solution, a mixed solution of a lipid bilayer membrane raw material and an organic solvent, and an electrolytic solution are sequentially injected into the microchannel 33. In order to make it possible to apply pressure to the electrolyte solution in the microchamber 32, a passage 36 that penetrates the wall 35 of the microchamber 32 is provided, and a lipid bilayer membrane is formed in the micropore 34 by adjusting the pressure in the microchamber 32 To do.
Japanese Patent Laying-Open No. 2006-312141 (page 11, FIG. 3) Okada Yasunobu "Patch clamp experiment technique" Yoshioka Shoten September 25, 1996 (p.133-139)

  In Patent Document 1, in order to reduce the thickness of the lipid bilayer membrane, the pressure of the microchamber 32 is adjusted, so that a pressurizing unit such as a syringe pump, a diaphragm pump, or a peristatic pump provided outside the microchamber 32 is used. Was used. Further, in order to transport the electrolyte solution and the mixed solution of the lipid bilayer membrane raw material and the organic solvent to the microchannel 33, a pressurization of a syringe pump, a diaphragm pump, a peristatic pump, or the like provided outside the microchannel 33 Means.

  However, each time the lipid bilayer membrane is formed, the microchamber 32 and the microchannel 33 have to be connected to the pressurizing means.

  An object of the present invention is to solve the above-mentioned conventional problems and to provide a method for easily forming a lipid bilayer even for beginners.

  The present invention that solves the conventional problem is a method of forming a lipid bilayer membrane using a lipid bilayer membrane formation apparatus, the lipid bilayer membrane formation apparatus comprising: a base material; and an interior of the base material A first chamber provided in the substrate, a second chamber provided in the substrate, and a hole connecting the first chamber and the outer peripheral surface of the substrate, the first chamber and the first chamber The two chambers are connected via a lipid bilayer formation part, and the cross-sectional area of the lipid bilayer formation part is greater than the cross-sectional area of the first chamber and the cross-sectional area of the second chamber. The distance between the first chamber and the center of rotation is smaller than the distance between the second chamber and the center of rotation, and the method can be applied to either the first chamber or the second chamber. 1st electrolyte filling electrolyte A raw material filling step in which a mixed liquid of a lipid bilayer membrane raw material and an organic solvent is disposed in the lipid bilayer membrane forming part, an electrolyte second filling step in which the other chamber fills the electrolytic solution, and the rotation center A centrifugal process of forming the lipid bilayer membrane in the lipid bilayer membrane formation part by sequentially centrifuging the lipid bilayer membrane formation apparatus as a rotation axis is included.

  The substrate is preferably a resin or glass.

  The cross-sectional area of the hole on the outer peripheral surface of the base material is preferably smaller than the cross-sectional area of the hole in the first chamber.

  It is preferable that the volume of the hole is larger than the volumes of the first chamber and the second chamber.

  The lipid bilayer membrane-forming part preferably has a water-repellent surface.

  The lipid bilayer membrane-forming part preferably has at least one constriction.

  The lipid bilayer membrane forming part preferably has at least one strut.

  The second chamber preferably has a hole connected to the outer peripheral surface of the base material.

  It is preferable that the first chamber and the second chamber have electrodes for detecting filling of the electrolytic solution and the mixed solution.

  The first chamber and the second chamber preferably have electrodes for detecting a membrane current flowing through the lipid bilayer.

  It is preferable that a plurality of the second chambers and the lipid bilayer-forming portions are arranged radially with respect to the rotation center.

  In the first electrolyte filling step, the second chamber is preferably filled with an electrolytic solution, and in the second electrolyte filling step, the first chamber is preferably filled with an electrolytic solution.

  In the first electrolyte filling step or the second electrolyte filling step, it is preferable to detect filling of the electrolytic solution using electrodes provided in the first chamber and the second chamber, respectively.

  In the raw material filling step, it is preferable that air bubbles be removed from the other chamber filled with the electrolyte and the lipid bilayer membrane forming portion where the mixed solution is arranged.

  It is preferable that the rotation speed of the lipid bilayer membrane formation apparatus in the electrolyte second filling step is smaller than the rotation speed of the lipid bilayer membrane formation apparatus in the centrifugation step.

  In the centrifugation step, it is preferable that the formation of a lipid bilayer is detected by an electrical signal obtained from an electrode provided in each of the first chamber and the second chamber.

  It is preferable to control the rotation speed and the rotation time of the lipid bilayer membrane formation device by means of electrical signals obtained from electrodes provided inside the first chamber and the second chamber, respectively.

  In the lipid bilayer membrane forming apparatus in which the second chamber and the lipid bilayer membrane formation section are arranged radially with respect to the rotation center, the plurality of lipid bilayer membranes created are electrically independent from each other. It is preferable.

  The lipid bilayer membrane forming device is preferably operated in an electromagnetic shield box.

  The lipid bilayer membrane formation method according to claim 1 is preferably used in an ion channel analyzer.

  With this configuration, the pressure for removing excess organic solvent from the mixed liquid of the lipid bilayer membrane raw material and the organic solvent can use the centrifugal force applied to the electrolyte itself, so external pressure means are provided. A lipid bilayer membrane can be easily formed without any problems.

  According to the method for forming a lipid bilayer membrane according to the present invention, first, the first chamber or the second chamber is filled with an electrolyte solution, and then the lipid bilayer membrane formation part is mixed with a lipid bilayer membrane raw material and an organic solvent. A lipid bilayer membrane can be easily formed by placing the solution, filling the other chamber with the electrolyte, and continuing to rotate the lipid bilayer membrane formation apparatus. Therefore, the cumbersome work of connecting the chamber and the pressurizing means each time a lipid bilayer membrane is formed as in the prior art does not have to be performed.

  Embodiments of the present invention will be described below with reference to the drawings.

  1 (a), (b), (c), and (d) are a perspective view and a cross-sectional view of a lipid bilayer membrane forming apparatus according to an embodiment of the present invention.

  In the present embodiment, as shown in FIG. 1A, a microchip 100 as a base material is manufactured by bonding a substrate 101 and an upper lid 102. The substrate 101 and the upper lid 102 are preferably made of resin but may be glass. The microchip 100 can be rotated about the rotation center 103 as a central axis.

  Inside the microchip 100, a first chamber 105 and a second chamber 107 are provided as containers for filling the electrolytic solution. The inner walls of the first chamber 105 and the second chamber 107 are preferably smooth. The center line average roughness (Ra) of the inner walls of the first chamber 105 and the second chamber 107 is preferably 100 nm or less, but is not particularly limited. Further, a hole 104 connected to the outer peripheral surface of the microchip 100 is provided at one end of the first chamber 105, and the first chamber 105 can be filled with the electrolytic solution from the hole 104. The position of the hole 104 is not particularly limited as long as it is one end of the first chamber 105, but is preferably closer to the rotation center 103.

  The first chamber 105 and the second chamber 107 are connected via a constriction 106 provided as a lipid bilayer membrane forming part.

1B, 1C, and 1D are cross-sectional views taken along the lines AB, CD, and EF in FIG. The cross-sectional areas 110, 111, and 112 represent the cross-sectional areas of the first chamber 105, the narrowest portion of the constriction 106, and the second chamber 107, respectively. Although more lipid bilayers sectional area 111 The smaller is prolonged stable form, since too small to handle the lipid bilayer membrane becomes difficult, preferably 0.0001 mm ² or more 0.1 mm ² or less.

  Further, the shape of the cross section of the constriction 106 may be a square as shown in FIG. 1C, or may be a rectangle, a circle, an ellipse or the like, but a symmetrical shape such as a circle or a square is more preferable.

  Furthermore, as shown in FIG. 1A, both the first chamber 105 and the second chamber 107 have a tapered shape, and the constriction 106 is formed thereby. That is, as the distance from the rotation center 103 increases, the cross-sectional area 110 of the first chamber 105 gradually decreases, and conversely, the cross-sectional area 112 of the second chamber 107 increases.

  The distance between the first chamber 105 and the rotation center 103 is arranged to be smaller than the distance between the second chamber 107 and the rotation center 103. Since the larger the distance between the first chamber 105 and the rotation center 103 is, the larger centrifugal force can be obtained, the distance is preferably 15 mm or more. Further, in order to facilitate the handling of the lipid bilayer membrane forming apparatus of the present invention, the distance between the second chamber 107 and the rotation center 103 is preferably 200 mm or less.

  The first chamber 105 and the second chamber 107 are provided with electrodes 109a and b, respectively. Using the electrodes 109a and 109b, the filling of the electrolyte and the formation of the lipid bilayer are detected as electrical signals. In addition, although the typical electrode was illustrated in Fig.1 (a), the position, shape, and number of an electrode are not restricted to this.

  Next, a procedure for forming a lipid bilayer membrane will be described. FIGS. 2A to 2E are operation diagrams of the lipid bilayer membrane forming apparatus according to the embodiment of the present invention.

  First, in the electrolyte first filling step, as shown in FIG. 2 (a), the electrolyte is injected from the hole 201 provided in the microchip 200 using the pipette 202, and the rotation is performed as shown in FIG. 2 (b). The microchip 200 is rotated around the center 203, and the electrolytic solution 204 is filled into the second chamber 205 by centrifugal force.

At this time, a 100 mM KCl aqueous solution is used as the electrolytic solution. The electrolytic solution is not limited to an aqueous KCl solution, and may be an aqueous solution such as NaCl or CaCl ₂ , or a buffer solution such as PBS (phosphate buffer solution) or HEPES. A plurality of electrolytes may be mixed. Further, an electrolytic solution generally used when performing an ion current measurement experiment flowing through the ion channel may be used. The concentration of the electrolytic solution is preferably 1 mM or more and 1 M or less. Further, although a pipette is used for injecting the electrolytic solution, a syringe, a tube, a spoid, an autosampler, or the like may be used.

  In the electrolyte first filling step, it is preferable to fill the electrolyte solution 204 so that no bubbles remain in the second chamber 205. Further, it is preferable to fill the region other than the second chamber 205 so that the electrolytic solution 204 does not remain.

  Next, in the raw material filling step, as shown in FIG. 2C, a liquid mixture 206 of the lipid bilayer membrane raw material and the organic solvent is injected from the hole 201, and the microchip 200 is rotated about the rotation center 203. The mixed liquid 206 is placed on the constriction 207 by centrifugal force.

  At this time, azolectin is preferred as a raw material for the lipid bilayer membrane. In the present invention, the raw material of the lipid bilayer membrane is not limited to azolectin, and may be other naturally derived lipids or synthetic lipids. Synthetic lipids are more preferred because they are easy to obtain highly pure and chemically stable. Specifically, it may be phospholipid diphytanoylphosphadylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, dipalmitoylphosphatidylcholine, or other phospholipids. The fatty acid portion of the lipid molecule is preferably a saturated or unsaturated fatty acid having 10 to 20 carbon atoms. These lipids may be used alone or in combination of two or more. The lipid concentration relative to the organic solvent is preferably 3 to 50 mg / mL.

  The organic solvent is preferably a nonpolar organic solvent. Although decane is particularly preferable, hexadecane or hexane may be used, or other saturated hydrocarbons may be used. A plurality of organic solvents may be mixed.

  Further, in the electrolyte second filling step, as shown in FIG. 2D, the electrolyte 208 injected from the hole 201 is filled into the first chamber 209 by centrifugal force. The electrolytic solution 208 may be the same type and concentration as the electrolytic solution 204 or may be different.

  Finally, in the centrifugation step, as shown in FIG. 2 (e), the mixed liquid 206 shown in FIG. 2 (c) is pressurized by the centrifugal force applied to the electrolyte 208, and the excess organic solvent is discharged to remove the lipid. A heavy film 210 is formed.

  Detection of the formation of the lipid bilayer membrane 210 can be performed by measuring the membrane current flowing through the lipid bilayer membrane 210 using the electrodes 211a and 211b. The membrane current can be measured by connecting a measuring instrument generally used in ion channel analysis, such as a preamplifier, an ammeter, and a patch clamp amplifier, to the electrodes 211a and 211b.

  According to such a configuration and operation procedure, a lipid bilayer membrane is formed using centrifugal force, so that a pump for removing excess organic solvent from the mixed solution becomes unnecessary, and a pump is formed each time a lipid bilayer membrane is formed. The lipid bilayer membrane can be easily formed.

  The feature of the present invention is that since the opening of the second chamber 107 is only the constriction 106 as shown in FIG. 1A, the electrolytic solution is filled through the constriction 106. Such a configuration is not found in the prior art. That is, the prior art including the painting method and the bonding method used the constriction 106 only as a means for holding a liquid mixture of a raw material of a lipid bilayer membrane and an organic solvent.

  However, in the present invention, the use of the constriction 106 also as the electrolyte solution transfer path eliminates the need to separately provide an electrolyte solution transfer path in the second chamber 107. As a result, it becomes possible to eliminate the need for pumps and valves for conveyance.

  Further, as shown in FIG. 1 (a), the opening of the entire apparatus can be only the hole 104, and the electrolyte solution and the mixed solution are injected through the hole 104. Such a configuration is not found in the prior art. That is, the conventional techniques such as the painting method and the bonding method have a plurality of openings for solution injection, pressure application, and electrode insertion, so that the solution may leak and scatter.

  However, the configuration of the present invention is effective for suppressing the leakage and scattering of the solution. In addition, according to the configuration of the present invention, the amount of reagents necessary for forming the lipid bilayer membrane, that is, the electrolyte solution and the raw material of the lipid bilayer membrane can be reduced. This is because, when forming the lipid bilayer membrane, it is sufficient to inject from the hole 104 by the minimum necessary amount. This also has the effect of reducing the amount of waste.

  In this embodiment mode, the opening of the entire apparatus may be provided in addition to the hole 104. For example, a hole may be provided at one end of the second chamber 107.

  Here, the procedure for forming a lipid bilayer when a hole is provided at one end of the second chamber 107 will be described with reference to FIGS. In addition, about the thing which has the structure and procedure similar to Fig.2 (a)-(e), the same code | symbol is attached | subjected and the description is abbreviate | omitted.

  First, in the electrolyte first filling step, as shown in FIG. 3A, an electrolyte solution is injected from the hole 212 provided in the second chamber 205 using the pipette 202, and as shown in FIG. The electrolytic solution 204 is filled into the second chamber 205. After this filling is completed, the hole 212 is preferably sealed so that the electrolyte does not leak from the hole 212.

  From the next raw material filling process to the centrifugation process, as shown in FIGS. 3C to 3E, the procedure is the same as in FIGS. 2C to 2E.

  When the lipid bilayer membrane is formed according to the procedure shown in FIGS. 3A to 3E, the centrifugal operation for filling the electrolytic solution is unnecessary in the first electrolyte filling step, which is preferable.

  In the present embodiment, the substrate is preferably resin or glass. The resin is preferably polyethylene terephthalate, vinyl chloride, polyethylene, polycarbonate, polypropylene, acrylic, cycloolefin, or the like. However, the substrate is not limited to these. Any member to which a general processing method such as injection molding, nanoimprinting, hot embossing, cutting, etching, photolithography, or screen printing can be applied may be used. Furthermore, a member having high translucency and low autofluorescence is more preferable so that the lipid bilayer formation step including the electrolyte filling step and the centrifugation step can be optically observed.

  The glass is preferably quartz glass, but other glasses such as borosilicate glass, soda glass, low melting point glass and the like may be used as long as they can be filled and retained with an electrolyte and an organic solvent. Alternatively, other inorganic materials such as silicon, silicon nitride, and alumina may be used.

In the present embodiment, the cross-sectional area of the hole 104 on the outer peripheral surface of the microchip 100 shown in FIG. 1A is preferably smaller than the cross-sectional area of the hole 104 in the first chamber 105. By making the hole 104 into a tapered shape, it is possible to suppress the electrolyte injected into the hole 104 from being scattered outside the microchip 100 when the microchip 100 is rotated. The smaller the cross-sectional area of the outer peripheral surface of the hole 104, the smaller the scattering of the electrolytic solution. On the other hand, if the sectional area is too small, the injection of the electrolytic solution is difficult and the gas-liquid exchange is not performed smoothly. It is difficult to fill the first chamber and the second chamber with the electrolyte. Cross-sectional area at the outer peripheral surface of the hole 104 is preferably 0.01 mm ² or more 1 mm ² or less. The cross-sectional area of the hole 104 in the first chamber 105 is preferably 1.5 mm ² or more and 4 mm ² or less.

  Further, in the present embodiment, the volume of the hole 104 shown in FIG. 1A is preferably smaller than the volumes of the first chamber 105 and the second chamber 107. The volume of the hole 104 is preferably 1.5 to 5 times the volume of the first chamber 105 and the second chamber 107, and more preferably 2 to 3 times. The same applies when a hole is provided in the second chamber 107.

  In the present embodiment, the constriction 106 shown in FIG. 1A preferably has a hydrophobic surface. In general, since the surface of the resin is hydrophobic, if the microchip 100 is formed of resin, the surface of the constriction 106 is also hydrophobic. However, in order to form a lipid bilayer more effectively, the surface is more hydrophobic. A polymer such as a fluororesin may be applied to the surface of the constriction 106. On the other hand, when the microchip 100 is formed of an inorganic material such as glass or silicon nitride, the surface of the constriction 106 is generally hydrophilic, and thus it is preferable to perform a water repellent treatment. HMDS (hexamethylene disilazane) may be coated, a Teflon (registered trademark) thin film may be coated, or a fluorocarbon film may be formed by plasma polymerization. In the present invention, the water repellent treatment method is not limited, and other known techniques relating to water repellent treatment may be used.

  Further, in the present embodiment, both the first chamber 105 and the second chamber 107 shown in FIG. 1A have a tapered shape, and the constriction 106 is formed thereby.

  That is, as the distance from the rotation center 103 increases, the cross-sectional area 110 of the first chamber 105 gradually decreases, and conversely, the cross-sectional area 111 of the second chamber 107 increases.

  The constriction 106 can be replaced with other shapes. FIG. 4 shows another example of the shape. As shown in FIG. 4, each of the first chamber 105 and the second chamber 107 has a rectangular shape, and a constriction 301 is formed thereby. It is possible to form a lipid bilayer even when the length 302 of the constriction 301 is very long, that is, in the case of a “channel” shape in a general biochip. The length 302 of the constriction 301 is preferably 0.01 mm or more and 1 mm or less because the position of the overlying membrane is not determined.

  In the present embodiment, a protrusion may be provided in the middle of the constriction 106 shown in FIG. That is, as shown in FIG. 5, the constriction group 402 may be formed by arranging a plurality of protrusions 401. 5 having the same configuration as that of the embodiment shown in FIG. 1A described above is denoted by the same reference numeral and description thereof is omitted.

  Furthermore, in this embodiment, the lipid bilayer membrane forming unit may be a column group 502 in which a plurality of columns 501 are arranged as shown in FIG. 6 having the same configuration as that of the embodiment shown in FIG. 1A described above is denoted by the same reference numeral and description thereof is omitted. The column shape is preferably a cylinder, but other shapes such as a square column, a triangular column, a hexagonal column, and a star column may be used. The number, size, and arrangement of the support columns are not limited.

  The lipid bilayer membrane forming part may be formed by combining the strut group 502 shown in FIG. 6 and the constriction 106 shown in FIG. 1 (a), or may be formed in combination with another shape of constriction. .

In the present embodiment, the substrate shown in FIG. 1A may be tube-shaped. FIG. 7 shows an example in which a tube 601 is used as a base material. 7 having the same configuration as that of the embodiment shown in FIG. 1A described above is denoted by the same reference numeral and description thereof is omitted. The material of the tube is preferably a resin, but may be glass or other inorganic materials. The diameter of the tube is preferably 0.01 mm or more and 3 mm or less, and more preferably 0.2 mm or more and 1 mm or less. Sectional area of the constriction 106 is preferably 0.0001 mm ² or more 0.1 mm ² or less. The cross-sectional area of the first chamber 105 is preferably at least twice the cross-sectional area of the narrowest portion of the constriction 106. The cross-sectional area of the second chamber 107 is preferably at least twice the cross-sectional area of the narrowest portion of the constriction 106.

  Furthermore, in the present embodiment, it is preferable that the filling of the electrolyte into the first chamber 105 and the second chamber 107 shown in FIG. 1A is electrically detected using the electrodes 109a and 109b. FIG. 8 shows a principle diagram for detecting filling of the electrolytic solution. For example, as shown in FIG. 8, the second chamber 107 is provided with a pair of electrodes 109c and 109d. If an ionic current flows when a voltage is applied between the electrode 109c and the electrode 109d, this indicates that the electrode 109c and the electrode 109d are immersed in the electrolytic solution 602. The same applies to the first chamber 105. In addition, it is more preferable to arrange the electrodes on the inner walls of the first chamber 105 and the second chamber 107 in a lattice shape so that it is possible to detect in detail how far the electrolyte is filled. Note that the shape, number, and arrangement of the electrodes are not limited to these, and they are included in the scope of the present invention as long as the same effect can be obtained.

  Furthermore, in the present embodiment, in the raw material filling step shown in FIG. 2 (c), it is preferable to remove air bubbles from the chamber filled with the electrolyte and the lipid bilayer forming part where the mixed solution is arranged. This is because bubbles may be mixed in the liquid in the electrolyte first filling step and the raw material filling step. In order to detect the removal of bubbles, the electrodes 109a and 109b shown in FIG. 1A may be used for electrical detection, or an optical observation unit may be provided outside the lipid bilayer membrane formation apparatus. It may be separately provided and optically detected such as light absorption, light scattering, and imaging.

  Moreover, in this Embodiment, the rotational speed of the lipid bilayer membrane formation apparatus in the electrolyte 2nd filling process shown in FIG.2 (d) is the lipid bilayer membrane formation apparatus in the centrifugation process shown in FIG.2 (e). It is preferable that it is smaller than the rotational speed. For example, the specific gravity of an aqueous solution of KCl that is an electrolytic solution is about 1, and the specific gravity of decane that is an organic solvent is about 0.7. Therefore, the liquid mixture of the lipid bilayer membrane raw material and the organic solvent tends to be detached from the lipid bilayer membrane formation portion by centrifugal force. Therefore, the rotation speed of the lipid bilayer membrane formation apparatus in the electrolyte second filling step is preferably a rotation speed at which the liquid mixture of the lipid bilayer membrane raw material and the organic solvent does not desorb from the lipid bilayer membrane formation section by centrifugal force. . On the other hand, the rotational speed of the lipid bilayer membrane formation apparatus in the centrifugation step is such that the mixture of the lipid bilayer membrane raw material and the organic solvent is not detached from the lipid bilayer membrane formation part by centrifugal force, and excess organic solvent is removed. A rotation speed that can be discharged is preferred.

  Further, in the present embodiment, in the centrifugation step shown in FIG. 2 (e), the lipid bilayer membrane is formed by electrical signals obtained from the electrodes 211a and 211b provided in the first chamber 209 and the second chamber 205, respectively. It is preferred to detect formation. The electric signal is preferably an electric current flowing through the lipid bilayer, but may be a voltage applied to both ends of the lipid bilayer or an electric capacity of the lipid bilayer. The electrical signal is preferably an AC signal, but may be a DC signal. Furthermore, it is preferable that the distance from the electrodes 211a, 211b to the preamplifier is short because noise can be reduced. It is more preferable that a preamplifier is provided in the lipid bilayer membrane forming apparatus to amplify an electric signal. In addition to this electrical detection, an optical observation unit may be separately provided outside the lipid bilayer membrane forming apparatus to perform optical detection such as light absorption, light scattering, and imaging.

  Further, in the present embodiment, lipid bilayer formation in each step is performed by electrical signals obtained from the electrodes 109a and 109b provided in the first chamber 105 and the second chamber 107 shown in FIG. It is preferable to control the rotation speed and rotation time of the apparatus. You may use the computer provided outside the lipid bilayer membrane formation apparatus for the analysis of an electrical signal. The rotation speed may be measured optically by providing an optical observation part outside the lipid bilayer membrane forming apparatus, or mechanically by a motor for rotating the lipid bilayer membrane forming apparatus. good.

  Moreover, in this Embodiment, it is preferable to operate a lipid bilayer membrane formation apparatus within an electromagnetic shielding box. It is preferable that a wide range of noise from high frequency to low frequency can be removed. Regarding the magnetic shield, the low frequency magnetic field attenuation (static magnetic field) is preferably 29 dB or more at a frequency of 0.1 Hz or less, preferably 50 dB at 1 Hz, preferably 80 dB or more at 25 Hz or more, and 40 dB at DC (direct current). Regarding the electromagnetic shield, the shielding rate is preferably 80 dB or more at a frequency of 100 Hz to 1 MHz, and preferably 60 dB or more at a frequency of 1 Hz to 1 MHz. In addition, this invention is not limited to this, Use of the electromagnetic shielding box which shows a general shield characteristic is included in the scope of the present invention. The motor for rotating the lipid bilayer membrane forming apparatus is preferably provided outside the electromagnetic shield box, but may be provided inside.

  Furthermore, in the practice of the present invention, it is preferable that a plurality of second chambers 107 and constrictions 106 shown in FIG. Thereby, a plurality of lipid bilayer membranes can be formed simultaneously. A plurality of microchips 100 shown in FIG. 1 (a) may be arranged, or the first chamber 105, the second chamber 107, the constriction 106, etc. may be arranged all together on a disk-shaped substrate. 9 (a), (b), and (c) show a front view, a cross-sectional view, and a device after the formation of the lipid bilayer membrane, which are collectively disposed on a disk-shaped substrate. The enlarged view of is shown. In addition, about the thing which has the structure similar to Fig.1 (a), the same code | symbol is attached | subjected and the description is abbreviate | omitted.

  9A is different from the embodiment shown in FIG. 1A in that a plurality of second chambers 107 and constrictions 106 are provided radially with respect to the rotation center 103.

  Note that, as shown in FIGS. 9A and 9B, a guide groove 701 may be provided on the bottom surface of the first chamber 105. Thereby, the electrolytic solution can be efficiently distributed and filled into the second chamber 107 and the first chamber 105.

  Furthermore, in the present embodiment, it is preferable that the plurality of lipid bilayer membranes created using the lipid bilayer membrane formation apparatus shown in FIG. FIG. 9C shows an enlarged view of a part of the apparatus for forming a lipid bilayer membrane. An appropriate amount can be filled in each chamber so that the electrolyte solutions 702a, b, and c do not contact each other. With this configuration, a plurality of lipid bilayers can be electrically independent without using an insulator such as air or an organic solution as in the prior art.

  Moreover, in this Embodiment, it is preferable that the lipid bilayer membrane formation method concerning this invention incorporates an ion channel in a lipid bilayer membrane, and uses it for an ion channel analyzer. Moreover, you may use for the functional analysis apparatus of an ion channel, the analysis apparatus of the interaction between a membrane protein-ion channel, a biosensing apparatus, etc.

(Example)
Next, a method for producing the lipid bilayer membrane forming apparatus shown in FIG. 1 configured as described above will be described. FIGS. 10A to 10D are process diagrams showing an example of a method for producing a lipid bilayer membrane forming apparatus.

  Polyethylene terephthalate (PET) was used for the substrate and the upper lid. As shown in FIG. 10A, a chamber 803 was formed on the upper lid 802 using a mold 801 by a hot embossing method. Hot embossing was carried out by holding for 3 minutes under heating and pressurization at 110 ° C. and 0.2 MPa. In this example of the manufacturing method, the mold is made of silicon by lithography and dry etching, but may be a mold made of stainless steel, quartz, silicon carbide, alumina, sapphire, diamond, or the like.

  The size of the upper lid was 22 mm in length, 22 mm in width, and 0.5 mm in thickness. The substrate was 26 mm long, 22 mm wide and 0.5 mm thick. Although not shown in FIG. 10A, a constriction was formed at the same time as the chamber. The depth of the chamber and the constriction was 100 μm. The depth of the constriction and the chamber need not be the same. The width of the constriction was 200 μm.

  Next, as shown in FIG. 10B, a hole 804 was formed using a drilling machine. The diameter of the hole in the opening was 1.5 mm. The method for opening the holes is not limited to the above drilling machine, and punching or end milling may be used. Furthermore, when the inner wall surface of the hole is made hydrophilic, the electrolyte solution has better wettability, and the lipid solution is more preferable because adhesion to the inner wall surface can be suppressed. Note that the order of the steps in FIGS. 10A and 10B may be reversed.

  Further, it is more preferable to form the chamber, the constriction, and the holes collectively by injection molding because they can be manufactured in large quantities at a low cost.

  Next, an electrode 806 was formed on the substrate 805 as shown in FIG. The electrode 806 is most preferably silver / silver chloride, but is not particularly limited as long as it is an electrode material generally used for electrochemical measurement, such as gold, platinum, and glassy carbon. The electrode forming method is preferably a photolithography method, but other known techniques known as electrode forming methods such as screen printing and ink jet printing may also be used.

  Finally, as shown in FIG. 10 (d), the upper lid 802 and the substrate 805 were held and joined for 5 minutes under heating and pressurization at 75 ° C. and 0.15 MPa. In this example of the manufacturing method, thermocompression bonding is used, but other known techniques known as a method for joining two substrates may be used.

  The operation of the lipid bilayer membrane production apparatus manufactured as described above will be described below with reference to FIG.

  First, as shown in FIG. 2A, 2 μL of an electrolyte solution was injected from a hole 201 provided in the microchip 200 by a pipette 202. At this time, a 100 mM KCl aqueous solution was used as the electrolytic solution. The amount and concentration of the electrolytic solution are not limited.

  Next, as shown in FIG. 2B, the microchip 200 was rotated at a rotation speed of 3000 rpm for 3 seconds with the rotation center 203 as a central axis, and the electrolytic solution 204 was filled into the second chamber 205 by centrifugal force. The higher the rotation speed, the faster the filling of the electrolyte solution is completed. Therefore, 3000 to 6000 rpm is preferable, but the present invention is not limited to this. The rotation time may be performed until completion of filling is detected. The distance between the second chamber 205 and the rotation center 203 was 35 mm.

  Next, as shown in FIG. 2 (c), 0.2 μL of a mixed solution of the lipid bilayer membrane raw material and the organic solvent was injected into the hole 201. At this time, a mixed solution of 50 mg of azolectin and 1 mL of decane was used as a lipid solution. The microchip 200 was rotated at a rotation speed of 3000 rpm for 3 seconds around the rotation center 203, and the mixed liquid 206 was placed in the constriction 207.

  Further, as shown in FIG. 2 (d), 1.5 μL of electrolyte was injected into the hole 201. Then, the microchip 200 was rotated at a rotation speed of 3000 rpm for 3 seconds with the rotation center 203 as the central axis, and the electrolyte 208 was filled in the first chamber 209.

  Finally, as shown in FIG. 3E, the microchip 200 was rotated. Centrifugal force applied to the electrolytic solution 208 pressurizes the mixed solution 206, and excess organic solvent is discharged to form the lipid bilayer membrane 210. The rotation speed at this time was 3500 rpm. At a rotational speed of 3000 rpm or less, the centrifugal force applied to the electrolytic solution 208 is insufficient, so that excess organic solvent cannot be discharged. Further, at a rotational speed of 4000 rpm or higher, the centrifugal force applied to the electrolyte solution 208 was too strong, so that the liquid mixture 206 could not be held in the constriction 207, and a lipid bilayer membrane was not formed.

  Since the lipid bilayer membrane formation apparatus concerning this invention forms a lipid bilayer membrane using a centrifugal force, it is not necessary to provide a pressurizing means outside. Therefore, it has the effect that it does not have to be connected to an external pressurizing means each time a lipid bilayer membrane is formed, and is useful as a simple method for forming a lipid bilayer membrane. Moreover, since this apparatus is manufactured with resin, it can be made disposable, simplifying the disposal of waste and reducing the amount of waste.

  If membrane proteins such as ion channels and receptors are incorporated into the lipid bilayer membrane prepared by the lipid bilayer membrane preparation method of the present invention, the basic structure analysis of membrane proteins, elucidation of functions, and correlation studies between membrane proteins and membrane proteins It can be applied to In addition, there is a possibility of industrial application to medical and pharmaceutical fields such as diagnosis of diseases caused by ion channels and screening for new drug development. In addition, if a specific molecular recognition property of a membrane protein is used, it can be applied to a biosensor.

The perspective view and sectional drawing of the lipid bilayer membrane formation apparatus in embodiment of this invention Operation diagram of lipid bilayer membrane forming apparatus in the same embodiment Operation diagram of another lipid bilayer membrane forming apparatus in the same embodiment The front view which shows the example of the other lipid bilayer formation part in the same embodiment The front view which shows the example of the other lipid bilayer formation part in the same embodiment The front view which shows the example of the other lipid bilayer formation part in the same embodiment The perspective view of the other lipid bilayer membrane formation apparatus in the same embodiment Principle diagram of electrolyte filling detection in the same embodiment Front view, sectional view and enlarged view of another lipid bilayer membrane forming apparatus in the same embodiment Process drawing of manufacturing method example of lipid bilayer membrane forming apparatus in the same embodiment Schematic diagram showing a conventional painting method Schematic diagram showing the conventional bonding method Schematic diagram of a conventional lipid bilayer membrane formation device

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Container 11 Flat plate 12 Electrolytic solution 13 Micropore 14 Mixed solution 15 Brush 20 Container 21 Flat plate 22 Micropore 23 Electrolytic solution 24 Inlet 25 Lipid molecule 26 Electrolytic solution 31 Substrate 32 Microchamber 33 Microchannel 34 Micropore 35 Wall 36 Passage 100 Microchip 101 Substrate 102 Upper lid 103 Center of rotation 104 Hole 105 First chamber 106 Constriction 107 Second chamber 109a to d Electrode 110 Cross sectional area of first chamber 111 Cross sectional area of narrowest portion of constriction 112 Cross sectional area of second chamber 200 Micro Tip 201 Hole 202 Pipette 203 Center of Rotation 204 Electrolyte 205 Second Chamber 206 Mixed Solution 207 Neck 208 Electrolyte 209 First Chamber 210 Lipid Bilayer 211a, b Electrode 212 Hole 301 Neck 302 length 401 projection 402 constricted group 501 posts 502 strut group 601 tube 602 electrolyte 701 guide groove 702a~c electrolyte 801 mold 802 upper cover 803 chamber 804 hole 805 substrate 806 electrode

Claims (20)

A method of forming a lipid bilayer using a lipid bilayer formation device,
The lipid bilayer membrane formation device is
A substrate;
A first chamber provided inside the substrate;
A second chamber provided inside the substrate;
A hole connecting the first chamber and the outer peripheral surface of the substrate;
The first chamber and the second chamber are connected via a lipid bilayer formation part,
The lipid bilayer membrane forming section has a cross-sectional area smaller than both the cross-sectional area of the first chamber and the cross-sectional area of the second chamber,
The distance between the first chamber and the rotation center is smaller than the distance between the second chamber and the rotation center,
The method
A first electrolyte filling step of filling one of the first chamber and the second chamber with an electrolyte;
Raw material filling step of arranging a mixed liquid of a raw material of lipid bilayer membrane and an organic solvent in the lipid bilayer membrane forming part,
Forming the lipid bilayer membrane in the lipid bilayer membrane formation part by centrifuging the lipid bilayer membrane formation device with the second chamber filling step of filling the other chamber with the electrolyte and the rotation center as the rotation axis A method for forming a lipid bilayer membrane, which in turn includes a centrifugation step. The method for forming a lipid bilayer according to claim 1, wherein the base material is resin or glass. 2. The method of forming a lipid bilayer according to claim 1, wherein a cross-sectional area of the hole in the outer peripheral surface of the base material is smaller than a cross-sectional area of the hole in the first chamber. The method for forming a lipid bilayer according to claim 1, wherein the pore volume is larger than the volume of the first chamber and the second chamber. The method for forming a lipid bilayer according to claim 1, wherein the lipid bilayer-forming part has a water-repellent surface. 6. The method of forming a lipid bilayer according to claim 5, wherein the lipid bilayer forming part has at least one constriction. The lipid bilayer membrane formation method according to claim 5, wherein the lipid bilayer membrane formation part has at least one support. The method for forming a lipid bilayer according to claim 1, wherein the second chamber has a hole connected to the outer peripheral surface of the base material. The method for forming a lipid bilayer according to claim 1, wherein the first chamber and the second chamber have electrodes for detecting filling of the electrolyte solution and the mixed solution. The method of forming a lipid bilayer according to claim 1, wherein the first chamber and the second chamber have electrodes for detecting a membrane current flowing in the lipid bilayer. 2. The lipid bilayer membrane formation method according to claim 1, wherein a plurality of the second chambers and the lipid bilayer membrane formation portions are arranged radially with respect to the rotation center. 2. The lipid bilayer formation according to claim 1, wherein in the first electrolyte filling step, the second chamber is filled with an electrolytic solution, and in the second electrolyte filling step, the first chamber is filled with an electrolytic solution. Method. The electrolyte first filling step or the second electrolyte filling step detects the filling of the electrolytic solution using electrodes respectively provided in the first chamber and the second chamber. 2. The method for forming a lipid bilayer membrane according to 1. 2. The lipid bilayer according to claim 1, wherein in the raw material filling step, air bubbles are removed from the other chamber filled with the electrolytic solution and the lipid bilayer forming portion where the mixed solution is arranged. Method for forming a heavy film. The lipid according to claim 1, wherein a rotation speed of the lipid bilayer membrane forming apparatus in the electrolyte second filling step is smaller than a rotation speed of the lipid bilayer membrane forming apparatus in the centrifugation step. Double film formation method. 2. The lipid according to claim 1, wherein in the centrifugation step, formation of a lipid bilayer is detected by an electric signal obtained from an electrode provided in each of the first chamber and the second chamber. Double film formation method. The rotation speed and the rotation time of the lipid bilayer membrane forming apparatus are controlled by electrical signals obtained from electrodes provided in the first chamber and the second chamber, respectively. The lipid bilayer membrane formation method as described. In the lipid bilayer membrane forming apparatus in which a plurality of the second chambers and the lipid bilayer membrane-forming portions are arranged radially with respect to the rotation center, the plurality of lipid bilayer membranes created are electrically independent from each other The method of forming a lipid bilayer membrane according to claim 1, wherein: The lipid bilayer membrane formation method according to claim 1, wherein the lipid bilayer membrane formation apparatus is operated in an electromagnetic shield box. An ion channel analyzer using the method for forming a lipid bilayer membrane according to claim 1.

Images