JP3769622B2 - Artificial lipid membrane formation method and lipid planar membrane formation apparatus therefor - Google Patents

Description

  The present invention relates to a method and apparatus for forming a lipid membrane, and more particularly, a lipid membrane formation method suitable for use in measuring the function and activity of a membrane protein, and a chip suitable for the method. Or it concerns a microarray.

  Conventionally, in order to examine the function and activity of physiologically active substances (hereinafter referred to as “membrane-related substances”) present on membrane proteins and other biological membranes (or inside and outside the membrane), lipid membranes are artificially used. The membrane-related substances such as membrane proteins are embedded in the artificial lipid membrane, and various measurements such as electrical characteristics and substance permeation characteristics of these membrane-related substances are performed. In addition, many attempts have been made to artificially construct lipid membranes as a basis for functional substances in order to use the functions of membrane-related substances as various sensors (biosensors).

  One of the artificial lipid membranes used for such measurements and for the construction of biosensors is the so-called “black membrane” (or “Black Membrane”), which is traditionally well known. It has been. To put it simply, the black membrane is provided with a small hole in a plastic plate, and in an aqueous solution, a lipid molecule dissolved in an organic solvent such as n-decane (lipid solution) is applied to the small hole of the plastic plate, It is formed as a planar film covering the small holes. This utilizes the property that lipid molecules spontaneously form a lipid bilayer membrane in an aqueous solution. As other artificial lipid membranes, LB membranes, liposomes and the like are known.

Various methods and apparatuses for constructing artificial lipid membranes as described above, and methods and apparatuses using those artificial membranes have been proposed and used. Such an example is described in, for example, the following Patent Document 1-5 or Non-Patent Document 1-3. In order to visualize a single membrane protein molecule under a fluorescence microscope and to measure the function and activity of the molecule, Ide et al. Formed a lipid flat membrane in a small hole opened in a plastic sheet, and formed the membrane into a microscope objective. An observation chamber was constructed so as to spread parallel to the focal plane of the lens, that is, the film surface could be observed under a microscope. And the electrical property of the film | membrane was measured, visualizing the motion of the molecule | numerator on the film | membrane (refer nonpatent literature 1, 2). Patent Document 5 proposes a lipid bilayer array that assumes application of a biosensor or the like.
JP 02-35941 A Japanese Patent Laid-Open No. 05-253467 Japanese Patent Application Laid-Open No. 07-241512 Special table 2002-505007 Special table 2003-511679 One person outside Ide, Biochemical and Biophysical Research Communications, 1999, 265, p.595-599 Three people outside Ide, Single Molecules, 2002, Volume 1, pages 33-42 Edited by Yasunobu Okada, New Patch Clamp Experiment Technology, July 5, 2001, Yoshioka Shoten Co., Ltd., p.209-244 Marc Madou, Fundamentals of Microfabrication, 1997, CRC Press LLC

  Among the various types of artificial lipid membranes described above, the black membrane is a planar membrane with both sides facing the aqueous phase (stretched only with the membrane in water), and only the electrodes are placed in the aqueous phase on both sides of the membrane. Thus, the electrical properties of the membrane or the membrane-related material embedded therein can be measured, and the response of the membrane to any stimulus can be sensed. Therefore, in the function measurement or sensor application of membrane-related substances, the black membrane is structurally more than a vesicle-like liposome or an LB membrane that is a planar membrane with one of its membrane surfaces attached to a substrate. It is advantageous. Further, as described above, since the black membrane is naturally formed by simply applying the lipid solution to the small pores in water, the forming operation is relatively simple.

  However, the formation of conventional black membranes or lipid membranes stretched in water actually requires skilled skills because of the simple formation operation, and always forms membranes in the same state. It is extremely difficult, and even if the forming operation is performed in the same manner, it often fails to form. This is because the amount and timing of giving the lipid solution to the small pores need to be finely adjusted. At present, for example, in the case of the above-mentioned method such as Ide, the operation is directly performed by a person who has successfully formed the membrane. Without mastery it is difficult to successfully form a film.

  If it is a simple forming operation, no skill is required, and anyone who has ordinary experimental operation techniques in this field can stably, ie, have little variation, a black membrane or a flat lipid membrane. If it can be formed, it is considered that the black membrane or the planar lipid membrane can be used for a wider range of experiments, inspections, sensors, and other devices. Furthermore, if stable formation of a black membrane or a lipid flat membrane is possible, a sample specimen in which a black membrane or a lipid flat membrane is formed in an array, that is, a “lipid planar membrane microarray” or “lipid planar membrane chip” is prepared. It is considered that it can be prepared, and various inspections or tests using an optical microscope, a spectroscopic device, and the like can be performed, and application to biosensors and the like will be facilitated in a wide range.

  Therefore, one problem to be solved by the present invention is to provide a novel method and apparatus therefor that can form a black membrane or a lipid flat membrane stretched in water stably or reproducibly without requiring skill. Is to provide.

  Another object of the present invention is to provide a method and apparatus as described above, and a method and apparatus for forming a lipid planar membrane that can be easily used in an optical microscope or other optical measuring apparatus. .

  Another object of the present invention is to provide a lipid membrane chip or microarray.

  Another object of the present invention is to provide a method for easily and stably forming a lipid membrane in a lipid membrane chip or a lipid membrane microarray.

  The above-mentioned problem is a method of forming a lipid planar membrane, and in the process of providing a first chamber and a second chamber separated from the first chamber by a partition wall, Providing at least one small hole in fluid communication between the chamber and the second chamber, filling the first chamber with the first aqueous solution, and filling the second chamber with the organic solution containing lipid; It includes the process of contacting the first aqueous solution with the organic solution through the small pores and the step of replacing the organic solution in the second chamber with the second aqueous solution, thereby forming a planar lipid membrane covering the small pores. This is achieved by a method characterized by:

  The conventional method for forming a black membrane is, as described briefly above, by applying a lipid solution with a brush or the like to a small hole in water, or giving it with a pipette or the like. "And then submerge the small holes in the water." In such a method, the application method of the lipid solution varies, and in order to form a membrane skillfully, a delicate “technique” is required, and thus the properties of the formed membrane vary and a stable membrane can be formed. Seems to have made it difficult.

  According to the present invention, first, the first chamber and the second chamber are respectively filled with the first aqueous solution and the lipid-containing organic solution, that is, the lipid solution, and the aqueous solution and the lipid solution are filled. Contact through a small hole. At this point, it is considered that the lipid molecules form at least a monomolecular film in the small pore that becomes the interface between the aqueous solution and the lipid solution. Thereafter, the lipid film is spontaneously formed by replacing the lipid solution with a second aqueous solution (which may or may not be the same as that filled in the first chamber). Such operations can be easily performed by those having ordinary experimental techniques in this field. Moreover, it is not necessary to adjust the “appropriate amount” of the lipid solution, which should be applied to the small holes as before, and thus the properties of the formed lipid membrane should be less varied than before. Can do. It should be understood that the lipid solution may be injected into the first chamber and the aqueous solution may be injected into the second chamber, and the terms “first” and “second” are used herein. It should be understood that the two chambers are merely used to distinguish the two chambers and are replaced with each other, and all the inventions expressed by substitution belong to the present invention (hereinafter the same).

  In the method of the present invention, the inventor further replaces the organic solution with air in the process of replacing the organic solution in the second chamber with the second aqueous solution, and then converts the second aqueous solution to the second aqueous solution. It has been found that a small hole can be formed more efficiently and stably when introduced into the chamber. According to the experiment, the second chamber is not directly replaced with the second aqueous solution from the lipid solution or the organic solution, but when the aqueous solution is introduced after being replaced with air, a more stable thin lipid membrane is obtained. Was found to be formed.

  Furthermore, in the method of the present invention described above, the first chamber may be configured as a part of the first flow path, and the second chamber may be configured as a part of the second flow path. This facilitates introduction of the solution into each chamber and replacement of the solution or air in the chamber, thereby further facilitating the forming operation. In addition, in the past, a lipid solution had to be given to a small hole by a human hand, but the formation operation using a pump or the like was automated by introducing the solution or air into the chamber using a flow path. Is also possible.

  The above-described method of the present invention is an apparatus for forming a lipid planar membrane, wherein the first channel and the second channel are at least partially sandwiched by a substantially flat partition wall. A second channel provided adjacent to at least a portion of the first channel, and at least one fluidly communicating the first channel and the second channel with a partition wall A small hole penetrates, whereby one of the first and second channels is filled with an aqueous solution, and the other of the first and second channels is filled with an organic solution containing lipid. Thus, the aqueous solution and the organic solution can be brought into contact with each other through the small pores, and thereafter, by replacing the organic solution with the aqueous solution, it can be carried out by an apparatus capable of forming a lipid planar membrane in the small pores. It should be understood that the portion corresponding to the chamber configured in the method of the present invention is a portion in the vicinity of the small holes of the partition walls in the first and second flow paths.

  According to the apparatus of the present invention described above, the solution may be flowed into the flow path by a human hand. However, for the solution delivery of the column or chamber usually used in this field, such as a syringe pump, By connecting the mechanical device to the flow path, it becomes possible to introduce or replace the solution or air while controlling the flow rate or flow rate. And if said lipid membrane formation apparatus uses mechanical instruments, such as a syringe pump, automatic and large-scale preparation (formation) of a lipid flat membrane like a black membrane will be attained.

  Further, in the method and apparatus of the present invention, according to the configuration in which a small hole for forming the lipid membrane is provided in the middle of the flow path, the aqueous solution facing the lipid membrane can be exchanged very easily and quickly after the lipid membrane is formed. To be able to do that. Then, by introducing an aqueous solution in which membrane proteins (or vesicles containing membrane proteins) and other membrane-related substances are dispersed into the channel, membrane proteins and other membrane-related substances are imparted to the lipid membrane formed in the small pores. In other words, it becomes easy to “embed a membrane protein in a lipid membrane”.

  Further, in the method of the present invention described above, the first chamber or flow path is provided between the first flat plate member and the second flat plate member laminated thereon, and the second chamber or flow path is provided in the second flat plate member. Provided between the member and the third flat plate member laminated on it, a partition is formed on at least a part of the second flat plate member, and a small hole extends from one surface of the second flat plate member to the other surface. You may make it provide so that it may penetrate. By doing in this way, the membrane | film | coat surface of a lipid membrane will be comprised in parallel with the surface of the 1st thru | or 3rd laminated | stacked planar member. If it does so, it will become possible to mount on the stage of an optical microscope, for example, and also a film surface can be observed now with an objective lens.

  Therefore, in the apparatus for forming a planar lipid membrane of the present invention, the first flow path is provided between the first flat plate member and the second flat plate member laminated thereon, and the second flow path is provided. Is provided between the second member and the third flat plate member laminated thereon, at least a part of the second flat plate member constitutes a partition, and the small hole is formed from one surface of the second flat plate member. You may comprise so that it may penetrate to the other surface. Furthermore, in order to optically capture the phenomenon on the film, at least one of the first and third flat plate members may be formed of a transparent material.

  Furthermore, a feature to be noted in the method of the present invention is that it is not necessary to provide each individual pore with a lipid solution. According to the present invention, by providing a plurality of small holes in the partition wall, it is possible to prepare a plurality of lipid membranes in each small hole at a time. And those small holes may be provided in the partition arbitrarily. Therefore, if the small holes are arranged in a lattice shape in the partition wall, for example, an array shape, a so-called “lipid membrane microarray” or “lipid membrane chip” can be formed. Thus, in the apparatus of the present invention, the partition wall may be provided with a plurality of small holes, and the plurality of small holes may be arranged in an array. In that case, one or both of the first and second flow paths may be provided with means for fluidly isolating adjacent small holes from each other. Then, for each small hole, it becomes possible to constitute an independent system in which the aqueous solution in contact with the lipid membrane formed therein does not mix with each other, and it is advantageous because experiments or measurements can be performed independently for each small hole. It is. Further, a “membrane protein chip” or a “membrane protein microarray” can be prepared by embedding a membrane protein in the lipid membrane after or together with the preparation of the lipid membrane chip.

  Further, since the lipid membrane formed in the present invention is configured in the same manner as the black membrane whose both faces the aqueous phase, at least one electrode is attached to each of the first and second flow paths. In addition, a voltage may be applied to the film with the small hole interposed therebetween. If a membrane-related substance such as a membrane protein is embedded in the lipid membrane, it becomes possible to measure the electrical characteristics or response of those membrane-related substances. In that case, a pair of electrodes is provided for each of the plurality of small holes (it should be understood that one electrode may be common) and one of the first and second flow paths. It is advantageous if both or both are provided with means for electrically insulating adjacent small holes from each other. By doing so, it becomes possible to apply electrical stimulation to the membrane independently for each small hole or to detect an electrical response, and to perform different measurements simultaneously with one chip (chips). Multi-channel).

  The lipid membrane prepared by the method and apparatus of the present invention can be formed in any size as long as it can be understood by those having ordinary knowledge in this field. When measuring a molecular level function or response of a substance such as single molecule observation, it may be formed in a size of micrometer order. In such a case, the structure of the flow path, the chamber, etc. in the apparatus of the present invention can advantageously employ a micro processing technique used for manufacturing a semiconductor circuit. In particular, the present inventor uses a silicon plate as a partition when forming a small hole, and etching the silicon plate along its crystal direction forms a structure suitable for the small hole, for example, having a taper at the edge. I found out that I can. Of course, the partition walls and small holes of the present invention may be configured in other ways, all of which should be understood to be included in the present invention.

  Furthermore, it has been found that the lipid membrane is better formed when the edge surface of the stoma is aggressively hydrophobic. At that time, when the edge of the small hole and the vicinity thereof or the whole partition wall is subjected to hydrophobic treatment, a hydrophobic coating such as parylene may be advantageously used.

  Recently, in order to examine the functions and activities of physiologically active substances such as proteins and DNA or cells, the "microarray" or "chip", that is, the physiologically active substances are fixed on the substrate at intervals of micrometer order. Attempts have been made to develop such specimens. The sample specimen prepared as a chip is also expected to be used as a rapid and accurate diagnostic means for a specific disease or disease. However, at present, as for membrane-related substances such as membrane proteins that do not exhibit activity unless embedded in a membrane, a sample prepared as a chip has hardly been proposed and has not been put into practical use. One of the reasons is that, as described above, it is difficult to stably form a black film or a flat film, and therefore it is very difficult to arrange the black film or the flat film in an array or to form a chip. It was difficult.

  According to the present invention, it is necessary to perform an adjustment directly by an experimenter's hand, such as adjusting an appropriate amount of a lipid solution applied to a small hole as in the conventional method for preparing a black membrane, and other skilled skills. It is possible to form a black film or a flat film stably or with good reproducibility. If a plurality of small holes are provided in the partition wall, a plurality of lipid membranes can be formed by a single forming operation. Therefore, a lipid membrane chip in which lipid membranes are arranged in an array can be prepared. It becomes.

  In the column of the embodiment described below, the apparatus of the present invention mainly describes the form of a preparation for observation with an optical microscope, but the present invention can also be applied to other types of sample specimens. That should be understood. In the following embodiment, the film surface, that is, the small hole is placed horizontally and parallel to the focal plane of the objective lens of the optical microscope so that the film surface can be observed through the objective lens. The apparatus of the invention may be configured so that the film surface is vertical, for example as a spectroscopic sample, and such an apparatus should be understood to be within the scope of the present invention.

  What should be noted in the method and apparatus of the present invention is that it is easy to replace the solution on both sides of the small hole by providing a small hole in which the lipid membrane is stretched in the middle of the flow path or a chamber constituting a part of the flow path. Thus, formation of a lipid membrane is further facilitated. According to such a feature, it is possible to automate the formation of the lipid membrane by connecting a device such as a syringe pump to the flow path and configuring the system to perform solution exchange with these devices. Mass production will also be possible. Even after the formation of the lipid membrane, the exchange of the solution on both sides of the lipid membrane is very simple. Therefore, the solution of the vesicle or liposome containing the membrane-related substance is circulated in the flow channel, so that the lipid membrane It is also possible to embed a membrane-related substance, and thus, for example, a “membrane protein chip” in which membrane proteins are arranged in an array can be prepared.

  Furthermore, in the apparatus of the present invention, as exemplified in the column of the embodiment, a lipid containing a lipid membrane or membrane-related substances arranged in an array by configuring a microchannel using a microfabrication technique or the like. The membrane can be fluidly isolated or electrically isolated. As a result, the arrayed lipid membranes can be configured as independent systems, and different experiments or measurements can be performed with one chip, that is, multi-channeling of the chip is achieved.

  Membrane-related substances such as membrane proteins have been found to be involved in many important phenomena in substance transport and information transmission inside and outside cells, or in various specific diseases or diseases in actual cells. Yes. By arranging such membrane-related substances in an array and forming a chip, it becomes possible to perform an unprecedented examination or test of the membrane-related substances, or diagnosis of diseases or diseases that could not be detected before. In addition, a membrane-related substance such as a membrane protein may have a useful function not found in existing semiconductor elements. The present invention can be used as a high-throughput detection tool for measuring and analyzing the functional activity of membrane-related substances such as membrane proteins, or as a novel apparatus utilizing the functions of membrane-related substances such as membrane proteins. It can be used as a sensitive membrane protein analyzer, ultra-sensitive multi-channel drug screening device, ultra-sensitive ion sensor, etc.

  Other objects and advantages of the present invention will become apparent from the following description of preferred embodiments of the present invention.

  Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. In the figure, the same reference numerals indicate the same parts.

The planar lipid membrane forming apparatus Figures 1 and 2A-E, according to the method of forming a lipid planar membrane of the present invention, there is provided a planar lipid bilayer forming apparatus 10 which is capable of forming a lipid membrane, in particular under an optical microscope A schematic diagram of an apparatus having the form of a slide configured to be observed is illustrated. The lipid flat film forming apparatus 10 includes a flat upper plate 12, a lower plate 14, and an intermediate plate 16 sandwiched between them. The upper plate 12 and the lower plate 14 may be formed from any glass, plastic, metal, or the like. When the lipid membrane is observed with another optical instrument such as an optical microscope to measure a phenomenon or a function seen on the membrane, the upper plate 12 and the lower plate 14 are either as a whole or below. It is preferable that only the vicinity of the small hole to be described is formed of a transparent material.

  The intermediate plate 16 is formed with a first flow path 18, a second flow path 20, and a partition wall 22, and at least one small hole 24 is drilled in the partition wall 22. As will be described below, a flat lipid membrane is formed in the small holes 24. The intermediate plate 16 may be formed of the same material as the upper plate 12 and the lower plate 14, but any material used in the field of micromachining technology capable of forming a structure such as a flow path or a partition wall. It is preferable that The first flow path 18 is formed in a groove shape on the lower side of the middle plate 16 and the second flow path 20 is formed on the upper side of the middle plate 16, and the middle plate 16 is interposed between the lower plate 14 and the upper plate 12. By being pinched, a passage through which a fluid is circulated is formed. In order to facilitate the introduction of the solution into the flow path, pipe joints 26 and 28 are provided at both ends of each flow path so that a tube or the like can be connected arbitrarily, and is connected to a liquid feed pump (not shown). It may be possible. The cross-sectional shape, width, and depth of the channel may be arbitrarily determined. However, when the apparatus 10 is configured as a preparation for optical microscopic observation, the width and depth are in millimeters or submillimeters. It can be an order. As shown, a branch channel 21 and a pipe joint 29 may additionally be provided in the channel. In this case, it is advantageous when various fluids including air are circulated sequentially.

  The first flow path 18 and the second flow path 20 each include a first chamber 30 and a second chamber 32 that are provided side by side and partitioned by a partition wall 22. The partition wall 22 is perforated with small holes 24 as described above. Thus, the first flow path 18 and the second flow path 20, or the first chamber 26 and the second chamber 28, have small holes. Through fluid communication. In the example of FIGS. 1 and 2A-C, the partition wall 22 has three small holes. However, the small hole may be one, and is arbitrarily arranged, for example, in a lattice pattern. (See FIG. 2E). The shape and size of the small holes may be arbitrarily selected. For example, under the experimental conditions in Examples as will be described in detail below, the diameter of the small holes that form the lipid membrane better and stably was in the range of several tens to several hundreds of μm. As shown in the drawing, the shape of the small hole is preferably provided with a taper 36a inward at the edge of the small hole. Further, the surface of the edge of the small hole is preferably hydrophobic, and may be optionally coated with a hydrophobic substance in order to make the surface hydrophobic.

  In the example of FIGS. 1 and 2A-D, the size of the first and second chambers 30, 32 is the same as the width of the first and second flow paths 18, 20, respectively. Only the portion may be of any size. However, as will be described later, in order to efficiently exchange the solution in the room, it is preferable that the width is equal to or smaller than the width of the flow path.

  Furthermore, electrodes 34, 36 may be provided in the vicinity or chamber of the small hole 24 of the device 10 to detect the electrical properties or electrical response of the lipid membrane. A pair of electrodes may be provided for each small hole, or a pair of electrodes may be provided for the entire chamber. It should be understood that the number or shape and dimensions of the electrodes may be arbitrarily selected by those skilled in the art according to the purpose of experimentation or measurement, all of which fall within the scope of the present invention.

  Further, when a plurality of small holes are provided in the partition wall 22, the lipid membrane formed in the adjacent small holes is fluidically isolated or electrically insulated to constitute an independent system. Therefore, means for fluidly or electrically shutting off the first and second flow paths may be provided for each small hole. The configuration of such means will be described below.

  In the illustrated example, the chamber in which the lipid membrane is formed and the flow path are integrally formed. However, although not shown in the figure, the apparatus 10 includes only the first and second chambers, the chamber is provided with a fluid inlet / outlet, and a flow path constituted by a tube or the like is connected to the chamber inlet / outlet. Such devices may also fall within the scope of the present invention.

Method for Producing Lipid Planar Film Forming Device In particular, the intermediate plate 16 of the above-described device 10 of the present invention can be advantageously formed by micromachining technology (see Non-Patent Document 4) as follows. In particular, a suitable shape of the small holes 24 can be formed by etching a silicon plate in the crystal direction. According to such a method, since the shape of the small hole is determined by the silicon crystal, it is easy to form the small hole having the same shape, which is extremely advantageous. Hereinafter, the process of forming the intermediate plate 16 will be described with reference to FIG.

(A) After both surfaces of the silicon wafer 38 are oxidized by heat to form the SiO ₂ film 40, a small hole 42 (about 50 μm wide, about 200 μm deep) is formed in the surface of the lower silicon plate in the figure by ICP-RIE etching. ) Is formed (FIG. 3A).

(B) S1830 photoresist was spin-coated at 3000 rpm 90 seconds using a spin coater (MIKASA SPINNER IH-D2), that is, rotated and uniformly coated thinly, and then irradiated with ultraviolet rays to form small holes. The pattern of the flow path and the chamber on the surface is exposed, and the resist exposed to ultraviolet rays is removed with a resist etchant. Thus, the resist on the surface corresponding to the flow path and the chamber is removed.

(C) When SiO ₂ is etched with BHF (buffered hydrofluoric acid) (FIG. 3B) and further reacted in a TMAH (tetramethylammonium hydroxide) solution, only Si is etched along the crystal direction, resulting in small holes. A depression 44 having a pyramid shape is formed in the portion where the is formed (FIG. 3C).

(D) Next, in the upper surface in the drawing, the flow path and the chamber are formed by ICP-RIE etching, and the small hole portion that is a pyramidal depression penetrates and has a small hole 24 having a taper at the edge. Is formed (FIG. 3D).

(E) Thereafter, the surface from which silicon is not exposed is oxidized, and further, the oxidized surface is covered with a hydrophobic substance (FIG. 3E). The hydrophobic coating 46 is preferably, for example, a thin film of Parylene (Specialty coating systems), but may be a coating with a material that can be arbitrarily selected by those skilled in the art.

(F) After that, the upper plate 12 and the lower plate 14 are fixed so as to sandwich the intermediate plate 16. For fixing, glass adhesive SU-8 or the like 48 may be used. At that time, the electrodes may be fixed at the same time (FIG. 3F).

  In the above production, a structure such as a flow path is formed only on the middle plate 16, but a similar molding process may be optionally applied to the upper plate and the lower plate, and an apparatus having such a configuration is also included in the present invention. Should be understood as belonging to the scope of

  Electron microscopic images of small holes formed by etching in the crystal direction of silicon are illustrated in FIGS. 4A and 4B (FIG. 4C is a schematic diagram of FIG. 4B). .)

Method for Forming Lipid Planar Membrane The method for forming a lipid planar membrane according to the present invention can be successfully performed by the apparatus shown in FIGS. 1 and 2 according to the following procedure. However, it should be understood that the method of the present invention can be carried out by other devices without departing from the concept and scope of the present invention, and a lipid membrane forming method using such other devices is also possible. It should be understood that it is within the scope of the present invention.

  Formation of the planar lipid membrane according to the present invention is performed as follows.

(A) An arbitrary aqueous solution 50 is injected into the first flow path 18 from the pipe joint 26 and filled in the first chamber 30. Preferably, the aqueous solution is introduced until at least the leading edge of the small hole. On the other hand, an organic solution in which lipid is dissolved, that is, a lipid solution 52, is injected into the second channel 20 from the pipe joint 28 and filled in the second chamber 32 (FIG. 5A). The lipid solution is also preferably introduced at least until it is at the leading edge of the small pore, thus bringing the aqueous solution and lipid solution into contact with the small pore. For the injection of the solution, a liquid pump such as a syringe pump may be arbitrarily used.

  The aqueous solution may be an aqueous solution used for experiments or measurements performed after forming the lipid film, or may be another. The lipid solution may be any phospholipid, glycolipid, or any other lipid that can form a lipid bilayer at room temperature, such as n-decane and chloroform, used by those skilled in the art to form a black membrane. It may be dissolved in an organic solvent. The lipid may be a lipid extracted from a natural product such as azolectin, or may be a synthetic lipid such as diphytanoylphosphatidylcholine. The lipid concentration may be, for example, several tens of mg / ml, and preferably about 20 mg / ml.

(B) Next, the lipid solution in the second channel 20 and the second chamber 32 is discharged by injecting air 54 into the second channel. At this point, as shown in FIG. 5B, a part of the lipid solution remains at the interface of the small pore aqueous solution.

(C) Next, the aqueous solution 56 is injected into the second channel 20 to push out the air 54 (FIG. 5C), and the second channel 20 and the second chamber 32 are replaced with the aqueous solution from the lipid solution through the air. (FIG. 5D). As a result, a planar lipid membrane 58 is formed in the small hole 24 as schematically illustrated. The flow rate when the aqueous solution is introduced may be arbitrarily set by the user, and may be several tens of μm / s, for example. The flow rate is determined by the cross-sectional area of the flow path and the flow velocity.

  In the process of (b) above, the lipid solution may be manipulated to be pushed out by an aqueous solution, but according to the experiments of the present inventors, once replaced with air, a lipid membrane is more stably formed. It was found that

  Various conditions for forming a lipid membrane in the method for forming a lipid membrane, such as the flow rate and flow rate of an aqueous solution, vary depending on the composition of the lipid solution, the ionic strength of the aqueous solution, the osmotic pressure, etc. In order to prepare a lipid membrane, a trial formation is first performed to examine suitable conditions. However, according to the method and apparatus of the present invention, the operation is performed only by adjusting the flow rate or the like of the aqueous solution. For example, unlike the conventional lipid membrane method, there is no operator's technical operation. It is to be understood that once such conditions are determined, lipid membranes can be stably prepared under the same conditions. In addition, by providing a plurality of small holes on the partition wall, a number of lipid membranes are formed by performing the steps (a) to (c) once, and a lipid membrane microarray is formed.

  Furthermore, after the lipid membrane was formed, a liposome containing membrane protein or a solution containing vesicles obtained by vesicle formation of a biological membrane obtained from a cell was injected into the first or second channel to form pores. It is possible to bring the membrane protein or membrane-related substance into the lipid membrane by contacting with the lipid membrane.

When a plurality of small holes are provided in the partition wall that blocks the first and second flow paths for each small hole, each of the formed lipid membranes forms a fluidly or electrically independent system. Means for blocking the flow path is shown in FIG.

  FIGS. 6A and 6B illustrate one embodiment of a configuration and method for fluidly or electrically blocking small holes in either or both of the first and second flow paths. It is shown. As understood from the figure, the channel is provided with a sub-channel 60 for injecting a substance that blocks the channel into each of the walls between the adjacent small holes. The substance that blocks the flow path may typically be air 62, but may be any organic solvent that does not mix with other aqueous solutions.

  As shown in FIG. 6 (A), the flow path for each small hole is blocked after the lipid film is formed in a series of small holes by the lipid film formation method described in relation to FIG. As shown in FIG. 6B, this is achieved by injecting a blocking substance such as air into the flow path.

  FIG. 6C illustrates another embodiment for blocking the flow path for each small hole, and in this embodiment, between the adjacent small holes, apart from the electrode 34 or 36. The bubble generating electrode 64 is provided in the channel or on the side of the channel. In this embodiment, after the lipid membrane is formed in each small hole by the above-described lipid membrane formation method, an electric current is passed through the bubble generating electrode to generate bubbles 70 in the aqueous solution in the flow path. By the bubbles 70, as shown in the figure, the flow path is blocked fluidly or electrically for each small hole. The bubble generation mechanism may be either (1) foaming by Joule heat or (2) hydrogen bubble generation by water electrolysis.

  It should be understood that the blocking means is preferably provided in each of the first and second flow paths, but the lipid membrane is substantially non-conductive to metal ions and water-soluble substances. Since it shows permeability, it may be sufficient to block only one of the first and second flow paths depending on the experiment or measurement, and such a configuration is also included in the present invention.

  Thus, the lipid membranes formed in the small pores are fluidly or electrically interrupted from each other, and each constitutes an independent system, thereby allowing measurement of chemical reactions or electrical measurement of individual lipid membranes. It becomes. In addition, when the membrane protein or the like is introduced into the lipid membrane, the above-described channel blocking operation is performed after the introduction of the membrane protein or the like.

  In order to verify the effectiveness of the present invention described above, the following experiment was conducted. It should be understood that the following examples illustrate the effectiveness of the present invention and do not limit the scope of the present invention.

Formation of a planar lipid membrane Using the apparatus 10 described above, a planar lipid membrane was formed in the small holes 24 by the procedure of the method for forming a planar lipid membrane of the present invention. Azolectin (Sigma Chemicals), a phospholipid derived from soybean, was dissolved in n-decane as an organic solvent, and the lipid concentration was 20 mg / ml. As the aqueous solution, 0.1 M KCl and 0.1 M glucose were used. The syringe solution KDS-200 (KD Scientific USA) was used to inject the second aqueous solution after the lipid solution filled in the second flow channel was purged with air.

  The width of the flow path and chamber of the apparatus 10 was 500 μm, and the depth was about 100 μm. The partition wall thickness was about 100 μm, and the aperture of the small holes was about 130 μm. The formation process of the film was observed with an optical microscope (IX-71 Olympus optics) using an objective lens of 40 times.

  FIG. 7A is a microscopic image of the state of small pores after the lipid solution has been expelled from the second chamber (see FIG. 5B). FIG. 7B is a microscopic image of the process of introducing the aqueous solution into the second chamber as shown in FIG. FIG. 7 (c) is a microscopic image of the formed lipid planar membrane, and a lipid bilayer membrane similar to the literature (see Non-Patent Documents 1 and 2) such as Ide et al. It was confirmed that was formed. (The translucent irregularly shaped objects observed around the small pores are lipid masses.)

  Although the above description has been made in connection with the embodiments of the present invention, it should be understood that many modifications and changes can be easily made by those skilled in the art. It will be understood that the invention is not limited to the embodiments illustrated in FIG. 1 and applies to various devices without departing from the inventive concept.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. The dotted line in the figure indicates the channel structure on the back surface. (A) Plan view, (B) Bottom view, (C) Cross-sectional view taken along line dd in (B), (D) ) A sectional view of the vicinity of the small hole is shown. (E) is a schematic diagram of an array of small holes in another embodiment. The dotted line in the figure indicates the channel structure on the back surface. The schematic diagram of the manufacture process of the lipid flat film formation apparatus by this invention. (A) A state where a small hole is made in a silicon wafer whose surface is oxidized. (B) Etching process of the surface with the small holes. (C) A state in which a pyramidal depression is formed in the silicon wafer by TMAH etching. (D) A state in which the top surface is etched and the small holes are penetrated. (E) A state in which the surface of the silicon wafer is oxidized and subjected to parylene coating. (F) A state in which the middle plate is sandwiched and fixed by the upper plate and the lower plate. (A) An electron microscope image of a partition wall in which small holes are formed. (B) Electron microscopic image of small holes. (C) The schematic diagram of the image of (B). The schematic diagram of the formation process of the lipid plane membrane by this invention. (A) A state in which the first channel is filled with an aqueous solution and the second channel is filled with a lipid solution. (B) A state in which the lipid solution is expelled from the second channel. (C) A process of introducing an aqueous solution into the second flow path. (D) State in which the second flow path is filled with an aqueous solution and the lipid flat membrane is formed in small pores. The schematic diagram of the blocking process of the flow path for every small hole after lipid membrane formation by this invention. (A) One embodiment of a means for blocking a flow path, in which a lipid membrane is formed in a series of small holes. (B) A state in which air is injected from the auxiliary flow channel into the flow channel and the flow channel is blocked for each small hole. (C) Another embodiment of the means for blocking the flow path, wherein after forming a lipid membrane in a series of small holes, bubbles are generated in the flow path by the bubble generating electrode, The flow path is blocked. An optical microscope image of small pores in the formation process of a planar lipid membrane. The black area at the edge of the image is the edge of the stoma, and the stoma opens in a generally square shape. (A) A state corresponding to FIG. (B) A state corresponding to FIG. (C) A state corresponding to FIG. A lipid planar membrane is formed.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Lipid plane membrane formation apparatus 12 ... Upper board 14 ... Lower board 16 ... Middle board 18 ... 1st flow path 20 ... 2nd flow path 22 ... Septum 24 ... Small hole or array of small holes 26, 28, 29 ... Pipe joint 30 ... First chamber 32 ... Second chamber 34, 36 ... Electrode 38 ... Silicon wafer 40 ... Silicon oxide film 42 ... Small hole 46 ... Hydrophobic coating 50, 56 ... Aqueous solution 52 ... Lipid solution 54 ... Air 58 ... Plate lipid membrane 60 ... Sub-channel 62 ... Blocking substance (air)
64 ... Bubble generating electrode 70 ... Bubble

Claims (17)

A method for forming a planar lipid membrane comprising:
In the process of providing a first chamber and a second chamber separated from the first chamber by a partition, the first chamber and the second chamber are in fluid communication with the partition. Providing at least one small hole,
Filling the first chamber with a first aqueous solution, filling the second chamber with an organic solution containing lipids, and contacting the first aqueous solution and the organic solution through the small holes;
Replacing the organic solution in the second chamber with a second aqueous solution, thereby forming a planar lipid membrane covering the small pores.   The method according to claim 1, further comprising the step of replacing the organic solution in the second chamber with a second aqueous solution, and then replacing the organic solution with air and then replacing the second aqueous solution with the second aqueous solution. A method characterized by being introduced indoors.   2. The method of claim 1, wherein the first chamber is part of a first flow path, the second chamber is part of a second flow path, and the organic in the second chamber. In the process of replacing the solution with the second aqueous solution, introducing the second aqueous solution into the second channel after extruding the organic solvent by introducing air into the second channel. Feature method.   2. The method of claim 1, wherein the edge of the small hole is tapered.   2. The method of claim 1, wherein the edge surface of the stoma is made hydrophobic.   2. The method according to claim 1, wherein the partition is formed of a silicon plate, and the small holes are formed by etching the silicon plate.   It is a method of Claim 1 or 3, Comprising: Said 1st chamber is provided between the 1st flat plate member and the 2nd flat plate member laminated | stacked on it, and said 2nd chamber is said 2nd member. The partition is provided between at least a part of the second flat plate member, and the small hole is formed from one surface of the second flat plate member to the other surface. A method characterized in that it is provided so as to penetrate through.   An apparatus for forming a lipid planar membrane, wherein a first channel and a second channel are at least partially adjacent to at least a part of the first channel with a partition wall in between. At least one small hole that fluidly communicates the first channel and the second channel with the partition wall, Filling one of the first and second flow paths with an aqueous solution and filling the other of the first and second flow paths with an organic solution containing a lipid, through the small holes, An apparatus in which an aqueous solution and the organic solution can be brought into contact with each other, and thereafter, the organic solution is replaced with an aqueous solution, whereby a lipid flat membrane is formed in the small holes.   9. The apparatus according to claim 8, wherein the partition wall is formed of a silicon plate, and the small holes are formed by etching the silicon plate along a crystal direction thereof.   9. The apparatus according to claim 8, wherein at least a part of the small holes and the partition walls are hydrophobically coated.   9. The apparatus according to claim 8, wherein a plurality of the small holes are provided in the partition wall.   The apparatus according to claim 11, wherein the plurality of small holes are arranged in an array.   12. The apparatus of claim 11, wherein at least one of the first and second flow paths has means for fluidly isolating adjacent small holes from each other.   9. The apparatus according to claim 8, wherein at least one electrode is attached to each of the first and second flow paths, and a voltage can be applied across the small hole. apparatus.   15. The apparatus according to claim 14, wherein a plurality of small holes are provided in the partition wall, and at least one of the first and second flow paths electrically isolates the adjacent small holes from each other. The apparatus characterized by having.   9. The apparatus according to claim 8, wherein the first flow path is provided between a first flat plate member and a second flat plate member laminated thereon, and the second flow path is the second member. And the third flat plate member laminated thereon, at least a part of the second flat plate member constitutes the partition, and the small hole extends from one surface of the second flat plate member to the other. A device characterized in that it is provided so as to penetrate through the surface. 17. The apparatus according to claim 16, wherein at least one of the first and third flat plate members is formed of a transparent material, and is configured so that a small hole can be observed with an optical microscope.