JP4953044B2 - Method and apparatus for forming lipid bilayer membrane - Google Patents

Description

  The present invention relates to a method and apparatus for forming a lipid bilayer membrane for membrane protein analysis used in fields such as biotechnology, biochips, membrane protein analysis, drug discovery screening, and biosensors.

Membrane proteins are present in cell membranes and play an important role in immune reactions and transport and excretion of substances inside and outside the cell. Therefore, elucidating the functions and characteristics of various membrane proteins one by one is the next generation. It has become an important issue for the development of new treatments and drug discovery methods.
Typical conventional methods for preparing planar lipid membranes for membrane protein analysis such as ion channels include planar membrane methods, that is, brush coating method and LB method (Longmuir-Blodgett method). In both methods, a lipid bilayer is formed in a small hole of several hundred microns opened in a Teflon (registered trademark) sheet or the like in a chamber filled with a buffer. The latter is a method of forming a planar lipid membrane by gradually raising the solution surface of the chambers on both sides of the Teflon sheet using the fact that a monolayer of lipid is formed on the liquid surface. is there.

FIG. 15 is a schematic diagram showing the planar lipid film formation method by the LB method.
In this figure, 101 is a Teflon sheet, 102 is a small hole opened in the Teflon sheet 101, 103 is a solution in which a monolayer of lipid 104 is formed on the surface, 105 is a buffer solution, The lipid membrane 106 is formed by gradually raising the surfaces of the solution 103 and the buffer solution 105 in the chamber.

Further, as Patent Document 6, the lower solution tank is surrounded by a bottom plate and a spacing member, and is formed below the upper solution tank. By reducing the pressure inside the lower solution tank, an artificial hole formed by a small hole is formed. A current measuring device having an artificial lipid bilayer membrane in which the lipid bilayer membrane is inflated and thinned to the lower solution tank side and is supported by the support layer in a thinned state has been proposed. However, the formation of the artificial lipid bilayer in that case was accompanied by difficulties as described later.
JP 02-35941 A Japanese Patent Laid-Open No. 05-253467 Japanese Patent Application Laid-Open No. 07-241512 Special Table 2002-505007 Japanese translation of PCT publication No. 2003-511679 Japanese Patent Laying-Open No. 2005-091308 H. Zhu et al. , “Global Analysis of Protein Activities Using Proteome Chips”, Science, Vol. 293, pp. 2101-2105, 2001. B. Alberts et al. "Molecular Biology of the Cell; 4th Ed.," Garland Science, 2002. C. Miller, ed. "Ion Channel Reconfiguration," Plenum Press, 1986. T.A. Ide and T.M. Yanagida, “An Artificial Lipid Formered on an Agarose-Coated Glass for Simulaneous Electrical and Optical Measurement of Single Ion.” Biophys. Res. Comm. , 265, pp. 595-599, 1999. T.A. Ide, Y .; Takeuchi and T.K. Yanagida, "Development of an Experimental Apparatus for Simulaneous Observation of Optical and Electrical Signals from Single Channel, 3 Channels." 33-42, 2002. J. et al. T.A. Groves, N.M. Ulman, and S.M. G. Boxer, “Micropatterning Fluid Lipid Layers on Solid Supports,” Science, Vol. 275, pp. 651-653. M.M. Mayer et al. , “Microfabricated Teflon Membranes for Low-Noise Recordings of Ion Channels in Planar Lipid Bayers,” Biophys. J. et al. , Vol. 85, pp. 2684-2695, 2003. Fertig et al. , “Microstructured Glass Chip for Ion-Channel Electrochemistry,” Phys. Rev. E, Vol. 64, 040901 (R), 2001. Hiroaki Suzuki, Hiroyuki Noji, Shoji Takeuchi, "Reconstitution of planar lipid membrane using microchannels", Proceedings of the 8th Chemistry and Micro-Nano System Research Meeting, 61, 2003

  However, both the brushing method and the LB method described above require a large chamber of about several centimeters, a large dead volume, and microscope observation is impossible. In addition, when a plurality of small holes are provided in the flow path by the above method and a plurality of planar films are formed at the same time, the adjacent small holes (planar films) are electrically connected to each other by the buffer solution in the flow path. Therefore, it is difficult to measure individual electrophysiology.

In addition, the number of lipid bilayer membranes that can be formed at one time is basically one, and the formation requires craftsmanship and poor reproducibility. Therefore, it has been impossible to make multi-channel analysis.
Therefore, the inventors of the present application have already formed the first and second microchannels, flowed the lipid solution into the second microchannel, and controlled the lipid solution to thereby obtain a planar lipid bilayer membrane. Have proposed a method and apparatus for forming an artificial lipid membrane for forming a membrane.

  According to this, first, the first microchannel is filled with a buffer solution (aqueous solution), then the second microchannel having small holes is filled with a lipid solution, and then the second microchannel is filled. The lipid solution is drained by injecting air into the tract. At this time, a part of the lipid solution remains at the interface of the small pore buffer solution. Next, the buffer solution is injected into the second microchannel to push out the air and replace the air with the buffer solution. Then, a planar lipid bilayer membrane is formed in the small pore.

The present invention further improves this method, makes it possible to control the film thickness and pressure, and dramatically improves reproducibility.
On the other hand, the function analysis method of membrane protein so far is generally a method of measuring membrane current. In particular, the patch clamp method is a method in which the tip of a glass tube is directly brought into contact with a cell membrane and sucked, and the amount of ions passing through an ion channel existing in the sucked inner membrane is measured as a membrane current. In principle, the life span, switching probability, and conductivity of membrane proteins can be clarified. However, in reality, various types of membrane proteins are mixed in the aspirated cell membrane, and one of them has been focused on. To argue, various ideas are required. In addition, membrane proteins involved in mass transport such as transporters generally cannot use this method because no measurable membrane current is generated.

  On the other hand, the above-described “planar film method” has attracted attention in recent years. This is a method of opening a small hole in a Teflon sheet and reconstituting the lipid bilayer in the same way as the cell membrane. Can be analyzed. For example, if electrodes are arranged above and below the membrane, the membrane current can be measured. Such a planar membrane method is an efficient method for analyzing membrane protein functions, but the products currently being sold leave the reconstitution process of the lipid bilayer membrane and the reproducibility of the reconstituted membrane. The stability is generally low. In addition, it is a difficult task to reconfigure a plurality of lipid membranes at the same time.

  In view of the above circumstances, the present invention provides a method and apparatus for forming a lipid bilayer membrane that can reconstruct a lipid membrane stably and reproducibly on a small chip using micro / nano processing technology. For the purpose.

In order to achieve the above object, the present invention provides
[1] In the method of forming a lipid bilayer membrane, a chamber is provided on the front surface of the substrate, a microchannel is provided on the back of the substrate, and a micropore penetrating the substrate between the chamber and the microchannel is provided at the bottom. A funnel-shaped hole having a diameter / height ratio of 100/21 to 82 is formed, a buffer medium is introduced into the chamber, and a buffer medium-organic containing lipid is contained in the microchannel. A lipid bilayer membrane is formed by thinning the lipid layer formed in the micropores by sequentially supplying a solvent-buffer medium and adjusting the pressure in the chamber by applying pressure to the buffer medium of the chamber. It is characterized by forming.

[2] The method for forming a lipid bilayer membrane according to [1] above, wherein a plurality of the micropores are formed.
[3] In the method for forming the above-mentioned [1] or [2], wherein the lipid bilayer membrane, characterized in that the pressure and the 2 00Pa-400Pa in the micropores 1 a diameter of 00Myuemu, before Symbol chamber.

[4] The method for forming a lipid bilayer membrane according to [3] above, wherein the success rate of the lipid bilayer membrane formation is 90% or more.
[5] The method for forming a lipid bilayer membrane according to [1], [3] or [4] above, wherein the micropores are independently arrayed to form a heterologous membrane protein.
[6] In the method for forming a lipid bilayer membrane according to any one of [1] to [5] above, a microelectrode is disposed on each of the chamber side and the microchannel side, and further connected to the microelectrode. A patch clamp amplifier is arranged, and the membrane current of the lipid bilayer membrane is measured.

[7] In the apparatus for forming a lipid bilayer membrane, a substrate, a chamber formed on the front surface of the substrate, a microchannel formed on the back of the substrate, and between the chamber and the microchannel A microhole penetrating the substrate is formed at the bottom , a funnel-shaped hole having a diameter / height ratio of the microhole of 100/21 to 82, and the chamber formed with the funnel-shaped hole as a unit. And a pressure adjusting means for adjusting the pressure.

[8] The apparatus for forming a lipid bilayer membrane according to [7], wherein a plurality of the micropores are arranged.
[9] The apparatus for forming a lipid bilayer membrane according to [7], comprising: a microelectrode disposed on each of the chamber side and the microchannel side; and a patch clamp amplifier connected to the microelectrode; It is possible to measure the membrane current of the double membrane.

  According to the present invention, a measurement platform capable of stably reconfiguring a lipid membrane on a small chip with high reproducibility is realized using micro / nano processing technology. By micro-processing, the size of the micropores is adjusted to stabilize the planar film. Also, in combination with a microchannel, the amount of lipid and liquid introduction pressure are controlled to improve reproducibility. Furthermore, a system capable of selectively arraying micropores and selectively measuring membrane currents and mass transport imaging of different types of membrane proteins is provided.

In the method for forming a lipid bilayer membrane of the present invention, a chamber is provided on the front surface of the substrate, a microchannel is provided on the back of the substrate, and a micropore passing through the substrate between the chamber and the microchannel is provided at the bottom. A funnel-shaped hole having a diameter / height ratio of 100/21 to 82 is formed, a buffer medium is introduced into the chamber, and a buffer medium-organic containing lipid is contained in the microchannel. A lipid bilayer membrane is formed by thinning the lipid layer formed in the micropores by sequentially supplying a solvent-buffer medium and adjusting the pressure in the chamber by applying pressure to the buffer medium of the chamber. Form.

Hereinafter, embodiments of the present invention will be described in detail.
FIG. 1 is a schematic view of an apparatus for forming a lipid bilayer membrane showing the principle of the present invention, and FIG. 2 is a schematic view showing an organic solvent containing lipid according to the present invention.
In this figure, 1 is a glass substrate (bottom plate), 2 is a microchannel, 3 is a substrate (chip), 4A is a funnel-shaped hole, 4B is a microhole formed at the bottom of the funnel-shaped hole, 5 Is a chamber, 6 is a lipid bilayer membrane, 7 is a buffer solution dropped from a microinjection device (not shown), 8 is a buffer solution in the microchannel 2, 9 is a patch clamp amplifier, 10 and 11 are microelectrodes, 12 is an objective lens, and 13 is an organic solvent containing lipid. In addition, it is desirable that the part of the micropore 4B is hydrophobic.

  As described above, the chamber 5 and the microchannel 2 are provided on the front and back of the substrate (chip) 3, and the lipid bilayer membrane 6 is reconfigured in the micropores 4 </ b> B penetrating the substrate (chip) 3. By alternately flowing an organic solvent 13 containing lipid and an aqueous solution as the buffer solution 8 through the microchannel 2, a thin film made of lipid is formed horizontally in the micropore 4B, and the formation of the thin film proceeds to form a lipid duplex. A film 6 is formed. The lipid bilayer membrane 6 can be directly observed with a microscope through the objective lens 12, and the area can be obtained, or the density of the protein introduced into the lipid bilayer membrane 6 can be obtained by fluorescence imaging. Further, the membrane current can be measured by the patch clamp amplifier 9. These pieces of information are information necessary for quantitatively determining the channel current value per molecule of membrane protein and the transport amount of the molecule.

FIG. 3 is a diagram showing a process for forming a lipid bilayer membrane according to the present invention.
First, as shown in FIG. 3A, a passage 5B for controlling the atmospheric pressure by a syringe pump (not shown) is formed in the wall 5A of the chamber 5.
Therefore, the buffer solution 7 is dropped from the microinjection device 16 into the chamber 5 while the air 15 is passed through the microchannel 2. Then, the interface of the buffer liquid 7 remains slightly exposed from the lower end of the micropore 4B due to surface tension.

Next, as shown in FIG. 3B, an organic solvent 13 containing lipid is allowed to flow through the microchannel 2.
Next, as shown in FIG. 3 (c), the organic solvent 13 containing lipid in the microchannel 2 is removed and air 15 is passed. Then, the lipid layer 18 remains attached to the surface of the buffer solution 7 at the lower end of the micropore 4B.
Next, as shown in FIG. 3 (d), the buffer solution 8 is passed through the microchannel 2.

  Next, as shown in FIG. 3E, the chamber 5 is covered with a lid 17 and sealed, and a fluid (here, air) is filled from a passage 5B formed in the wall 5A of the chamber 5 to fill the inside of the chamber. Adjust pressure. Then, the buffer layer 8 is pushed down as the atmospheric pressure increases, and the lipid layer 18 becomes thin (see the upper part of FIG. 7). Thereby, as shown in FIG.3 (f), the lipid bilayer membrane 6 is formed.

As conditions for improving the reproducibility and stability of the lipid bilayer membrane 6 formed in this way, the design of the micropores, the pressure for contacting the two buffer solutions (aqueous solution), the speed, the amount of lipid, etc., form the membrane. It has been found from previous studies that this is an important element.
As a measure for increasing the stability, first, by reducing the diameter of the micropores, a lipid bilayer membrane that is more stable and difficult to break can be formed. Conventionally, if the diameter of the pore is small, it is difficult for the lipid molecular layer to be thinned and it is difficult to obtain a double membrane. However, in the present invention, the amount of lipid can be reduced by a microchannel, so it is optimal for a thin film. A large amount can be extracted. Furthermore, a glass tube is used or a solution switching system is introduced so that there is no solution fluctuation causing a pressure difference. Another solution is to introduce fibrous molecules that have a raft structure into the planar membrane, which essentially improves the stabilization of the membrane.

FIG. 4 is a schematic diagram of the apparatus for forming a lipid bilayer membrane according to the first embodiment of the present invention, and FIG. 5 is a diagram showing a funnel-shaped hole and a micropore formed at the bottom thereof. Is an overall view of the funnel-shaped hole, and FIG. 5B is an enlarged view of the minute hole.
In these drawings, 20 is an apparatus for forming a lipid bilayer, 21 is an upper chamber, 22A is a funnel-shaped hole, 22B is a micropore formed at the bottom of the funnel-shaped hole 22A, and 23 is a microchannel. is there.

  In the present invention, as an apparatus 20 for forming a lipid bilayer, an acrylic plastic (PMMA) plate is subjected to micromachining to produce microchannels 23 and micropores 22B, and the lipid bilayer is reconfigured therein. As described above, since the micro flow path and the chamber are transparent, easy to machine, and made of electrically insulated PMMA, it is easy to perform microscopic observation and membrane current measurement. In the horizontal device, when the diameter and height of the micropores 22B are adjusted and the pressure is gradually applied through the chamber 21, the formation rate of the lipid bilayer membrane of 90% or more at maximum is succeeded. Incidentally, it was 10% or less in the conventional method.

FIG. 6 is a view showing a lipid planar membrane formed in the micropores at the bottom of the funnel-shaped hole, and FIG. 6 (a) shows the state of the lipid layer before formation of the lipid bilayer membrane, FIG. 6 (b). Indicates the state after lipid bilayer formation.
FIG. 7 is a diagram showing the pressure and formation process of the lipid bilayer in the chamber. FIG. 7 (1) applies 20 Pa, FIG. 7 (2) applies 170 Pa, and FIG. 7 (3) applies 210 Pa. The formation state of the lipid bilayer is shown. From these figures, it can be seen that the lipid bilayer membrane is formed by applying 210 Pa when the lipid layer is pushed down from the center and becomes thinner as the pressure increases.

Here, the influence of the height H of the micropores 22B [see FIG. 7 (1)] on the success rate of lipid bilayer formation was examined.
FIG. 8 is a graph showing the success rate of formation of the lipid layer and lipid bilayer membrane.
From this figure, it can be seen that when the height H of the micropores is 47 μm, a success rate of 90% or more can be achieved. In addition, the black graph in FIG. 8 shows the rate at which a thick lipid layer is formed, and the gray graph shows the success rate of lipid bilayer formation.

Thus, the reason why the success rate of lipid bilayer formation has been dramatically improved by the present invention is as follows.
(1) As shown in the upper part of FIG. 7, a certain amount of lipid solution (lipid layer) having a volume proportional to the height H remains on the inner wall of the micropore having a certain height H (a micropore having a diameter of 100 μm). The optimum amount of height H when the experiment was conducted was about 50 μm).
(2) Further, as described in FIG. 3D, the pressure adjustment is to be performed precisely.

FIG. 9 is a characteristic diagram of the initial lipid layer thickness and micropore height H. FIG.
In this figure, x is the raw data of each test, and the solid line is a straight line fitted to it. As can be seen from this figure, there is a large variation of about ± 20 μm, but the initial film thickness is almost proportional to the height H of the micropores.
The case where the lipid layer does not become a bilayer is usually broken when the membrane becomes thin, just as a soap bubble breaks.

FIG. 10 is a characteristic diagram of the pressure in the chamber and the initial lipid layer thickness when the lipid layer becomes a bilayer (circle) and when the lipid layer breaks without becoming a bilayer (square).
When the double membrane is successfully formed, the applied pressure is almost less than 400 Pa regardless of the initial thickness. When the pressure is 400 Pa or higher, the lipid layer is greatly curved and is more likely to break.

From these figures, it can be seen that the optimum value of the height H of the micropores is approximately 50 μm. When the height H of the micropores is 20 μm or less, the lipid layer is broken even when the buffer is introduced or when the pressure is slightly increased. If the height H of the micropores is 60 μm or more, a large pressure is required for thinning the film and it will be broken in the final thinning process.
Next, a second embodiment of the present invention will be described.

FIG. 11 is a view showing a chamber in which a plurality of minute holes are formed at the bottom of a funnel-type hole according to a second embodiment of the present invention. FIG. 11 (a) is an overall view, and FIG. An enlarged view of a plurality of micro holes, FIG. 11C is a perspective view showing one of the plurality of micro holes. In addition, it is desirable that the micropores are hydrophobic.
As is clear from this figure, four micropores 31 to 34 are formed in one chamber (funnel-shaped hole) 30 so that a plurality of planar lipid membranes can be formed at the same time. It is composed.

FIG. 12 is a view showing a planar lipid membrane thus obtained (the micropore height H is 43 μm), FIG. 12 (a) shows the state of the initial thick lipid layer, and FIG. 12 (b). Fig. 12 (a) shows a fluorescent image of a fluorescent lipid molecule in the state of Fig. 12 (a), Fig. 12 (c) shows the state of the planar bilayer membrane in the final state, and Fig. 12 (d) shows the state of Fig. 12 (c). It is a figure which shows the fluorescence image of the fluorescent lipid molecule in the state of ().
As shown in FIG. 12B, in a state where a thick lipid film is formed, almost uniform fluorescence is seen in all four holes, so that it can be seen that the amount of the lipid solution arranged is uniform. In FIG. 12 (d), the upper chamber is pressurized, the center is thinned, almost no fluorescence is seen, and the lipid film is thick only in the surrounding part that is the bulk phase. It is done.

FIG. 13 is a diagram (enlarged view of FIG. 12 (c)) showing lipid membranes simultaneously formed in four micropores and their confirmation states, respectively, and FIG. 13 (1) is shown in FIG. 12 (c). 13 (2) is the upper right of FIG. 12 (c), FIG. 13 (3) is the lower left of FIG. 12 (c), and FIG. 13 (4) is the lower right lipid of FIG. 12 (c). A double membrane is shown.
FIG. 14 shows that in the first embodiment of the present invention (that is, in the single-pore device of FIG. 5), when a channel protein called gramicidin is incorporated into a lipid bilayer membrane and a voltage (80 mV) is applied to both sides of the membrane. The current passing through gramicidin is shown. At this time, KCl is added to the aqueous phase. Gramicidin forms a channel that passes through the membrane when the monomer enters each side of the bilayer membrane, but the gramicidin incorporated on the front and back sides combine to form a dimer. Although the binding occurs stochastically, the step-like passing current also seen in FIG. 14 is obtained by capturing the current passing through the phenomenon of dimerization of gramicidin at a single molecule level. Since the phenomenon that gramicidin forms a channel by dimerization occurs only when the lipid membrane is a bilayer, it has been proved that the membrane formed thereby is truly a lipid bilayer. It also shows that the device of the present invention can withstand channel current measurement of a single protein molecule.

  In addition, this invention is not limited to the said Example, Based on the meaning of this invention, a various deformation | transformation is possible and these are not excluded from the scope of the present invention.

  Membrane proteins play an important role in physiological functions such as drug response, energy conversion, immune reaction, mass transport, and information transmission. Many of the membrane proteins are main targets for drug discovery, and the market size of drugs related to a series of receptor proteins called GPCR (G-protein coupled receptor; G protein coupled receptor) is large. For this reason, arraying of membrane proteins on the chip is expected, but there has been no report of efficiently reconstituting a lipid planar membrane into an array. In addition, there is no device that can measure the membrane current as in the physiological condition setting. Therefore, the development of the membrane protein function measuring system of the present invention is extremely useful and can be a breakthrough in the field of drug discovery and treatment.

  For example, in the Human Genome Project, all GPCR genes have already been identified, and the number of targets that can be practically limited is limited. Therefore, there is an urgent need to arrange these in an array on the chip and examine the response to the drug for each GPCR. Besides these, membrane proteins are also the main next-generation drug targets, such as a series of membrane proteins called ABC transporters that are responsible for drug resistance of cancer cells. The development of this system can contribute to rapid drug development by incorporating proteins as target membranes for such drug discovery.

It is a schematic diagram of the apparatus for forming a lipid bilayer membrane showing the principle of the present invention. It is a schematic diagram which shows the organic solvent containing the lipid concerning this invention. It is a figure which shows the formation process of the lipid bilayer membrane concerning this invention. It is a schematic diagram of the apparatus for forming a lipid bilayer membrane showing the first embodiment of the present invention. It is a figure which shows the funnel type | mold hole which shows 1st Example of this invention, and the micropore formed in the bottom part. It is a figure which shows the lipid plane membrane formed in the micropore of the bottom part of the funnel-shaped hole which shows 1st Example of this invention. It is a figure which shows the formation process of the pressure and lipid bilayer membrane in the chamber which shows 1st Example of this invention. It is a figure which shows the success rate of formation of the lipid layer and lipid bilayer membrane which show 1st Example of this invention. FIG. 3 is a characteristic diagram of the initial lipid membrane thickness and micropore height showing the first embodiment of the present invention. It is a characteristic view of the pressure in the chamber which shows 1st Example of this invention, and the thickness of the initial lipid layer. It is a figure which shows the chamber in which the several micropore was formed in the bottom part of the funnel type | mold hole which shows 2nd Example of this invention. It is a figure which shows the planar lipid membrane obtained by 2nd Example of this invention. It is a figure which shows the planar lipid membrane formed in the single micropore by 2nd Example of this invention, and its confirmation state. It is a figure which shows the characteristic of the planar lipid membrane of 1st Example of this invention. It is a schematic diagram which shows the conventional planar lipid membrane formation method by LB method.

1 Glass substrate (bottom plate)
2,23 Micro flow path 3 Substrate (chip)
4A, 22A, 30 Funnel-shaped hole 4B, 22B Micropore 5 Chamber 5A Chamber wall 5B Passage 6 Lipid bilayer membrane 7 Buffer solution dropped from microinjection device (not shown) 8 Buffer solution in microchannel 9 Patch clamp amplifier 10, 11 Microelectrode 12 Objective lens 13 Organic solvent containing lipid 15 Air 16 Microinjection device 17 Lid 18 Lipid layer 20 Lipid bilayer formation device 21 Upper chamber 30 One chamber 31-34 Four micro Hole

Claims (9)

A chamber is provided on the front surface of the substrate, and a microchannel is provided on the back of the substrate. A microhole penetrating the substrate between the chamber and the microchannel is formed at the bottom, and the diameter / height of the microhole is A funnel-shaped hole having a ratio of 100/21 to 82 is provided, a buffer medium is introduced into the chamber, a buffer medium-an organic solvent containing a lipid-a buffer medium is sequentially supplied to the microchannel, and the chamber A lipid bilayer membrane characterized in that a lipid bilayer membrane is formed by thinning the lipid layer formed in the micropores by adjusting the pressure in the chamber by applying pressure to the buffer medium of Forming method.   2. The method of forming a lipid bilayer according to claim 1, wherein a plurality of the micropores are formed. In forming method according to claim 1 or 2, wherein the lipid bilayer membrane, the microporous 1 a diameter of 00Myuemu, prior SL forming method of the lipid bilayer membrane, characterized in that the pressure in the chamber to 2 00Pa-400Pa .   4. The method for forming a lipid bilayer according to claim 3, wherein the success rate of the formation of the lipid bilayer is 90% or more.   5. The method for forming a lipid bilayer membrane according to claim 1, 3 or 4, wherein the micropores are arrayed independently to form a heterogeneous membrane protein. 6. The method of forming a lipid bilayer membrane according to claim 1, wherein a microelectrode is disposed on each of the chamber side and the microchannel side, and a patch clamp amplifier connected to the microelectrode is further disposed. and, the method of forming the lipid bilayer membrane and measuring membrane currents of the lipid bilayer. (A) a substrate;
(B) a chamber formed on the surface of the substrate;
(C) a microchannel formed on the back of the substrate;
(D) a funnel-shaped hole having a diameter / height ratio of 100/21 to 82 in which a microhole penetrating the substrate between the chamber and the microchannel is formed in the bottom;
(E) A device for forming a lipid bilayer, comprising pressure adjusting means for adjusting the pressure in the chamber formed with the funnel-shaped hole as a unit.   8. The apparatus for forming a lipid bilayer membrane according to claim 7, wherein a plurality of the micropores are arranged.   The apparatus for forming a lipid bilayer according to claim 7, comprising: a microelectrode disposed on each of the chamber side and the microchannel side; and a patch clamp amplifier connected to the microelectrode. An apparatus for forming a lipid bilayer membrane, characterized in that a membrane current can be measured.