JP5159808B2 - Lipid bilayer substrate - Google Patents

Description

  The present invention relates to a lipid bilayer membrane substrate.

  In measuring the function of membrane proteins expressed on the cell membrane, the detection of changes in the concentration of intracellular second messengers in the cytoplasm, as well as the inflow of ions from the outside of the cell into the cell, the outflow of ions from the inside of the cell to the outside of the cell, and the like. Is done.

  A membrane protein that causes an inflow of ions from the outside of the cell to the inside of the cell and an outflow of ions from the inside of the cell to the outside of the cell is called an ion channel membrane protein. Among them, a membrane protein that controls opening and closing of a channel by selectively binding a molecule (hereinafter referred to as “ligand”) to the ion channel membrane protein is called an ion channel receptor. Among ion channel receptors, there are calcium ions, sodium ions, potassium ions, chloride ions, and the like, as well as substances that permeate molecules larger than the aforementioned ions called organic ions under specific conditions. As an effective method for measuring the function of such an ion channel, there is an electrophysiological method, which has been widely applied (for example, see Non-Patent Document 1).

  In addition, a calcium ion derived from the endoplasmic reticulum is well known as a factor called the above-mentioned intracellular second messenger. Since these second messengers are released by the activation of various enzymes in the cell and metabolizing the molecules in the cell, the membrane protein involved in this reaction is called a metabotropic membrane protein. Those having a ligand are called metabotropic receptors. G protein-coupled receptor (GPCR) is well known as a representative metabotropic receptor. Metabotropic receptors are often expressed in electrically inactivated cells. In such a case, the electrophysiological method is unsuitable for measurement, and the activation of the metabotropic receptor is measured using the second messenger, that is, a fluorescent reagent that binds to calcium ions, using this intensity as an index.

  Thus, the function of the membrane protein can be measured using the change in intracellular ion concentration as an index. Membrane proteins (especially receptors) are said to account for about 45% as targets for drug discovery in 2000, and it is very important to accurately measure the occurrence of various diseases and the functions of membrane proteins related to their treatment. is there. Of the top 20 drug sales in the US in 2000, membrane proteins (including GPCRs and ion channels) account for about 50% (see Non-Patent Document 2, for example). Therefore, a system for measuring the function of various membrane proteins is very useful.

  However, there are usually multiple membrane proteins other than the target membrane protein on the cell membrane, and even when stimulated with a certain drug, a mechanism called transactivation to another membrane protein adjacent to the target membrane protein. In some cases, information transmission may be performed, and it may be unclear whether the obtained reaction is only through the target membrane protein. Therefore, an in vitro measurement system using the purified membrane protein is necessary.

  A functional measurement substrate having an artificially produced hole and having a generated membrane protein disposed thereon is expected to satisfy such a requirement (for example, see Non-Patent Document 3). In addition, a structure having pores artificially produced on a solid substrate is also expected to be applied to nanobiodevices utilizing the function of membrane proteins (for example, see Non-Patent Document 4).

E. Neher and B. Sakaman, Nature 260, 799 (1976) Drews J. Science, 287: 1960-1964 (2000) Y. Shinozaki et al. PLoS Biology Volume 7, Issue 5, e1000103 (2009) K. Sumitomo et al. NTT Technical Review Vol. 4, No.9 pp40-47

As described above, in order to perform stable measurement of membrane protein function electrophysiologically or by utilizing changes in fluorescence intensity, the lipid bilayer membrane that reconstitutes the membrane protein is stably retained. There is a need.
In order to measure the function of a small amount of membrane protein with high sensitivity and quantitatively, it is only necessary to create a micropore having a predetermined size and to cover it with a lipid bilayer stably. It is even better if a plurality of minute holes can be produced.

However, until now, when a minute hole was provided on the substrate, it was difficult to cover it with a lipid bilayer membrane.
Therefore, the present invention provides a lipid bilayer membrane substrate in which minute pores provided in the substrate are reliably covered with the lipid bilayer membrane.

The present invention employs the following means in order to solve the above problems.
According to the first aspect of the present invention, there is provided a thin film layer having an overhang portion provided with a hole in the substrate, made of a material different from the substrate and extending in a direction of narrowing the opening of the hole. The lipid bilayer substrate is characterized in that the opening is covered with the lipid bilayer by adhesion of the lipid bilayer to the surface of the thin film layer .
The invention according to claim 2 is characterized in that, in the invention according to claim 1, the hole is formed in a concave shape having a bottom surface, and fluorescent molecules are confined inside the hole .
The invention according to claim 3 is the invention according to claim 1 or 2, characterized in that the shape of the opening is circular and the inner diameter thereof is 100 nm to 10 μm.
The invention according to claim 4 is the invention according to claim 1 or 2, wherein the shape of the opening is a quadrangle, and the length of one side thereof is 100 nm to 10 μm.
The invention according to claim 5 is the invention according to any one of claims 1 to 4, wherein a plurality of the holes covered with the lipid bilayer membrane are provided in the substrate. And

The invention according to claim 6 is the invention according to any one of claims 1 to 5, wherein a membrane protein is arranged in a portion covering the opening in the lipid bilayer membrane. Features.
The invention according to claim 7 is the invention according to claim 1, or any one of claims 3 to 6, wherein an electrode is provided inside the hole.

According to the present invention, by providing the overhang portion at the opening of the pore, the pore can be stably covered with the lipid bilayer membrane.
When the substrate has a thin film layer on the surface side, the overhang portion can be formed by etching the thin film layer.
When a plurality of pores are provided in the substrate, it is possible to realize that the plurality of pores are covered with the lipid bilayer membrane stably at a time.
As described above, when a plurality of pores are provided, when used to measure the function of a membrane protein by measuring the change in the emission intensity of fluorescent molecules arranged inside the pores, the sensitivity and the quantification can be improved. Can be realized.
When an electrode is provided in the hole, functional measurement by the patch clamp method (see Non-Patent Document 1) becomes possible.
In addition, by reconstituting membrane proteins in the part of the crosslinked lipid bilayer membrane covering the pores, it can be applied to the measurement of ion permeation of ion channel membrane proteins and permeation of molecules other than ions. It can also be applied to the measurement of the activity of metabotropic membrane proteins. Further, application to high-throughput screening in the pharmaceutical field is expected.

It is a conceptual diagram which shows the cross section of the membrane protein function measurement board | substrate in Embodiment 1 of this invention. It is a conceptual diagram which shows the cross section of the membrane protein function measurement board | substrate in Embodiment 2 of this invention. In the membrane protein function measuring substrate of Example 1 corresponding to Embodiment 1, it is a conceptual diagram showing a cross section when forming a hole. It is the electron micrograph which image | photographed the hole part from the board | substrate upper part at the same time as the said hole part formation. In the membrane protein function measuring substrate of Example 1, it is a conceptual diagram which shows the cross section at the time of arrange | positioning a fluorescent molecule inside a hole. In the membrane protein function measurement board | substrate of Example 1, it is an electron micrograph image | photographed from the board | substrate upper part at the time of arrange | positioning a fluorescent molecule in the inside of a hole. It is the electron micrograph which image | photographed the membrane protein function measurement board | substrate of Example 1 which has an overhang part from upper direction. It is the electron micrograph which image | photographed the membrane protein function measurement board | substrate of the comparative example which does not have an overhang part from upper direction. It is an electron micrograph of the section in the membrane protein function measurement substrate of Example 2 corresponding to Embodiment 2. It is a figure which shows the electrode pattern in the membrane protein function measurement board | substrate of Example 2. FIG. It is the figure which showed a mode that a lipid bilayer membrane fell in an opening part, when not having an overhang part.

Hereinafter, embodiments of a lipid bilayer membrane substrate according to the present invention will be described with reference to the drawings of FIGS.
The lipid bilayer substrate of the present invention is a substrate used for measurement of the function of membrane protein utilizing fluorescence and electrophysiological measurement (patch clamp method etc.), and has a hole, and the opening of the hole Is covered with a lipid bilayer. In addition, it is characterized in that it has an “overhang” shape for stably holding the lipid bilayer membrane at the opening of the pore. In other words, it is a lipid bilayer membrane substrate in which a hole is provided in the substrate and the opening of the hole is covered with a lipid bilayer, and the overhang extends in the direction of narrowing the opening to the opening of the hole. A portion is provided. The fluorescence intensity can be measured with a fluorescence microscope. Electrophysiological measurement can be performed using a patch clamp device or the like.

[Embodiment 1]
In FIG. 1, the conceptual diagram of Embodiment 1 of the lipid bilayer membrane substrate which concerns on this invention is shown. The lipid bilayer membrane substrate in Embodiment 1 is an embodiment as a lipid bilayer membrane substrate (hereinafter referred to as membrane protein function measurement substrate 1) suitable for measuring the function of membrane protein using fluorescence.
As shown in FIG. 1, the membrane protein function measurement substrate 1 has a hole 20 formed in the substrate body 10, and an opening (opening) 21 of the hole 20 has an overhang portion (eave portion) 11 a. Is provided. The opening 21 of the pore 20 is covered with a lipid bilayer membrane 30 containing a membrane protein 31. A fluorescent substance (fluorescent molecule) 22 is disposed inside the hole 20.

Hereinafter, each configuration will be described in detail.
The material of the substrate body 10 is not limited as long as it does not hinder the observation of the fluorescence intensity of the hole 20 by the fluorescence microscope. Examples of the material of the substrate body 10 include silicon, glass, and plastic.
The substrate body 10 has a hole 20. The number of holes 20 is not particularly limited, and is preferably 1 to 10,000. The shape of the hole 20 is a concave hole having a bottom surface.
The shape of the opening 21 of the hole 20 is preferably circular or square from the viewpoint of stably forming the lipid bilayer membrane. The diameter of the circular opening 21 or one side of the rectangular opening 21 is 100 nm to 10 μm. The reason why the lower limit is 100 nm is that the size of the membrane protein is about 10 to 20 nm, and a size that can arrange the membrane protein of this size is required. The reason why the upper limit is 10 μm is that, as will be described later, the lipid bilayer membrane 30 is formed by the development of giant lipid vesicles, and the size of the giant vesicles is distributed in large numbers of several tens of micrometers.

Moreover, the depth of the hole part 20 should just have the depth which the lipid bilayer membrane 30 does not contact the bottom face of the hole part 20, and should just design according to the magnitude | size of the opening part 21. FIG.
As a method for forming the hole 20 in the substrate body 10, for example, a fine processing technique such as a photolithography method, an electron beam lithography method, or a dry etching method can be applied.

  On the surface of the substrate body 10, a thin film layer 11 for forming an overhang portion 11 a in the opening 21 of the hole 20 is provided. The overhang portion 11a is formed to extend in the direction of narrowing the opening of the opening portion 21 of the hole portion 20 so as to extend the upper surface of the substrate body 10. The material of the thin film layer 11 may be the same as or different from the material of the substrate body 10 as long as the lipid bilayer 30 is attached. Since a selective etching method can be applied in the manufacturing process of the overhang portion 11a, it is desirable to use a different material. The thickness of the thin film layer 11 is desirably 50 nm to 500 nm.

Examples of the lipid molecules of the lipid bilayer membrane 30 include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), and phosphatidic acid (PA). ), Phosphatidylglycerol (PG), sphingolipid and the like. These lipid molecules may use only 1 type and may use 2 or more types.
As a method for forming the lipid bilayer membrane 30, for example, the lipid bilayer membrane 30 is obtained by spreading a giant lipid vesicle having a diameter of 10 μm or more on a substrate. When this formation method is employed, when a plurality of holes 20 are provided in the substrate body 10, the plurality of holes 20 can be covered with the lipid bilayer membrane 30 at once and stably.

  Typical methods for producing a vesicle structure include a static hydration method and an electro-swelling method (electric field forming method). The method for producing the vesicle is not particularly limited, but it is preferable to adopt the electric field forming method because it is easy to produce a huge vesicle and the reaction time and the simplicity of the reaction process. The electric field forming method is a method of forming giant vesicles in an aqueous solution by applying an alternating electric field after thinning phospholipid molecules on an electrode such as indium tin oxide (ITO). In order to obtain vesicles of uniform size, it is preferable to form a uniform thin film of phospholipid molecules with a thickness of several tens of nanometers to several micrometers on an ITO substrate, and the alternating electric field is about several hundred mV to 2V. Application conditions are preferred. This is because, when the electric field strength is lower than the electric field range, the yield of vesicles is low, and when the electric field strength is higher than the electric field range, there is a possibility that the vesicles are structurally destroyed or water is electrolyzed, and the vesicles cannot be produced.

A known method can be used as a method for reconstituting the membrane protein 31 to be a function measurement target in the lipid bilayer membrane 30, and examples thereof include a vesicle fusion method. Specifically, by adding a vesicle containing a membrane protein 30 called a proteoliposome onto the lipid bilayer membrane 30, the proteoliposome and the lipid bilayer membrane 30 are fused, and the membrane protein 31 becomes a lipid bilayer. Reconstituted in the membrane 30.
A fluorescent material 22 for measuring the function of the membrane protein 31 is disposed inside the hole 20. The function of the membrane protein 31 can be measured by, for example, observing the change in the fluorescence intensity with a fluorescence microscope.

[Embodiment 2]
In FIG. 2, the conceptual diagram of Embodiment 2 of the lipid bilayer membrane substrate which concerns on this invention is shown. The lipid bilayer membrane substrate in Embodiment 2 is an aspect as a lipid bilayer membrane substrate (hereinafter referred to as membrane protein function measurement substrate 2) suitable for electrophysiologically measuring membrane protein functions.
The membrane protein function measurement substrate 2 of Embodiment 2 has an electrode 40 inside the hole 20. Except for this feature, the membrane protein function measurement substrate 1 of the first embodiment is the same as the first embodiment. Differences from the membrane protein function measurement substrate 1 of Embodiment 1 will be described below.

In the membrane protein function measuring substrate 1 of the first embodiment described above, the fluorescent substance 22 is disposed inside the hole portion 20. However, in the membrane protein function measuring substrate 2 of the second embodiment, the fluorescent substance 22 is provided inside the hole portion 20. Do not place.
In the membrane protein function measurement substrate 2 of the second embodiment, the electrode 40 is embedded in the inside of the hole 20 in the production. As a material of the electrode 40, silver / silver chloride, gold, platinum or the like can be considered.
In the membrane protein function measuring substrate 2 of Embodiment 2, since the electrode 40 is embedded, the substrate body 10 needs to be excellent in insulation. Therefore, it is considered that silicon oxide, silicon nitride, alumina, tantalum oxide, resist film, etc. can be adopted as the material of the substrate body 10.

Also in the membrane protein function measuring substrate 2 of the second embodiment, the lipid bimolecule in the portion covering the opening 21 of the pore 20 with the lipid bimolecular membrane 30 and covering the opening 21 as in the first embodiment. By reconstituting the membrane protein 31 in the membrane 30, the function of the membrane protein can be measured.
In measuring the function of the membrane protein, the counter electrode 41 is disposed on the outside 23 of the hole 20. The electrode 40 and the counter electrode 41 are connected to a patch clamp measurement device.
For example, when an ion channel protein is arranged as the membrane protein 31, the function of the membrane protein can be measured by measuring an ionic current that has passed through the ion channel protein with a patch clamp measurement device.

[Example 1]
Next, an example of creating the membrane protein function measurement substrate 1 of Embodiment 1 will be specifically described.
The hole formation model in Example 1 is shown in FIG.
A silicon substrate was used as the substrate body 10. A silicon oxide film layer (thin film layer) 11 having a thickness of 200 nm was formed on the substrate body 10 by an electron cyclotron resonance plasma (ECR plasma) deposition method. Further, a hole 20 having a square opening (2 μm × 2 μm) was formed by using a photolithography method and a dry etching method. The depth was 1 μm. Further, the substrate body 10 under the silicon oxide film layer 11 is selectively etched with a potassium hydroxide solution (10 wt%) to form overhang portions 11 a at the four corners of the opening 21 of the hole 20. did. An electron microscope (SEM) photograph of the hole 20 formed at this time is shown in FIG.

Next, a lipid bimolecular film 30 was formed by spreading a huge vesicle on the substrate body 10 having the hole 20 produced as described above.
Here, the huge vesicle was produced as follows. A mixed chloroform solution (concentration 2.5 mM) of diphytanyl phosphatidylcholine (DPhPC) (80 mol%) and cholesterol (20 mol%) was prepared. Subsequently, 200 μL of the chloroform solution was uniformly applied on an indium tin oxide (ITO) substrate (a substrate in which ITO having a thickness of 100 nm was thinned on SiO ₂ , size 40 × 40 mm, 50 to 100 Ω / cm). . The substrate was dried under reduced pressure at room temperature for 2 hours to completely remove the chloroform solvent, thereby forming a uniform phospholipid thin film on the ITO substrate. On top of that, a silicone rubber having a window portion (having a window portion in which a silicone rubber having an outer dimension of 30 × 30 mm and a thickness of 1 mm is cut in a size of 20 × 20 mm) is closely attached, and 200 mM of the window portion is placed. 400 μL of a sucrose aqueous solution was dropped. Further, an ITO substrate was placed on the top, and the solution in the silicone rubber window was sandwiched between the ITO substrates and sealed. Subsequently, a clip electrode was joined to the ITO substrate, and an alternating electric field (sine wave, 1 V, 10 Hz) was applied on a hot plate at 60 ° C. for 2 hours, thereby producing a huge vesicle by an electric field forming method.

  Then, 100 μL of a solution containing fluorescent molecules 22 (a mixed solution of 200 mM glucose and 100 μM calcein) was dropped onto the substrate body 10 having the hole 20 shown in FIGS. Further, 5 μL of the giant vesicle solution was dropped into the solution and allowed to stand for 10 minutes to precipitate the giant vesicle. Further, 5 μL of 100 mM calcium chloride solution was dropped. After standing for another 10 minutes, most of the giant vesicles were developed on the substrate.

Furthermore, the fluorescent molecule (calcein) 22 was removed from the exterior 23 by replacing the solution of the exterior 23 of the hole 20 with a 200 mM glucose solution. FIG. 5 shows the model diagram. Moreover, the fluorescence micrograph at that time is shown in FIG.
Bright fluorescence is observed from the hole 20a covered with the lipid bilayer membrane 30, and it can be seen that the fluorescent molecules 22 are stably trapped inside the hole 20a. On the other hand, fluorescent molecules are washed away from the hole 20b not covered with the lipid bilayer membrane 30, and fluorescence is not observed.

7 and 8 show a comparison of fluorescence observation results when the overhang portion 11a is provided in the opening 21 of the hole portion 20 (FIG. 7) and when the overhang portion 11a is not provided (FIG. 8). . In any case, the opening 21 is a square having a side of 1 μm.
When the overhang part 11a is not provided, as shown in FIG. 8, it can be seen that a large number of hole parts 20b not covered with the lipid bilayer membrane 30 occupy and the fluorescent molecules 22 are not confined.
On the other hand, when the overhang part 11a is provided, as shown in FIG. 7, the hole part 20a covered with the lipid bilayer 30 occupies a large number except for the outer peripheral part of the developed lipid bilayer 30. You can see that It can be seen that by providing the overhang portion 11a, the fluorescent molecules 22 have been successfully confined stably in many holes 20.

Membrane protein 31 is reconstituted on the “substrate having minute pores 20 covered with lipid bilayer membrane 30” produced in this way, and the intensity change of fluorescent molecules 22 arranged inside pores 20 is changed. By measuring, the function of the membrane protein 31 can be measured.
And it becomes possible to improve the sensitivity and precision of the function measurement of the membrane protein 31 by covering the plurality of pores 20 with the lipid bilayer membrane 30 stably.

In addition, when the overhang part 11a is provided in the opening part 21 of the hole part 20, the reason why the opening part 21 can be covered with the lipid bilayer membrane 30 is estimated as follows.
When a giant lipid vesicle is deployed on a substrate, there must be a slight attraction between the lipid membrane and the substrate, and the giant vesicle is deformed and deployed by the attraction acting between the substrate and the lipid membrane. The resulting lipid bilayer membrane. Further, in order to stably hold the developed lipid bilayer membrane, an attractive force between the lipid bilayer membrane and the substrate is required.

However, as shown in FIG. 11, when the overhanging portion is not provided in the opening portion 21 of the hole portion 20, the lipid covering the top of the minute hole portion 20 with the attractive force between the substrate body 10. When the bilayer membrane 30 is held, it is considered that the lipid bilayer membrane 30 falls into the inside of the hole 20 along the side surface of the hole 20 due to the attractive force. This is considered to make it difficult to cover the fine pores with the lipid bilayer membrane 30.
In the present invention, by providing the overhang portion 11 a in the opening 21 of the hole 20, it is possible to prevent the lipid bilayer membrane 30 from falling into the inside of the hole 20 along the side surface of the hole 20. Guessed.

  In addition, as a method of controlling the attractive force between the lipid bilayer membrane 30 inside the pore 20 and the substrate (side surface of the pore) to prevent the drop into the pore 20, the material is changed or the surface is modified. It can be considered. For example, the inside of the hole 20 may be hydrophobized by silane coupling treatment. However, it is not easy to modify only the inside of the hole 20, and it becomes difficult to fill the inside of the hole 20 with the solution when it is made hydrophobic. Therefore, providing an overhang portion in the opening as in the present invention is an effective means for forming a minute pore portion stably covered with a lipid bilayer membrane.

[Example 2]
Next, an example of creating the membrane protein function measurement substrate 2 for electrophysiology measurement of Embodiment 2 described above will be specifically described.
FIG. 9 shows an electron microscope (SEM) photograph of a cross section during the production of the membrane protein function measuring substrate 2 of Example 2 (before the opening 21 of the hole 20 is covered with the lipid bilayer membrane 30). With reference to FIG. 9, the example of the preparation methods of the membrane protein function measurement board | substrate 2 of Example 2 is demonstrated.

A Si wafer (14) covered with an insulating layer 13 (here, a thermal oxide film having a thickness of 200 nm) was used as a substrate.
An electrode layer (electrode) 40 is first deposited on this substrate. Here, the electrode pattern as shown in FIG. 10 was formed by patterning by photolithography and depositing gold (60 nm). The electrode layer 40 is connected to a sufficiently large pad portion (400 μm square) and can be connected to an electrophysiological measurement device.
Further, a 1 μm thick silicon nitride film is deposited as an insulating layer 12 on the electrode layer 40 by plasma CVD.
Further, a silicon oxide film is deposited on the insulating layer 12 to a thickness of 200 nm as the overhang forming layer (thin film layer) 11 by sputtering.
In the second embodiment, the overhang forming layer 11 is formed on the substrate body 10 constituted by the Si wafer 14 covered with the insulating layer 13 and the insulating layer 12.

The hole 20 and the opening 21 are formed in the substrate body 10 having the overhang shape forming layer 11 formed as described above by using the resist film 50 by the photolithography method and the dry etching method. The etching for forming the hole is performed until it reaches the electrode layer 40.
In the process of this dry etching, an overhang portion 11a as shown in FIG. 9 is formed by utilizing the selectivity between the silicon oxide film (overhang shape forming layer 11) and the silicon nitride film (insulating layer 12). did.
After forming the hole 20 and the opening 21, the resist film 50 was washed and removed to produce a substrate having the electrode 40 in the hole 20.
A plurality of holes 20 may be formed in each electrode 40, but since all the holes 20 need to be covered with the lipid bilayer membrane 30, one hole 20 is formed in one electrode 40. It is desirable to do.

As in the case of Example 1 described above, a giant lipid membrane vesicle is developed on the substrate provided with the electrode 40 in the hole 20 thus formed, thereby opening the opening 21 of the hole 20. Covered with a lipid bilayer 30.
Furthermore, the membrane protein function measurement substrate 2 can be prepared by reconstituting the membrane protein by the vesicle fusion method in the portion covering the opening 21 in the lipid bilayer membrane, and the ion channel function of the membrane protein is measured. It becomes possible.

1 Membrane protein function measurement substrate (lipid bilayer substrate)
2 Membrane protein function measurement substrate (lipid bilayer substrate)
10 Substrate body (substrate)
11 Thin film layer (silicon oxide film layer, overhang shape forming layer)
11a Overhang portion 12 Insulating layer 13 Insulating layer 14 Si wafer (substrate)
20 Hole 20a Hole 20b covered with lipid bilayer 20b Hole 21 not covered with lipid bilayer Opening (opening)
22 Fluorescent substances (fluorescent molecules)
23 Outside 30 pores Bilayer membrane 31 Membrane protein 40 Electrode (electrode layer)
41 Counter electrode 50 Resist film

Claims (7)

A hole portion is provided in the substrate, and a thin film layer having an overhang portion formed of a material different from that of the substrate and extending in a direction of narrowing the opening of the hole portion is provided on the surface of the substrate,
A lipid bilayer membrane substrate characterized in that the opening is covered with the lipid bilayer membrane by adhesion of the lipid bilayer membrane to the surface side of the thin film layer . The lipid bilayer membrane substrate according to claim 1, wherein the pore is formed in a concave shape having a bottom surface, and fluorescent molecules are confined inside the pore .   The lipid bilayer membrane substrate according to claim 1 or 2, wherein the opening has a circular shape and an inner diameter of 100 nm to 10 µm.   The lipid bilayer membrane substrate according to claim 1 or 2, wherein the shape of the opening is a quadrangle, and the length of one side thereof is 100 nm to 10 µm.   The lipid bilayer membrane substrate according to any one of claims 1 to 4, wherein a plurality of the holes covered with the lipid bilayer membrane are provided in the substrate.   The lipid bilayer substrate according to any one of claims 1 to 5, wherein a membrane protein is disposed in a portion of the lipid bilayer membrane covering the opening. The lipid bilayer membrane substrate according to any one of claims 1 or 3 to 6, further comprising an electrode inside the pore.