JP2019037213A - Lipid bimolecular membrane substrate - Google Patents

Abstract

To provide a lipid bimolecular membrane substrate in which ion inflow is suppressed.SOLUTION: A lipid bimolecular membrane substrate has a substrate including a hole part. An opening of the hole part is coated with a lipid bimolecular membrane. The outermost surface of the lipid bimolecular membrane substrate that is not coated with the lipid bimolecular membrane is coated with protein.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a lipid bilayer membrane substrate.

  The movement of so-called nanobiotechnology, which combines nanotechnology microfabrication technology and biotechnology that handles biomolecules such as DNA and proteins, is rapidly expanding. Nanobiotechnology is expected to be applied not only to basic research fields but also to various fields such as medicine and drug discovery. As a trend of basic molecular biological research, attempts have been made to control the structure of membrane proteins present in cell membranes from the post-genomic flow and to utilize and elucidate their functions.

  Membrane proteins are responsible for transport of substances necessary for life support and signal transduction, and are biomolecules greatly related to physiological functions such as the occurrence of various diseases, drug responses and immune reactions. Currently, it is known that about 60% or more of commercially available drugs target membrane proteins. However, since membrane proteins are diverse and difficult to handle as compared to DNA, there is a problem that it takes enormous time and cost for functional analysis. Therefore, if an ultra-small biochip in which membrane proteins are arrayed on a semiconductor substrate can be realized, the functions of many membrane proteins can be analyzed simultaneously and at high speed. Many effects such as reduction of

  As a method for analyzing a membrane protein on a substrate, there is a method in which cells themselves are arranged on a substrate and a function is measured by a patch clamp method. However, there are usually multiple membrane proteins other than the target membrane protein on the cell membrane, and it is unclear whether the response obtained by stimulation with a certain drug is due to the target membrane protein. There was a problem. Therefore, the need for an in vitro measurement system using purified membrane protein is increasing.

  As a typical membrane protein arrangement method in an in vitro system, a membrane is formed on a black membrane formed by dissolving lipid molecules in an organic solvent such as n-decane, and applying the lipid solution to small holes provided on the substrate in an aqueous solution. Examples include a method of fusing proteins (see, for example, Non-Patent Document 1). In this method, it has been pointed out that the residual organic solvent and heterogeneity in the formed black film may affect the physiological activity of the membrane protein. Further, the stability of the black film is low, and the lifetime is usually about several hours.

  In order to improve these problems, the inventors firstly made a thin film having an artificially provided hole and an overhang formed so as to extend in the direction of narrowing the width of the opening of the hole. A substrate comprising a layer was produced. Next, a substrate was proposed in which a giant lipid membrane vesicle was developed in the opening of the pore to reconstruct an artificial lipid bilayer membrane containing a membrane protein (see, for example, Non-Patent Document 2). Since the lipid bilayer substrate produced in this way uses a giant lipid membrane vesicle, it does not contain an organic solvent, and the lifetime of the lipid bilayer membrane is much longer than that of the black membrane (several days). It has also been confirmed by optical techniques (fluorescence observation) that the reconstituted membrane protein has activity.

“Latest Patch Clamp Experiment Technology” edited by Yasunobu Okada, pp. 158-159 (2001, Yoshioka Shoten) Sumitomo K et al., “Ca2 + ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate.”, Biosensors and Bioelectronics, Vol. 31, p445-450, 2012. Johnson SJ et al., “Structure of an adsorbed dimyristoylphosphatidylcholine bilayer measured with specular reflection of neutrons.”, Biophysical Journal, Vol. 59, p289-294, 1991.

  As described in Non-Patent Document 2, the function of the membrane protein was successfully analyzed by measuring by an optical method using a substrate reconstructed from an artificial lipid bilayer membrane containing a membrane protein. However, even in the absence of membrane protein, results suggesting ion inflow from the outside into the pores were observed. It is known that a thin water layer having a thickness of about 1 to 2 nm exists between the substrate and the lipid bilayer membrane (see, for example, Non-Patent Document 3), and ion diffusion through this water layer is an ion. It is thought to be the cause of inflow. This ion inflow was not so much a problem for relatively short optical measurements. However, in the electrophysiological measurement that measures the channel signal at the molecular level, it is necessary to measure a minute current with a background noise level (several pA), so this ion inflow is a problem that must be solved.

  In view of the above circumstances, the present invention provides a lipid bilayer membrane substrate in which ion inflow is suppressed.

That is, the present invention includes the following aspects.
The lipid bilayer membrane substrate according to the first aspect of the present invention is a lipid bilayer membrane substrate comprising a substrate provided with pores, the openings of the pores being covered with a lipid bilayer membrane, wherein the lipid The outermost surface of the lipid bilayer membrane substrate not covered with the bilayer membrane is coated with protein.

The lipid bilayer membrane substrate according to the first aspect may further include a thin film layer having an overhang portion formed on the surface of the substrate so as to extend in a direction of narrowing the width of the opening of the hole portion. Good.
In the lipid bilayer membrane substrate according to the first aspect, a plurality of the holes covered with the lipid bilayer membrane may be provided in the substrate.
The lipid bilayer membrane substrate according to the first aspect may further include an electrode at the bottom of the hole.
The electrode may be an electrode composed of silver and silver chloride (silver-silver chloride electrode).
The protein may be bovine serum albumin.

  According to the said aspect, the lipid bilayer membrane substrate by which ion inflow was suppressed can be provided.

It is the conceptual diagram which showed an example of the lipid bilayer membrane substrate in this embodiment. It is the conceptual diagram which showed an example of the lipid bilayer membrane substrate in this embodiment. It is the conceptual diagram which showed an example of the lipid bilayer membrane substrate in this embodiment. It is the conceptual diagram which showed an example of the lipid bilayer membrane substrate in this embodiment. 2 is a fluorescence image of a lipid bilayer substrate encapsulating calcein in Example 1. FIG. 2 is a fluorescence image of a lipid bilayer membrane substrate in a charged state (blue light excitation (B excitation) and green light excitation (G excitation)) subjected to blocking by BSA in Example 1. FIG. (A) is a lipid bilayer membrane substrate using an anionic lipid membrane, (b) is a lipid bilayer membrane substrate using a neutral lipid bilayer membrane, and (c) is a cationic lipid bilayer membrane. It is a lipid bilayer membrane substrate using 2 is a fluorescence image obtained by observing a change with time of a lipid bilayer substrate in Test Example 1. The upper row is a lipid bilayer membrane substrate that is blocked with BSA (a lipid bilayer membrane substrate that uses the neutral lipid membrane of Example 1), and the lower row is a lipid bilayer membrane substrate that is not blocked with BSA (Comparative Example 1). ). 4 is a graph plotting changes over time in fluorescence intensity of a lipid bilayer membrane substrate using neutral lipid membranes of Example 1 and Comparative Example 1 in Test Example 1. FIG. It is the schematic diagram explaining the mechanism which can be estimated about the ion inflow from the hole covered with the lipid bilayer membrane. 6 is a fluorescence image obtained by observing a change with time of a lipid bilayer membrane substrate in Example 2. 6 is a graph plotting changes with time in fluorescence intensity of a lipid bilayer substrate in Example 2. FIG. 6 is a schematic diagram of a cross section of a lipid bilayer membrane substrate in Example 3 during production (before the opening 21 of the hole 20 is covered with the lipid bilayer membrane 30). FIG. FIG. 10 is a schematic diagram of a cross section during the production of a lipid bilayer membrane substrate in Example 4 (before the opening 21 of the hole 20 is covered with the lipid bilayer membrane 30). It is an electron microscopic image inside a hole part in the middle of preparation of a lipid bilayer membrane substrate in Example 4 (stage before covering opening 21 of hole part 20 with lipid bilayer film 30).

  Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

≪Liquid bilayer substrate≫
A lipid bilayer membrane substrate according to an embodiment of the present invention includes a substrate provided with pores, and the openings of the pores are covered with the lipid bilayer membrane. In the lipid bilayer membrane substrate of the present embodiment, the outermost surface of the lipid bilayer membrane substrate that is not coated with the lipid bilayer membrane is coated with protein.

Since the channel current of the membrane protein is at the same level as the background noise, it has been difficult to determine whether the obtained response is a channel current or due to ion inflow.
On the other hand, in the lipid bilayer membrane substrate of this embodiment, ion inflow is suppressed. Therefore, by using the lipid bilayer membrane substrate of this embodiment, it is possible to measure the function of membrane proteins at the molecular level. Furthermore, in the lipid bilayer membrane substrate of this embodiment, by providing a plurality of holes and making an array, application to high-throughput screening in the drug discovery field is expected.

<First Embodiment>
FIG. 1 is a conceptual diagram showing an example of a lipid bilayer membrane substrate in the present embodiment. The lipid bilayer membrane substrate in this embodiment is an embodiment as a lipid bilayer membrane substrate suitable for measurement of the function of a membrane protein using an optical technique (fluorescence measurement) (hereinafter referred to as “lipid bilayer membrane substrate 1”). ").

  As shown in FIG. 1, the lipid bilayer membrane substrate 1 has holes 20 formed in the substrate 10. Further, the opening 21 of the hole 20 is covered with a lipid bilayer membrane 30. On the other hand, the outermost surface of the lipid bilayer substrate 1 that is not covered with the lipid bilayer membrane 30 is covered with the protein 15. In the lipid bilayer membrane substrate 1, one hole portion 20 covered with the lipid bilayer membrane 30 may be provided in the substrate 14, or two or more holes may be provided. When a plurality of pores 20 covered with the lipid bilayer membrane 30 are provided on the substrate 14, the functions of a plurality of membrane proteins of the same or different types can be measured simultaneously.

In order to increase the efficiency of covering the pores 20 with the lipid bilayer membrane, the lipid bilayer membrane substrate 1 is an overhang portion (such as an overhang portion) formed so as to extend in the direction of narrowing the width of the aperture (opening) 21 of the pore 20. It is preferable to include a thin film layer 11 having an eaves portion 11a.
A fluorescent material 22 is disposed inside the hole 20.
Hereinafter, each configuration will be described in detail.

○ Substrate The material of the substrate 10 may be any material that does not interfere with the fluorescence observation of the hole 20 by the fluorescence microscope. Examples of the material of the substrate 10 include silicon oxide, silicon nitride, quartz, mica, and glass.

(Hole)
The substrate 10 has a hole 20. The number of the hole parts 20 is not specifically limited, For example, it is preferable that it is 1-10000 pieces. The shape of the hole 20 is a concave hole having a bottom surface.

  The shape of the opening 21 (opening 21) of the hole 20 is preferably circular or square from the viewpoint of stably forming the lipid bilayer membrane. Further, the diameter of the circular opening 21 or one side of the square opening 21 is preferably 100 nm to 10 μm. When it is 100 nm or more, since the size of the membrane protein is about 10 to 20 nm, the membrane protein can be arranged. In the case of 10 μm, the pore can be covered with a giant lipid membrane vesicle.

  Moreover, the depth of the hole part 20 should just be the depth which the lipid bilayer membrane 30 does not contact the bottom face of the hole part 20, and should just design suitably according to the magnitude | size of the opening part 21. FIG.

  As a method for forming the hole 20 in the substrate 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.

Thin Film Layer A thin film layer 11 is provided on the surface of the substrate 10. In order to increase the covering efficiency of the pores 20 with the lipid bilayer membrane, the thin film layer 11 in the lipid bilayer membrane substrate 1 preferably has an overhang portion (eave portion) 11a. The overhang portion 11a is formed so as to extend in the direction of narrowing the width of the opening 21 of the hole 20 so as to extend the top surface of the substrate 10. In the overhang portion 11a, in addition to the attractive force between the lipid bilayer membrane 30 and the inner wall of the hole 20, the lipid bending stress acting when the lipid bilayer 30 falls along the side wall can be increased. Therefore, the lipid bilayer membrane 30 can be stably supported without falling into the pores 20.

  The material of the thin film layer 11 may be the same as or different from the material of the substrate 10 as long as the lipid bilayer 30 is attached. In the process of manufacturing the overhang portion 11a, it is preferable to use a different material because a selective etching method can be applied. The thickness of the thin film layer 11 is preferably 50 nm to 500 nm.

○ Lipid bilayer In this specification, “lipid bilayer” refers to a lipid molecule having a hydrophilic functional group at one end and a hydrophobic fatty acid at the other end. It means a film in which a functional group is arranged outside and a hydrophobic fatty acid is arranged inside to form a double layer structure. Further, the term “giant lipid membrane vesyl” means that the above-mentioned fat bilayer membrane is converted into vesicles in an aqueous solution, and the average particle size thereof is on the order of μm or more.

Examples of lipid molecules of the lipid bilayer membrane 30 include cationic lipid molecules such as 1,2-dioleoluyl-3-trimethylammonium propane and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine; diphytanyl Phosphatidylcholine (DPhPC), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidic acid (PA), phosphatidylglycerol (PG), etc. Mention may be made of neutral or anionic lipid molecules such as phospholipids and sphingolipids. These lipid molecules may use only 1 type and may use 2 or more types. Moreover, you may mix sterols, such as cholesterol and ergosterol, with the lipid film comprised by these lipids.
Among them, the lipid bilayer 30 is preferably a neutral lipid molecule having no charge, since it can suppress adsorption of a protein to be described later on the lipid bilayer, and diphytanyl phosphatidylcholine. It is more preferable that

  Examples of the method of forming the lipid bilayer membrane 30 include a method of obtaining the lipid bilayer membrane 30 by spreading a giant lipid vesicle having a diameter of 10 μm or more on a substrate. When this formation method is adopted, when a plurality of hole portions 20 are provided in the substrate 10, the plurality of hole portions 20 can be covered with the lipid bilayer membrane 30 at once and stably.

  As a representative technique for producing giant lipid membrane vesicles, the stationary hydration method (Reference 1: Akashi K, et al., “Preparation of Giant Liposomes in Physiological Conditions and Their Characterization under an Optical Microscope.”, Biophysical Journal., Vol. 71, p3242-3250, 1996.) and electric field formation method (Reference 2: Dimitrov DS et al., “Lipid swelling and pulse formation mediated by electric fields.”, Bioelectrochemistry and Bioenergetics, Vol. 19, p323-336, 1988.).

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 a giant lipid membrane vesicle in an aqueous solution by applying an alternating electric field after thinning lipid 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 lipid molecules with a thickness of several tens of nanometers to several micrometers on an ITO substrate, and an alternating electric field of about several hundred mV to 2 V is applied. Conditions are preferred. When the AC electric field is not less than the above lower limit, the yield of vesicles can be further increased. On the other hand, when the AC electric field is not more than the above upper limit value, it is possible to further prevent the structural destruction of the vesicle and the electrolysis of water from occurring.

(Membrane protein)
As shown in FIG. 2, the lipid bilayer membrane 30 may include a membrane protein 31. In the lipid bilayer membrane substrate of this embodiment shown in FIG. 2, for example, the function of the membrane protein 31 can be measured by observing the change in fluorescence intensity with a fluorescence microscope. As a method of arranging the membrane protein 31 on the lipid bilayer membrane 30, a known method such as a vesicle fusion method can be applied.

○ Protein Examples of the protein 15 include bovine serum albumin (BSA), casein, and globulin. Further, skim milk containing casein or serum containing globulin may be used. In view of the uniformity of the protein membrane and the low cost, the type of protein 15 is preferably BSA.

  The above protein such as BSA is usually used for suppressing non-specific adsorption of protein or the like. On the other hand, in the lipid bilayer membrane substrate of the present embodiment, as shown in Examples described later, it is considered that the layer made of the protein plays a role as a block layer for ion diffusion. That is, the ion inflow can be suppressed by blocking the outer peripheral portion of the lipid bilayer membrane serving as an interface for the ion inflow from the outer liquid portion with the protein such as BSA.

  As a method for blocking the outermost surface of the lipid bilayer substrate not covered with the lipid bilayer membrane 30 with the protein 15, for example, the lipid bilayer substrate coated with the lipid bilayer membrane 30 is immersed in an aqueous protein solution. (Hereinafter sometimes referred to as “immersion method”), a method of applying a protein aqueous solution to the lipid bilayer substrate after coating with the lipid bilayer membrane 30 (hereinafter also referred to as “coating method”) Etc. Especially, since the outermost surface of the lipid bilayer membrane substrate not covered with the lipid bilayer membrane 30 can be uniformly coated with the protein 15, the method of blocking with the protein 15 is preferably an immersion method.

  In the case of the immersion method, the concentration of the aqueous protein solution is preferably 0.01% by mass or more and 0.1% by mass or less in order to prevent excessive adsorption. In order to prevent protein denaturation, the pH is preferably adjusted to around 7. In addition, from the viewpoint of uniform protein film formation, the immersion time is desirably 10 to 60 minutes.

○ Fluorescent substances (fluorescent molecules)
In addition, a fluorescent material 22 may be disposed inside the hole 20 in order to measure changes in the ion concentration in the hole.
Examples of the “fluorescent substance” include a fluorescent probe whose fluorescence characteristics change by binding to calcium ions, a fluorescent probe whose fluorescence intensity changes depending on pH, and a fluorescent probe whose fluorescence intensity changes in proportion to the chlorine ion concentration Etc.
Specific examples of the fluorescent substance include, for example, 1- [6-Amino-2- (5-carboxy-2-oxazolyl) -5-benzofuranyoxy] -2- (2-amino-5-methylphenoxy) ethane-N, N. , N ′, N′-tetraacetic acid, pentapotassium salt (reagent name: Fluo2), 1- [2-Amino-5- (2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl) phenoxy]- 2- (2-amino-5-methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid (reagent name: Fluo3), 1- [2-Amino-5- (2,7-difluoro-6- acetoxyme thoxy-3-oxo-9-xanthenyl) phenoxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid, tetra (acetoxymethyl) ester (reagent name: Fluo4- AM), 2 ′, 7′-Bis (carboxyethyl) -4 or 5-carbofluorescein (reagent name: BCECF), N-ethoxycarbonylmethyl-6-methoxyquinoline bromide (reagent name: MQAE), and the like.

Second Embodiment
The lipid bimolecular membrane substrate according to one embodiment of the present invention further has an electrode at the bottom of the hole.

  According to the lipid bilayer membrane substrate of this embodiment, the function of membrane protein can be measured electrophysiologically.

  FIG. 3 is a conceptual diagram showing an example of a lipid bilayer membrane substrate in the present embodiment. The lipid bimolecular membrane substrate 2 of the present embodiment is different from the first embodiment in that the electrode 40 is provided inside the hole 20. About the part of the same aspect as 1st embodiment, the same code | symbol is attached | subjected and description is abbreviate | omitted and the point different from 1st embodiment is demonstrated below.

○ Electrode In the lipid bilayer membrane substrate 2 of the present embodiment, the electrode 40 is embedded in the hole 20.
Examples of the material of the electrode 40 include silver, silver chloride, a silver surface processed with silver chloride (silver-silver chloride), gold, platinum, and the like. Among these, silver-silver chloride is preferable.
In addition, when the electrode material is gold, the electric double layer formed on the gold surface in the electrolyte solution often hinders the measurement of the channel current of minute membrane proteins. Therefore, the electrode 40 is preferably an unpolarized electrode made of silver-silver chloride or the like. The electrode 40 may have a structure in which an unpolarized electrode 60 made of silver-silver chloride or the like is laminated on an electrode 40 made of gold or the like, as shown in an example described later (see FIG. 13). Good.

  In measuring the function of the membrane protein, the counter electrode 41 may be disposed outside the hole 20. The electrode 40 and the counter electrode 41 can be connected to an electrophysiological measurement system such as a patch clamp measurement device.

  Further, in the lipid bilayer membrane substrate 2 of the present embodiment, the outermost surface of the lipid bilayer membrane substrate that is not coated with the lipid bilayer membrane is coated with the protein 15 as in the first embodiment.

  As shown in FIG. 4, the lipid bilayer membrane 30 may include a membrane protein 31 as in the first embodiment described above. In the lipid bilayer membrane substrate of this embodiment shown in FIG. 4, for example, by measuring an ionic current that has passed through the membrane protein 31 with a patch clamp measurement device, the function of the membrane protein can be measured.

  It is preferable that the lipid bilayer membrane substrate 2 of the present embodiment does not include the fluorescent substance 22 inside the hole 20.

  The embodiment of the present invention has been described in detail with reference to the drawings. However, the specific configuration is not limited to this embodiment, and includes designs and the like that do not depart from the gist of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to a following example.

[Example 1] Production of lipid bilayer membrane substrate A lipid bilayer membrane substrate having the structure shown in Fig. 1 was produced.

1. First, a silicon substrate was used for the substrate body 10. A thin film layer 11 made of a silicon oxide film layer having a thickness of 120 nm was formed on the upper surface of the substrate body 10 by a thermal oxidation method. Further, a hole 20 having a circular opening (diameter 2 μm or 4 μm) was formed by photolithography and dry etching. The depth was 1 μm. Further, the substrate body 10 under the thin film layer 11 made of the silicon oxide film layer is selectively etched using a potassium hydroxide solution (10% by weight), so that the four corners of the opening 21 of the hole 20 are overlaid. The hang part 11a was formed.

2. Production of Lipid Bilayer Membrane (Giant Lipid Membrane Vesicle) The lipid bilayer membrane 30 was formed of three types of lipid bilayer membranes having different charge states at around pH 7 as follows.

(1) Preparation of lipid-containing solution A mixed chloroform solution (concentration 2.5 mM) of diphytanyl phosphatidylcholine (DPhPC) (80 mol%) and cholesterol (20 mol%) was prepared as a neutral lipid bilayer membrane. A mixed chloroform solution of DPhPC (70 mol%), 1,2-dioleyl-sn-glycero-3-ethylphosphocholine (EDOPC) (10 mol%) and cholesterol (20 mol%) as a cationic lipid bilayer membrane (Concentration 2.5 mM) was prepared. A mixed chloroform solution (concentration 2.5 mM) of DPhPC (70 mol%), dioleyl phosphatidylserine (DOPS) (10 mol%) and cholesterol (20 mol%) was prepared as an anionic lipid bilayer membrane. At this time, 0.5 mol% of a lipid with rhodamine or nitrobenzooxadiazole (NBD) was added for fluorescence observation.

(2) Preparation of lipid bilayer membrane Next, prepared on (1) on an ITO substrate (a substrate in which ITO with a film thickness of 100 nm was thinned on SiO ₂ , size 40 × 40 mm, 50-100 Ω / cm). 200 μL of each chloroform solution was uniformly applied. 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. 500 μL of a sucrose aqueous solution was added dropwise. Further, the ITO substrate was placed on the top thereof so as not to enter bubbles, and the solution in the silicone rubber window was sandwiched between the ITO substrates. Next, a clip electrode is joined to the ITO substrate, and an AC electric field (sinusoidal wave, 1 V, 10 Hz) is applied for 2 hours in a constant temperature bath at 50 ° C., thereby forming a lipid bilayer membrane (giant lipid membrane vesicle by an electric field forming method). ) Sucrose dispersion.

3. Production of lipid bilayer membrane substrate Next, a solution containing fluo-4 as a fluorescent molecule 22 (200 mM glucose, 20 μM fluo-4, 1 mM EDTA, on the substrate body 10 having the hole 20 produced in “1.”) 100 μL of a mixed solution of 20 mM potassium chloride) was added dropwise. fluo-4 is a calcium ion indicator, and is a compound that emits green fluorescence in the presence of calcium ions. Furthermore, 2 μL of the sucrose dispersion of the giant lipid membrane vesicle prepared in (2) of “2.” is dropped on the substrate and left to stand for 5 minutes to develop the giant lipid membrane vesicle on the substrate. A lipid bilayer substrate coated with the membrane 30 was obtained. Further, the fluorescent molecules 22 were removed from the external solution 23 by replacing the external solution 23 in the hole 20 with a 200 mM glucose solution.

  FIG. 5 shows a fluorescence image when calcein is enclosed instead of fluo-4 in order to show that the fluorescent molecule 22 is stably trapped inside the hole 20. Bright green fluorescence was observed from the hole 20 a covered with the lipid bilayer membrane 30. As a result, it was confirmed that the fluorescent molecules 22 were stably trapped inside the hole 20a. On the other hand, fluorescent molecules were washed away from the hole 20b not covered with the lipid bilayer membrane 30, and fluorescence was not observed.

4). Next, bovine serum albumin (BSA) is coated as protein 15 on the outermost surface of the lipid bilayer membrane substrate not coated with the lipid bilayer membrane using the method shown below. It was.

(1) Preparation of BSA solution The BSA solution was prepared so that it might become 0.1 weight% BSA, 200 mM glucose, and 10 mM Hepes Buffer (pH 7.0).

(2) Immersion of lipid bilayer membrane substrate in BSA solution Next, the lipid bilayer membrane substrate was immersed in the BSA solution prepared in (1) for 1 hour. After immersion, it was washed 10 times with 200 mM glucose solution, and BSA A lipid bilayer substrate subjected to blocking was obtained. FIG. 6 shows fluorescence images of three types of lipid bilayer membrane substrates having different charge states after blocking. For fluorescence observation, lipids labeled with 0.5 mol% of NBD were contained in the lipid bilayer membrane, and BSA fluorescently labeled with 1 mol% of rhodamine was contained in BSA. In FIG. 6, (a) is a lipid bilayer membrane substrate using an anionic lipid membrane, (b) is a lipid bilayer membrane substrate using a neutral lipid bilayer membrane, and (c) is This is a lipid bilayer membrane substrate using a cationic lipid bilayer membrane.

  From FIG. 6, in the case of blue light excitation (B excitation), green fluorescence derived from a lipid bilayer was observed. In the case of green light excitation (G excitation), red fluorescence derived from bovine serum albumin was observed on the outermost surface of the lipid bilayer membrane substrate not covered with the lipid bilayer membrane in any substrate. Furthermore, in the lipid bilayer membrane substrate using the anionic lipid bilayer membrane and the cationic lipid bilayer membrane, red fluorescence was also observed on the lipid bilayer membrane. This is considered to be because BSA molecules are adsorbed on the lipid bilayer membrane in the anionic lipid bilayer membrane and the cationic lipid bilayer membrane.

  From this result, it became clear that a neutral lipid membrane having no electric charge is particularly suitable as the lipid bilayer membrane used in the lipid bilayer membrane substrate.

[Comparative Example 1]
A lipid bilayer membrane using a neutral lipid membrane using the same method as “1.” to “3.” in Example 1 except that blocking with BSA in “4.” in Example 1 is not performed. A substrate was produced.

[Test Example 1] Measurement of ion inflow For the lipid bilayer substrates of Example 1 and Comparative Example 1, an aqueous calcium chloride solution was added to the outside 23, and the time-dependent change in fluorescence intensity inside the pores was measured using a fluorescence microscope. did. The results are shown in FIG. Here, as typical observation results, fluorescence images immediately after adding the calcium chloride aqueous solution (t = 0), 10 minutes, 20 minutes, and 30 minutes are shown. 7 is a lipid bilayer membrane substrate with a BSA coating (lipid bilayer membrane substrate using a neutral lipid membrane of Example 1), and the lower row in FIG. 7 is a lipid bilayer membrane substrate without a BSA coating ( The result of Comparative Example 1) is shown. Each of the images is composed of a circular lipid bilayer membrane 30, a hole portion 20a covered with the lipid bilayer membrane, and a hole portion 20b not covered with the lipid bilayer membrane.

  Immediately after the calcium chloride aqueous solution was added (t = 0), almost no change in fluorescence intensity was observed on any lipid bilayer membrane substrate. In the lipid bilayer membrane substrate not subjected to blocking with BSA of Comparative Example 1, fluo-4 fluorescence indicating inflow of calcium ions was observed from the pore 20a covered with the lipid bilayer membrane in about 10 minutes. On the other hand, in the lipid bilayer membrane substrate subjected to blocking with BSA, no significant change in fluorescence intensity was observed even after 30 minutes.

FIG. 8 shows a graph plotting changes with time in fluorescence intensity of the lipid bilayer membrane substrates of Example 1 and Comparative Example 1.
From FIG. 8, the lipid bilayer membrane substrate blocked with BSA of Example 1 has very little ion inflow compared to the lipid bilayer membrane substrate not blocked with BSA of Comparative Example 1. Was confirmed.

  FIG. 9 is a schematic diagram illustrating a mechanism that can be presumed with respect to the inflow of ions from the pores covered with the lipid bilayer membrane. Conventionally, it is known that a thin water layer having a thickness of 1 to 2 nm exists between a lipid bilayer membrane and a substrate surface. It is thought that calcium ions diffuse through this water layer and ion inflow occurs. BSA is usually used to suppress non-specific adsorption of proteins and the like, but it is considered that the lipid bilayer membrane substrate of this embodiment plays a role as a block layer for ion diffusion. That is, the outer periphery of the lipid bilayer membrane serves as an interface for ion inflow from the outer liquid portion, and it is assumed that the ion inflow was suppressed by blocking the outer periphery of the lipid bilayer membrane with BSA. It was done.

[Example 2] Preparation of membrane protein-containing lipid bilayer substrate A lipid bilayer substrate having the structure shown in Fig. 2 was prepared.

1. Production of Substrate A substrate was produced in the same manner as in “1.” of Example 1.

2. Preparation of lipid bilayer membrane (giant lipid membrane vesicle) Then, using the same method as “2.” in Example 1, sucrose dispersion of lipid bilayer membrane (giant lipid membrane vesicle) using a neutral lipid membrane A liquid was prepared.

3. Production of Lipid Bilayer Substrate Next, a lipid bilayer membrane substrate was produced using the same method as “3.” in Example 1.

4). Blocking with BSA Next, using the same method as “4.” in Example 1, a lipid bilayer membrane substrate subjected to blocking with BSA was obtained.

5. Embedding Membrane Protein Next, α-hemolysin as a model channel protein was introduced into the lipid bilayer membrane of the lipid bilayer membrane substrate. It is known that α-hemolysin molecules are 7-merized in lipid bilayers to form transmembrane pores. Therefore, under observation with a fluorescence microscope, first, an aqueous calcium chloride solution is added to the outside 23, and after 20 minutes, an α-hemolysin solution is added to the outer liquid part of the lipid bilayer substrate so that the final concentration is 2 nM, Transmembrane holes were formed. The time course of the lipid bilayer membrane substrate is shown in FIG.

  From FIG. 10, it was confirmed that no fluorescence indicating ion inflow from the pore interior 20a was observed even after 20 minutes, and there was no ion inflow in the lipid bilayer membrane substrate. When α-hemolysin was added after 20 minutes, green fluorescence indicating the inflow of calcium ions from the inside 20a of the pores was observed after about 50 minutes. This suggests that calcium ions have flowed into the pore interior 20a through the transmembrane pore formed by α-hemolysin.

FIG. 11 shows a graph plotting changes over time in the fluorescence intensity of the lipid bilayer membrane substrate.
From FIG. 11, a rapid increase in fluorescence intensity was observed in about 2500 seconds. This is considered to be due to calcium ion transport by α-hemolysin.
From these results, it was confirmed that the function measurement of the membrane protein can be performed by using the lipid bilayer membrane substrate of the present embodiment.

[Example 3] Production of lipid bilayer membrane substrate for electrophysiological measurement A lipid bilayer membrane substrate having the structure shown in Fig. 3 was produced.
FIG. 12 is a schematic diagram of a cross section during the production of the lipid bilayer membrane substrate (a stage before the opening 21 of the hole 20 is covered with the lipid bilayer membrane 30). With reference to FIG. 12, a method for producing a lipid bilayer substrate for electrophysiological measurement will be described.

1. Production of Substrate A Si wafer covered with an insulating layer 13 (a thermal oxide film having a thickness of 120 nm was used) was used as the substrate 14. An electrode layer 40 (electrode 40) having a thickness of about 60 nm was formed on the Si wafer 14 by depositing gold 60 nm and patterning it by photolithography. This electrode layer 40 is connected to a sufficiently large pad portion (300 μm square), and can be connected to an electrophysiological measuring device such as a patch clamp measuring instrument by ordinary wire bonding.
Further, a 1 μm thick silicon nitride film was deposited as an insulating layer 12 on the electrode layer 40 by plasma CVD. Further, a 200 nm thick silicon oxide film was deposited on the insulating layer 12 as a thin film layer 11 which is an overhang shape layer by a sputtering method.
A hole 20 and an opening 21 were formed on the substrate 10 thus formed by using a resist film 50 by a photolithography method and a dry etching method. The etching for forming the hole was performed until it reached the electrode layer 40.

  In this dry etching process, by utilizing the selectivity between the thin film layer 11 which is an overhang shape layer made of a silicon oxide film and the insulating layer 12 made of a silicon nitride film, an overhang as shown in FIG. Part 11a was formed. After forming the hole 20 and the opening 21, the resist film 50 was washed and removed to obtain a substrate (the lipid bilayer membrane substrate 3 in the process of production) provided with the electrode 40 at the bottom of the hole 20.

2. Preparation of lipid bilayer membrane (giant lipid membrane vesicle) Then, using the same method as “2.” in Example 1, sucrose dispersion of lipid bilayer membrane (giant lipid membrane vesicle) using a neutral lipid membrane A liquid was prepared.

3. Production of Lipid Bilayer Substrate Next, a lipid bilayer membrane substrate was produced using the same method as “3.” in Example 1.

4). Blocking with BSA Next, using the same method as “4.” in Example 1, a lipid bilayer membrane substrate subjected to blocking with BSA was obtained.

5. Embedment of membrane protein Furthermore, a lipid bilayer membrane substrate having the structure shown in FIG. 4 was prepared by introducing a membrane protein. Specifically, the membrane protein 31 was reconstituted by a vesicle fusion method in a portion covering the opening 21 in the lipid bilayer membrane 30. Thereby, when a voltage is applied between the electrode 40 and the counter electrode 41, the ionic current through the membrane protein can be detected by measuring the current flowing between them.

[Example 4] Preparation of lipid bilayer membrane substrate having an unpolarized electrode layer on the electrode FIG. 13 shows a process during the production of the lipid bilayer membrane substrate (before the opening 21 of the pore 20 is covered with the lipid bilayer membrane 30). FIG. With reference to FIG. 13, a method for producing a lipid bilayer membrane substrate for electrophysiology comprising an unpolarized electrode layer on an electrode will be described.

1. Production of Substrate A substrate was produced using the same method as “1.” in Example 3 until a substrate having the electrode 40 at the bottom of the hole 20 was obtained. Note that a silver-silver chloride electrode was employed as the nonpolarizing electrode portion 60. First, 200 μL of silver plating solution (Precious Fab Ag4710, manufactured by Tanaka Kikinzoku Co., Ltd.) was dropped onto the hole. Subsequently, the silver wire connected to the manual prober was put in the plating solution, and the electrode part 40 was also connected to the manual prober. Subsequently, the surface of the electrode part 40 was silver-plated by supplying a constant current (0.1 to 0.5 nA) between the silver wire and the electrode 40 for 1 minute. Thereafter, the substrate was immersed in a chlorine bleach solution for 2 minutes to chlorinate the silver surface to obtain a substrate (a lipid bilayer membrane substrate 4 in the process of production) provided with a silver-silver chloride electrode. FIG. 14 shows an electron microscope image inside the hole.

  From FIG. 14, it was confirmed that a silver-silver chloride electrode portion was formed at the bottom of the hole. Moreover, since the silver-silver chloride surface has unevenness | corrugation and the surface area is increasing, it turned out that it is a more preferable shape when performing electrophysiological measurement.

2. Preparation of lipid bilayer membrane (giant lipid membrane vesicle) Then, using the same method as “2.” in Example 1, sucrose dispersion of lipid bilayer membrane (giant lipid membrane vesicle) using a neutral lipid membrane A liquid was prepared.

3. Production of Lipid Bilayer Substrate Next, a lipid bilayer membrane substrate was produced using the same method as “3.” in Example 1.

4). Blocking with BSA Next, using the same method as “4.” in Example 1, a lipid bilayer membrane substrate subjected to blocking with BSA was obtained.

5. Embedment of membrane protein Furthermore, a lipid bilayer substrate having a membrane protein was prepared by introducing the membrane protein. Specifically, a membrane protein was reconstituted by a vesicle fusion method in a portion covering the opening in the lipid bilayer membrane. Thereby, when a voltage is applied between an electrode and a counter electrode, an ionic current via a membrane protein can be detected by measuring a current flowing between the electrodes.

  In the lipid bilayer membrane substrate of this embodiment, ion inflow is suppressed. Therefore, by using the lipid bilayer membrane substrate of this embodiment, it is possible to measure the function of membrane proteins at the molecular level.

1 Lipid bilayer substrate 1
2 Lipid bilayer substrate 2
3 Lipid bilayer substrate 3 in the process of preparation
4 Lipid bilayer substrate 4 in the process of production
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 Silicon wafer (substrate)
15 Protein 20 Pore 20a Pore covered with lipid bilayer 20b Pore not covered with lipid bilayer 21 Opening (opening)
22 Fluorescent substances (fluorescent molecules)
23 External liquid portion of pore 30 Lipid bilayer membrane 31 Membrane protein 40 Electrode (electrode layer)
41 Counter electrode 50 Resist film 60 Non-polarized electrode

Claims (6)

A lipid bilayer membrane substrate comprising a substrate provided with pores, wherein the apertures of the pores are coated with a lipid bilayer membrane,
A lipid bilayer membrane substrate in which an outermost surface of the lipid bilayer membrane substrate not covered with the lipid bilayer membrane is coated with a protein.   The lipid bilayer membrane substrate according to claim 1, further comprising a thin film layer having an overhang portion formed so as to extend in a direction of narrowing the width of the opening of the hole portion on the surface of the substrate.   The lipid bilayer membrane substrate according to claim 1 or 2, wherein a plurality of the holes covered with the lipid bilayer membrane are provided in the substrate.   Furthermore, the lipid bilayer membrane substrate as described in any one of Claims 1-3 which has an electrode in the bottom part of the said hole.   The lipid bilayer membrane substrate according to claim 4, wherein the electrode is an electrode composed of silver and silver chloride (silver-silver chloride electrode).   The lipid bilayer membrane substrate according to any one of claims 1 to 5, wherein the protein is bovine serum albumin.