JP2014178121A - Hydrogel array substrate and manufacturing method of the substrate, and bimolecular lipid membrane array substrate and manufacturing method of the substrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a hydrogel array substrate and a manufacturing method of the substrate which are capable of developing vesicles without using metal cation and arranging a bimolecular lipid membrane on hole portions filled with hydrogel, and a bimolecular lipid membrane array substrate in which the bimolecular lipid membrane is arranged on the hole portions and a manufacturing method of the membrane.SOLUTION: There are provided: [1] a hydrogel array substrate which includes at least a substrate 10, a plurality of hole portions 20 arrayed on a surface of the substrate, and hydrogel 21 having electric charges which are filled in the hole portions; [2] the hydrogel array substrate in which the hole portions filled with hydrogel having positive electric charges and the hole portions filled with hydrogel having negative electric charges are severally arrayed on the same substrate; and [3] a bimolecular lipid membrane array substrate 4 in which the bimolecular lipid membrane 30 having electric charges opposite from the electric charges of the hydrogel filled in the hole portion is arranged so as to cover an opening portion 25 of the hole portion.

Description

  The present invention relates to a hydrogel array substrate, a method for producing the substrate, a lipid bilayer array substrate, and a method for producing the substrate.

  With the development of nanotechnology to date, the level of microfabrication has reached the order of 10 nanometers, and biochips in which biomolecules are arrayed on a substrate have been actively produced using the microfabrication technology. ing. Drug screening for confirming the influence of a drug on a living body, which is an application of a biochip, is an indispensable technique for drug discovery development. Membrane proteins are attracting attention as targets for drug discovery, and biochips in which membrane proteins related to the occurrence and treatment of various diseases are arrayed can be expected as high-throughput drug screening substrates (Non-patent Document 1).

  As a method of arraying biomolecules on a substrate, there is a method of immobilizing biomolecules on a substrate through physical adsorption or chemical bonding. However, in particular, when a protein is immobilized on a substrate, the protein may be denatured by any of the physical adsorption and chemical bonding methods. In particular, since the membrane protein retains its activity in the lipid bilayer membrane, there is a problem that immobilizing the membrane protein isolated from the lipid bilayer membrane on the substrate significantly impairs its activity. . Therefore, there is a demand for a technique for arraying (arranging and arranging biomolecules, in particular, membrane proteins) while keeping the activity of the membrane proteins.

  As a conventional method for immobilizing membrane proteins, a black membrane formed by dissolving lipid molecules in an organic solvent such as n-decane and applying the organic solvent to a small hole provided in a plastic plate or the like in an aqueous solution. A method for fusing membrane proteins (Non-patent Document 2) is known. In this black film formation method, it has been pointed out that the organic solvent remaining in the formed black film and the non-uniformity of the black film may affect the function and form of the membrane protein.

  In order to improve these problems, the present inventors have proposed a method of using a substrate having a hole on the substrate and having an overhang shape at the opening of the hole (Non-Patent Document 3). . Specifically, a lipid bilayer membrane containing a membrane protein is prepared by filling the hole with a hydrogel and expanding a vesicle (giant vesicle) which is a spherical lipid bilayer membrane on the hydrogel. Has proposed a substrate that is placed on top of a hydrogel.

  In the substrate proposed previously, since the hydrogel filled in the hole can support the lipid bilayer membrane, the influence of the lipid bilayer membrane on osmotic pressure and temperature change can be reduced. In addition, since the lipid bilayer can be arranged on the substrate by spreading the vesicle on the substrate, the conventional black film forming method can solve the problem that the organic solvent remains on the lipid bilayer.

Drews J. Science, 287: 1960-1964 (2000) "New Patch Clamp Experiment Method" edited by Yasunobu Okada (2001, Yoshioka Shoten) “Gelated microhole array mimicking cytoplasmic structure” Tanaka et al. (2012, 33rd JSAP Autumn Meeting, No. 12p-H4-5)

  The method of disposing a lipid bilayer on the hydrogel filled in the hole portion includes disposing an aqueous solution containing vesicles on the hydrogel, and then adding a metal cation such as calcium ion or sodium ion to the aqueous solution. By adding the inside, an electrostatic attractive force is applied between the vesicle and the substrate, and the vesicle is developed in a state where the vesicle is in close contact with the substrate surface and the hydrogel.

  However, in the upper part of the hole filled with the hydrogel, the metal cation diffuses inside the hydrogel, so that the electrostatic attractive force obtained on the hydrogel is higher than the electrostatic attractive force obtained on the substrate surface. Will be weaker. As a result, there is a problem that the development of vesicles hardly occurs and the formation efficiency of the lipid bilayer film is deteriorated. Furthermore, the metal cation added to develop the vesicle changes the ionic composition in the hole and further increases the ionic strength in the hole. There was a problem of affecting the function of the membrane protein.

  The present invention has been made in view of the above circumstances, and is a hydrogel capable of developing a vesicle without using a metal cation and disposing a lipid bilayer membrane on a hole filled with the hydrogel. An object of the present invention is to provide an array substrate and a manufacturing method thereof, and a lipid bilayer array substrate in which a lipid bilayer is disposed on the hole and a manufacturing method thereof.

[1] A hydrogel array substrate comprising at least a substrate, a plurality of holes arranged on the surface of the substrate, and a hydrogel having a charge filled in the holes.

[2] The hole filled with a hydrogel having a positive charge and the hole filled with a hydrogel having a negative charge are respectively arranged on the same substrate. 1].
According to this configuration, hydrogels having different electrical properties can be arrayed on the same substrate.

[3] A lipid bilayer membrane array substrate comprising the lipid bilayer membrane provided on the hydrogel array substrate according to [1] or [2], wherein the charge of the hydrogel filled in the hole portion A lipid bilayer membrane substrate, wherein a lipid bilayer membrane having a charge opposite to that of the hole portion is arranged so as to cover the opening of the hole.

  It is preferable that the hydrogel and the lipid bilayer membrane are in contact with each other at the opening of the lipid bilayer membrane array substrate. More preferably, the entire upper surface (upper part) of the hydrogel exposed in the opening is in contact with the lipid bilayer membrane. With this configuration, the membrane structure of the lipid bilayer membrane is supported by the hydrogel, whereby the membrane structure can be more stably maintained.

  The hydrogel is preferably a chemical gel in which polymers are crosslinked by covalent bonds. By using a chemical gel as the hydrogel, the lipid bilayer can be more stably supported, and moisture, a fluorescent substance, and the like can be stably held in the chemical gel. Moreover, since various functions and properties can be imparted to the chemical gel, hydrogels having different functions and properties can be patterned (arranged) on the same substrate.

  The hydrogel may contain a fluorescent substance such as a fluorescent molecule (fluorescent dye). For example, by including different types of fluorescent molecules in the hydrogel filled in each hole, the hole and the hydrogel pattern arranged on the substrate can be confirmed by fluorescence observation. Furthermore, by arranging a lipid bilayer membrane and a membrane protein on the hole and hydrogel, the function of the membrane protein can be measured by fluorescence observation.

  An electrode may be disposed in the hole. With respect to a plurality of holes on the substrate, electrodes that are electrically independent from each other may be disposed in each hole, or electrodes that are electrically connected to each other may be individually arranged. You may arrange | position in a hole. By disposing the electrode in the hole, the environment in the hole can be electrically measured (monitored). Furthermore, the function of the membrane protein can be electrically measured by disposing a lipid bilayer membrane and a membrane protein on the hole and the hydrogel.

[4] The method for producing a hydrogel array substrate according to [1] or [2], wherein a filling step of filling the hole with a solution containing a monomer constituting the hydrogel, and in the solution And a gelling step of gelling the monomer to form the hydrogel in the hole.

[5] The method for manufacturing a hydrogel array substrate according to [1] or [2], wherein the first solution containing the monomer constituting the first hydrogel is filled in the plurality of holes. Filling and gelling (polymerizing) the monomer in the first solution in the first group of the plurality of holes to form the first hydrogel in the first group of holes And a second filling step of filling a second group of holes different from the first group out of the plurality of holes with a second solution containing a monomer constituting the second hydrogel. And a second gelation step of gelling (polymerizing) the monomer in the second solution in the second group of holes to form a second hydrogel in the second group of holes. A method for producing a hydrogel array substrate, comprising:

  According to the manufacturing method, among a plurality of holes provided on the same substrate, for example, 1 to 100 holes constituting an arbitrary group (first group) are filled with the first hydrogel, For example, 1 to 500 holes constituting another arbitrary group (second group) different from the previous group can be filled with the second hydrogel. Here, the case where the plurality of holes provided on the substrate are divided into the first group and the second group has been described, but it is further divided into a large number of groups, for example, the first group to the tenth group. Good. If different types of hydrogels are filled in the holes divided into 10 groups (10 groups), 10 types of hydrogels can be patterned on the same substrate. In addition, since the said 1st group and the 2nd group are contained in the many groups, the manufacturing method at the time of dividing into a group larger than 2 groups corresponds to the manufacturing method as described in said [5].

[6] The photopolymerization initiator is contained in a solution containing the monomer, and the monomer is gelled (polymerized) by irradiating the hole filled with the solution with light. 4] or the method for producing a hydrogel array substrate according to [5].

  The hydrogel is preferably a photocurable gel that gels upon irradiation with light. If a photocurable gel is used, it is easy to make the solution in an arbitrary hole portion gelled at an arbitrary timing among the plurality of hole portions on the substrate. Moreover, if two or more types of photocurable gels are used, arbitrary types of photocurable gels can be formed in arbitrary hole portions among the plurality of hole portions arranged at high density on the substrate. That is, the hydrogel array can be patterned. In order to irradiate light to the local area of the substrate with high accuracy and high efficiency, a photomask used in the formation of a conventional resist film may be used. By using a photomask, it is possible to pattern all the holes on the substrate with a single light irradiation.

[7] A solution containing a vesicle is placed on each step of the method for producing a hydrogel array substrate according to [4] or [5] above, and the hole filled with the hydrogel, and the vesicle And a lipid bilayer membrane forming step of spreading the vesicle on or near the opening of the hole and covering the opening with a lipid bilayer. A method for manufacturing an array substrate.

[8] The method for producing a lipid bilayer array substrate according to [7], wherein the vesicle composed of lipid molecules having a charge opposite to that of the hydrogel is used.

  When the charge of hydrogel and vesicle is different from each other, electrostatic attractive force (interaction) occurs between the two on the substrate, so that the efficiency of vesicle deployment is improved and the lipid bilayer is placed on the hole and hydrogel. Efficiency can be improved significantly.

  In the present invention, the size of the vesicle is not particularly limited. Here, the term “vesicle” is defined as a term including a commonly used “giant vesicle”. In general, a vesicle having a relatively large diameter is referred to as a giant vesicle, but the determination of whether or not the vesicle is giant depends on the subjectivity of the experimenter. Therefore, in the present specification and claims, unless otherwise specified, Does not distinguish huge vesicles.

  In the method for producing a lipid bilayer array substrate of the present invention, it is preferable to use a vesicle having a large diameter (that is, a giant vesicle) in order to efficiently cover the opening of the hole.

  If the hydrogel substrate of the present invention is used, since the charged hydrogel is contained in the hole portion, the vesicle is settled on or near the hole portion, and the hydrogel and the vesicle are allowed to interact with each other. The vesicle can be developed and the lipid bilayer membrane can be disposed on the hole without using a metal cation that has been conventionally required. As a result, the metal cation penetrates into the hydrogel in the hole, and there is no problem that affects subsequent use. In addition, the hydrogel array substrate according to the present invention has an improved efficiency of deploying vesicles compared to conventional products filled with a hydrogel having no charge. Can be improved (efficiency of covering the hole with the lipid bilayer membrane). By filling any kind of hydrogel into the hole at an arbitrary position, the hydrogel can be arrayed on the substrate in an arbitrary pattern.

  According to the lipid bilayer membrane array substrate of the present invention, the hydrogel can stably support the membrane structure of the lipid bilayer membrane. For this reason, even when the size (diameter) of the opening of the hole is larger than before, the opening can be covered with the lipid bilayer membrane. In addition, by supporting the lipid bilayer membrane with the hydrogel, the stability of the lipid bilayer membrane is improved, and the lipid bimolecule to chemical changes such as osmotic pressure and pH, and physical impact such as vibration The resistance of the film can be improved. By arranging, for example, a membrane protein on a lipid bilayer membrane with improved membrane stability, functional analysis of the membrane protein can be performed stably, and highly reliable data can be obtained.

According to the method for producing a hydrogel array substrate of the present invention, the hole can be easily filled with hydrogel.
According to the method for producing a lipid bilayer array substrate of the present invention, since the hydrogel has a charge, the vesicle can be developed more efficiently than before and the lipid bilayer membrane can be easily arranged on the substrate.

1 is a schematic cross-sectional view of a first embodiment of a substrate according to the present invention. It is a schematic cross section of a second embodiment of a substrate according to the present invention. It is a schematic cross section of a third embodiment of a substrate according to the present invention. It is a schematic cross section of 4th embodiment of the board | substrate concerning this invention. It is a schematic cross section of 5th embodiment of the board | substrate concerning this invention. It is a schematic cross section of 6th embodiment of the board | substrate concerning this invention. It is a schematic cross section which shows a mode that the said upper part is removed by pushing the upper part of the hydrogel 21 protruding from the hole part 20 in the direction parallel to the board | substrate upper surface 11a. 2 is a fluorescence observation image of the upper surface of a lipid bilayer array substrate prepared in Example 1. FIG. (A) is the result of observing only the fluorescence emitted by calcein, and (b) is the result of observing the fluorescence emitted by both calcein and rhodamine. 6 is a fluorescence observation image of the upper surface of the hydrogel array substrate produced in Example 2. FIG. (A) is the result of observing only the fluorescence emitted by calcein, and (b) is the result of observing the fluorescence emitted by both calcein and rhodamine. 6 is a schematic diagram showing a state of a lipid bilayer array formed on the upper surface of a lipid bilayer array substrate produced in Example 4. FIG.

  Hereinafter, the present invention will be described with reference to the drawings based on preferred embodiments, but the present invention is not limited to such embodiments.

<< Hydrogel Array Substrate; First Embodiment >>
The hydrogel array substrate 1 shown in FIG. 1 is illustrated as a substrate of the first embodiment of the present invention. The hydrogel array substrate 1 includes a substrate body (substrate) 10, a plurality of holes (microcavities) 20 arranged on the surface 11 a of the substrate, and a hydrogel 21 having a charge filled in the holes 20. At least.

  The material of the substrate body 10 is not particularly limited, and examples thereof include silicon, glass, and plastic. Moreover, the thickness and shape of the substrate body 10 are appropriately adjusted according to the application. The thickness of the substrate body 10 can be set to 0.1 μm to 10 mm, for example. Examples of the shape of the substrate body 10 include a flat plate shape.

  When a hydrophobic material is used as the material of the substrate body 10, it is preferable that a hydrophilic layer (hydrophilic layer) 11 is disposed on the surface (upper surface) 10 a of the substrate body 10. A hydrophilic layer 11 is formed on the hydrogel array substrate 1 of the present embodiment. When a hydrophilic material such as glass is used for the substrate body 10, the hydrophilic layer 11 may not be disposed.

  The shape of the opening 25 of the hole 20 provided in the hydrogel array substrate 1 is not particularly limited. Examples of the shape when the opening 25 is viewed from the upper surface side of the substrate include shapes such as a circle, an ellipse, a triangle, a rectangle, and a polygon.

The diameter (diameter) of the opening 25 of the hole 20 or the longest length of the polygonal diagonal lines constituting the opening 25 or the diameter of the circumscribed circle of the polygon is not particularly limited, but is 0.1 μm to 10 μm. The degree is preferred.
By setting the lower limit of the above range to 0.1 μm or more, the hydrogel 21 can be easily filled into the hole 20. In a fourth embodiment to be described later, a sufficient amount of membrane protein 31 can be disposed in the lipid bilayer membrane 30 disposed above the hole portion 20, and the function of the membrane protein can be measured more easily. .
By setting the upper limit of 10 μm or less in the above range, it is possible to stably hold the hydrogel 21 inside the hole 20 and reliably prevent the hydrogel 21 from dropping from the hole 20.

  The depth of the hole 20 is not particularly limited, and may be appropriately designed according to the size of the opening 25 and the volume of the hydrogel 21 filled in the hole 20. The depth can be, for example, 0.1 μm to 10 μm.

  The number of holes 20 provided in the substrate body 10 is not particularly limited, and may be, for example, 1 to 10,000.

  The hydrogel 21 filled in the hole 20 is not particularly limited as long as it has a charge in the aqueous solution. It is preferable that the hydrogel 21 does not inhibit the formation of the lipid bilayer membrane 30 described later. The charge may be a positive charge or a negative charge. Moreover, as a charge which the functional group of the polymer which comprises the hydrogel 21 has, 1-3 positive charges or -1 to -3 negative charges are preferable.

  The hydrogel 21 is preferably a hydrophilic polymer. Examples of the hydrophilic polymer include (meth) acrylic resins. Examples of the acrylic resin include an acrylic resin obtained by polymerizing only one kind of charged monomer, acrylic acid having a negative charge or dimethylaminopropylmethacrylamide (DMAPMA) having a positive charge.

  In addition, the monomer having a charge and one or more neutral acrylic monomers such as acrylamide, N, N′-dimethylacrylamide or N-isopropylacrylamide are used as monomers, and these monomers are copolymerized. In addition, an acrylic resin crosslinked with a polyfunctional monomer such as methylene bis (meth) acrylamide or polyethylene glycol di (meth) acrylate can be used as the hydrogel 21. Moreover, you may comprise the hydrogel 21 using the derivative | guide_body of the said monomer.

  As one of the monomers constituting the hydrogel 21, a monomer having no charge may be used in combination with another monomer having a charge.

  The water content of the hydrogel 21 is not particularly limited and is appropriately adjusted according to the application. What is necessary is just to adjust content of the water contained in the said hydrogel 21 with respect to the total weight of the hydrogel 21, for example in the range of 1 to 99 weight%. The content is preferably 50 to 99% by weight, more preferably 70 to 95% by weight, and still more preferably 85 to 95% by weight. With these preferable water contents, the hydrogel 21 can imitate the cytoplasm or extracellular matrix of the cells constituting the living body.

  In the hydrogel array substrate 1, the volume of the hydrogel 21 in the hole portion with respect to the total volume of the hole portion 20 is preferably 50 to 100%, more preferably 80 to 100%, and more preferably 90 to 100%. More preferably. The hydrogel 21 is preferably filled from the bottom of the hole 20 to the opening 25 (height of the upper surface 11a). By filling up to the opening 25, the hydrogel 21 can support the lipid bilayer membrane 30 at the height of the upper surface 11a.

  The upper surface of the hydrogel 21 in the opening 25 of the hole 20 (the portion where the hydrogel 21 is exposed to the upper surface 11a side of the substrate) may be flat or not flat. For example, the upper surface may swell upward. The upper surface of the hydrogel 21 may bulge upward or be depressed due to the influence of surface tension or the like. If the degree of non-flatness is slight, there is almost no problem in forming the lipid bilayer 30 on the hydrogel 21.

  Each hole 20 provided in the hydrogel array substrate 1 may be filled with the same type of hydrogel 21 or may be filled with different types of hydrogel 21. For example, the hole 20 provided in the first region of the hydrogel array substrate 1 is filled with a cationic hydrogel having a positive charge, and the hole 20 provided in the second region of the same substrate The structure filled with the anionic hydrogel which has a negative charge can be illustrated.

  In this way, different types of lipid bilayers can be arranged in each region by placing or patterning hydrogels having different charges on the same substrate in each region. Specifically, in a single hydrogel array substrate, a lipid bilayer membrane having a negative charge is arranged above the first hole filled with a hydrogel having a positive charge, and the hydrogel having a negative charge is disposed. Examples thereof include a lipid bilayer membrane array substrate in which a lipid bilayer membrane having a positive charge is arranged on the upper part of a second hole filled with a gel. Details of the lipid bilayer membrane array substrate in which the lipid bilayer membrane is arranged on the hydrogel will be described later.

<< Method for producing hydrogel array substrate >>
As a method for manufacturing a hydrogel array substrate according to the present invention, a method for manufacturing the hydrogel array substrate 1 of the first embodiment will be exemplified.

  First, the hydrophilic layer (hydrophilic layer) 11 is formed on the surface 10a of the substrate body 10 made of a desired material. The method for forming the hydrophilic layer 11 is not particularly limited. For example, when the substrate body 10 is made of silicon, a method of forming a silicon oxide layer (hydrophilic layer) by thermal oxidation is exemplified. When the substrate body 10 is made of plastic, An example is formation of a hydrophilic layer by plasma cleaning.

  The method for forming the hole 20 in the substrate body 10 having the hydrophilic layer 11 formed on the surface 10a is not particularly limited. For example, a fine processing technique such as a photolithography method, an electron beam lithography method, or a dry etching method is applied. Can do. Specifically, after a predetermined resist pattern is formed on the hydrophilic layer 11, a desired number of holes 20 having a desired size can be formed by performing dry etching or wet etching. In FIG. 1, only one hole 20 is drawn for the sake of simplicity, but an array in which a plurality of holes 20 are arranged is actually formed.

  The method of filling the hole 20 with the hydrogel 21 is not particularly limited. For example, the hole 20 is filled with a solution containing the monomer of the hydrogel 21 before gelation, and a polymerization initiator is added as necessary. After the addition, before gelling, the solution (hydrogel solution) is dropped on the surface 11a of the substrate, the hole 20 is filled with the solution, and the solution in the hole 20 is gelled. Can be mentioned.

  As said hydrogel solution, the aqueous solution containing the monomer mentioned later is mentioned, for example. After the gelation, the hydrogel array substrate 1 of the first embodiment is obtained by removing the excess hydrogel 21 protruding from the hole 20. In order to prevent the hydrogel 21 from drying, the solution L may be placed on the surface 11a of the substrate, or the entire surface 11a of the substrate may be sealed by attaching a resin film or the like.

  The method for initiating a polymerization reaction for synthesizing the hydrogel 21 by polymerizing the monomer is not particularly limited, and examples thereof include a method in which a polymerization initiator is added to the composition containing the monomer for polymerization. Examples of the polymerization initiator and the polymerization method thereof include a method of adding ammonium persulfate (APS) and N, N, N ′, N′-tetramethylethylenediamine (TEMED) to an aqueous solution containing the monomer, an azobisisobutronitrile (AIBN), and the like. Examples thereof include a method of thermally decomposing in the composition, and a method of adding a photoreactive polymerization initiator such as riboflavin to the composition and irradiating with light.

  Since the polymer obtained by polymerization in an aqueous solution contains water, it can be used as it is as the hydrogel 21. Moreover, in order to change the composition of the aqueous solution contained in the hydrogel 21, an aqueous solution having a desired composition may be impregnated into the polymer. A polymer obtained by polymerization in a solution not containing water can be used as the hydrogel 21 by impregnating the polymer with a desired solution.

  In the case where different types of hydrogels 21 are filled into the individual hole portions 20 of the hydrogel array substrate 1, it is preferable to apply a polymerization method (gelation method) by light irradiation. The gelation method by light irradiation is not particularly limited, and examples thereof include a method of gelling only an arbitrary hole using a laser beam and a photomask.

  As a specific example of the gelation method, first, a first solution containing a monomer constituting the first hydrogel is dropped on the entire substrate surface 11a provided with a plurality of holes 20, and a plurality of holes are formed. Is filled with the first solution. Next, only a desired hole is irradiated with light using a photomask or the like to form the first hydrogel only in the desired hole. Subsequently, the first solution in the hole not irradiated with light is washed away, the second solution containing the monomer constituting the second hydrogel is dropped, and the hole in which the first hydrogel is not formed Fill the second solution. By irradiating the second solution in the hole with light using a photomask or the like, the second hydrogel is formed in the desired hole where the first hydrogel is not formed. In this way, by applying a stepwise polymerization method by light irradiation, various hydrogels can be formed in individual holes even in an array in which a plurality of holes are arranged at high density.

  What is necessary is just to adjust the water content of the hydrogel 21 suitably according to the kind etc. of the membrane protein to analyze the use and function. By examining the concentration of the monomer constituting the hydrogel 21 in the solution and the polymerization method thereof, it is possible to form the hydrogel 21 having an optimal water content according to the application. For example, when the hole 20 is filled with the hydrogel 21 composed of acrylamide and DMAPMA, the total concentration of the two monomers in the solution containing these two monomers is 1 with respect to the total mass of the solution. The method of adjusting at -30 weight% is mentioned. This concentration is preferably 3 to 20% by weight, more preferably 5 to 15% by weight.

  As a monomer constituting the hydrogel 21, one type of monomer may be used alone, or two or more types of monomers may be used in combination.

  When the number of monomers constituting the hydrogel 21 is two or more, the respective distribution ratios (blending ratios) are not particularly limited, and may be appropriately adjusted according to desired properties to be imparted to the hydrogel. For the purpose of producing a lipid bilayer array substrate to be described later, it is preferable that the weight ratio of the charged monomer to the total weight of all monomers is 1 to 20% by weight.

  As a manufacturing method of the hydrogel array substrate concerning this invention, the method of performing the filling process and gelation process which are demonstrated below in this order is preferable. Further, other steps than the two steps mentioned here may be added unless departing from the gist of the present invention.

[Filling process]
The filling step is a step of pouring a solution containing a monomer constituting the hydrogel 21 onto the upper surface 11a of the substrate 10 on which the hydrophilic layer 11 is disposed, and filling the solution into the hole 20 provided on the upper surface 11a of the substrate. It is.

  The monomer constituting the hydrogel 21 is a monomer that has not been polymerized (unpolymerized). The solvent for dissolving or dispersing the monomer is not particularly limited, and an aqueous solvent is preferable. The monomer concentration is appropriately adjusted according to the hardness and water content of the hydrogel 21.

  The amount of the solution poured onto the upper surface 11a of the substrate 10 is not particularly limited as long as the hole 20 can be filled, but the amount that can overflow the upper surface 11a, that is, the solution above the upper surface 11a. It is preferable to pour an amount that can be accumulated.

  The method of pouring the solution onto the upper surface 11a of the substrate 10 is not particularly limited, and after dropping the solution onto the upper surface 11a with a pipette or the like, by subjecting the substrate and the solution to ultrasonic treatment or vacuum treatment as necessary, It is possible to discharge bubbles from the hole 20 and fill the hole 20 with the solution.

[Gelling process]
The gelation step is a step of forming the hydrogel 21 in the hole 20 by gelling the monomer of the hydrogel 21 in the solution.
The method for polymerizing the monomer is as described above. When the solution containing the monomer of the hydrogel 21 is outside the hole 20 after the filling step, the following two methods (methods A and B) can be exemplified. Method A is a method in which the solution outside the hole 20 is removed and the monomer of the hydrogel 21 is gelled only inside the hole 20. Method B is a method in which the monomer of the hydrogel 21 is gelled inside and outside the hole 20 without removing the solution containing the monomer of the hydrogel 21 outside the hole 20. In the former case (in the case of method A), after replacing the external solution of the hole 20 with a solvent in which, for example, the monomer of the hydrogel 21 and water do not dissolve (do not flow out of the hole 20), the inside of the hole 20 Examples of the method for gelling the solution remaining in (1). In the latter case (method B), the upper part of the hydrogel 21 that has gelled outside (above) the hole 20 is removed by an appropriate method such as slowly peeling off, and the hydrogel 21 is only inside the hole 20. Can be left (see FIG. 7). The removal of the upper portion of the hydrogel 21 is preferably performed in a state where the upper surface 11a of the substrate is covered with an appropriate external solution L such as a physiological salt solution.

<< Hydrogel Array Substrate; Second Embodiment >>
As shown in FIG. 2, the hydrogel array substrate 2 is mentioned as 2nd embodiment of this invention. In addition to the configuration of the first embodiment, the hydrogel array substrate 2 includes a fluorescent material 22 (fluorescent molecules 22) in a hydrogel 21 inside the hole 20.
Other than this configuration, since it is the same as the hydrogel array substrate 1 of the first embodiment described above, the same portions are denoted by the same reference numerals and description thereof is omitted.

  The fluorescent material 22 is preferably a water-soluble fluorescent molecule that emits fluorescence when a state change inside the hole 20 occurs. The fluorescent molecule is more preferably a fluorescent molecule whose fluorescence intensity changes as the ion concentration inside the hole 20 changes. Specifically, for example, Fluo-4, Fura-2, Fluo-3 and the like capable of detecting a change in calcium ion concentration can be exemplified.

<< Hydrogel array substrate; Third embodiment >>
As shown in FIG. 3, the hydrogel array substrate 3 is mentioned as 3rd embodiment of this invention. In addition to the configuration of the first embodiment, the hydrogel array substrate 3 includes an electrode 40 in the hole 20 and a counter electrode 41 in the external solution L in the hole 20.
Other than this configuration, since it is the same as the hydrogel array substrate 1 of the first embodiment described above, the same portions are denoted by the same reference numerals and description thereof is omitted.

  As an arrangement method of the electrode 40, for example, when the hydrogel array substrate 3 is manufactured, the metal layer 40a is formed by a film forming method such as sputtering, and the metal layer 40a is appropriately patterned by etching or the like. The electrode 40 may be embedded and disposed so as to be exposed inside the hole 20 by covering the region excluding the portion exposed inside the hole 20 with the insulating layer 13 (second insulating layer 13). it can.

  When the metal layer 40a is formed by being embedded in the substrate body 10, the metal layer 40a needs to be electrically insulated. Examples of the insulating method include a method in which an insulator such as silicon oxide, silicon nitride, alumina, tantalum oxide, or resin is used as a material for the substrate body. When the substrate body 10 is a semiconductor or a conductor, a first insulating layer 12 (substrate insulating layer 12) is formed on the upper surface 10a of the substrate body 10, and a metal layer is formed on the upper surface of the first insulating layer. 40 may be arranged. The material of the first insulating layer 12 and the second insulating layer 13 is not particularly limited, and examples thereof include silicon oxide, silicon nitride, alumina, and tantalum oxide. The material which comprises the 1st insulating layer 12 and the 2nd insulating layer 13 may be the same, and may differ. Examples of the material of the metal layer 40a (electrode 40) include silver, silver chloride, gold, and platinum.

<< Lipid Bilayer Array Substrate; Fourth Embodiment >>
As shown in FIG. 4, as a fourth embodiment of the present invention, a lipid bilayer array substrate 4 is exemplified. In addition to the configuration of the first embodiment, the lipid bilayer membrane array substrate 4 is provided with a lipid bilayer membrane 30 so as to cover the upper part of the hydrogel 21 filled in the hole 20.
Other than this configuration, since it is the same as the hydrogel array substrate 1 of the first embodiment described above, the same portions are denoted by the same reference numerals and description thereof is omitted.
Below, the point from which the lipid bilayer membrane array substrate 4 of 4th embodiment differs from the hydrogel array substrate 1 of 1st embodiment is demonstrated.

  The type of lipid molecule constituting the lipid bilayer membrane 30 is not particularly limited as long as it can form a lipid bilayer membrane (lipid bilayer membrane). For example, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidyl Examples include serine (PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidic acid (PA), phosphatidylglycerol (PG), and sphingolipid. These lipids may be used alone or in combination of two or more.

  The lipid bilayer membrane 30 is preferably a lipid bilayer membrane composed of lipid molecules having a charge opposite to that of the hydrogel 21 in the hole 20. Specifically, when the hydrogel 21 has a positive charge, it is preferable to dispose a lipid bilayer membrane having a negative charge on the hydrogel 21, and when the hydrogel 21 has a negative charge, It is preferable to arrange a lipid bilayer membrane having a positive charge on the hydrogel 21. Thus, when the charges of the hydrogel 21 and the lipid bilayer membrane 30 are opposite, the lipid bilayer membrane 30 can be easily disposed on the hydrogel 21 by electrostatic interaction between the two, The lipid bilayer membrane 30 can be more stably held on the hydrogel 21.

  If the arrangement and stabilization of the lipid bilayer membrane by the electrostatic interaction are utilized, the positively charged hydrogel and the negatively charged hydrogel each have arbitrary holes in a single substrate having a plurality of holes. Further, a lipid bilayer array substrate is obtained in which lipid bilayer membranes filled in the portions and having a charge opposite to the charge of the hydrogel in each hole portion are arranged on the hydrogel in each hole portion. In this case, a membrane protein array substrate in which a plurality of types of membrane proteins are arranged (immobilized) at predetermined positions by containing different types of membrane proteins for each lipid bilayer membrane having different charges. Is obtained.

Examples of lipid bilayer membranes having a positive charge in a solution such as an aqueous solution include synthetic cationic lipids such as N- (1- (2,3-dioleooxy) propyl) -N, N, N-trimethyl-ammonium chloride, Examples thereof include a lipid bilayer composed of diphytanyl phosphatidylcholine (DPhPC) or the like.
Examples of the lipid bilayer membrane having a negative charge in a solution such as an aqueous solution include a lipid bilayer membrane composed of phosphatidylserine (PS) or the like.

  The lipid bilayer membrane may contain cholesterol molecules. Moreover, the lipid molecule constituting the lipid bilayer membrane may be one type or two or more types. The lipid bilayer membrane may contain neutral lipid molecules having no charge in the solution. Examples of the neutral lipid molecule include phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingolipid, and the like.

  Examples of the method of forming the lipid bilayer membrane 30 include a method of developing a vesicle (giant vesicle (giant lipid vesicle)) having a diameter of 10 μm or more on the hydrogel 21 and the surface 11a of the substrate.

  Typical methods for forming vesicles 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 an electric field forming method from the viewpoint of easy production of a huge vesicle and simple reaction time and reaction process. The electric field forming method is a method of forming a vesicle in an aqueous solution by applying an alternating electric field after forming a thin film of phospholipid on an electrode such as indium tin oxide (ITO). In order to obtain vesicles of uniform size, it is preferable to form a film made of uniform phospholipid molecules having a thickness of several tens of nm to several μm on the ITO substrate.

  The water contained in the hydrogel 21 may contain a pH buffer, various ions, an antioxidant, a surfactant and the like as long as the lipid bilayer membrane 30 is stably maintained. It is preferable that the water contained in the hydrogel 21 contains a component necessary for the function of the membrane protein 31 to be optimally exhibited.

  In the present embodiment, the membrane protein 31 is disposed on the lipid bilayer membrane 30 that covers the upper part (upper surface) of the hydrogel 21 filled in the hole 20. Therefore, “lipid bilayer array substrate 4” according to the present invention can be read as “membrane protein array substrate 4”. The lipid bilayer membrane 30 is covered with an external solution L outside the hole 20. For example, the function of the membrane protein 31 can be analyzed by adding a drug candidate substance or the like to the external solution L.

  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 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 the lipid bilayer membrane 30. Reconfigured inside.

  The membrane protein 31 fused to the lipid bilayer membrane 30 is a function that is held in the lipid bilayer membrane 30 and changes the ion concentration inside and outside the hole 20 with the lipid bilayer membrane 30 as a boundary. A membrane protein having is preferred. Examples of such membrane protein 31 include an ion channel type receptor.

  Examples of ion channel receptors include intercellular information transmission, temperature sensing, TRP (transient receptor potential) channels involved in inflammation and pain, intercellular information transmission and pain-related ATP receptors, and intercellular information transmission. Serotonin receptor involved in sexuality and emotion, NMDA receptor involved in intercellular communication and excitatory neurotransmission, AMPA receptor involved in intercellular communication and excitatory neurotransmission, intercellular communication and excitatory neurotransmission And kainic acid receptor involved in cell transfer, GABA receptor involved in intercellular signal transmission and inhibitory neurotransmission, and the like.

When the electrolyte solution is disposed inside and outside the hole 20, the electrolyte solution is preferably one in which the lipid bilayer membrane 30 in which the membrane protein 31 is embedded is stably held. Examples of the electrolyte solution include an aqueous solution containing 100 mM NaCl, 5 mM KCl, 2 mM CaCl ₂ , and 10 mM Tris-HCl (pH 7.4). The composition of the electrolyte solution disposed inside and outside may be the same or different.

<< Method for producing lipid bilayer array substrate >>
As a method for producing a lipid bilayer array substrate according to the present invention, a method for producing the lipid bilayer array substrate 4 of the fourth embodiment shown in FIG. 4 will be exemplified.
The production method is preferably a method in which the filling step, the gelation step, and the lipid bilayer formation step are performed in this order. In addition, other steps other than the three steps listed here may be added without departing from the spirit of the present invention.

  Since the filling step and the gelation step in the method for producing the lipid bilayer array substrate are the same as the filling step and the gelation step in the method for producing the hydrogel array substrate described above, description thereof is omitted here.

[Lipid bilayer formation process]
In the lipid bilayer formation step, a vesicle is added to the solution L on the upper surface 11a of the substrate 10, the vesicle is settled on the upper surface 11a of the substrate 10, the vesicle is developed in the vicinity of the hole 20, and the hole This is a step of covering 20 openings 25 with a lipid bilayer membrane 30.

  As the vesicle, those prepared by the method described above can be used. The size (diameter) of the vesicle is preferably sufficiently larger than the opening 25. For example, vesicles having a size of 10 μm to 100 μm (giant vesicles) can be used, but the size is not limited to this range, and vesicles having other sizes may be used.

  The method for adding the vesicle to the solution L on the upper surface 11a of the substrate 10 is not particularly limited, and examples thereof include a method of preparing a vesicle dispersion containing vesicles and adding the vesicle dispersion to the solution L. .

  The method for causing the vesicles added to the solution L to settle so as to contact the upper surface 11a is not particularly limited. Usually, since the specific gravity of the vesicle is heavier than that of the solution L, after adding the vesicle to the solution L and waiting for it to settle naturally, the vesicle can be settled on the upper surface 11a of the substrate.

  As a standard of the concentration of the vesicle in the vesicle dispersion, it is preferable that the added vesicle has such a concentration that it occupies a wide area of the upper surface 11a of the substrate 10, for example, an area of 80% or more of the upper surface 11a. . The concentration of the vesicle dispersion can be adjusted to, for example, a concentration of 0.1 mM to 10 mM, but is not limited to this concentration range, and may be used at other concentrations.

<< Lipid Bilayer Array Substrate; Fifth Embodiment >>
As shown in FIG. 5, a lipid bilayer array substrate 5 is mentioned as a fifth embodiment of the present invention. In addition to the configuration of the fourth embodiment, the lipid bimolecular membrane array substrate 5 includes a fluorescent substance 22 (fluorescent molecule 22) in the hydrogel 21 inside the hole 20.
Other than this configuration, since it is the same as the hydrogel array substrate 4 of the fourth embodiment described above, the same parts are denoted by the same reference numerals and description thereof is omitted. Note that the lower surface 30 a of the lipid bilayer membrane of the present embodiment is in contact with the upper surface of the hydrogel 21 in the same manner as the lower surface of the lipid bilayer membrane of the hydrogel array substrate 1 of the first embodiment.

  If the lipid bilayer membrane array substrate 5 of the present embodiment is used, for example, an ion permeable membrane protein 31 is arranged on the lipid bilayer membrane 30 and the hydrogel 21 in the hole 20 is interposed through the membrane protein 31. It can be detected by a fluorescence microscope or the like that the fluorescence intensity of the fluorescent molecules 22 contained in the hydrogel 21 has changed when ions have flowed into or when the ions have flowed out of the hydrogel 21. That is, the function of the membrane protein 31 can be detected as a change in the fluorescence intensity of the fluorescent molecules in the hydrogel 21.

<< Lipid Bilayer Array Substrate; Sixth Embodiment >>
As shown in FIG. 6, a lipid bilayer array substrate 6 is mentioned as a sixth embodiment of the present invention. In addition to the configuration of the third embodiment, the lipid bilayer membrane array substrate 6 has a lipid bilayer membrane 30 including a membrane protein 31 disposed on the hole 20 and the hydrogel 21.
Other than this configuration, since it is the same as the hydrogel array substrate 3 of the third embodiment described above, the same portions are denoted by the same reference numerals and description thereof is omitted.

  By using the lipid bilayer array substrate 6 of the present embodiment, for example, when an ion channel protein is arranged as the membrane protein 31, an ionic current that has passed through the ion channel protein is converted into the electrode 40 and / or the counter electrode. By measuring with an electrometer connected to 41, the function of the membrane protein 31 can be measured by an electrophysiological technique. That is, the function of the membrane protein 31 can be detected as a potential difference inside or outside the hole 20 or a change in current value.

  As described above, when producing the lipid bilayer membrane array substrate of the present invention, the hydrogel can be produced without adding a metal cation by applying an electrostatic attraction between the lipid bilayer membrane and the hydrogel. A lipid bilayer can be easily formed on the filled hole. As a result, the ionic composition in the hole 20 and the ionic composition of the external solution L that are separated by the lipid bilayer membrane can be arbitrarily set. In addition, by using a photocurable gel and a photomask, it is possible to produce a hydrogel array substrate in which a positively charged hydrogel and a negatively charged hydrogel are arrayed on the same substrate in an arbitrary pattern. it can. If this hydrogel array substrate is used, a lipid bilayer membrane having a positive charge and a lipid bilayer membrane having a negative charge can be easily produced. Further, when forming a lipid bilayer membrane array, a membrane protein array substrate useful for drug screening can be provided simply and efficiently by using a vesicle into which a membrane protein has been introduced.

  EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited by these examples.

Example 1
Below, the specific example of preparation of the lipid bilayer membrane array board | substrate 4 shown in FIG. 4 is shown.
<Formation of hole>
A silicon substrate was used as the substrate body 10. A silicon oxide film layer (hydrophilic layer) 11 having a thickness of 120 nm was formed on the upper surface 10a of the substrate body 10 by a thermal oxidation method. Further, a plurality of hole portions 20 having circular openings 25 (diameter 4 μm) were formed by using a photolithography method and a dry etching method. The depth of each hole 20 was 1 μm.

<Preparation of huge vesicle>
The lipid bilayer membrane 30 was formed as follows.
Chloroform in a ratio of DPhPC: DOPS: cholesterol = 7: 2: 1 (molar ratio) so that the total concentration of diphytanylphosphatidylcholine (DPhPC), dioleylphosphatidylserine (DOPS) and cholesterol is 2.5 mM. A dissolved mixed chloroform solution was obtained. Furthermore, a mixed chloroform solution was prepared by dissolving rhodamine-dihexadecanylphosphatidylethanolamine (Rhod-DHPE), which is a fluorescent labeling agent, in this mixed chloroform solution so as to be 25 μM.

Subsequently, 200 μL of the mixed chloroform solution is formed on a substrate (ITO substrate) (size: 40 × 40 mm, resistance: 50-100 Ω / sq) on which a thin film made of indium tin oxide having a thickness of 100 nm is formed on SiO _2. Was applied uniformly. This substrate was dried under reduced pressure at room temperature for 2 hours to completely remove chloroform as a solvent, thereby forming a uniform phospholipid thin film on the ITO substrate. On the phospholipid thin film, silicone rubber having a window portion was placed in close contact, and 200 μL of 200 mM sucrose aqueous solution was dropped inside the window portion. Furthermore, another ITO substrate was placed on top of the silicone rubber, and the sucrose aqueous solution inside the silicone rubber window was sandwiched between the two ITO substrates and sealed. The silicone rubber having the window portion includes a window portion formed by punching out silicone rubber having an outer size of 30 × 30 mm and a thickness of 1 mm in a size of 20 × 20 mm.

  Next, a clip electrode is joined to each ITO substrate, and an alternating electric field (sine wave, 1 V, 10 Hz) is applied on a hot plate at 60 ° C. for 2 hours, thereby forming a large vesicle having a negative charge by an electric field forming method. A dispersion containing (hereinafter referred to as an anionic giant vesicle) was prepared.

<Formation of hydrogel>
As the monomer aqueous solution of the hydrogel 21, acrylamide (12 wt%) as the first monomer, dimethylaminopropylmethacrylamide (DMAPMA) (3 wt%) as the second monomer, bisacrylamide (of the two monomers) Monomer aqueous solution (hereinafter referred to as cationic monomer aqueous solution) obtained by dissolving calcein (100 μM) in a 200 mM glucose aqueous solution so as to have a concentration in parentheses, respectively. .) Was prepared. Thereafter, immediately before use, a small amount of APS and TEMED was further added to prepare an aqueous solution for filling.

  The filling aqueous solution was dropped onto the upper surface 11a of the substrate 10 to fill the plurality of holes 20 and sufficiently overflow above the upper surface 11a. The mixture was allowed to stand for 15 minutes or longer to form a hydrogel 21 composed of a polymer obtained by polymerizing monomers and further crosslinked by covalent bonds. Thereafter, the external solution L is replaced with a 200 mM glucose aqueous solution, and as shown in the schematic diagram of FIG. 7, the excess hydrogel 21 that has gelled outside the hole 20 is placed in the horizontal direction (in the direction of the arrow in FIG. 7). ), The excess hydrogel was removed and only the inside of the hole 20 was filled with the hydrogel 21. Next, the dispersion containing the anionic giant vesicle is added to the external solution L, and allowed to stand at room temperature for 10 minutes or more in an environment not containing the metal cation (external solution L), and then the excess anionic giant vesicle is added. Removed. FIG. 8 shows the result of fluorescence observation of the lipid bilayer array substrate in this state.

  FIG. 8a shows the fluorescence from calcein only, and FIG. 8b shows the fluorescence from both calcein and rhodamine. 8a and 8b are the same field of view. In the fluorescence image of FIG. 8 a, not only from the hole 20 b (20) filled with the hydrogel 21 and covered with the lipid bilayer membrane 30, but also filled with the hydrogel 21 and the lipid bilayer membrane. Fluorescence derived from calcein contained in the hydrogel is also observed from the hole 20a (20) not covered with 30. From this result, even when the hydrogel 21 is not covered by the lipid bilayer membrane 30, the fluorescent molecule 22 is stably held in the hydrogel 21, and the fluorescent molecule 22 diffuses from the inside of the hole 20 to the external solution. It can be seen that this is suppressed.

  From the fluorescence image of FIG. 8b, it can be seen that the lipid bilayer membrane 30 is formed so as to cover the hole 20b (20). Further, it was observed that the fluorescence emitted from the hole 20b has a stronger intensity than the fluorescence emitted from the hole 20a. From this result, the inside of the hole 20a and the hole 20b is filled with the hydrogel 21, the opening of the hole 20a is not covered with the lipid bilayer membrane 30, and the opening of the hole 20b is lipid bimolecular. It can be seen that the film 30 is covered.

  In the lipid bilayer array substrate 4 produced by the above method, the lipid bilayer array substrate according to the present invention can be used as the membrane protein array substrate by arranging the membrane protein 31 on the lipid bilayer membrane 30. . 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.

Example 2
In Example 1, the case where the hydrogel 21 has a positive charge and the lipid bilayer membrane 30 has a negative charge was illustrated. In Example 2, the case where the charge of the hydrogel 21 and the lipid bilayer membrane 30 is the same is illustrated.
In the cationic monomer aqueous solution that is the material of the hydrogel 21, the total concentration of the first monomer and the second monomer is changed to 15% by weight, and DOPS is changed to ethyl dioleylphosphatidylcholine (EDOPC). Except for this, an attempt was made to prepare a lipid bilayer array substrate by the same method as in Example 1.

  FIG. 9 shows the result of fluorescence observation of the hydrogel array substrate in which cationic giant vesicles were precipitated on the substrate. FIG. 9a shows the fluorescence from calcein only, and FIG. 9b shows the fluorescence from both calcein and rhodamine. 9a and 9b are the same field of view. In the fluorescence image of FIG. 9a, similarly to Example 1, fluorescence derived from calcein is observed from the hole 20a (20) filled with the hydrogel 21.

  In the fluorescent image of FIG. 9b, the cationic giant vesicle V is present on the substrate in the shape of a vesicle even though it is disposed on the hole 20a. That is, the cationic giant vesicle V was not developed and was not disposed on the substrate as a lipid bilayer. This is probably because the cationic hydrogel and the cationic giant vesicle have the same charge, and electrostatic repulsion occurred between them, and the giant vesicle could not be developed on the substrate. From this result, in an environment where there is no metal cation, electrostatic attraction between the hydrogel and the giant vesicle is required to deploy the giant vesicle and place the lipid bilayer on the substrate. I understand.

Example 3
A production example of the lipid bilayer array substrate 6 shown in FIG. 6 will be specifically described below.

  First, a metal layer 40a is deposited in the order of titanium, gold, and platinum on the Si wafer 10 covered with an insulating layer made of a thermal oxide film having a thickness of 200 nm, and the metal layer 40a is patterned by photolithography. As a result, an electrode 40 having a thickness of about 60 nm was formed. The electrode 40 is connected to a pad portion (not shown) having a size of 300 μm square, for example, and can be connected to an external electrophysiological measurement device.

Next, 1 μm of a silicon nitride film was deposited as an insulating layer 13 on the Si wafer 10 on which the metal layer 40a was formed by a plasma CVD method. A silicon oxide film 11 was deposited on the insulating layer 13 with a thickness of 200 nm by sputtering.
A hole 20 was formed in the thus formed substrate body 10 by photolithography and dry etching. This etching was performed until the electrode 40 (metal layer 40a) was exposed on the bottom surface of the hole 20. After forming the hole 20, the resist film was removed and washed to obtain a substrate having the electrode 40 in the hole 20.

  In this embodiment, the number ratio between the hole 20 and the electrode 40 is preferably 1: 1. A plurality of holes 20 may be provided for one electrode 40, but in this configuration, it is necessary to cover all the holes 20 with the lipid bilayer membrane 30 when measuring with the one electrode 40. Therefore, measurement preparation and measurement conditions are complicated. Therefore, it is desirable to form one electrode 40 that is electrically independent from the other electrodes in one hole 20.

  As in the case of Example 1, the cationic hydrogel 21 is filled on the substrate provided with the electrode 40 in the hole portion 20, the anionic giant vesicle is developed, and the lipid bilayer membrane 30 is disposed. The opening of the hole 20 was covered with a lipid bilayer membrane 30. That is, a huge vesicle could be developed without using a metal cation. The lower surface 30 a of the lipid bilayer membrane was in contact with the upper surface of the hydrogel 21. Furthermore, by arranging α-hemolysin as a membrane protein 31 in the lipid bilayer membrane 30 and measuring a current flowing between the electrode 40 and the counter electrode 41 when the voltage is applied, ions via α-hemolysin are measured. Can be quantitatively detected.

Example 4
As the cationic monomer aqueous solution, the same aqueous solution as in Example 1 was prepared. Moreover, the anionic monomer aqueous solution which has the same composition as the said cationic aqueous solution except having changed the DMAPMA which is the 2nd monomer which comprises the cationic aqueous solution of Example 1 to acrylic acid was prepared. Using each monomer aqueous solution, a plurality of holes filled with a cationic hydrogel and a plurality of holes filled with an anionic hydrogel were arrayed on the same substrate.

Dispersion A in which cationic giant vesicles were dispersed and Dispersion B in which anionic giant vesicles were dispersed were prepared by the electric field forming method described above.
By spreading the giant vesicles contained in each dispersion on an anionic hydrogel and a cationic hydrogel filled in a plurality of holes in the same substrate, the hydrogel charge and the lipid bilayer charge Were attracted by electrostatic interaction. By this method, a lipid having a cationic lipid bilayer membrane disposed on a hole filled with an anionic hydrogel, and an anionic lipid bilayer membrane disposed on a hole filled with a cationic hydrogel. A bilayer array substrate (hereinafter sometimes referred to as a patterned array substrate) could be easily produced. This will be described in more detail below.

  First, in the same manner as in Example 1, a substrate in which holes were formed was prepared. On this substrate, an aqueous solution obtained by adding riboflavin to a cationic monomer aqueous solution so as to have a final concentration of 10 μg / ml was dropped. Next, using a confocal microscope, arbitrary portions on the substrate were locally irradiated with UV light to gel the irradiated portion. Thereafter, the substrate was rinsed with a 200 mM glucose aqueous solution to remove the unreacted cationic monomer aqueous solution.

  Next, on this substrate, an aqueous solution in which riboflavin is added to an anionic monomer aqueous solution so as to have a final concentration of 10 μg / ml is dropped, and UV light is irradiated locally at an arbitrary position, whereby the anionic monomer is applied. An anionic hydrogel was formed in the hole that was gelled and not filled with the cationic hydrogel. Thereafter, the substrate was rinsed with a 200 mM glucose aqueous solution to remove unreacted anionic monomers.

  Dispersion B containing an anionic giant vesicle was added to the external solution on the obtained hydrogel substrate, and allowed to stand for 10 minutes or longer. Then, after substituting the external solution L with 200 mM glucose aqueous solution, the dispersion liquid A containing a cationic giant vesicle was added, and it left still at room temperature for 10 minutes or more. Thereafter, the external solution L having a predetermined composition was substituted, and the substrate was rinsed. As a result, due to the electrostatic attraction of the hydrogel and the giant vesicle, an anionic lipid bilayer membrane is disposed on the hole filled with the cationic hydrogel, and on the hole filled with the anionic hydrogel. A lipid bilayer membrane array substrate on which a cationic lipid bilayer membrane was arranged could be produced. That is, a huge vesicle could be developed without using a metal cation. FIG. 10 shows a schematic diagram of the substrate.

  In such a lipid bilayer array substrate, a membrane protein array substrate can be easily prepared by inserting different membrane proteins into an anionic giant vesicle and a cationic giant vesicle, respectively.

  The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.

  The substrate according to the present invention is widely applicable in the field of functional measurement of biomolecules.

1-3 ... Hydrogel array substrate, 4-6 ... Lipid bilayer array substrate (membrane protein array substrate), 10 ... Substrate body, 10a ... Upper surface of substrate body, 11 ... Hydrophilic layer, 11a ... Upper surface of hydrophilic layer ( Top surface of substrate), 12 ... first insulating layer, 13 ... second insulating layer, 20 ... hole, 20a ... hole not covered with lipid bilayer, 20b ... covered with lipid bilayer 21 ... hydrogel, 22 ... fluorescent molecule, 30 ... lipid bilayer membrane (lipid bilayer membrane), 30a ... lower surface of lipid bilayer membrane, 31 ... membrane protein, 40 ... electrode, 40a ... metal layer ( Electrode layer), 41 ... counter electrode, L ... external solution, V ... cationic giant vesicle, P ... direction of pushing the gel

Claims (8)

A substrate,
A plurality of holes arranged on the surface of the substrate;
A hydrogel having a charge filled in the hole;
A hydrogel array substrate comprising: A hole filled with a hydrogel having a positive charge;
A hole filled with a hydrogel having a negative charge;
The hydrogel array substrate according to claim 1, wherein each is arranged on the same substrate. A lipid bilayer array substrate comprising a lipid bilayer membrane on the hydrogel array substrate according to claim 1 or 2,
A lipid bilayer membrane, wherein a lipid bilayer membrane having a charge opposite to the charge of the hydrogel filled in the hole is arranged so as to cover the opening of the hole substrate. A method for producing a hydrogel array substrate according to claim 1 or 2,
A filling step of filling the hole with a solution containing a monomer constituting the hydrogel;
Gelling the monomer in the solution to form the hydrogel in the hole; and
A method for producing a hydrogel array substrate, comprising: A method for producing a hydrogel array substrate according to claim 1 or 2,
A first filling step of filling the plurality of holes with a first solution containing a monomer constituting the first hydrogel;
The first gelation step of gelling the monomer in the first solution in the first group of holes among the plurality of holes to form a first hydrogel in the first group of holes,
A second filling step of filling a second group of holes different from the first group among the plurality of holes with a second solution containing a monomer constituting the second hydrogel;
A second gelation step of gelling the monomer in the second solution in the second group of holes to form a second hydrogel in the second group of holes;
A method for producing a hydrogel array substrate, comprising: 6. The hydrogel according to claim 4 or 5, wherein a photopolymerization initiator is contained in the solution containing the monomer, and the monomer is gelled by irradiating light to the hole filled with the solution. A method for producing a gel array substrate. Each process which the manufacturing method of the hydrogel array substrate according to claim 4 or 5 has,
A solution containing vesicles is placed on the holes filled with the hydrogel, the vesicles are allowed to settle, the vesicles are developed on or near the openings of the holes, and the openings are made lipid-free. A process of forming a lipid bilayer covering with a molecular membrane;
A method for producing a lipid bilayer array substrate, comprising: The method for producing a lipid bilayer array substrate according to claim 7, wherein the vesicle composed of lipid molecules having a charge opposite to that of the hydrogel is used.