Biosensor and method for manufacturing the same

Abstract

To provide a biosensor that can detect an ion flowing through a molecule within a lipid bilayer membrane with high sensitivity and can pattern and integrate the lipid bilayer membrane at an arbitrary position.SOLUTION: Provided are: a biosensor comprising a solid substrate, a graphene film provided on the solid substrate, a linker molecule provided on the graphene film, a hydrophilic polymer attached to the linker molecule, and a lipid bilayer membrane molecule bonded to the hydrophilic polymer; and a method for producing the same.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a biosensor for examining the behavior of molecules in a biological membrane and a method for producing the same. [Background technology]

[0002] Active research is being conducted on sensor devices that examine the behavior of membrane proteins in artificial biomembranes on the surface of tiny microchips, as well as biodevices that utilize the functions of membrane proteins.

[0003] As an example of such a microdevice, Non-Patent Document 1 discloses a microchip having a fluid artificial lipid bilayer membrane formed on the solid surface thereof, which mimics a biological membrane necessary for maintaining the function of membrane proteins.

[0004] Meanwhile, graphene, a two-dimensional layered material, has attracted attention as a functional carbon material in the field of electronic devices. Graphene is known for its physical strength, extremely high thermal conductivity, and electron mobility, as well as its chemical and thermal stability. These characteristics make graphene a promising next-generation electronic material with potential applications in a variety of device technologies, including electrodes, chemical sensors, and biosensors.

[0005] As a method for producing graphene, for example, Patent Document 1 discloses a method for holding a graphene film, which includes an adhesive layer formation step of forming an adhesive layer made of aromatic small molecules on one or both main surfaces of a solid substrate, and a graphene film transfer step of transferring a graphene film grown on a metal substrate onto the adhesive layer. This technology provides a graphene film holding method that enables graphene to be held efficiently and easily with minimal damage during the production process, and also aims to provide a structure including a graphene film that is minimally damaged during the production process. Furthermore, for example, Non-Patent Documents 2 to 4 disclose techniques for hydrophilizing graphene oxide. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2021-5601 [Non-patent literature]

[0007] [Non-Patent Document 1] Groves et al., Acc. Chem. Res. 2002, 35, 149-157. [Non-patent document 2] Huang et al., Adv. Funct. Mater. 2015, 25, 5809-5815. [Non-patent document 3] Wang et al., Polymer 205 (2020), 122851. [Non-patent document 4] Mao et al., RSC Adv., 2016, 6, 111632-111639. Summary of the Invention [Problem to be solved by the invention]

[0008] The inventors focused on the properties of graphene and lipid bilayer membranes. Graphene, a two-dimensional layered material, has excellent electrical properties and is expected to have a wide range of applications as an electrode for biosensors. Forming a lipid bilayer membrane on this graphene and introducing a membrane protein enables highly sensitive detection of ions flowing through the membrane protein, which is expected to enable quantitative evaluation. Furthermore, if lipid bilayer membranes can be patterned and integrated at arbitrary locations, it is expected that a multi-channel simultaneous measurement system can be constructed.

[0009] However, due to the hydrophobic nature of graphene surfaces, it has been difficult to form bilayer structures and maintain fluidity on hydrophilic surfaces using lipid membranes. For example, as shown in Non-Patent Documents 2-4, there have been reports of hydrophilic treatments for graphene oxide. However, for single-layer graphene, there has been a lack of technology for supporting graphene on solid substrates so that it can withstand wet chemical reaction processes such as surface modification (exposure to various chemicals and solvents, washing, drying, etc.). Furthermore, because graphene is a hydrophobic membrane, it is particularly susceptible to damage in aqueous environments, such as those used for hydrophilic polymer modification, and is prone to peeling from solid substrates. While graphene oxide has functional groups that allow it to stably disperse in aqueous solutions, graphene membranes lack such functional groups, making solution dispersion methods unsuitable.

[0010] In view of the above circumstances, the present invention aims to provide a biosensor that is capable of highly sensitive detection of ions flowing through molecules within a lipid bilayer membrane and that allows the lipid bilayer membrane to be patterned and integrated at any position. [Means for solving the problem]

[0011] One aspect of the present invention is a biosensor including a solid substrate, a graphene film provided on the solid substrate, a linker molecule provided on the graphene film, a hydrophilic polymer bound to the linker molecule, and a lipid bilayer membrane bound to the hydrophilic polymer.

[0012] One aspect of the present invention is a method for manufacturing a biosensor, including a graphene-forming step of providing a graphene film on a solid substrate, a linker-forming step of providing linker molecules on the graphene film, a hydrophilic polymer-forming step of bonding a hydrophilic polymer to the linker molecules, and a lipid bilayer-forming step of bonding a lipid bilayer to the hydrophilic polymer. [Effects of the Invention]

[0013] The present invention makes it possible to provide a biosensor that is capable of highly sensitive detection of ions flowing through molecules within a lipid bilayer membrane and that allows the lipid bilayer membrane to be patterned and integrated at any desired position. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a schematic side view of a biosensor according to a first embodiment of the present invention. [Figure 2] FIG. 2 is a schematic side view of a biosensor according to a second embodiment of the present invention. [Figure 3] 10A to 10C are side schematic views showing a method for manufacturing a biosensor according to a second embodiment. [Figure 4] FIG. 1 is a graph showing the fluorescence recovery curve derived from rhodamine of a lipid bilayer membrane formed on a polyethyleneimine-modified graphene substrate. DETAILED DESCRIPTION OF THE INVENTION

[0015] The biosensor and the manufacturing method thereof according to the present invention will be described below with reference to embodiments, although the present invention is not limited to the following embodiments.

[0016] First Embodiment (Configuration of the biosensor of this embodiment) 1 is a schematic side view of a biosensor 1 according to this embodiment. The biosensor 1 includes a solid substrate 2, a graphene film 3, a linker molecule 4, a hydrophilic polymer 5, and a lipid bilayer membrane 6.

[0017] The solid substrate 2 is a substrate on which a device is formed. It is also a component on which the graphene film 3 is formed. The material of the solid substrate 2 can be selected from plastic, glass, quartz, silicon, and the like. The solid substrate 2 preferably has a flat surface. Specifically, a silicon substrate with an oxide film such as SiO2 can be used, in which an oxide film is formed on the side where the bond with the graphene film 3 is formed. In the embodiment shown in the figure, a glass plate with a nearly flat surface is used as the solid substrate 2.

[0018] The graphene film 3 is a layer made of graphene. Graphene is a two-dimensionally arranged SP 2 It is a substance in which bonded carbon forms a monoatomic film, and the thickness of one graphene film is one atomic layer, or approximately 1 nm. The graphene constituting one layer of the graphene film 3 may be a single continuous film or a patchwork of multiple films. The number of graphene layers constituting the graphene film 3 is not particularly limited, but is preferably about 1 to 5 layers.

[0019] The graphene film 3 can be provided on the solid substrate 2 by a conventionally known method. In this embodiment, the graphene film 3 is grown on another metal substrate by a chemical vapor deposition method (CVD method) or the like and transferred to the solid substrate. The graphene film 3 may be provided in a configuration described in Patent Document 1, for example.

[0020] The linker molecule 4 is a molecule for adhering the graphene film 3 and the hydrophilic polymer 5. The adhesion may be by chemical bonding or physical adhesion. The linker molecule 4 preferably has a structure that allows it to adhere to both the hydrophobic graphene film 3 and the hydrophilic hydrophilic polymer 5. For example, a compound having both a hydrophobic backbone and a hydrophilic group can be used as such a compound.

[0021] The linker molecules 4 are provided on at least a portion of the graphene membrane 3. The location where the linker molecules 4 are provided can be appropriately selected depending on the location where the hydrophilic polymer 5 (described later) is provided, i.e., the state where the lipid bilayer membrane 6 is provided. In the example shown in the figure, only the location where the hydrophilic polymer 5 is provided is shown, but the linker molecules 4 may be provided so as to cover a wider area of ​​the graphene membrane 3.

[0022] A molecule having a pyrene skeleton may be used as the linker molecule 4. A molecule having a pyrene skeleton is a molecule that includes a pyrene structure in a part thereof. By using a molecule having a pyrene skeleton as linker molecule 4, the pyrene structure consisting of multiple six-membered rings is 2 It has a high affinity with the structure of graphene, which is bonded to form a so-called hexagonal lattice structure, and has the effect of easily adhering to graphene. In this embodiment, pyrenebutanoic acid is used as the molecule having a pyrene skeleton.

[0023] The structure in which the linker molecules 4 are provided on the graphene film 3, i.e., the structure in which the graphene film 3 and the linker molecules 4 are bonded together, can be appropriately selected depending on the structure of the linker molecules 4. For example, when pyrenebutanoic acid is used as the linker molecule 4, the butanoic acid structure side of pyrenebutanoic acid may be activated as described below, and then the solution may be prepared in an organic solvent, and the solution may be dropped onto the graphene film 3 to bond the linker molecule 4.

[0024] The hydrophilic polymer 5 is a polymer having hydrophilic properties. The compound constituting the hydrophilic polymer 5 can be appropriately selected, and may be, for example, a hydrocarbon polymer or a peptide polymer, and may be a homopolymer or a copolymer. Examples of these include polyethyleneimine and polyethylene glycol as hydrocarbon polymers. As peptide polymers, proteins or polypeptides derived from proteins can be used. More specifically, the protein-derived polymer may be avidin, biotin, or a fragment thereof.

[0025] By providing the hydrophilic polymer 5, the graphene membrane 3 and the lipid bilayer membrane 6 are adhered to each other at a distance corresponding to the molecular weight of the hydrophilic polymer 5. That is, in the biosensor of this embodiment, the graphene membrane 3 and the lipid bilayer membrane 6 have an intermolecular distance corresponding to the size of the hydrophilic polymer 5. Therefore, the structure including the graphene membrane 3 and the lipid bilayer membrane 6 can be maintained more stably than when molecules of different properties, the hydrophobic graphene membrane 3 and the hydrophilic lipid bilayer membrane 6, are in close contact with each other without an intermolecular distance.

[0026] The molecular weight of the hydrophilic polymer 5 can be selected from, for example, 600 to 750000. When the hydrophilic polymer 5 is in this range, the intermolecular distance between the hydrophobic graphene membrane 3 and the hydrophilic lipid bilayer membrane 6 becomes appropriate.

[0027] The means for binding the hydrophilic polymer 5 to the linker molecule 4 can be appropriately selected depending on the structure of these molecules. In this embodiment, the COOH group of pyrenebutanoic acid in the linker molecule 4 is activated with succinimide and then bound to the hydrophilic polymer 5.

[0028] The lipid bilayer membrane 6 is a membrane formed by arranging two lipid molecules 62 as a constituent unit. Specifically, as shown in the figure, two lipid molecules 62, each having a hydrophilic and a hydrophobic portion, form a constituent unit with their hydrophobic portions facing each other, and these constituent units are arranged approximately parallel to form a membrane. In other words, two membranes in which lipid molecules 62 are arranged are stacked with their hydrophobic portions facing each other. Due to this structure, the surfaces of the lipid bilayer membrane 6 are hydrophilic on both sides of the membrane. The lipid bilayer membrane 6 may contain other components than the lipid molecules. For example, it may have a membrane protein 61 that penetrates one or both of the two overlapping membranes. In the example shown in the figure, the membrane protein 61 penetrates both of the two membranes.

[0029] The lipid bilayer membrane 6 can be appropriately selected depending on the type of lipid molecules 62. For example, it is preferable to use unsaturated lipid molecules such as dioleoylphosphatidylcholine (DOPC) or saturated lipid molecules such as dipalmitoylphosphatidylcholine (DPPC) and cholesterol as constituent materials. The type of lipid molecules 62 may be one type or a mixture of multiple types.

[0030] As will be described later, the lipid bilayer membrane 6 is formed, for example, by spreading a lipid membrane vesicle composed of lipid molecules 62 on hydrophilic polymer-modified graphene.

[0031] (Effects of the biosensor of this embodiment) The biosensor 1 of this embodiment includes the graphene membrane 3 and the lipid bilayer membrane 6, and therefore is capable of highly sensitive detection of ions flowing through molecules in the lipid bilayer membrane.

[0032] In the biosensor 1 of this embodiment, the graphene membrane 3 and the lipid bilayer membrane 6 are held together via the linker molecules 4 and the hydrophilic polymer 5, so this configuration can be suitably formed during manufacturing and is easy to maintain after manufacturing. Conventionally, it has been difficult to form or maintain a configuration including the graphene membrane 3 and the lipid bilayer membrane 6 because the graphene membrane 3 is hydrophobic and the lipid bilayer membrane 6 is hydrophilic. In this embodiment, by providing the linker molecules 4 and the hydrophilic polymer 5, it is possible to form a structure that holds the graphene membrane 3 and the lipid bilayer membrane 6 together and to arrange the lipid bilayer membrane 6 so that it maintains fluidity.

[0033] This embodiment is a fundamental technology for realizing a new electronic device for bioanalysis that combines two types of two-dimensional materials, a graphene membrane 3 and a lipid bilayer membrane 6, and is expected to be applied as a research tool related to bio- and medical care, as well as to industrial applications in the same fields.

[0034] <Second embodiment> 2 is a schematic side view of a biosensor 1A according to this embodiment. In the biosensor 1A, the graphene membrane 3 has a hydrophobic region 3a where no lipid bilayer membrane 6 is provided, and a hydrophilic region 3b where a lipid bilayer membrane 6 is provided. The same components as those in the first embodiment are designated by the same reference numerals, and a description thereof will be omitted.

[0035] In this embodiment, the graphene of the biosensor 1A film A resist layer, which will be described later in the manufacturing method, is provided on 3, and a specific pattern is formed on the resist layer by lithography or the like, and then linker molecules 4 are provided in the patterned portions. Thereafter, the resist layer is removed.

[0036] That is, since a hydrophilic polymer 5 and a lipid bilayer 6 are formed in the areas where the linker molecules 4 are provided, the areas where the patterns are provided become hydrophilic areas 3b, and the areas where they are not provided become hydrophobic areas 3a.

[0037] In this embodiment, in addition to the effects of the first embodiment, the linker molecules 4 can be formed to form a pattern on the graphene membrane 3, thereby allowing the hydrophilic polymer 5 and lipid bilayer membrane 6 to be provided at any desired location on the graphene membrane 3. This allows the biosensor 1A to be provided with a graphene membrane 3 having both hydrophobic regions 3a and hydrophilic regions 3b, and can be applied to analyses depending on the properties of the graphene membrane 3. The lipid bilayer membrane 6 can be arranged in any desired pattern, allowing for analyses depending on the shape of the lipid bilayer membrane 6.

[0038] (Method for manufacturing the biosensor of this embodiment) Next, a method for manufacturing the biosensor of the second embodiment will be described. Figure 2 is a schematic side view showing the method for manufacturing the biosensor of this embodiment. The manufacturing process of the biosensor of this embodiment includes a graphene formation step of providing a graphene film on a solid substrate, a linker formation step of providing linker molecules on the graphene film, a hydrophilic polymer formation step of bonding a hydrophilic polymer to the linker molecules, and a lipid bilayer formation step of bonding a lipid bilayer to the hydrophilic polymer. Furthermore, in this embodiment, the graphene formation step is followed by a resist layer formation step of forming a resist layer on the graphene layer, a pattern formation step of forming a pattern on the resist layer, and the hydrophilic polymer formation step is followed by a resist layer removal step of removing the resist layer.

[0039] The graphene formation step (not shown) can be performed by a conventionally known method depending on the solid substrate 2 and the configuration of the graphene film 3 to be formed. As described above, in this embodiment, the graphene film 3 grown by chemical vapor deposition (CVD) or the like is transferred onto another metal substrate.

[0040] In particular, the graphene film 3 may be provided in a configuration described in, for example, Patent Document 1. This method provides a graphene solid substrate in which the graphene film 3 is less likely to peel off when subjected to a surface treatment process involving water, such as treatment with a hydrophilic polymer 5. This makes it possible to produce a solid substrate 2 and a graphene film 3 of a size (several mm square or larger) that can be used practically as a device such as an electrode.

[0041] Next, in this embodiment, a resist layer forming step is performed to form a resist layer 31 on the graphene film 3 in a region where the linker molecules 4 are not provided. 2(a), in this embodiment, the resist layer 31 is formed so as to cover substantially the entire surface of the graphene film 3. The resist layer 31 may be formed using a conventionally known method for forming an electron beam resist.

[0042] Next, as shown in FIG. 2(b), a pattern formation step is performed in which a pattern 32 is formed in the resist layer 31. The pattern 32 can be formed by, for example, lithography. Specifically, photolithography or electron beam lithography can be used as the lithography method. The shape of the pattern 32 may be drawn by lithography, and the drawn area may be removed with a developer to form the pattern 32 as an area where the resist layer 31 has been removed.

[0043] Next, as shown in Figure 3(c), a linker formation step is performed in which linker molecules 4 are provided on the graphene film 3, and a hydrophilic polymer formation step is performed in which a hydrophilic polymer 5 is bonded to the linker molecules 4. The figure shows the state after the linker formation step and the hydrophilic polymer formation step (also called a hydration treatment because the hydrophilic polymer 5 is provided on the hydrophobic graphene film 3) have been performed.

[0044] In the linker formation step, the linker molecule 4 may be provided on the graphene film 3 by any suitable means depending on the structure of the linker molecule 4. For example, when pyrenebutanoic acid is used as the linker molecule 4, the linker molecule 4 may be dissolved in an organic solvent such as dimethylformamide, dropped onto the graphene film 3, and allowed to stand to react, after which the organic solvent may be removed. Furthermore, in order to bond the hydrophilic polymer 5 to the linker molecule 4 in the hydrophilic polymer formation step, the linker molecule 4 may be activated and then provided on the graphene film 3. This operation can be performed by activating the COOH group of pyrenebutanoic acid with succinimide to form a pyrenebutanoic acid succinimide ester, for example.

[0045] In the hydrophilic polymer formation step, a means for binding the hydrophilic polymer 5 to the linker molecule 4 can be appropriately selected depending on the structure of these molecules. In this embodiment, the hydrophilic polymer 5 may be bound to the activated linker molecule 4 by adding an aqueous solution of the hydrophilic polymer 5 to the solid substrate 2 on which the linker molecule 4 is provided.

[0046] 3(d), a resist layer removal step is performed to remove the resist layer 31. The resist layer 31 can be removed by chemical treatment using an organic solvent or physical treatment using ultrasound, or by a combination of these. Examples of the organic solvent that can be used include acetone and methyl ethyl ketone.

[0047] Next, as shown in FIG. 3(e), a lipid bilayer membrane formation step is carried out in which a lipid bilayer membrane is bonded to the hydrophilic polymer 5. In the example shown in the figure, the lipid bilayer membrane is formed by forming a pattern on the graphene. film Since it does not cover the entire surface of 3 and the sides are broken, it has the shape of a lipid membrane vesicle in which lipids are associated to form a bag-like bilayer membrane. In this embodiment, a lipid membrane vesicle is prepared as a lipid bilayer membrane on a separate electrode substrate, and then spread on the solid substrate 2, thereby bonding the lipid bilayer membrane 6.

[0048] Typical methods for producing lipid membrane vesicles include the static hydration method and the electric field generation method. There are no particular restrictions on the method for producing vesicles, but it is preferable to use the electric field generation method because it is easy to produce large vesicles and the reaction time and reaction process are simple. The electric field formation method is a technique in which a thin film of lipid molecules is formed on an electrode such as indium tin oxide (ITO), and then an alternating current electric field is applied to form giant lipid membrane vesicles in an aqueous solution. 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 the ITO substrate, and it is also preferable to apply an alternating current electric field of approximately several hundred millivolts to 2 volts. This is because electric field strengths lower than this electric field range result in low vesicle yields, and electric field strengths higher than this electric field range may destroy the vesicle structure or electrolyze water, making it impossible to produce vesicles.

[0049] The lipid membrane vesicles can be spread on the solid substrate 2 by adding the lipid membrane vesicle dispersion onto the solid substrate 2. Specifically, a neutral buffer solution is dropped onto substantially the entire surface of the solid substrate 2, and then the lipid membrane vesicle dispersion is dropped into the solution and allowed to stand, thereby spreading the lipid membrane vesicles on the substrate. The lipid membrane vesicles, which have a spherical structure, collide with the substrate, destroying the spherical structure to form a bilayer membrane, and the hydrophilic groups on the surface bind to the hydrophilic polymer 5. In this case, a reagent that promotes the destruction of the spherical structure may be used, such as a divalent cation such as calcium or a peptide that opens holes in the membrane.

[0050] According to the manufacturing method of this embodiment, the hydrophilic polymer 5 is provided by photolithography or electron beam lithography, thereby providing the hydrophilic polymer 5 at any desired position on the graphene film 3. That is, a lipid bilayer membrane 6 (described later) is bonded to the hydrophilic polymer 5, providing the lipid bilayer membrane 6 at the desired position. By forming a resist layer 31 and a pattern 32 on the graphene film 3, the linker molecules 4 and the hydrophilic polymer 5 can provide hydrophobic portions on the graphene film 3. As a result, hydrophobic and hydrophilic surface regions are formed on the graphene film 3, and the lipid bilayer membrane is patterned on the graphene film 3. As a result, inspection of the lipid bilayer membrane 6 in any desired pattern shape is possible.

[0051] 1, for example, one lipid bilayer 6 is bonded to a plurality of hydrophilic polymers 5, and as a result, the lipid bilayer 6 is configured to cover substantially the entire surface of the graphene membrane 3. Alternatively, the lipid bilayer 6 may be configured to cover only a portion of the graphene membrane 3. This configuration can be adjusted by adjusting the arrangement pattern of the hydrophilic polymers 5, such as the mutual distance between them.

[0052] Furthermore, although the manufacturing method of this embodiment is a manufacturing method of the biosensor 1A of the second embodiment, it can also be a manufacturing method of the biosensor 1 of the first embodiment if the resist layer forming process, the pattern forming process, and the resist layer removing process are omitted. That is, when it is not necessary to pattern the positions where the hydrophilic polymer 5 or the lipid bilayer 6 is to be provided, the resist layer formation, pattern formation, or resist layer removal steps may be omitted, and the linker formation step and hydrophilic polymer formation step may be performed directly on the graphene film 3.

[0053] Although an embodiment of the present invention has been described above in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and includes designs within the scope of the gist of the present invention. [Example]

[0054] The effects of the present invention will be made clearer by the following examples and comparative examples. Note that the present invention is not limited to the following examples, and can be practiced by making appropriate changes within the scope of the present invention.

[0055] <Preparation of hydrophilic polymer-modified graphene substrate> The solid substrate is 18 x 18 mm 2 Graphene deposited by chemical vapor deposition on copper foil was transferred onto the surface of the cover glass. Next, pyrenebutanoic acid was introduced as a linker molecule (adhesion molecule) to fix the hydrophilic polymer to the graphene surface. A 0.5 mM dimethylformamide solution of pyrenebutanoic acid succinimide ester, in which the COOH group of pyrenebutanoic acid was activated with succinimide, was added dropwise to the graphene surface and allowed to stand for 1 hour. After that, the graphene surface was washed with dimethylformamide and air-dried.

[0056] Next, an aqueous solution containing 0.3% polyethyleneimine, a hydrophilic polymer, was dropped onto the graphene. To evaluate the hydrophilic effect of polyethyleneimine, the contact angle of graphene surfaces was measured with and without polyethyleneimine modification. The contact angle changed from 75-80° without modification to 55-60° with modification. This result confirmed that modification increases hydrophilicity.

[0057] <Preparation of giant lipid membrane vesicles and their application to substrates> The lipid bilayer membrane was formed as follows. A chloroform solution of dioleoylphosphatidylcholine (DOPC) was prepared. The solution contained 0.1 mol% rhodamine-dipalmitoylphosphatidylethanolamine (Rhod-DOPE) as a fluorescent labeling agent. Next, 200 μL of the chloroform solution was uniformly applied to an ITO substrate (a 100 nm-thick ITO thin film on a glass substrate, 40 × 40 mm, 50–100 Λ). The substrate was dried under reduced pressure at room temperature for 2 hours to completely remove the chloroform solvent, forming a uniform lipid molecular thin film on the ITO substrate. A silicone rubber sheet with a window (30 × 30 mm outer dimensions, 1 mm thick silicone rubber with a 20 × 20 mm window) was placed on top of the ITO substrate in close contact with the silicone rubber sheet, and 500 μL of 200 mM sucrose solution was dripped onto the window. An ITO substrate was then placed on top of the sucrose solution, taking care to prevent air bubbles from forming, and the solution in the silicone rubber window was sandwiched between the ITO substrates. A clip electrode was then attached to the ITO substrate, and an AC electric field (sine wave, 1 V, 10 Hz) was applied at room temperature for two hours, forming giant lipid membrane vesicles dispersed in the sucrose solution using the electric field formation method. Fluorescence observation using an excitation wavelength of 561 nm revealed the fluorescence of rhodamine molecules uniformly present within the lipid bilayer, allowing evaluation of the lipid bilayer.

[0058] Next, PBS buffer (pH 7.4) was dropped onto the polyethyleneimine-modified graphene substrate. The giant lipid vesicle dispersion was then dropped into the solution and allowed to stand, spreading the giant lipid vesicles onto the substrate. The spherical vesicles collided with the substrate, destroying their spherical structure and forming a bilayer membrane.

[0059] The maintenance of fluidity of the formed lipid bilayer membrane was confirmed using the photobleaching fluorescence recovery method, which focuses a laser on a specific area, bleaching that area, and then observes the recovery of the fluorescence in that area. The membrane maintains fluidity in areas where the bleached surrounding molecules can move. Figure 4 shows the fluorescence recovery curve of rhodamine from a lipid bilayer formed on a polyethyleneimine-modified graphene substrate. After bleaching, the fluorescence intensity recovered, indicating that the formed lipid bilayer maintained its fluidity. These results demonstrate that this method makes it possible to maintain a stable and fluid lipid bilayer on the graphene surface. [Industrial Applicability]

[0060] According to the present invention, there is provided a biosensor including a solid substrate, a graphene film provided on the solid substrate, a linker molecule provided on the graphene film, a hydrophilic polymer bound to the linker molecule, and a lipid bilayer membrane molecule bound to the hydrophilic polymer, and a method for manufacturing the biosensor. [Explanation of symbols]

[0061] 1,1A...biosensor, 2...solid substrate, 3...graphene film, 3a...hydrophobic region, 3b...hydrophilic region, 4...linker molecule, 5...hydrophilic polymer, 6...lipid bilayer membrane, 61...membrane protein, 31...resist layer, 32...pattern, 62...lipid molecule

Claims

1. solid substrate, a graphene film provided on the solid substrate; a linker molecule provided on the graphene film; a hydrophilic polymer attached to the linker molecule; and a lipid bilayer membrane bound to the hydrophilic polymer; Equipped with the linker molecule is pyrenebutanoic acid; the hydrophilic polymer is polyethyleneimine, polyethylene glycol, avidin or biotin, or a polypeptide derived from avidin or biotin; A biosensor comprising the graphene film as an electrode.

2. a graphene formation step of providing a graphene film on a solid substrate; a linker formation step of providing linker molecules on the graphene film; a hydrophilic polymer forming step of attaching a hydrophilic polymer to the linker molecule; and a lipid bilayer membrane forming step of binding a lipid bilayer membrane to the hydrophilic polymer; Equipped with the linker molecule is pyrenebutanoic acid; the hydrophilic polymer is polyethyleneimine, polyethylene glycol, avidin or biotin, or a polypeptide derived from avidin or biotin; A method for manufacturing a biosensor, which configures the graphene film as an electrode.

3. Following the graphene formation step, a resist layer forming step of forming a resist layer on the graphene film; a pattern forming step of forming a pattern on the resist layer; and Following the hydrophilic polymer forming step, a resist layer removing step of removing the resist layer; The method for manufacturing a biosensor according to claim 2 , comprising:

4. The method for manufacturing a biosensor according to claim 3 , wherein the pattern forming step is performed by a lithography method.

5. 4. The method for producing a biosensor according to claim 2, wherein the lipid bilayer membrane forming step is carried out by reacting lipid membrane vesicles prepared by an electrolytic formation method.

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