Graphene grid, method for manufacturing graphene grid, and method for analyzing structure of structure analysis target substance

Abstract

To provide a graphene grid capable of suppressing or preventing uneven distribution, deviation of orientation or the like of a structure analysis object substance, capturing the structure analysis object substance with high efficiency, and performing structure analysis of the structure analysis object substance with high resolution, for example, in structure analysis or the like by a cryo-electron microscope.SOLUTION: According to an aspect of the present invention, there is provided a graphene grid having a structure in which a functional group is introduced onto a surface of graphene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a graphene grid, a method for manufacturing a graphene grid, and a method for structural analysis of a substance to be structurally analyzed. [Background technology]

[0002] Cryo-electron microscopes are used for structural analysis of substances such as proteins (Patent Document 1, etc.).

[0003] For structural analysis of target substances (such as proteins) using cryo-electron microscopy, a carbon grid with a support film made of graphene or amorphous carbon attached is used to capture the target substance. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-250721 Summary of the Invention [Problem to be solved by the invention]

[0005] However, when target substances for structural analysis such as proteins are captured on carbon grids, there is a risk that the target substances may be unevenly distributed (localized) or oriented on the carbon grid, or that a sufficient number of target substances may not be captured, which may hinder structural analysis.In addition, there are cases where it is difficult to perform structural analysis of target substances at high resolution.

[0006] Therefore, an object of the present invention is to provide a graphene grid, a method for manufacturing a graphene grid, and a substance to be structurally analyzed that can suppress or prevent uneven distribution, bias in orientation, etc. of a substance to be structurally analyzed in, for example, structural analysis using a cryo-electron microscope, and that can capture the substance to be structurally analyzed with high efficiency and enable structural analysis of the substance to be structurally analyzed with high resolution. [Means for solving the problem]

[0007] To achieve the above object, the graphene grid of the present invention is a graphene grid having a structure in which functional groups are introduced onto the graphene surface.

[0008] The method for producing a graphene grid according to the present invention (hereinafter sometimes simply referred to as the "production method of the present invention") comprises the steps of: a surface treatment step of modifying the graphene surface by reacting the graphene surface with halogen oxide radicals; and a functional group introduction step of introducing functional groups onto the modified surface.

[0009] The method for structural analysis of a substance to be structurally analyzed according to the present invention comprises: A method for structural analysis of a substance to be structurally analyzed using a cryo-electron microscope, comprising: a structural analysis grid preparation step and a structural analysis step, In the structural analysis grid preparation step, a substance to be structurally analyzed is bound to the graphene grid of the present invention or a graphene grid manufactured by the manufacturing method of the present invention, or a cryo-electron microscope grid obtained by binding a substance to be structurally analyzed by a cryo-electron microscope to the graphene grid of the present invention, or a cryo-electron microscope grid manufactured by a manufacturing method for a cryo-electron microscope grid including a structural analysis substance binding step of further binding a substance to be structurally analyzed by a cryo-electron microscope to the graphene grid manufactured by the manufacturing method of the present invention, In the structural analysis step, the structural analysis target substance bound to the graphene grid is structurally analyzed using a cryo-electron microscope. The structural analysis method is characterized by the above. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a graphene grid, a method for manufacturing a graphene grid, and a substance to be structurally analyzed, which can suppress or prevent uneven distribution, bias in orientation, etc. of a substance to be structurally analyzed in structural analysis using a cryo-electron microscope, can capture the substance to be structurally analyzed with high efficiency, and can perform structural analysis of the substance to be structurally analyzed with high resolution. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a diagram schematically illustrating an example of an apparatus for carrying out the surface treatment step using a reaction in a gas phase. [Figure 2] FIG. 2 is a graph showing the results of XPS measurement of graphene before and after the surface treatment step (oxidation reaction) in the reference example. [Figure 3] FIG. 3 is a graph showing the results of measuring the fluorescence intensity of graphene bound to rhodamine B in a reference example. [Figure 4] FIG. 4 is a schematic diagram of an apparatus used to perform the surface treatment step using a gas-phase reaction in a reference example, and a diagram showing the contact angle of water on the graphene surface before and after the surface treatment step. [Figure 5] FIG. 5 is a scheme showing an outline of the surface modification process of the graphene grid and the functional group introduction process in the examples. [Figure 6] Figure 6 shows a scheme outlining the graphene grid surface modification process, functional group introduction process, and β-galactosidase binding (structural analysis target substance binding process) in the example, as well as a photograph of the graphene grid surface bound with β-galactosidase, observed using a negative staining method. [Figure 7] FIG. 7 is a photograph of the surface of a grid for cryo-electron microscopy, produced in the example, with β-galactosidase bound to the surface of the graphene grid, observed with a cryo-electron microscope. [Figure 8] FIG. 8 shows data obtained by structural analysis using a cryo-electron microscope of β-galactosidase bound to the surface of a grid for a cryo-electron microscope in an example. [Figure 9]FIG. 9 shows data obtained by structural analysis using a cryo-electron microscope of β-galactosidase bound to the surface of a grid for a cryo-electron microscope in an example. [Figure 10] FIG. 10 shows data obtained by structural analysis using a cryo-electron microscope of β-galactosidase bound to the surface of a grid for a cryo-electron microscope in an example. [Figure 11] FIG. 11 is a photograph of the surface of a graphene grid to which GroEL was bound, observed by negative staining, in an example. [Figure 12] FIG. 12 shows data obtained by structural analysis using a cryo-electron microscope of GroEL bound to the surface of a grid for a cryo-electron microscope in an example. [Figure 13] FIG. 13 shows data obtained by structural analysis using a cryo-electron microscope of GroEL bound to the surface of a grid for a cryo-electron microscope in an example. [Figure 14] FIG. 14 shows data obtained by structural analysis using a cryo-electron microscope of GroEL bound to the surface of a grid for a cryo-electron microscope in an example. [Figure 15] FIG. 15 shows data obtained by structural analysis using a cryo-electron microscope of GroEL bound to the surface of a grid for a cryo-electron microscope in an example. [Figure 16] FIG. 16 shows data obtained by structural analysis using a cryo-electron microscope of GroEL bound to the surface of a grid for a cryo-electron microscope in an example. [Figure 17] FIG. 17 is a table summarizing the results of structural analysis of β-galactosidase or GroEL using a cryo-electron microscope in the Examples. [Figure 18] FIG. 18 shows data obtained by structural analysis using a cryo-electron microscope of V1-ATPase bound to the surface of a grid for a cryo-electron microscope in an example. [Figure 19] FIG. 19 shows data obtained by structural analysis using a cryo-electron microscope of GroEL bound to the surface of a grid for a cryo-electron microscope in an example. [Figure 20] FIG. 20 shows data obtained by structural analysis using a cryo-electron microscope of V1-ATPase bound to the surface of a grid for a cryo-electron microscope in an example. [Figure 21] FIG. 21 shows an example of the measurement results of graphene by AFM before and after the surface treatment step (oxidation). [Figure 22] FIG. 22 shows an example of functional group introduction in an example. [Figure 23] FIG. 23 is a diagram schematically illustrating an example of a surface treatment process using a liquid reaction system. [Figure 24] FIG. 24 is a photograph of the surface of a graphene grid, produced in the example, to which β-galactosidase was bound, observed by a negative staining method. [Figure 25] FIG. 25 is a photograph of the surface of a graphene grid to which GroEL was bound, observed by negative staining, in an example. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following description.

[0013] The graphene grid of the present invention may have a structure in which, for example, at least one substituent selected from the group consisting of a hydroxyl group, a carboxyl group, and an aldehyde group is introduced onto the graphene surface, and further, the functional group is introduced by a reaction of the substituent.

[0014] In the graphene grid of the present invention, for example, the functional group may be at least one selected from the group consisting of a hydroxy group, a carboxy group, an aldehyde group (formyl group), a carbonyl group, an ether bond, an ester bond, an amino group, an imino group, a sulfonyl group, a sulfonyloxy group, and a fluoroalkyl group.

[0015] In the graphene grid of the present invention, for example, the functional group may be an ether bond.

[0016] In the graphene grid of the present invention, for example, the functional group may be an epoxy group.

[0017] The graphene grid of the present invention may be, for example, a grid for cryo-electron microscopy obtained by bonding a substance to be subjected to structural analysis by a cryo-electron microscope to the graphene grid of the present invention.

[0018] The grid for cryo-electron microscopy of the present invention may be a graphene grid to which a substance to be structurally analyzed by a cryo-electron microscope is bonded, or may be a graphene grid to which a substance to be structurally analyzed by a cryo-electron microscope is not bonded.

[0019] In the method for producing a graphene grid according to the present invention (the production method of the present invention), for example, in the functional group introduction step, at least one functional group selected from the group consisting of a hydroxy group, a carboxy group, an aldehyde group (formyl group), a carbonyl group, an ether bond, an ester bond, an amino group, an imino group, a sulfonyl group, a sulfonyloxy group, and a fluoroalkyl group may be introduced as the functional group.

[0020] In the production method of the present invention, for example, in the functional group introduction step, an ether bond may be introduced as the functional group.

[0021] In the production method of the present invention, for example, in the functional group introduction step, an epoxy group may be introduced as the functional group.

[0022] In the production method of the present invention, for example, the halogen oxide radical may be a chlorine dioxide radical.

[0023] In the production method of the present invention, for example, the surface treatment step may oxidize the graphene surface, and the substance with the modified graphene surface may be a substance with an oxidized graphene surface.

[0024] In the production method of the present invention, for example, in the surface treatment step, the reaction system may or may not be irradiated with light.

[0025] In the production method of the present invention, for example, the reaction system in the surface treatment step may be a gas reaction system or a liquid reaction system.

[0026] The manufacturing method of the present invention may be, for example, a method for manufacturing a grid for a cryo-electron microscope, further comprising a step of binding a substance to be structurally analyzed by a cryo-electron microscope to the graphene grid manufactured by the manufacturing method of the present invention.

[0027] The cryo-electron microscope grid manufactured by the manufacturing method of the present invention may be a graphene grid to which a substance to be structurally analyzed by a cryo-electron microscope is bonded, or may be a graphene grid to which a substance to be structurally analyzed by a cryo-electron microscope is not bonded.

[0028] In the method for structural analysis of a substance to be structurally analyzed according to the present invention, for example, the substance to be structurally analyzed may be an organic substance.

[0029] In the method for structural analysis of a substance to be structurally analyzed according to the present invention, for example, the organic substance may be a protein.

[0030] The graphene grid of the present invention may be, for example, a grid for instrumental analysis.

[0031] The graphene grid of the present invention may be, for example, a grid for an electron microscope.

[0032] The graphene grid of the present invention may be, for example, a grid for a cryo-electron microscope.

[0033] Furthermore, the uses of the graphene grid of the present invention are not limited to grids for instrumental analysis, grids for electron microscopes, and grids for cryo-electron microscopes. For example, the graphene grid may be used in bioreactors, sensors (e.g., biosensors such as enzyme sensors), microreactors, etc., as well as grids that can be used for detection reagents when antibodies or various proteins are linked to beads or the like, or for affinity column chromatography.

[0034] The cryo-electron microscope grid of the present invention can be used in a method for structural analysis of a substance using a cryo-electron microscope. The specific method of use is not particularly limited, but may be, for example, the same method as that used for a general cryo-electron microscope grid. The substance to be structurally analyzed is also not particularly limited, but may be, for example, at least one selected from the group consisting of proteins, antibodies, nucleic acids, viruses, ribosomes, mitochondria, ion channels, enzymes, and enzyme complexes. The protein serving as the substance to be structurally analyzed is not particularly limited, but may be, for example, an enzyme, a chaperonin protein, an iron storage protein, an antibody-antigen complex, a viral surface protein, etc. The enzyme is not particularly limited, but may be, for example, β-galactosidase, V1-ATPase, etc. The chaperonin protein is not particularly limited, but may be, for example, GroEL, etc. The iron storage protein is not particularly limited, but may be, for example, ferritin, etc. The substance to be structurally analyzed may be, for example, a biological substance (a biological substance or an identical substance), but is not limited thereto and may be any substance.

[0035] In the present invention, the surface treatment step can modify the graphene surface. In this specification, the surface treatment step may be referred to as a "modification treatment" or a "modification method." When the graphene is oxidized by the surface treatment step, the surface treatment step can be said to be a method for oxidizing the graphene.

[0036] In the present invention, the salt is not particularly limited and may be, for example, an acid addition salt or a base addition salt. The acid that forms the acid addition salt may be, for example, an inorganic acid or an organic acid, and the base that forms the base addition salt may be, for example, an inorganic base or an organic base. The inorganic acid is not particularly limited and examples thereof include sulfuric acid, phosphoric acid, hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, hypofluorite acid, hypochlorous acid, hypobromous acid, hypoiodous acid, fluorite acid, chlorous acid, bromous acid, iodous acid, fluoric acid, chloric acid, bromic acid, iodic acid, perfluoric acid, perchloric acid, perbromic acid, and periodic acid. The organic acid is not particularly limited and examples thereof include p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. The inorganic base is not particularly limited, and examples thereof include ammonium hydroxide, alkali metal hydroxides, alkaline earth metal hydroxides, carbonates, and hydrogen carbonates, and more specific examples thereof include sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, calcium hydroxide, and calcium carbonate. The organic base is not particularly limited, and examples thereof include ethanolamine, triethylamine, and tris(hydroxymethyl)aminomethane.

[0037] Hereinafter, the embodiments of the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following embodiments.

[0038] [1. Surface treatment process (method for producing a material with a modified graphene surface)] As described above, the method for producing a graphene grid according to the present invention (the production method of the present invention) is characterized by comprising a surface treatment step of reacting a graphene surface with a halogen oxide radical, and a functional group introduction step of introducing a functional group into the modified surface. The surface treatment step can also be considered a method for producing a material with a modified graphene surface. The surface treatment step will be specifically described below using examples.

[0039] (1) Graphene The graphene is a type of carbon allotrope. Examples of carbon allotropes other than graphene include diamond-like carbon (hereinafter sometimes abbreviated as "DLC"), carbon nanotubes, fullerenes, nanodiamonds, graphite, diamond, and carbon fibers. As described above, the manufacturing method of the present invention is characterized by including a surface treatment step of reacting the graphene surface with halogen oxide radicals, and a functional group introduction step of introducing functional groups into the modified surface. Furthermore, the graphene grid of the present invention is, as described above, a graphene grid having a structure in which functional groups are introduced onto the graphene surface.

[0040] Graphene is extremely thin compared to other carbon allotropes, and therefore has the advantage of easily achieving high resolution when used for instrumental analysis, such as in a grid for a cryo-electron microscope. Specifically, since graphene is theoretically only one atom thick, when used in a grid for a cryo-electron microscope, for example, the signal-to-noise ratio tends to be high. Therefore, according to the present invention, as described above, it is possible to perform structural analysis of a target substance with high resolution.

[0041] In the present invention, the graphene may or may not contain elements other than carbon. Examples of the other elements include hydrogen, oxygen, nitrogen, sulfur, boron, silicon, phosphorus, and various metal elements. The graphene may have a skeleton formed only by bonds between carbon atoms, with the other atoms bonded to its surface. For example, the graphene may have groups such as hydroxyl groups, methyl groups, carboxy groups, aldehyde groups, carbonyl groups, ether bonds, and ester bonds present on its surface. When the graphene contains the other elements, the content of the other elements is not particularly limited, and may be, for example, 40% or less, 20% or less, or 5% or less in terms of the number of atoms, or may be, for example, 0.01% or more, 0.1% or more, or 1% or more in terms of the number of atoms.

[0042] According to the surface treatment step of the present invention, for example, as described below, the surface of graphene can be oxidized to introduce functional groups such as hydroxyl groups and carboxy groups. The mechanism behind this is not clear, but it is speculated that, for example, methyl groups on the graphene surface are oxidized and converted into hydroxymethyl groups, carboxy groups, etc. However, this explanation is merely an exemplary speculation, and the present invention is not limited by this explanation.

[0043] (2) Halogen oxide radicals In the present invention, the halogen oxide radicals are contained in the reaction system of the surface treatment step. For example, the halogen oxide radicals may be generated in the reaction system and contained in the reaction system, or the halogen oxide radicals may be generated separately and contained in the reaction system. The method for generating the halogen oxide radicals is not particularly limited. Specific examples of the generation of the halogen oxide radicals will be described later.

[0044] The halogen oxide radicals may be, for example, any one type or a combination of two or more types, and can be appropriately selected depending on, for example, the type of graphene to be modified, reaction conditions, etc.

[0045] The halogen oxide radical is, for example, FO · (oxygen difluoride radical), F2O2 · (dioxygen difluoride radical), ClO2 · (chlorine dioxide radical), BrO2 · (bromine dioxide radical), I2O5 · Examples include halogen oxide radicals such as iodine (V) oxide.

[0046] (3) Reaction system The reaction system in the surface treatment step is a reaction system containing the graphene and the halogen oxide radicals. As described above, the reaction system may be, for example, a gas reaction system or a liquid reaction system. In the surface treatment step, the reaction system may or may not be irradiated with light. That is, the graphene and the halogen oxide radicals can react without irradiating the graphene with light. Not irradiating the graphene with light can provide effects such as improved safety and cost reduction. For example, halogen oxide radicals may be generated by light irradiation in a radical generation reaction system separate from the reaction system in the surface treatment step, and light irradiation may not be performed in the reaction system in the surface treatment step. As described above, the method for generating the halogen oxide radicals is not particularly limited, and may or may not involve light irradiation.

[0047] (3A) Gas Reaction System When the reaction system is a gas reaction system, for example, the graphene is placed in the gas reaction system containing the halogen oxide radicals and irradiated with light. However, in the present invention, the surface treatment step is not limited thereto. For example, the surface treatment step may be performed without light irradiation as long as the graphene surface can be reacted with the halogen oxide radicals. The gas reaction system may contain the radicals, and the type of gas phase in the gas reaction system is not particularly limited, and may be air, nitrogen, a rare gas, oxygen, or the like.

[0048] In the present invention, for example, the halogen oxide radicals may be introduced into the gaseous reaction system before or simultaneously with the surface treatment step, or the halogen oxide radicals may be generated in the gaseous reaction system. In the former case, for example, a gas containing the halogen oxide radicals may be introduced into the gas phase. In the latter case, for example, as described below, the halogen oxide radicals may be generated in a liquid-phase radical-generating reaction system and then transferred to the gas phase for introduction.

[0049] For example, when the halogen oxide radical is the chlorine dioxide radical, the chlorine dioxide radical can be present in the gas phase by introducing chlorine dioxide gas into the gas phase. The chlorine dioxide radical may be generated in the gas phase by, for example, an electrochemical method.

[0050] (3B) Liquid reaction system When the reaction system is a liquid reaction system, it contains, for example, an organic phase. The liquid reaction system may be, for example, a one-phase reaction system containing only the organic phase, or a two-phase reaction system containing the organic phase and an aqueous phase. In the case of a one-phase reaction system containing only an organic phase, for example, as described below, an aqueous phase containing a source of the halogen oxide radicals may be separately prepared, the halogen oxide radicals may be generated in the aqueous phase, and then the organic phase may be mixed with the aqueous phase to dissolve (extract) the halogen oxide radicals in the aqueous phase into the organic phase.

[0051] (3B-1) Organic phase As described above, the organic phase has the graphene disposed therein, and is, for example, a phase of an organic solvent containing the halogen oxide radical and having the graphene disposed therein.

[0052] The organic solvent is not particularly limited. For example, only one type of organic solvent may be used, or multiple types may be used in combination. In the present invention, examples of the organic solvent include halogenated solvents and fluorous solvents, as described above. When the liquid reaction system is a two-phase reaction system, the organic solvent is preferably, for example, a solvent that can form the two-phase system, i.e., a solvent that separates from the aqueous solvent that constitutes the aqueous phase, as described below, or a solvent that is poorly soluble or insoluble in the aqueous solvent.

[0053] A "halogenated solvent" refers to, for example, a solvent in which all or most of the hydrogen atoms of a hydrocarbon have been substituted with halogen. The halogenated solvent may be, for example, a solvent in which 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the number of hydrogen atoms of the hydrocarbon have been substituted with halogen. The halogenated solvent is not particularly limited, and examples thereof include methylene chloride, chloroform, carbon tetrachloride, carbon tetrabromide, and the fluorous solvents described below.

[0054] A "fluorous solvent" is a type of halogenated solvent, e.g., a solvent in which all or most of the hydrogen atoms of a hydrocarbon are substituted with fluorine atoms. The fluorous solvent may be, for example, a solvent in which 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the total number of hydrogen atoms of a hydrocarbon are substituted with fluorine atoms. The use of a fluorous solvent in the present invention has the advantage that, for example, side reactions can be suppressed or prevented due to the low reactivity of the solvent itself. Examples of side reactions include oxidation reactions of the solvent, hydrogen abstraction reactions or halogenation reactions (e.g., chlorination reactions) of the solvent by radicals, and reactions between radicals derived from raw material compounds and the solvent (e.g., reactions between ethyl radicals and the solvent when the hydrocarbon groups on the side chains or ends of the graphene are ethyl groups). The fluorous solvent is poorly miscible with water, making it suitable for forming the two-phase reaction system.

[0055] Examples of the fluorous solvent include solvents represented by the following chemical formulas (F1) to (F6), and among these, for example, CF3(CF2)4CF3 in which n=4 in the following chemical formula (F1) is preferred.

[0056] [ka]

[0057] The boiling point of the organic solvent is not particularly limited. The organic solvent can be appropriately selected depending on, for example, the temperature conditions of the surface treatment step. When the reaction temperature is set to a high temperature in the surface treatment step, a high-boiling-point solvent can be selected as the organic solvent. Note that, as will be described later, the present invention does not require heating and can be carried out, for example, at room temperature and atmospheric pressure. In such a case, the organic solvent does not need to be a high-boiling-point solvent, and a solvent with a not-so-high boiling point can be used from the viewpoint of ease of handling.

[0058] The organic phase may contain, for example, only the graphene, the halogen oxide radical, and the organic solvent, or may further contain other components. The other components are not particularly limited and may include, for example, a Bronsted acid, a Lewis acid, and oxygen (O). In the organic phase, the other components may be, for example, dissolved in the organic solvent or may not be dissolved. In the latter case, the other components may be, for example, dispersed in the organic solvent or may be precipitated.

[0059] As described above, the organic phase contains the halogen oxide radicals. The halogen oxide radicals can be contained in the organic phase by, for example, generating them outside the organic phase and extracting them with the organic phase. That is, when the reaction system is a one-phase reaction system containing only an organic phase, for example, the halogen oxide radicals can be generated separately outside the organic phase, which is the reaction system, and the generated halogen oxide radicals can be extracted with the organic phase. The organic phase containing the extracted halogen oxide radicals can be subjected to the surface treatment process as the reaction system. The generation of the halogen oxide radicals can be carried out, for example, in a separately prepared aqueous phase, as described below. On the other hand, when the liquid reaction system is a two-phase reaction system containing the organic phase and the aqueous phase, for example, the halogen oxide radicals can be generated in the aqueous phase, and the generated halogen oxide radicals can be extracted from the aqueous phase with the organic phase. The aqueous phase and the organic phase containing the halogen oxide radicals can be subjected to the surface treatment process as the two-phase reaction system.

[0060] When the graphene is a shaped body, it is preferable that the shaped body is fixed in the organic phase so that a portion to be surface-treated is immersed in the organic phase and is not exposed from the organic phase, for example, in terms of efficiency of a reaction treatment described later.

[0061] (3B-2) Water phase The aqueous phase is, for example, a phase of an aqueous solvent. The aqueous solvent is, for example, a solvent that is separated from the solvent used in the organic phase. Examples of the aqueous solvent include water such as H2O and D2O.

[0062] The aqueous phase may contain optional components such as a Lewis acid, a Bronsted acid, a radical generating source, etc., as described below. In the aqueous phase, these optional components may be dissolved in the aqueous solvent, or may not be dissolved. In the latter case, the optional components may be dispersed in the aqueous solvent, or may be precipitated.

[0063] (4) Surface treatment process As described above, the surface treatment step is a step of reacting the graphene surface with the halogen oxide radicals. In the surface treatment step, for example, the reaction system of the reaction may or may not be irradiated with light. Hereinafter, a method of irradiating the reaction system with light will be mainly described, but the present invention is not limited thereto. As described above, it is only necessary to cause the graphene surface to react with the halogen oxide radicals, and the surface treatment step may be performed without light irradiation. In that case, for example, in the following description, the surface treatment step may be performed without light irradiation. As described above, not irradiating the graphene with light provides effects such as improved safety and cost reduction.

[0064] The graphene is disposed in the reaction system, and the graphene can be modified. Specifically, according to the present invention, the graphene can be easily modified in the presence of the halogen oxide radicals. According to the present invention, the degree of modification of the graphene (e.g., the degree of modification by oxidation or the like) can be easily adjusted, for example, by adjusting the amount of the halogen oxide radicals or the length of time of light irradiation. Therefore, for example, decomposition of the graphene caused by excessive oxidation or the like can be prevented, and for example, impairment of the inherent properties of the graphene can be avoided.

[0065] In the surface treatment step, if a methyl group is present on the graphene surface, the methyl group (-CH3) is oxidized to at least one of, for example, a hydroxymethyl group (-CH2OH), a formyl group (-CHO), and a carboxyl group (-COOH). The following mechanism is presumed to explain this: That is, light irradiation converts the halogen oxide radical (for example, a chlorine dioxide radical) into the halogen radical (for example, a chlorine radical (Cl · )) and the oxygen molecules are generated. Then, the methyl group (-CH3) on the graphene surface is converted into the halogen radical (for example, a chlorine radical (Cl · )) acts as a hydrogen abstracting agent and forms a carboradical (-CH2 · ), and then the oxygen molecule (e.g., O2) acts as an oxidizing agent to form a hydroxymethyl group (-CH2OH). The hydroxymethyl group (-CH2OH) can be further oxidized to formyl group (-CHO) or carboxy group (-COOH).

[0066] In the surface treatment step, if an ethyl group is present on the graphene surface, the ethyl group (-CH2CH3) is oxidized to, for example, a hydroxyethyl group (-CH2CH2OH), an acetaldehyde group (-CH2CHO), or a carboxymethyl group (-CH2COOH).

[0067] Furthermore, for example, when a methylene group (-CH2-) is contained on the graphene surface, the methylene group is oxidized to, for example, a hydroxymethylene group (-CHOH-), a carbonyl group (-CO-), or the like.

[0068] In the surface treatment step, the conditions for light irradiation are not particularly limited. The wavelength of the irradiation light is not particularly limited, and the lower limit is, for example, 200 nm or more and the upper limit is, for example, 800 nm or less. The light irradiation time is not particularly limited, and the lower limit is, for example, 1 second or more and the upper limit is, for example, 1,000 hours. The reaction temperature is not particularly limited, and the lower limit is, for example, -20°C or more and the upper limit is, for example, 100°C or less or 40°C or less, and the range is, for example, 0 to 100°C or 0 to 40°C. The atmospheric pressure during the reaction is not particularly limited, and the lower limit is, for example, 0.1 MPa or more and the upper limit is, for example, 100 MPa or less, 10 MPa or less, or 0.5 MPa or less, and the range is, for example, 0.1 to 100 MPa, 0.1 to 10 MPa, or 0.1 to 0.5 MPa. Examples of reaction conditions for the surface treatment step include a temperature of 0 to 100°C or 0 to 40°C and a pressure of 0.1 to 0.5 MPa. As described above, the surface treatment step can be performed even without light irradiation. According to the present invention, the surface treatment step or all of the steps including the surface treatment step can be performed at room temperature (room temperature) and atmospheric pressure (atmospheric pressure) without heating, pressurizing, depressurizing, or the like. "Room temperature" is not particularly limited and is, for example, 5 to 35°C. Therefore, the present invention is applicable even if the graphene contains graphene with low heat resistance. According to the present invention, the surface treatment step or all of the steps including the surface treatment step can be performed in the atmosphere without inert gas replacement, for example.

[0069] The light source for the light irradiation is not particularly limited, and for example, visible light contained in natural light such as sunlight can be used. The use of natural light allows, for example, easy excitation. Furthermore, as the light source, for example, a xenon lamp, a halogen lamp, a fluorescent lamp, a mercury lamp, an LED lamp, or the like can be used instead of or in addition to the natural light. In the light irradiation, for example, a filter that cuts off wavelengths other than the required wavelength can also be used as appropriate.

[0070] In the present invention, by irradiating only a desired region of the graphene with light, only the desired region can be modified. There are no particular limitations on the method for controlling such selective light irradiation, and for example, only the desired region may be irradiated with light, or only the region not to be irradiated may be masked and the entire region may be irradiated with light.

[0071] When the reaction system is a liquid reaction system, for example, at least the organic phase may be irradiated with light in the surface treatment step. In the case of a one-phase reaction system consisting of only the organic phase, for example, the surface treatment step can be carried out by irradiating the one-phase reaction system with light. In the case of a two-phase reaction system containing the organic phase and the aqueous phase, for example, light may be irradiated only to the organic phase, or light may be irradiated to the two-phase reaction system. In the case of a liquid reaction system, for example, light may be irradiated to the liquid reaction system while the liquid reaction system is in contact with air, and in the case of a two-phase reaction system, light may be irradiated to the aqueous phase with oxygen dissolved therein.

[0072] According to the present invention, in the surface treatment step, for example, the halogen radical (e.g., chlorine atom radical Cl) can be obtained by a very simple method of simply irradiating the surface with light in the presence of the halogen oxide radical. · ) and oxygen molecules O2 are generated, which react with the graphene (for example, an oxidation reaction), thereby modifying the graphene. Furthermore, the graphene can be easily and efficiently changed and modified, even under extremely mild conditions, such as room temperature and normal pressure.

[0073] According to the present invention, a substance in which the graphene surface is modified can be obtained without using, for example, a toxic heavy metal catalyst, etc. Therefore, as described above, the reaction can be carried out under, for example, extremely mild conditions, and the graphene surface can be efficiently modified by a method that imposes an extremely small load on the environment.

[0074] Examples of methods for oxidizing the surface of graphene include a method using a strong oxidizing agent such as potassium permanganate (KMnO), a gas-phase plasma treatment method, and a method using ultraviolet (UV) light irradiation in an ozone atmosphere. However, these methods not only oxidize the surface of graphene but also sever carbon-carbon bonds in the graphene skeleton, potentially decomposing the graphene. In contrast, the surface treatment step of the present invention allows the reaction to be carried out under mild reaction conditions as described above, thereby suppressing or preventing severance of carbon-carbon bonds in the graphene skeleton. Furthermore, the surface treatment step of the present invention facilitates control of reaction conditions. Specifically, the reaction in the surface treatment step can be stopped at will by, for example, stopping light irradiation or the supply of the halogen oxide radicals. This, for example, further suppresses severance of carbon-carbon bonds in the graphene skeleton and makes it easier to control the progress of the reaction on the graphene surface. Therefore, the present invention allows the graphene surface to be efficiently modified without decomposing the graphene. The fact that graphene is less deteriorated (decomposed, etc.) after the surface treatment step can be confirmed, for example, by observing the graphene surface after the surface treatment step with an atomic force microscope (AFM). Furthermore, while oxidation by liquid-phase treatment using potassium permanganate or ultraviolet (UV) irradiation in an ozone atmosphere takes several tens of minutes, the surface treatment step of the present invention makes it possible to easily modify (oxidize) the graphene surface while maintaining its structure by, for example, gas-phase treatment (dry process) in a short time (within a few minutes). This allows, for example, functional groups to be effectively introduced in the subsequent functional group introduction step. However, this explanation is merely an example, and the present invention is not limited thereto. For example, the reaction system in the surface treatment step of the present invention is not limited to a gas reaction system (gas-phase treatment) as described above, but may also be a liquid reaction system.

[0075] (5) Halogen oxide radical generation process The present invention may further include, for example, a halogen oxide radical generating step of generating the halogen oxide radical. The halogen oxide radical generating step may be carried out, for example, before the surface treatment step or simultaneously with the surface treatment step. The method for generating the halogen oxide radical is not particularly limited.

[0076] The halogen oxide radical generating step may use, for example, a radical generating reaction system to generate the halogen oxide radicals. The reaction system in the surface treatment step may be, for example, the gas reaction system (gas phase) or the liquid reaction system (liquid phase). The radical generating reaction system may be used as the liquid reaction system in the surface treatment step after generating the halogen oxide radicals.

[0077] When the reaction system of the surface treatment step is the gas reaction system, for example, the radical generation reaction system may be prepared separately from the reaction system of the surface treatment step. The radical generation reaction system may be, for example, an aqueous phase containing the halogen oxide radical generation source. The aqueous phase may contain, for example, the halogen oxide radical generation source, and in the halogen oxide radical generation step, the halogen oxide radicals are generated from the halogen oxide radical generation source. The aqueous phase may be, for example, an aqueous solvent phase, and the aqueous solvent may be the same as described above. When the halogen oxide radicals generated in the aqueous phase are hydrophobic, for example, a two-phase reaction system containing the organic phase and the aqueous phase can be used to transfer the halogen oxide radicals to the organic phase. As described above, when the surface treatment step is performed in the gas reaction system, the halogen oxide radical generation reaction system may be, for example, an aqueous phase only, or a two-phase reaction system containing an aqueous phase and an organic phase. When the halogen oxide radicals are hydrophobic, for example, the radicals generated in the aqueous phase can be transferred directly to the gas phase, and therefore the reaction system for generating radicals may consist of only the aqueous phase.

[0078] The reaction system for the surface treatment step and the radical-generating reaction system may be, for example, the reaction system shown in FIG. 1 . Specifically, as shown in FIG. 1 , a radical-generating reaction system 5 is first placed in a petri dish 3. The radical-generating reaction system 5 may be, for example, an aqueous phase containing a source of halogen oxide radicals. The source of halogen oxide radicals is not particularly limited, but may be, for example, as described below. Meanwhile, graphene 6 is placed in another petri dish 4. These petri dishes 3 and 4 are placed in another larger petri dish 1, and the petri dish 1 is covered with a lid 2 to prevent leakage of the internal gas. Thereafter, the radical-generating reaction system 5 in the petri dish 3 is irradiated with light. This light irradiation generates halogen oxide radical gas in the petri dish 3, and the halogen oxide radical gas flows into the petri dish 4 and reacts with the graphene 6. In this manner, the surface treatment step can be performed. In addition, in FIG. 1, the sources of halogen oxide radicals in the radical generating reaction system 5 are sodium chlorite aqueous solution (NaClO2aq) and hydrochloric acid (HCl), and the halogen oxide radicals generated by the reaction between them are chlorine dioxide radicals (ClO2 · However, as described above, in the present invention, the source of halogen oxide radicals and halogen oxide radicals are not limited to these.

[0079] When the reaction system of the surface treatment step is the liquid reaction system and includes the aqueous phase, the aqueous phase may be the radical-generating reaction system, for example. The aqueous phase may be the same as the radical-generating reaction system when the reaction system of the surface treatment step is the gas reaction system, for example. When the halogen oxide radicals generated in the aqueous phase are hydrophobic, the halogen oxide radicals can be transferred to the organic phase by, for example, forming a two-phase reaction system including the organic phase and the aqueous phase.

[0080] The source of the halogen oxide radical (radical generation source) is not particularly limited and can be appropriately selected depending on, for example, the type of the halogen oxide radical. The source of the halogen oxide radical may be, for example, one type, or a combination of multiple types.

[0081] The source of the halogen oxide radical is, for example, a compound containing oxygen and a halogen, and specific examples include, for example, a halous acid (HXO2) or a salt thereof. The salt of the halous acid is not particularly limited, and examples thereof include metal salts, and examples of the metal salts include alkali metal salts, alkaline earth metal salts, and rare earth salts. The source of the halogen oxide radical may also be, for example, a compound containing oxygen, a halogen, and a Group 1 element (e.g., at least one selected from the group consisting of H, Li, Na, K, Rb, and Cs), and examples thereof include, for example, the halous acid or an alkali metal salt thereof. When the halogen oxide radical is the chlorine dioxide radical, its source is not particularly limited and may be, for example, chlorous acid (HClO) or a salt thereof. Specific examples include sodium chlorite (NaClO), lithium chlorite (LiClO), potassium chlorite (KClO), magnesium chlorite (Mg(ClO)), and calcium chlorite (Ca(ClO)). Among these, sodium chlorite (NaClO) is preferred from the standpoints of cost, ease of handling, and the like. For example, similar salts can also be used as sources of other halogen oxide radicals. Other sources include, for example, bromates such as sodium bromite, and iodates such as sodium iodite.

[0082] In the aqueous phase, the concentration of the source is not particularly limited. When the source is the compound, the concentration, when converted into the halogen oxide ion concentration, is, for example, 0.0001 mol / L or more at the lower limit and 1 mol / L or less at the upper limit. When the concentration, when converted into the number of moles of the halogen oxide ions, is, for example, 1 / 100,000 times or more and 1,000 times or less of the number of moles of the raw material. When the source is a halogen acid or halogen acid salt (e.g., chlorous acid or halogen acid salt), the concentration is, for example, 1 / 100,000 times or more of the number of moles of the raw material, and 1,000 times or less of the number of moles of the raw material. -When converted into a concentration of halous acid ions (e.g., chlorous acid ions (ClO2)), for example, the lower limit is 0.0001 mol / L or more and the upper limit is 1 mol / L or less. - When converted to the number of moles of the raw material, for example, the lower limit is 1 / 100,000 times or more the number of moles of the raw material, and the upper limit is 1,000 times or less. For other sources, for example, the above concentrations can also be applied.

[0083] The aqueous phase may further contain at least one of a Lewis acid and a Bronsted acid, which may be reacted with the halogen oxide ions to generate the halogen oxide radicals. The at least one of the Lewis acid and the Bronsted acid may be, for example, at least one of a Lewis acid and a Bronsted acid containing a Group 1 element. The halogen oxide ions may be, for example, chlorite ions (ClO2 - ) The aqueous phase may contain, for example, only one of the Lewis acid and the Bronsted acid, or both, or one substance may serve as both the Lewis acid and the Bronsted acid. Only one type of Lewis acid or one type of Bronsted acid may be used, or multiple types may be used in combination. In the present invention, the "Lewis acid" refers to, for example, a substance that acts as a Lewis acid with respect to the source of halogen oxide radicals.

[0084] In the aqueous phase, the concentration of at least one of the Lewis acid and the Bronsted acid is not particularly limited and can be appropriately set depending on, for example, the type of graphene to be modified, etc. The lower limit of the concentration is, for example, 0.0001 mol / L or more and the upper limit is 1 mol / L or less.

[0085] The Bronsted acid is not particularly limited and may be, for example, an inorganic acid or an organic acid, and specific examples include trifluoromethanesulfonic acid, trifluoroacetic acid, acetic acid, hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid. ais, for example, 10 or less. a The lower limit of is not particularly limited, and is, for example, −10 or more.

[0086] The aqueous phase preferably contains, for example, the halogen oxide ion and the Bronsted acid, and is, for example, an aqueous phase in which the compound and the Bronsted acid (e.g., hydrochloric acid) are dissolved in an aqueous solvent. As a specific example, when the halogen oxide radical is a chlorine dioxide radical, the aqueous phase preferably contains, for example, chlorite ion (ClO - ) and a Bronsted acid, for example, an aqueous phase in which the sodium chlorite (NaClO2) and the Bronsted acid (for example, hydrochloric acid) are dissolved in an aqueous solvent is preferred.

[0087] In the aqueous phase, for example, the Lewis acid, the Bronsted acid, the radical generating source, etc. may be dissolved or insoluble in the aqueous solvent. In the latter case, they may be dispersed or precipitated in the aqueous solvent.

[0088] The halogen oxide radical generation step is not particularly limited, and for example, by adding a source of halogen oxide radicals to the aqueous solvent, the halogen oxide radicals (e.g., chlorite ions) can be spontaneously generated from the halogen oxide ions (e.g., chlorite ions). The aqueous phase preferably contains the source dissolved in the aqueous solvent, and is preferably allowed to stand. In the halogen oxide radical generation step, the generation of the halogen oxide radicals can be further promoted by, for example, further allowing at least one of the Lewis acid and the Bronsted acid to coexist in the aqueous phase. In the halogen oxide radical generation step, the halogen oxide radicals can be generated by, for example, irradiating the aqueous phase with light. However, the halogen oxide radicals can also be generated without light irradiation, for example, by simply allowing the aqueous phase to stand.

[0089] The mechanism by which the halogen oxide radicals are generated from the halogen oxide ions in the aqueous phase is presumed to be similar to that shown in Fig. 15 (a two-phase liquid phase reaction system consisting of an organic phase and an aqueous phase), which will be described later. However, this explanation is merely an example and does not limit the present invention in any way.

[0090] When the reaction system is the liquid reaction system and is a two-phase reaction system including the organic phase and the aqueous phase, after generating the halogen oxide radicals as described above, the liquid reaction system may be subjected to the surface treatment step as described above. The halogen oxide radicals generated from the source in the aqueous phase of the reaction system are poorly soluble in water and therefore dissolve in the organic phase of the reaction system. For example, the liquid reaction system in which the halogen oxide radicals have been generated may be further irradiated with light to perform the surface treatment step of modifying the surface of the graphene. In this case, for example, the halogen oxide radical generation step and the surface treatment step can be performed consecutively by irradiating the liquid reaction system with light. In the present invention, performing the halogen oxide radical generation step and the surface treatment step in the two-phase reaction system may, for example, achieve better reaction efficiency.

[0091] On the other hand, when the reaction system in the above-described surface treatment step is the liquid reaction system and is a one-phase reaction system containing only the organic phase, for example, the halogen oxide radicals are generated in the aqueous phase by the above-described method, the generated halogen oxide radicals are dissolved (extracted) in the organic phase, the aqueous phase is removed, and the organic phase containing the halogen oxide radicals is subjected to the surface treatment step as the one-phase reaction system.

[0092] FIG. 23 is a schematic diagram illustrating an example of the halogen oxide radical generation step and the surface treatment step using the two-phase reaction system. In FIG. 23, the chlorine dioxide radical is shown as a specific example of the halogen oxide radical, but the present invention is not limited to these examples. As shown in FIG. 23, the reaction system is configured such that two layers, an aqueous layer (the aqueous phase) and an organic layer (the organic phase), are separated in a reaction vessel and are in contact with each other only at the interface. The upper layer is the aqueous layer (the aqueous phase) 12, and the lower layer is the organic layer (the organic phase) 11. Although FIG. 23 is a cross-sectional view, hatching of the aqueous layer 12 and the organic layer 11 has been omitted for clarity. As shown in FIG. 23, chlorite ions (ClO2) in the aqueous layer (aqueous phase) 12 are separated from each other at the interface. - ) reacts with acid to form chlorine dioxide radicals (ClO2 · ) is generated. Chlorine dioxide radicals (ClO2 · ) is hardly soluble in water, so it dissolves in the organic layer 11. Next, chlorine dioxide radicals (ClO2 · The organic layer 11 containing chlorine dioxide radicals (ClO2 · ) decomposes to form chlorine radicals (Cl · ) and oxygen molecules (O2) are generated. As a result, graphene in the organic layer (organic phase) 11 is oxidized and the surface is modified. However, FIG. 23 is merely an example and does not limit the present invention in any way.

[0093] In FIG. 23 , the aqueous layer 12 is the upper layer and the organic layer 11 is the lower layer. However, for example, if the organic layer 11 has a lower density (specific gravity), the organic layer 11 becomes the upper layer. For example, the graphene may be immobilized in the reaction vessel so that it is disposed in the upper organic layer. In this case, an immobilizing unit for immobilizing the graphene may be provided, for example, in the reaction vessel or outside the reaction vessel. In the latter case, for example, the graphene may be suspended from the outside and immersed in the organic layer.

[0094] While the two-phase reaction system is illustrated in FIG. 23 , in the manufacturing method of the present invention, the surface treatment step may be performed in a single-phase reaction system using only an organic phase. In this case, for example, an aqueous phase containing a source of halogen oxide radicals is separately prepared, and the halogen oxide radicals are generated in the aqueous phase. The organic phase is then mixed with the aqueous phase, and the halogen oxide radicals in the aqueous phase are dissolved (extracted) into the organic phase. The aqueous phase and the organic phase are then separated, the organic phase is recovered, and the graphene is disposed therein. This forms a single-phase reaction system, and the surface treatment step is performed by light irradiation alone in the presence of the halogen oxide radicals. Furthermore, when the reaction system in the surface treatment step is a gas reaction system, as described above, the halogen oxide radicals may be generated in the aqueous phase, and then the surface treatment step may be performed in the gas reaction system.

[0095] Examples of methods for oxidizing the surface of graphene include a method using a strong oxidizing agent such as potassium permanganate (KMnO), a gas-phase plasma treatment method, and a method using ultraviolet (UV) light irradiation in an ozone atmosphere. However, these methods not only oxidize the surface of graphene but also sever carbon-carbon bonds in the graphene skeleton, potentially decomposing the graphene. In contrast, the surface treatment step of the present invention allows the reaction to be carried out under mild reaction conditions as described above, thereby suppressing or preventing severance of carbon-carbon bonds in the graphene skeleton. Furthermore, the surface treatment step of the present invention facilitates control of reaction conditions. Specifically, the reaction in the surface treatment step can be stopped at will by, for example, stopping light irradiation or the supply of the halogen oxide radicals. This, for example, further suppresses severance of carbon-carbon bonds in the graphene skeleton and makes it easier to control the progress of the reaction on the graphene surface. Therefore, the present invention allows the graphene surface to be efficiently modified without decomposing the graphene. The fact that graphene is less deteriorated (decomposed, etc.) after the surface treatment step can be confirmed, for example, by observing the graphene surface after the surface treatment step with an atomic force microscope (AFM). Furthermore, while oxidation by liquid-phase treatment using potassium permanganate or ultraviolet (UV) irradiation in an ozone atmosphere takes several tens of minutes, the surface treatment step of the present invention makes it possible to easily modify (oxidize) the graphene surface while maintaining its structure by, for example, gas-phase treatment (dry process) in a short time (within a few minutes). This allows, for example, functional groups to be effectively introduced in the subsequent functional group introduction step. However, this explanation is merely an example, and the present invention is not limited thereto. For example, the reaction system in the surface treatment step of the present invention is not limited to a gas reaction system (gas-phase treatment) as described above, but may also be a liquid reaction system.

[0096] In the method for producing a graphene grid according to the present invention, the surface treatment step can efficiently modify the graphene surface without decomposing the graphene, as described above. This allows, for example, as described below, the substance to be structurally analyzed to be firmly fixed to the surface of the cryo-electron microscope at a high concentration, thereby suppressing or preventing uneven distribution (localization, e.g., aggregation) and bias in orientation of the substance to be structurally analyzed. This can dramatically reduce the time required for screening to optimize measurement conditions for a cryo-electron microscope compared to conventional methods. It can also shorten the time required for collecting data for structural analysis.

[0097] [2. Graphene grid manufacturing method] As described above, the method for producing a graphene grid according to the present invention (the production method of the present invention) is a method for producing a graphene grid formed from a substance having functional groups introduced onto the surface of the graphene, characterized by comprising: a surface treatment step of modifying the graphene surface by reacting the graphene surface with halogen oxide radicals; and a functional group introduction step of introducing functional groups onto the modified surface.

[0098] By introducing the functional group, various functions can be imparted to the graphene. Specifically, for example, as shown in the examples described later, the functional group makes it easier to capture a substance (e.g., a protein) that is the subject of structural analysis using a cryo-electron microscope. In addition, for example, by appropriately selecting the type of the functional group, any function depending on the type of the functional group can be imparted to the graphene.

[0099] In the functional group introduction step, for example, a functional group can be introduced into a graphene surface that has been oxidized (modified) to introduce a substituent such as a hydroxyl group, a carboxyl group, or an aldehyde group (formyl group) by reacting the substituent such as a hydroxyl group, a carboxyl group, or an aldehyde group with another substance (for example, a condensation reaction). This allows, for example, the introduction of any functional group that can be introduced by a reaction with the substituent (for example, a hydroxyl group, a carboxyl group, an aldehyde group, etc.) introduced into the graphene surface.

[0100] Examples of the functional group introduction step include those described in the Examples below. Specifically, for example, as described in the Examples below, the hydrogen of the hydroxyl group introduced onto the graphene surface can be substituted with a 2,3-epoxypropyl group, and then an addition reaction can be carried out between the epoxy group and a protein or 1H,1H-undecafluorohexylamine, etc. Note that 1H,1H-undecafluorohexylamine is not limited thereto, and any other amine may be used. The amine is not particularly limited, and may be, for example, a primary amine or a secondary amine. Furthermore, the present invention is not limited thereto, and any amine may be used as long as it does not deviate from the scope of the present invention.

[0101] The functional group that can be introduced in the functional group introduction step is not particularly limited. For example, as described above, any functional group that can be introduced by a reaction with the substituent (e.g., hydroxyl group, carboxy group, aldehyde group, etc.) introduced onto the graphene surface can be introduced. The functional group introduced in the functional group introduction step may be, for example, at least one selected from the group consisting of a hydroxy group, a carboxy group, an aldehyde group, a carbonyl group, an ether bond, an ester bond, an amino group, an imino group, a sulfonyl group, a sulfonyloxy group, and a fluoroalkyl group. The fluoroalkyl group may be a group in which some or all of the hydrogen atoms of an alkyl group are substituted with fluorine atoms, such as a perfluoroalkyl group in which all of the hydrogen atoms of an alkyl group are substituted with fluorine atoms, or may be, for example, a trifluoromethyl group. The functional group may also be, for example, a combination of two or more functional groups, such as a trifluoroacetyl group, which is a combination of a trifluoromethyl group and an ester bond, or a trifluoromethylsulfonyl group, which is a combination of a trifluoromethyl group and a sulfonyl group. In the present invention, the number of carbon atoms in the "alkyl group" is not particularly limited and may be, for example, 1 to 24, 1 to 18, 1 to 12, or 1 to 6, and the "alkyl group" may be linear or branched. The same applies to groups in which at least one hydrogen atom of an alkyl group is substituted (e.g., a fluoroalkyl group). Specific functional groups to be introduced in the functional group introduction step include succinimidyl, isothiocyano, sulfonyl chloride, carboxylic acid chloride, ethylene oxide, alkyl chloride, carboxylic acid anhydride, maleimide, and hydrazide functional groups, which can be introduced onto the graphene surface by reacting them with substituents on the graphene surface, such as hydroxyl, carboxy, and aldehyde groups. The functional groups introduced in this manner can react with groups such as amino, sulfhydryl (-SH, also referred to as mercapto, or thiol), and aldehyde groups, and thus can bind and capture compounds having these groups (e.g., proteins).

[0102] The reaction conditions in the functional group introduction step are not particularly limited and can be set arbitrarily. The reaction conditions may be the same as or similar to existing reaction conditions for similar or similar reactions. For example, when a functional group is introduced by a reaction with a hydroxyl group or a carboxyl group, reaction conditions similar to or similar to the reaction conditions for existing reactions with a hydroxyl group or a carboxyl group may be used.

[0103] The graphene grid produced by the production method of the present invention can be used, for example, as a grid for a cryo-electron microscope. For example, during structural analysis using a cryo-electron microscope, a substance to be structurally analyzed by the cryo-electron microscope can be bound to the cryo-electron microscope grid (graphene grid) produced by the production method of the present invention. The cryo-electron microscope grid produced by the production method of the present invention may be a graphene grid bound to a substance to be structurally analyzed by the cryo-electron microscope, or may be a graphene grid not bound to a substance to be structurally analyzed by the cryo-electron microscope.

[0104] [3. Graphene grids (grids for cryo-electron microscopes, etc.)] As described above, the graphene grid of the present invention is a graphene grid having a structure in which functional groups are introduced onto the graphene surface. The graphene grid of the present invention can be used, for example, as a grid for cryo-electron microscopy. For example, during structural analysis using a cryo-electron microscope, the cryo-electron microscopy grid (graphene grid) of the present invention can be used by bonding a substance to be structurally analyzed by the cryo-electron microscope. Furthermore, as described above, the graphene grid of the present invention may be, for example, a cryo-electron microscopy grid in which a substance to be structurally analyzed by the cryo-electron microscope is bonded to the graphene grid of the present invention. The cryo-electron microscopy grid of the present invention may be a graphene grid to which a substance to be structurally analyzed by the cryo-electron microscope is bonded, or a graphene grid to which no substance to be structurally analyzed by the cryo-electron microscope is bonded.

[0105] The method for producing a graphene grid of the present invention is not particularly limited, but the graphene grid can be produced, for example, by the above-mentioned method for producing a graphene grid of the present invention, which includes the surface treatment step and the functional group introduction step, as described above.

[0106] Furthermore, the graphene grid of the present invention can be produced by, for example, omitting the surface treatment step and carrying out the functional group introduction step on the graphene. More specifically, for example, if the graphene surface that has not been subjected to the surface treatment step has substituents such as hydroxyl groups or carboxyl groups, the graphene grid of the present invention can be produced by carrying out the functional group introduction step on these substituents.

[0107] The graphene grid of the present invention can suppress or prevent uneven distribution, bias in orientation, etc. of the substance to be structurally analyzed by, for example, cryo-electron microscopy by binding the substance to the functional group. The bond between the functional group and the substance to be structurally analyzed is not particularly limited, and examples thereof include a covalent bond, an ionic bond, a metal coordination bond, a host-guest interaction, and a hydrogen bond, with a covalent bond being particularly preferred in terms of bond strength.

[0108] In the graphene grid of the present invention, covalently binding a substance to be analyzed by a cryo-electron microscope or the like to the functional group provides the following advantages. First, by covalently binding the substance to be analyzed, the substance is not easily removed even when the surface of the cryo-electron microscope is washed. Therefore, unnecessary impurities (e.g., proteins denatured at the air-liquid interface) that are not covalently bound to the surface of the cryo-electron microscope can be washed away by the washing, enabling highly accurate structural analysis. Second, even if the substance to be analyzed is difficult to concentrate (e.g., a protein), the substance can be introduced to the surface of the cryo-electron microscope at a high concentration by repeatedly forming a covalent bond to the functional group. This can dramatically reduce the time required for, for example, optimizing the measurement conditions for a cryo-electron microscope or collecting data for structural analysis. Third, covalent bonding allows the substance to be firmly fixed to the surface of the cryo-electron microscope, thereby suppressing or preventing uneven distribution (localization, e.g., aggregation) and orientation bias of the substance. This allows for high-accuracy and high-resolution structural analysis of the substance. Fourth, for example, by possessing the first to third features, as demonstrated in the examples described below, structural analysis can be performed with high accuracy and high resolution without introducing tags or the like into the substance to be analyzed (e.g., proteins). This eliminates the need for a step of introducing tags or the like into the substance to be analyzed, allowing for the simple production of a measurement sample (a cryo-electron microscope grid of the present invention to which the substance to be analyzed is bound) in a short time. Thus, according to the present invention, for example, the time required to prepare a measurement sample for a cryo-electron microscope can be dramatically reduced compared to conventional methods, and the measurement sample can be prepared with easier procedures than conventional methods. However, the present invention is not limited to methods in which tags or the like are not introduced into the substance to be analyzed; structural analysis can also be performed by introducing tags or the like into the substance to be analyzed (e.g., proteins).

[0109] Conventional carbon grids for cryo-electron microscopes have a weak bond between the grid and a substance to be structurally analyzed. As described above, this can lead to uneven distribution (localization) and biased orientation of the substance to be structurally analyzed on the carbon grid, which can interfere with structural analysis. In contrast, the graphene grid of the present invention can strongly bond the substance to be structurally analyzed, as described above. This can suppress or prevent uneven distribution, biased orientation, and the like of the substance to be structurally analyzed.

[0110] The method for binding the target substance to the functional group is not particularly limited. For example, depending on the combination of the functional group and the target substance, reaction conditions similar to or equivalent to those of known similar reactions can be appropriately set. For example, the graphene grid of the present invention having the functional group may be immersed in a solution (e.g., an aqueous solution) of the target substance for structural analysis for an appropriate period of time, but the method is not limited to this.

[0111] The target substance for structural analysis is not particularly limited and may be the same as or different from the target substance for structural analysis in a general cryo-electron microscope. Examples of target substances for structural analysis include proteins, antibodies, nucleic acids, viruses, ribosomes, mitochondria, ion channels, enzymes, and enzyme complexes. Examples of proteins include membrane proteins, water-soluble proteins, and glycoproteins classified by physical properties, as well as enzyme proteins, structural proteins, transcription factors, transport proteins, storage proteins, contractile proteins, and defense proteins classified by function. Examples include apoferritin, which is used as a standard protein for analysis, as well as ribosomes, proteasomes, RNA polymerases, capsids, GPCRs, photosystem complexes, ATP synthases, and antibody complexes. Tubulin, which is composed of these proteins, can also be used as the target substance for structural analysis in the present invention. Furthermore, examples of target substances for structural analysis in the present invention include tissues themselves, such as muscle, collagen, and flagella. Furthermore, for example, living organisms themselves or tissue surfaces can be used as the target substances for structural analysis in the present invention by using antibodies or other labeled proteins.

[0112] As described above, the graphene grid of the present invention can suppress or prevent uneven distribution, bias in orientation, and the like of the substance to be structurally analyzed. Furthermore, the cryo-electron microscope grid of the present invention enables structural analysis of substances that were difficult to analyze using conventional cryo-electron microscope grids. For example, membrane proteins and the like are difficult to crystallize, making three-dimensional structural analysis difficult using conventional cryo-electron microscope grids. In contrast, the graphene grid of the present invention can strongly bind and stabilize the structure of substances to be structurally analyzed, such as membrane proteins, thereby enabling structural analysis, including three-dimensional structural analysis. Furthermore, the graphene grid of the present invention also enables, for example, single-particle analysis of proteins.

[0113] The method of using the graphene grid of the present invention is not particularly limited, and may be the same as the method of using a general cryo-electron microscope grid. For example, as with a general cryo-electron microscope grid, the captured substance to be analyzed can be observed under a microscope using the ice embedding method, thereby enabling structural analysis.

[0114] In the present invention, for example, as described above, structural analysis can be performed with high accuracy and high resolution without adding tags, etc. to proteins. However, as described above, the present invention is not limited to the embodiment in which tags, etc. are not introduced into the substance to be structurally analyzed, and structural analysis may be performed by introducing tags, etc. into the substance to be structurally analyzed (e.g., protein).

[0115] Furthermore, the use of the graphene grid of the present invention is not limited to grids for cryo-electron microscopes, but can be used for a wide range of applications, such as bioreactors, sensors (e.g., biosensors such as enzyme sensors), and microreactors, as well as detection reagents and affinity column chromatography in which antibodies or various proteins are linked to beads or the like. [Example]

[0116] Examples of the present invention will be described below, but the present invention is not limited to the following examples.

[0117] [Reference example 1] As described below, the surface of graphene was modified by the surface treatment process of the present invention to prepare a material having a modified graphene surface.

[0118] In this example, the surface treatment step was carried out using a reaction in a gas phase.

[0119] First, as shown in Figure 1, 20 mL of deionized water was added to a 5 cm diameter Petri dish 3, and sodium chlorite NaClO2 (300 mg) and a 36 mass % HCl aqueous solution (100 μL) were dissolved therein to prepare a hydrochloric acid acidified NaClO2 aqueous solution (reaction system for radical generation) 5. Meanwhile, 10 mg of graphene 6 was placed in a 3 cm diameter Petri dish 4. These Petri dishes 3 and 4 were placed in an 11 cm diameter Petri dish 1. Furthermore, a lid 2 was placed on the Petri dish 1 to prevent leakage of the internal gas. Thereafter, at room temperature, LED light with a wavelength of 365 nm was irradiated from above the Petri dish 1 at a light intensity of 10 mW / cm onto the hydrochloric acid acidified NaClO2 aqueous solution 5 in the Petri dish 3. 2 The graphene 6 was irradiated with light for 3, 10, 30, or 60 minutes. This light irradiation activated the ClO2 radical gas generated by the reaction between hydrochloric acid and NaClO2 in Petri dish 3 and flowed into Petri dish 4, where it reacted with graphene 6. This surface treatment process oxidized the surface of graphene 6. The oxidized graphene 6 was dried under reduced pressure for 24 hours to obtain the target product (a material with a modified surface of graphene 6).

[0120] In this example, powdered graphene (FUJIFILM Wako Pure Chemical Industries, Ltd.: product name "06-0313") was used as graphene 6 to produce a substance in which the surface of graphene 6 was modified.

[0121] X-ray photoelectron spectroscopy (XPS) measurements were performed on the graphene 6 before and after the surface treatment step (oxidation reaction), and the progress of the oxidation reaction was confirmed by detecting the energy peak position of C1s (1s orbital of carbon atom). The results of the XPS measurements are shown in the graphs in Figure 2. In each graph, the horizontal axis represents binding energy (eV) and the vertical axis represents the relative value of peak intensity (cps).

[0122] As mentioned above, Figure 2 shows the results of XPS measurements of graphene before and after the surface treatment process (oxidation reaction). As shown in the figure, there was no significant change after 30 minutes of reaction, but after 60 minutes of reaction, the C=C bond peak was attenuated and the C-OH bond peak and C-O-C bond peak were increased, confirming oxidation of the graphene surface. The results of the elemental analysis by XPS are also shown in Table 1 below. As shown in Table 1, the ratio of O atoms increased after the oxidation reaction, confirming oxidation of the graphene surface.

[0123] [Table 1]

[0124] The surface conditions of the graphene before and after the surface treatment step (oxidation reaction) were observed using an AFM (atomic force microscope). Specifically, the surface morphology was evaluated using a scanning probe microscope (Shimadzu Corporation, product name SPM-9500J3, cantilever: Olympus Corporation, product name OMCL-AC200TS-C3). The results are shown in FIG. 21. As shown in the figure, the AFM evaluation revealed that no damage to the graphene due to the surface treatment step (oxidation reaction) was observed, and no deterioration of the graphene was confirmed.

[0125] The structure of the graphene surface after oxidation in this reference example is presumed to be, for example, as shown in the following chemical formula E1, although this structure is only one example of the presumed structure.

[0126] [ka]

[0127] [Reference example 2] Rhodamine B was bound to the graphene of Reference Example 1 by the following method, and the fluorescence intensity was measured.

[0128] First, 6 mg of rhodamine B was added to a test tube. Next, 2 mL of deionized water was added, and then 0.1 M acetic acid solution was added to adjust the pH to 6.5. Two of these were prepared, and graphene before and after 60 minutes of oxidation reaction was added to each, followed by stirring at room temperature for 15 minutes. After the reaction, each sample was washed twice with 3 mL of deionized water and dried under reduced pressure for 24 hours. The fluorescence intensity of each sample was then measured using a UV-vis spectrometer in the range of 560–660 nm with an excitation light of 555 nm.

[0129] Figure 3 shows the results of measuring the fluorescence intensity of graphene. In this figure, the horizontal axis represents wavelength (nm) and the vertical axis represents peak intensity (relative value). As shown in the figure, the peak intensity around 575 nm of graphene after the oxidation reaction increased, confirming that rhodamine B had been bound. This suggests that hydroxyl groups, carboxyl groups, or both had been introduced to the surface of graphene after the oxidation reaction.

[0130] [Example 1] A graphene grid was prepared, and the surface was modified (oxidized) by a surface treatment process in which the graphene grid was reacted with chlorine dioxide radicals. A functional group introduction process was then carried out to introduce functional groups onto the surface of the surface-modified (oxidized) graphene grid, producing a graphene grid with functional groups. Furthermore, a protein, which was the target substance for structural analysis using a cryo-electron microscope, was bonded (bound) to the graphene grid with functional groups.

[0131] [Graphene grid production] A polymethyl methacrylate (PMMA)-graphene-Cu trilayer sheet was floated on the surface of a 0.5 mol / L aqueous solution of ammonium persulfate with the Cu side facing the water. The sheet was then left to stand for 30 minutes to dissolve the Cu layer. The PMMA-graphene sheet, obtained after removing the Cu layer, was washed by floating it in ultrapure water twice for 10 minutes each time. A TEM Mo grid (Quantifoil Micro Tools GmbH, QUANTIFOIL) and an Au grid (Quantifoil Micro Tools GmbH, QUANTIFOIL) were then placed at the bottom of a membrane stretcher (Oken Shoji Co., Ltd., product name: Collodion membrane stretcher) filled with ultrapure water. The PMMA-graphene sheet was then floated on the water surface of the membrane stretcher and gently drained to adhere the PMMA-graphene sheet to the grid. The grid was then dried at room temperature for 1 hour and then heated at 130°C for 20 minutes. The grid was then immersed in acetone at 60°C for 60 minutes, chloroform at room temperature (approximately 25°C) for 30 minutes, acetic acid for 3 hours, and isopropyl alcohol for 10 minutes twice each to remove the PMMA. Finally, the grid was heated at 100°C for 10 minutes to produce a graphene grid in which the surface of the Mo or Au grid was coated with graphene.

[0132] [Reaction of graphene grid with chlorine dioxide radicals (surface treatment process)] The graphene grids manufactured from the Mo grids or Au grids were each heat-treated at 130°C for 15 minutes. The heat-treated graphene grids were then subjected to a surface treatment process (surface oxidation) using chlorine dioxide radicals (ClO2 radicals) in the same manner as described in Reference Example 1 and FIG. 1 . However, in this example, the surface treatment process (surface oxidation) was performed while the graphene grid was covered with aluminum foil to prevent direct exposure to LED light (UV light). The reaction time (light irradiation time) was 10 minutes, and the reaction temperature was room temperature, the same as in Example 1. XPS measurements were performed on the graphene grids before and after the surface treatment process, as in Example 1. After the surface treatment process, attenuation of the C=C bond peak and increases in the C-OH bond peak and C-O-C bond peak were observed, and the ratio of O atoms also increased, confirming oxidation of the graphene surface.

[0133] [Functional group introduction process] According to the following scheme 1, functional groups were introduced onto the surface of the graphene grid after the surface treatment step (functional group introduction step).

[0134] [ka]

[0135] Specifically, the functional group introduction step was carried out as follows. First, the graphene grid after the surface treatment step was immersed in a 2 mmol / L DMSO solution of polyethylene glycol 2-aminoethyl ether acetic acid (average molecular weight 2100) and left to stand overnight at room temperature. As a result, the epoxide on the graphene grid surface reacted with the polyethylene glycol 2-aminoethyl ether acetic acid to introduce functional groups, as shown in Scheme 1.

[0136] Furthermore, the graphene grid that had undergone the functionalization step was bonded (conjugated) with a protein, which was the target substance for structural analysis by cryo-electron microscopy. Specifically, the graphene grid was immersed in an aqueous solution containing 5 mmol / L N-hydroxysulfosuccinimide (Sulfo-NHS) and 5 mmol / L EDC for 15 minutes, and then in an aqueous solution containing 0.2 mg / mL β-galactosidase (B-gal) for 60 minutes. As shown in Scheme 1, the protein was bonded by reacting with the amino groups present in β-galactosidase (B-gal).

[0137] [Reference example 3] As described below, the surface of the graphene grid was modified by the surface treatment process of the present invention to prepare a material having a surface-modified graphene grid.

[0138] First, sodium chlorite NaClO2 (200 mg) was dissolved in 20 mL of ultrapure water, and then 37 mass % HCl aqueous solution (100 μL) was added to prepare a 1 mass % hydrochloric acid acidified NaClO2 aqueous solution (reaction system for radical generation). Next, as shown in Figure 4, the hydrochloric acid acidified NaClO2 aqueous solution was added to the outer container of a 10 cm x 10 cm glass double container (Petri dish). Meanwhile, a cover glass was placed in the inner container of the glass double container, and the graphene grid produced by the above method was placed on top of it. Furthermore, the glass double container was covered with a lid to prevent leakage of the internal gas. Thereafter, at room temperature, LED light with a wavelength of 365 nm and a light intensity of 20 mW / cm was applied to the hydrochloric acid acidified NaClO2 aqueous solution in the glass double container from above the glass double container. 2The graphene grid was irradiated with light for 10 minutes. As shown in FIG. 4, aluminum foil was placed over the lid of the inner container to prevent direct light exposure to the graphene grid. This light irradiation activated ClO radical gas, which was generated by the reaction between hydrochloric acid and NaClO2 in Petri dish 3 and flowed into Petri dish 4, and reacted with graphene 6. This surface treatment process oxidized the surface of the graphene grid. The oxidized graphene grid was dried under reduced pressure for 24 hours to obtain the target product (a material with a modified surface of the graphene grid).

[0139] As shown in the lower part of Figure 4, a water droplet was dropped on the graphene grid surface before and after the surface treatment process (surface oxidation). Photographs of the droplets were taken, and the water contact angle was measured visually. The water contact angle was 83° before the surface treatment process and 51° immediately after the surface treatment process. This indicates that the water contact angle decreased due to the surface treatment process, confirming that the hydrophilicity of the graphene grid surface had increased. Furthermore, when the water contact angle was measured again using the same method one hour after the surface treatment process, it was 59°, as shown in the figure. This indicates that the hydrophilicity of the graphene grid surface was maintained even one hour after the surface treatment process.

[0140] [Example 2] A graphene grid was prepared, and then subjected to a surface treatment process in which the graphene grid was reacted with chlorine dioxide radicals to modify (oxidize) the surface, producing a graphene grid with a modified (oxidized) surface. A functional group introduction process was then performed to introduce functional groups onto the surface of the surface-modified (oxidized) graphene grid, producing a graphene grid with functional groups introduced. Furthermore, a protein, which is a substance to be structurally analyzed using a cryo-electron microscope, was bonded (bound) to the graphene grid with functional groups introduced. This process is outlined in the schemes of Figures 5 and 6. This process will be described in more detail below.

[0141] [Graphene grid production] A graphene grid in which the surface of an Au grid was covered with graphene was produced in exactly the same manner as in the "Production of a Graphene Grid" described in Example 1.

[0142] [Reaction of graphene grid with chlorine dioxide radicals (surface treatment process)] The graphene grid manufactured from the Au grid was subjected to the surface treatment (surface oxidation) in exactly the same manner as in the "reaction of graphene grid with chlorine dioxide radicals (surface treatment)" described in Example 1. XPS measurements were performed on the graphene grid in the same manner as in Example 1. After the surface treatment, attenuation of the C=C bond peak and increases in the C-OH bond peak and C-O-C bond peak were observed, and furthermore, the ratio of O atoms increased, confirming oxidation of the graphene surface.

[0143] [Functional group introduction process] Epichlorohydrin was reacted with the graphene grid surface after the surface treatment process to introduce functional groups (functional group introduction process). Specifically, the graphene grid after the surface treatment process was first fixed with tweezers, and a 1% by volume epichlorohydrin aqueous solution (3 μL) was placed on the graphene side and allowed to stand at room temperature for 5 minutes. The grid was then washed three times with 5 μL of HEPES buffer (25 mM HEPES pH 8.0, 50 mM NaCl, 2 mM MgCl2, 1 mM TCEP) to wash away excess epichlorohydrin. This resulted in a graphene grid with 2,3-epoxypropyl groups introduced into the graphene grid.

[0144] Furthermore, a protein, the target substance for structural analysis by cryo-electron microscopy, was conjugated (bound) to the graphene grid that had undergone the functionalization process. Specifically, 3 μL of HEPES buffer containing 0.2 mg / mL of β-galactosidase (B-gal) was placed on the graphene grid with the 2,3-epoxypropyl groups and allowed to stand at room temperature for 5 minutes. The 2,3-epoxypropyl groups on the graphene grid surface reacted with the amino groups present in the β-galactosidase (B-gal) to bind the protein. The grid was then washed three times with 5 μL of HEPES buffer to remove excess β-galactosidase (B-gal). While the functionalization process and β-galactosidase binding are presumed to have occurred according to the scheme shown at the top of Figure 6, other reactions may have also occurred. For example, in the scheme at the top of Figure 6, hydroxyl groups (OH) on the surface of the graphene grid react with epichlorohydrin, but it is also possible that a substituent other than hydroxyl groups (e.g., carboxyl groups) reacted with epichlorohydrin.

[0145] The graphene grid surface with the β-galactosidase bound thereto was observed at magnifications of 15,000x, 30,000x, and 60,000x using a negative staining method with a 2% aqueous uranyl acetate solution. As shown in the photograph at the bottom of Figure 6, it was confirmed that β-galactosidase was bound to the graphene grid surface.

[0146] [Example 3] An electron microscope grid (usable as a cryo-electron microscope grid) with β-galactosidase bound to the graphene grid surface was produced using the same method as in Example 2. Furthermore, an electron microscope grid with β-galactosidase bound to the graphene grid surface was produced using the same method as in Example 2, except that the concentration of the aqueous β-galactosidase solution was changed from 0.2 mg / mL to 0.5 mg / mL or 1.2 mg / mL, or the concentration of the aqueous β-galactosidase solution was changed to 0.5 mg / mL and further treated with 1 M Tris. The 1M Tris treatment involved placing 3 μL of 1M trishydroxymethylaminomethane (Tris) pH 8.0 aqueous solution on the graphene grid after the epichlorohydrin functionalization step, allowing the grid to stand at room temperature for 5 minutes, and then washing three times with 5 μL of HEPES buffer. This treatment allowed the excess Tris to react with the 2,3-epoxypropyl groups on the graphene grid, blocking subsequent reaction with the amino groups of β-galactosidase. Furthermore, the resulting β-galactosidase-bound electron microscope grids were flash-frozen in liquid ethane and observed at 50,000x magnification using a cryo-electron microscope (JEOL Ltd., Cryo ARM 300). As shown in Figure 7, β-galactosidase was bound only to the graphene grid surface that had not been treated with 1M Tris.

[0147] Furthermore, using the electron microscope grid with β-galactosidase bound to the graphene grid surface produced as described above, image data was collected using a cryo-electron microscope (manufactured by JEOL Ltd., trade name Cryo ARM 300), and based on the obtained data set, structural analysis of the β-galactosidase bound to the surface was performed using structural analysis software RELION (trade name). As a result, as shown in Figures 8 to 10, structural analysis of β-galactosidase was successful with high resolutions of 2.49 Å from 288,402 particles extracted from 1,000 images and 2.24 Å from 1,411,632 particles extracted from 4,128 images. Note that 1 Å is defined as 0.1 nm (10 -10 Furthermore, by optimizing the analysis method, we succeeded in analyzing the structure of β-galactosidase at an even higher resolution of 1.81 Å from 231,395 particles extracted from 3,242 images.

[0148] [Example 4] The graphene grid was subjected to a surface treatment process (oxidation reaction with chlorine dioxide radicals) and a functional group introduction process (introduction of epoxy groups by reaction with epichlorohydrin) using the same method as in Example 2. Then, 40 μL of 1H,1H-Undecafluorohexylamine was added and stirred for 60 minutes to allow the reaction to proceed, resulting in a grid with fluoroalkylamino groups introduced into the graphene. The resulting grid was washed twice with deionized water and dried under reduced pressure for 24 hours.

[0149] The functional group introduction reaction in this example is presumed to be as shown in the scheme at the top of Figure 22. As shown, it is believed that the hydrogen atoms of the hydroxyl groups on the nanodiamond surface were replaced with 2,3-epoxypropyl groups, and then an addition reaction occurred between the epoxy groups and 1H,1H-undecafluorohexylamine. In addition, samples were prepared in the same manner as above, except that either or both of the surface treatment process (oxidation reaction with chlorine dioxide radicals) and the functional group introduction process (introduction of epoxy groups by reaction with epichlorohydrin) were omitted. XPS measurements were then performed on each sample, along with the grids used in this example. The XPS measurement results are shown in the graph and table at the bottom of Figure 22. As shown in the figure, only the grid of this example (Sample 5), which underwent both the surface treatment process (oxidation reaction with chlorine dioxide radicals) and the functional group introduction process (introduction of epoxy groups by reaction with epichlorohydrin), showed the presence of fluorine atoms on the surface, confirming that a reaction with 1H,1H-undecafluorohexylamine (bonding to the grid surface) had occurred.

[0150] [Example 5] Electron microscope grids with GroEL bound to their surface (usable as cryo-electron microscope grids) were fabricated using the same method as in Example 2, except that: (1) a four-layer polymethyl methacrylate (PMMA)-graphene-Cu-graphene sheet was used; (2) the graphene on the non-PMMA side was removed using water-resistant abrasive paper (P3000); (3) the PMMA removal process involved immersion in acetic acid twice for 2 hours each and isopropyl alcohol for 10 minutes, rather than acetone and chloroform; and (4) a HEPES buffer (25 mM HEPES, pH 8.0, 50 mM NaCl) containing 0.3 mg / mL GroEL was used instead of the β-galactosidase buffer. GroEL is a chaperonin protein and is a target protein for cryo-electron microscopy. Furthermore, electron microscope grids were produced in the same manner, except that treatment with an epichlorohydrin aqueous solution (introduction of the functional epoxy group) was not performed. The surfaces of these two types of electron microscope grids were observed using a negative staining method with a 2% uranyl acetate aqueous solution. The results are shown in the photographs in Figure 11. As shown in Figure 11, GroEL bound to the surface of electron microscope grid (1), which was treated with an epichlorohydrin aqueous solution (introduction of the functional epoxy group), was observed. In contrast, no GroEL was observed at all in grid (2), which was not treated with an epichlorohydrin aqueous solution (introduction of the functional epoxy group).

[0151] [Example 6] An electron microscope grid (usable as a cryo-electron microscope grid) with GroEL bound to its graphene surface was produced using the same manufacturing method as in Example 2, except that a HEPES buffer (25 mM HEPES pH 8.0, 50 mM NaCl) containing 3.2 mg / mL GroEL was used instead of the β-galactosidase-containing buffer. Furthermore, image data was collected using a cryo-electron microscope (JEOL Ltd., trade name: Cryo ARM 300) using this electron microscope grid with GroEL bound to its graphene surface. Based on the obtained data set, structural analysis of the GroEL bound to the surface was performed using structural analysis software RELION (trade name). As a result, as shown in Figures 12-16 and 19, we successfully performed structural analysis of GroEL at an extremely high resolution of 2.18 Å from 115,424 particles extracted from 531 images. As of August 2020, the highest resolution GroEL map registered in the Electron Microscopy Data Bank (EM Data Bank) was 3.26 Å, which is a significant improvement over the previous resolution. The main reason for this is that, as shown in Figure 16, the proportion of lateral directions in the distribution of molecular projection angles has increased significantly, eliminating the bias in orientation in the up-down direction (along the seven-fold axis), which was a problem in analysis using conventional methods. Furthermore, by optimizing the analysis method, we succeeded in analyzing the GroEL structure at an even higher resolution of 1.99 Å from 158,485 particles extracted from 504 images.

[0152] The results of structural analysis of β-galactosidase or GroEL in Examples 3 and 6, performed using a cryo-electron microscope (manufactured by JEOL Ltd., trade name Cryo ARM 300), are summarized in the table of FIG.

[0153] [Example 7] An electron microscope grid (usable as a cryo-EM grid) with V1-ATPase bound to its surface was prepared using the same method as in Example 2, except that a 1.0 mg / mL V1-ATPase solution was used instead of the β-galactosidase-containing buffer. V1-ATPase is an enzyme and a protein that can be analyzed by cryo-EM. Furthermore, using this electron microscope grid with V1-ATPase bound to its surface, image data was collected using a cryo-EM microscope (manufactured by JEOL Ltd., trade name: Cryo ARM 300). Based on the obtained data set, structural analysis of the V1-ATPase bound to the surface was performed using the structural analysis software RELION (trade name). As shown in Figures 18 and 20, the structure of V1-ATPase was successfully analyzed at an extremely high resolution of approximately 2.6 Å from approximately 300,000 extracted particles.

[0154] [Example 8] Using the same method as in Example 2, the graphene grid was subjected to a surface treatment step (oxidation reaction using chlorine dioxide radicals) and a functional group introduction step (introduction of epoxy groups by reaction with epichlorohydrin). In this manner, a graphene grid (upper left in Scheme 2 below) with functional groups (epoxy groups) introduced therein was produced. Then, according to Scheme 2 below, a graphene grid (upper right in Scheme 2 below) with maleimide groups introduced therein was obtained. Furthermore, according to the lower scheme in Scheme 2 below, a protein (β-galactosidase), which was the target substance for structural analysis using a cryo-electron microscope, was conjugated (bound) to the graphene grid.

[0155] [ka]

[0156] Specifically, the reaction of Scheme 2 was carried out as follows. First, as described above, a graphene grid was subjected to a surface treatment process (oxidation reaction using chlorine dioxide radicals) and a functional group introduction process (introduction of epoxy groups by reaction with epichlorohydrin) using the same method as in Example 2 to produce a graphene grid with functional groups (epoxy groups) introduced thereto (upper left in Scheme 2). Next, 3 μL of a 56 mM aqueous solution of N-(2-aminoethyl)maleimide hydrochloride (top center of Scheme 2) at pH 9.0 was placed on the graphene grid with functional groups (epoxy groups) introduced thereto and allowed to stand at room temperature for 5 minutes. The grid was then washed three times with distilled water (5 μL) to wash away excess N-(2-aminoethyl)maleimide hydrochloride. Thus, the 2,3-epoxypropyl groups introduced onto the graphene grid surface reacted with the amino groups present in N-(2-aminoethyl)maleimide hydrochloride to produce a graphene grid with maleimide groups introduced thereto (upper right in Scheme 2).

[0157] Furthermore, a protein (β-galactosidase), which was the target substance for structural analysis by cryo-electron microscopy, was conjugated (bound) to the maleimide-introduced graphene grid. Specifically, a Tris buffer (25 mM Tris pH 7.0, 50 mM NaCl, 2 mM MgCl2, 1 mM dithiothreitol (DTT)) containing 0.5 mg / mL of β-galactosidase (B-gal) was placed on the maleimide-introduced graphene grid and allowed to stand at room temperature for 5 minutes. This allowed the maleimide groups introduced onto the graphene grid surface to react with the sulfhydryl groups present in the β-galactosidase (B-gal). The grid was then washed three times with 5 μL of the Tris buffer to wash away excess β-galactosidase (B-gal). In this way, a graphene grid bound to β-galactosidase (bottom right of Scheme 2) was obtained.

[0158] The graphene grid surface to which the β-galactosidase was bound was observed at a magnification of 15,000 times using a negative staining method with a 2% aqueous uranyl acetate solution. As shown in the photograph in Figure 24, it was confirmed that β-galactosidase was bound to the graphene grid surface.

[0159] [Example 9] The graphene grid was subjected to a surface treatment process (oxidation reaction using chlorine dioxide radicals) and a functional group introduction process (introduction of epoxy groups by reaction with epichlorohydrin) using the same method as in Example 2. In this way, a graphene grid (upper left in Scheme 3 below) with functional groups (epoxy groups) introduced was produced. This graphene grid was then reacted with NH2-PEG4-NH2 and then with epichlorohydrin, as shown in Scheme 3 below, and then bonded (bonded) to a protein (GroEL), the target substance for structural analysis using a cryo-electron microscope.

[0160] [ka]

[0161] Specifically, the reaction of Scheme 3 was carried out as follows. First, as described above, a graphene grid was subjected to a surface treatment process (oxidation reaction using chlorine dioxide radicals) and a functional group introduction process (introduction of epoxy groups by reaction with epichlorohydrin) using the same method as in Example 2 to produce a graphene grid with functional groups (epoxy groups) introduced thereto (upper left in Scheme 3). Next, a 42 mM NH2-PEG4-NH2 aqueous solution (3 μL) was placed on the graphene grid with functional groups (epoxy groups) introduced thereto and allowed to stand at room temperature for 5 minutes. The grid was then washed three times with distilled water (5 μL) to wash away excess NH2-PEG4-NH2. This resulted in a graphene grid in which the 2,3-epoxypropyl groups introduced to the graphene grid surface reacted with the amino groups present in the NH2-PEG4-NH2 and bonded thereto. Furthermore, a 1% by volume aqueous solution of epichlorohydrin (3 μL) was placed on the graphene surface and allowed to stand at room temperature for 5 minutes, after which the grid was washed three times with distilled water (5 μL) to wash away excess epichlorohydrin. This allowed the amino groups introduced onto the graphene grid surface to react with the epoxy groups present in the epichlorohydrin, yielding a graphene grid with 2,3-epoxypropyl groups and PEG chains (lower left in Scheme 3).

[0162] Furthermore, a protein, which is a target substance for structural analysis by cryo-electron microscopy, was conjugated (bound) to the graphene grid that had undergone the functionalization process. Specifically, a HEPES buffer (25 mM HEPES pH 8.0, 50 mM NaCl) containing 0.3 mg / mL of GroEL was placed on the graphene grid with the 2,3-epoxypropyl and PEG chains introduced thereto and allowed to stand at room temperature for 5 minutes. This allowed the epoxy groups introduced onto the graphene grid surface to react with the amino groups present in GroEL, resulting in binding. The grid was then washed three times with 5 μL of the HEPES buffer to wash away excess GroEL. In this way, a graphene grid (bottom right of Scheme 3) to which a protein (GroEL), a target substance for structural analysis by cryo-electron microscopy, was conjugated (bound).

[0163] Furthermore, when the surface of the graphene grid to which the GroEL was bound was observed at a magnification of 15,000 times using a negative staining method with a 2% aqueous uranyl acetate solution, it was confirmed that GroEL was bound to the surface of the graphene grid, as shown in the photograph at the bottom of Figure 25.

[0164] [Example 10] The surface of the monolayer graphene (graphene grid) on the copper substrate was modified (oxidized) by a surface treatment step of reacting it with chlorine dioxide radicals in exactly the same manner as in the "Reaction of Graphene Grid with Chlorine Dioxide Radicals (Surface Treatment Step)" described in Example 1, except that single-layer graphene (ACS Materials, product name "CVCU1S22") was used instead of the graphene grid produced from the Au grid in Example 1. Furthermore, a functional group introduction step was performed in which new functional groups were introduced in the gas phase to the hydroxyl groups on the surface of the surface-modified (oxidized) graphene, producing a graphene grid with the functional groups introduced (functional group introduction step) according to the following Scheme 4.

[0165] [ka]

[0166] [Reaction of graphene after the surface treatment step with trifluoroacetic anhydride] Specifically, the reaction in the upper part of Scheme 4 was carried out as follows. First, a cover glass was placed in a 6 cm × 6 cm glass dish, and the graphene on the copper substrate (graphene grid) after the surface treatment step was placed on top of it. Furthermore, a 3 cm × 3 cm glass dish containing trifluoroacetic anhydride (molecular weight 210) (2 mL) was placed inside the glass dish, and the dish was covered and allowed to stand at room temperature for 5 hours. As a result, as shown in the upper part of Scheme 4, the hydroxy groups on the graphene surface reacted with the trifluoroacetic anhydride to introduce trifluoroacetyl groups. In this way, a graphene grid into which trifluoroacetyl groups were introduced as functional groups was produced (functional group introduction step).

[0167] [Reaction of graphene after the surface treatment step with trifluoromethanesulfonic anhydride] Specifically, the reaction in the lower part of Scheme 4 was carried out as follows. First, a cover glass was placed in a 6 cm × 6 cm glass dish, and the graphene on the copper substrate (graphene grid) after the surface treatment step was placed on top of it. Furthermore, a 3 cm × 3 cm glass dish containing trifluoromethanesulfonic anhydride (molecular weight 282) (2 mL) was placed inside the glass dish, and the dish was covered and allowed to stand at room temperature for 5 hours. As a result, as shown in Scheme 1, the hydroxyl groups on the graphene surface reacted with the trifluoromethanesulfonic anhydride to introduce trifluoromethylsulfonyl groups. In this way, a graphene grid having trifluoromethylsulfonyl groups introduced as functional groups was produced (functional group introduction step).

[0168] The graphene on the copper substrate was subjected to elemental analysis by X-ray photoelectron spectroscopy (XPS) before and after the surface treatment step (oxidation reaction), after the reaction with trifluoroacetic anhydride, and after the reaction with trifluoromethanesulfonic anhydride. The elemental analysis results by XPS are shown in Table 2 below. As shown in Table 2 below, the ratio of the number of F atoms increased after the reaction with trifluoroacetic anhydride, and the ratio of the number of F atoms to the number of S atoms increased after the reaction with trifluoromethanesulfonic anhydride. This also confirmed the functionalization of the graphene surface.

[0169] [Table 2]

[0170] As demonstrated by the above examples, the present invention enables the preparation of measurement samples for cryo-electron microscopy in an extremely short time and with ease. Furthermore, as demonstrated by the above examples, the present invention enables the structural analysis of various proteins, such as β-galactosidase, GroEL, and V1-ATPase, with extremely high accuracy and high resolution without the addition of tags.

[0171] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above embodiments. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention. [Industrial Applicability]

[0172] As described above, the present invention provides a cryo-electron microscope grid, a method for manufacturing a cryo-electron microscope grid, and a method for structural analysis of a substance to be structurally analyzed, which are capable of suppressing or preventing uneven distribution, bias in orientation, etc. of a substance to be structurally analyzed in structural analysis by a cryo-electron microscope. According to the present invention, a cryo-electron microscope grid having a substance to be structurally analyzed, such as a protein, bound thereto can be manufactured in an extremely short time, and the substance to be structurally analyzed can be structurally analyzed with extremely high resolution, and therefore the present invention is of great industrial value. [Explanation of symbols]

[0173] 1 Petri dish 2 lid 3 Petri dishes 4 Petri dishes 5. Reaction system for radical generation 6 Carbon allotropes 11 Organic layer (organic phase) 12 Water layer (aqueous phase)

Claims

1. A graphene grid with a structure in which functional groups are introduced onto the graphene surface.

2. 2. The graphene grid according to claim 1, wherein at least one substituent selected from the group consisting of a hydroxyl group, a carboxyl group, and an aldehyde group is introduced onto a surface of the graphene, and the functional group is further introduced by a reaction of the substituent.

3. 3. The graphene grid according to claim 1, wherein the functional group is at least one selected from the group consisting of a hydroxy group, a carboxy group, an aldehyde group, a carbonyl group, an ether bond, an ester bond, an amino group, an imino group, a sulfonyl group, a sulfonyloxy group, and a fluoroalkyl group.

4. The graphene grid according to claim 1 or 2, wherein the functional group is an ether bond.

5. The graphene grid according to claim 1 or 2, wherein the functional group is an epoxy group.

6. A grid for cryo-electron microscopy, comprising the graphene grid according to claim 1 and a substance to be subjected to structural analysis by a cryo-electron microscope bound to the grid.

7. a surface treatment step of modifying the graphene surface by reacting the graphene surface with halogen oxide radicals; and a functional group introduction step of introducing functional groups onto the modified surface.

8. 8. The method according to claim 7, wherein in the functional group introduction step, at least one functional group selected from the group consisting of a hydroxy group, a carboxy group, an aldehyde group, a carbonyl group, an ether bond, an ester bond, an amino group, an imino group, a sulfonyl group, a sulfonyloxy group, and a fluoroalkyl group is introduced as the functional group.

9. The method according to claim 7 , wherein an ether bond is introduced as the functional group in the functional group introduction step.

10. The method according to claim 7 , wherein in the functional group introduction step, an epoxy group is introduced as the functional group.

11. 11. The method according to claim 7, wherein the halogen oxide radical is a chlorine dioxide radical.

12. 12. A method for manufacturing a grid for a cryo-electron microscope, further comprising a step of bonding a substance to be structurally analyzed by a cryo-electron microscope to the graphene grid manufactured by the manufacturing method according to any one of claims 7 to 11.

13. A method for structural analysis of a substance to be structurally analyzed using a cryo-electron microscope, comprising: a structural analysis grid preparation step and a structural analysis step, In the structural analysis grid preparation step, the substance to be structurally analyzed is bound to the graphene grid according to any one of claims 1 to 5 or a graphene grid manufactured by the manufacturing method according to any one of claims 7 to 11, or a grid for a cryo-electron microscope according to claim 6 or a grid for a cryo-electron microscope manufactured by the manufacturing method according to claim 12 is prepared, In the structural analysis step, the structural analysis target substance bound to the graphene grid is structurally analyzed using a cryo-electron microscope. A structural analysis method characterized by:

14. 14. The structural analysis method according to claim 13, wherein the substance to be structurally analyzed is an organic substance.

15. 15. The structural analysis method according to claim 14, wherein the organic substance is a protein.

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