Graphene linker compound and field effect transistor biosensor

Abstract

The present invention pertains to: a compound which is represented by formula (I) (in the formula, A, B, Z, and R1 to R6 are as described in the description and claims); a linker agent composition which contains the linker compound and a specific solvent; a composite of the linker compound and a graphene nanosheet; and a GFET biosensor which is provided with the composite.

Description

Graphene linker compounds and field-effect transistor biosensors

[0001] The present invention relates to a novel linker compound that enables surface modification of two-dimensional materials such as graphene, a linker composition comprising the linker compound and a specific solvent, a composite of the linker compound and graphene nanosheets, and a graphene field-effect transistor biosensor comprising the composite.

[0002] Methods and systems for the highly sensitive and rapid detection of target substances such as proteins, viruses, and bacteria are important in medical diagnosis, environmental monitoring, and bio-research, and various detection methods and means are being investigated. Many target substances have specific characteristics in terms of surface shape, protein, and charge, and selective detection methods utilizing these specific characteristics have been proposed. Optical means and electrical means using field-effect transistors (FETs) have been proposed as detection means, but electrical means using FETs in particular are expected to be highly sensitive and provide a simple system.

[0003] Graphene, a representative two-dimensional material, consists of a single layer of carbon atoms arranged in a hexagonal lattice. Graphene possesses several unique material properties, including electrical conductivity, stability, and uniformity, making it highly suitable for sensor applications. In recent years, there has been a surge in the development of graphene field-effect transistors (GFETs) using graphene. For example, methods have been reported for manufacturing sensor devices that immobilize modified molecules (i.e., linker compounds) containing functional and anchor portions, as well as these modified molecules, on the surface of two-dimensional materials for the purpose of sensing and capturing target molecules (see, for example, Patent Document 1).

[0004] Japanese Patent Publication No. 2023-125621

[0005] The introduction of functional moieties enabling the sensing and capture of target molecules on the surface of two-dimensional materials typified by graphene is very important in the functionalization process of biosensor development. For the development of more sensitive and reliable biosensors, new linker compounds with high immobilization efficiency on two-dimensional materials are required. The present invention aims to provide a novel linker compound enabling surface modification of two-dimensional materials, a linker agent composition containing the linker compound and a specific solvent, a complex of the linker compound and a graphene nanosheet, and a GFET biosensor equipped with the complex.

[0006] As a result of intensive studies to solve the above problems, the present inventors have found a new linker compound with high immobilization efficiency on two-dimensional materials and completed the present invention. That is, the present invention relates to the following.

[0007] 1. The following formula (I): [In the formula, A is a single bond or C １－１０ alkylene group (C ２ of the above alkylene groups, at least one -CH ２ - unit may be replaced by an atomic group selected from the group consisting of an amide bond, an ester bond and an ether bond), B is a single bond, an amide bond, an ester bond, an ether bond or C １－１０ alkylene group (C ２ of the above alkylene groups, at least one -CH ２ - unit may be replaced by a phenylene group which may be substituted, an atomic group selected from the group consisting of an amide bond, an ester bond and an ether bond), Z is a condensed polycyclic hydrocarbon group containing three or more benzene rings, or a condensed heterocyclic group containing one or more benzene rings and one or more heterocycles (containing one or more heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom or a sulfur atom), R １ to R ６ are each independently a hydrogen atom, C １－１０1. A compound represented by a group selected from the group consisting of alkyl groups and phenyl groups (excluding 1-pyreneacetic acid (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)phenyl ester). 2. A compound as described in 1. above, wherein A is a single bond. 3. A is a single -CH ２ - C where the unit is replaced by an ester bond or amide bond ２－１０ The compound described in 1. above, which is an alkylene group.

[0008] 4. A compound according to any one of 1 to 3 above, wherein B is a single bond. 5. B is a single -CH ２ - C where the unit is replaced by an ester bond or amide bond ２－１０ A compound according to any one of 1. to 3. above, wherein B is an alkylene group. 6. B is at least two -CH ２ - A C atom in which each unit is independently replaced by an atomic group selected from the group consisting of a phenylene group, an amide bond, an ester bond, and an ether bond, which may be substituted. ４－１０ A compound according to any one of the above 1. to 3., which is an alkylene group.

[0009] 7. A compound according to any one of 1 to 6 above, wherein Z is an anthryl group, a tetracenyl group, a pentacenyl group, a pyrenyl group, a perilenyl group, a coronenyl group, or a phenoxadinyl group. 8. A compound according to any one of 1 to 7 above, wherein Z is a 1-pyrenyl group. 9. R １ ~R ６ A compound according to any one of items 1 to 8 above, wherein the compound is a hydrogen atom.

[0010] 10. Formulas 1-14 below: A compound selected from the group consisting of structures represented by 11. A linker composition characterized by comprising a compound according to any one of 1 to 10 above and at least one solvent selected from the group consisting of ethylene glycol monomethyl ether, propylene glycol monomethyl ether, propylene glycol methyl ether acetate and ethylene glycol monobutyl ether, and being in solution form. A linker / graphene nanosheet composite obtained by immobilizing the compound according to any one of 1 to 10 above onto a graphene nanosheet. A GFET biosensor comprising the linker / graphene nanosheet composite described in 12 above.

[0011] The compounds of the present invention exhibit high immobilization efficiency onto nanosheet-like materials and are suitable as linker compounds for use in GFET biosensors.

[0012] This graph shows the FET transfer characteristics measured 30 minutes after adding 1 μL of a 4 mM MCS solution of PM-1 obtained in Example 1 to the gate liquid, and compared with the initial transfer characteristics. This graph shows the FET transfer characteristics measured 30 minutes after adding 1 μL of a 4 mM MCS solution of PM-2 obtained in Example 2 to the gate liquid, and compared with the initial transfer characteristics. This graph shows the FET transfer characteristics measured 30 minutes after adding 1 μL of a 4 mM MCS solution of PM-3 obtained in Example 3 to the gate liquid, and compared with the initial transfer characteristics. This graph shows the FET transfer characteristics measured 30 minutes after adding 1 μL of a 4 mM MCS solution of PM-4 obtained in Example 4 to the gate liquid, and compared with the initial transfer characteristics.

[0013] The following describes the linker compound of the present invention, the linker / nanosheet composite using the linker compound, and the GFET sensor comprising the composite. Note that the following description is illustrative and descriptive only and does not limit the claimed invention.

[0014] <Linker Compound> The linker compound of the present invention is of the following formula (I): [In the formula, A is a single bond or C １－１０ Alkylene group (C ２ The alkylene groups above include at least one -CH２ - The unit may be replaced by an atomic group selected from the group consisting of amide bonds, ester bonds, and ether bonds), and B is a single bond, amide bond, ester bond, ether bond, or C １－１０ Alkylene group (C ２ The alkylene groups above include at least one -CH ２ - The unit may be replaced by an atomic group selected from the group consisting of a substituted phenylene group, an amide bond, an ester bond, and an ether bond), Z is a condensed polycyclic hydrocarbon group containing three or more benzene rings, or a condensed heterocyclic group containing one or more benzene rings and one or more heterorings (containing one or more heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms, or sulfur atoms), R １ ~R ６ These are, independently, hydrogen atoms and C １－１０ The compound is represented by a group selected from the group consisting of alkyl groups and phenyl groups (excluding 1-pyreneacetic acid (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)phenyl ester).

[0015] "C １－１０ Examples of alkyl groups include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group (including various isomers), etc.

[0016] "C １－１０ "alkylene group" (C １－１０Examples of alkanediyl groups include methylene group, ethylene group, propylene group, trimethylene group, tetramethylene group, 1-methylpropylene group, 2-methylpropylene group, dimethylethylene group, ethylethylene group, pentamethylene group, 1-methyltetramethylene group, 2-methyltetramethylene group, 1,1-dimethyl-trimethylene group, 1,2-dimethyl-trimethylene group, 2,2-dimethyl-trimethylene group, 1-ethyl-trimethylene group, hexamethylene group, heptamethylene group, octamethylene group, nonamethylene group, decamethylene group (including various isomers), etc.

[0017] An "amide bond" means -NHC(=O)- or -C(=O)NH-, an "ester bond" means -OC(=O)- or -C(=O)O-, and an "ether bond" means -O-.

[0018] "Optionally substituted phenylene group" means an (unsubstituted) 1,4-phenylene group, a 1,3-phenylene group, or a 1,2-phenylene group, or any substituent, for example, C １－１０ This refers to a 1,4-phenylene group, a 1,3-phenylene group, or a 1,2-phenylene group substituted with at least one substituent selected from the group consisting of alkyl groups and phenyl groups.

[0019] One preferred embodiment of the present invention is a compound in which A is a single bond in formula (I) above (i.e., the maleimide ring and the benzene ring are directly bonded).

[0020] Another preferred embodiment of the present invention is in which, in formula (I) above, A is C １－１０ It is an alkylene group, or one -CH group ２ - C where the unit is replaced by an amide bond or ester bond ２－１０ The compound is an alkylene group, preferably C １－５ It is an alkylene group, or one -CH group ２ - C where the unit is replaced by an amide bond or ester bond ２－５ It is a compound containing an alkylene group.

[0021] "One-CH" ２- C where the unit is replaced by an amide bond or ester bond ２－１０ "Alkylene group" refers to C ２－１０ One of the -CH groups contained in the alkylene group ２ - A group in which the unit is replaced by an amide bond (i.e., -NHC(=O)- or -C(=O)NH-) or an ester bond (i.e., -OC(=O)- or -C(=O)O-), specifically as shown in the following formula: The base is represented by the formula [wherein m and n are integers from 0 to 9 (wherein m + n is in the range of 1 to 9), preferably each being an integer from 0 to 4 (wherein m + n is in the range of 1 to 4)]. A specific embodiment of this is -CH ２ CH ２ NHC(=O)-,-CH ２ CH ２ C(=O)NH-, -CH ２ CH ２ OC(=O)-,-CH ２ CH ２ C(=O)O-, -NHC(=O)CH ２ CH ２ -, -C(=O)NHCH ２ CH ２ -, -OC(=O)CH ２ CH ２ -, or -C(=O)OCH ２ CH ２ - are some examples.

[0022] In a particularly preferred embodiment, A is a single bond, -(CH ２ ) １－５ -, -C(=O)O(CH ２ ) １－４ -, or -C(=O)NH(CH ２ ) １－４ - is the case.

[0023] One preferred embodiment of the present invention is a compound in which B is a single bond in formula (I) above (i.e., Z is directly bonded to the benzene ring).

[0024] Another preferred embodiment of the present invention is in which, in formula (I) above, B is C １－１０ It is an alkylene group, or one -CH group ２- C where the unit is replaced by an amide bond or ester bond ２－１０ The compound is an alkylene group, preferably C １－５ It is an alkylene group, or one -CH group ２ - C where the unit is replaced by an amide bond or ester bond ２－５ It is a compound with an alkylene group. ２ - C where the unit is replaced by an amide bond or ester bond ２－１０ The meaning and specific form of the "alkylene group" are as described above.

[0025] A further preferred embodiment of the present invention is in which, in formula (I) above, B is at least two -CH ２ - A C atom in which each unit is independently replaced by an atomic group selected from the group consisting of a phenylene group, an amide bond, an ester bond, and an ether bond, which may be substituted. ４－１０ It is a compound containing an alkylene group.

[0026] "At least two -CH ２ - A C atom in which each unit is independently replaced by an atomic group selected from the group consisting of a phenylene group, an amide bond, an ester bond, and an ether bond, which may be substituted. ４－１０ "Alkylene group" refers to C ４－１０ The alkylene group contains two or more, preferably two to four -CH groups ２ - The units may be independently substituted with phenylene groups (for example, -C) ６ H ４ A group that is replaced by an atomic group selected from the group consisting of -, amide bonds (i.e., -NHC(=O)- or -C(=O)NH-), ester bonds (i.e., -O-C(=O)- or -C(=O)-O-), and ether bonds (i.e., -O-), provided that the substitution is within a chemically acceptable range, for example, the following formula: Examples of groups represented by the formula [wherein Ph means a phenylene group that may be substituted, and m and n each mean integers from 0 to 8 (wherein m + n is in the range of 1 to 8)] include the following:

[0027] In a particularly preferred embodiment, B is a single bond, -(CH ２ ), １－１０ -, -C(=O)O-, -C(=O)NH-, -(CH ２ ), １－９ -C(=O)O-, -(OCH ２ CH ２ ), １－２ -OC(=O)-(CH ２ ), １－２ -C(=O)O-, -(CH ２ ), １－４ -C(=O)NH-(CH ２ ), １－４ -C(=O)O-, or -(CH ２ ), １－７ -C(=O)O-Ph-C(=O)O- and the like.

[0028] The compound of the above formula (I) of the present invention is a linker compound that forms a complex with a nanosheet material, preferably a graphene nanosheet. The linker compound (I) is immobilized on the nanosheet material via the Z moiety (so-called anchor moiety) to form a complex. This immobilization is generally considered to be via non-covalent bonds such as π-π stacking interactions and hydrophobic interactions between aromatic molecules. Therefore, in the above formula (I), Z is not particularly limited as long as it forms a non-covalent bond with the nanosheet material and is immobilized, but is a condensed polycyclic hydrocarbon group containing three or more benzene rings, or a condensed heterocyclic group containing one or more benzene rings and one or more heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms, or sulfur atoms.

[0029] One of the preferred embodiments of the present invention is a compound in which Z in the above formula (I) is a condensed polycyclic hydrocarbon group containing three or more benzene rings. The "condensed polycyclic hydrocarbon group containing three or more benzene rings" is a monovalent group of a condensed polycyclic hydrocarbon containing three or more, preferably 3 to 8, more preferably 4 to 8, and even more preferably 4 to 6 benzene rings. Examples include anthryl group, tetracenyl group, pentacenyl group, phenanthryl group, triphenylenyl group, pyrenyl group, chrysenyl group, perylenyl group, coronenyl group, ovalenyl group, and the like.

[0030] In a particularly preferred embodiment, Z is an anthryl group, a tetracenyl group, a pentacenyl group, a pyrenyl group, a perylenyl group, or a coronenyl group having a more planar structure, and more particularly, a 1-pyrenyl group.

[0031] Another preferred embodiment of the present invention is a compound in which, in the above formula (I), Z is a condensed heterocyclic group containing one or more benzene rings and one or more heterocycles (containing one or more heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom, or a sulfur atom). The "condensed heterocyclic group containing one or more benzene rings and one or more heterocycles (containing one or more heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom, or a sulfur atom)" means one or more, preferably 1 to 4, more preferably 1 to 3, even more preferably 1 or 2 benzene rings and one or more, preferably 1 to 3, more preferably 1 to 2, even more preferably 1 heterocycle (containing one or more heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom, or a sulfur atom) condensed monovalent group of a condensed heterocyclic ring, and examples include the following formula (Z １ ), (Z ４ ): [In the formula Z １ , at least one of -X １１ -, -X １２ -, and -X １３ - is independently selected from the group consisting of -N=, -NH-, -S-, and -O-, and the rest is -CH ２ - or -CH=, provided that the combination of -X １１ -, -X １２ -, and -X １３ - is chemically acceptable; in the formula Z ２ , at least one of -X ２１ -, -X ２２ -, -X ２３ -, and -X ２４ - is independently selected from the group consisting of -N=, -NH-, -S-, and -O-, and the rest is -CH ２ - or -CH=, provided that the combination of -X ２１ -, -X ２２ -, -X ２３ -, and -X ２４The combination of - is chemically acceptable; Formula Z ３ Medium, -X ３１ - is selected from the group consisting of -NH-, -S-, and -O-; Formula Z ４ Medium, -X ４１ - and -X ４２ At least one of the - is independently selected from the group consisting of -N=, -NH-, -S-, and -O-, and the rest are -CH ２ - or -CH =, however, -X ４１ - and -X ４２ The combination of - is chemically acceptable. This is a monovalent group of a condensed heterocycle represented by .

[0032] In a particularly preferred embodiment, Z is an indolyl group, isoindolyl group, indazolyl group, benzotriazolyl group, benzoxazolyl group, benzothiazolyl group, benzofuranyl group, isobenzofuranyl group, benzothiophenyl group, quinolyl group, isoquinolyl group, synnolinyl group, phthalazinyl group, quinazolinyl group, quinoxalinyl group, clomenyl group, isoclomenyl group, carbazolyl group, acridinyl group, phenazinyl group, xanthenyl group, phenoxazinyl group, or phenothiazinyl group, and more particularly, phenoxazine-10-yl group.

[0033] One preferred embodiment of the present invention is that in formula (I) above, R １ ~R ６ It is a compound in which the atom is a hydrogen atom.

[0034] One preferred embodiment of the present invention is that the compound of formula (I) is a compound represented by the following formulas 1 to 14.

[0035] In a particularly preferred embodiment, the compound of formula (I) is the following compound.

[0036] <Method for synthesizing linker compounds> The compound represented by formula (I) above in the present invention can be produced by known methods. Typical methods for producing the compound of the present invention are shown below, but are not limited to them. Furthermore, the compound represented by formula (I) above in the present invention is not limited to compounds produced by the following production methods.

[0037] In the manufacturing method described below, if a compound contains substructures (e.g., hydroxyl, amino, etc.) that inhibit the desired reaction or undergo side reactions, the target product can be obtained by introducing protecting groups to these substructures, carrying out the desired reaction, and then removing the protecting groups. These reactions for introducing and removing protecting groups can be carried out in accordance with methods commonly used in organic synthesis (for example, methods described in Protective Groups in Organic Synthesis, 4th Edition, by TW Greene and PGM Wuts, John Wiley & Sons Inc. (2006), etc.).

[0038] For example, in the compound represented by the above formula (I), B is -B １ Compounds that are -C(=O)O- can be produced according to the method shown in Scheme 1 below.

[0039] In the formula, A, Z, R １ ~R ６ As stated above, B １ is a single bond, -(CH ２ ) １－９ -, - (OCH ２ CH ２ ) １－２ -OC(=O)-(CH ２ ) １－２ -, - (CH ２ ) １－４ -C(=O)NH-(CH ２ ) １－４ - or - (CH ２ ) １－７ It is -C(=O)O-Ph-.

[0040] Scheme 1 involves reacting compound (i) in a solvent with a chlorinating agent and optionally a catalyst to form an acid chloride, and then reacting the acid chloride obtained in the reaction system with compound (ii) in the presence of a base, thereby forming a compound represented by formula (I) where B is -B １ This is a method for obtaining compound (Ia) which is -C(=O)O-. A part of compound (i), for example B １ However, single bonds or -(CH ２ ) １－９ - The compound is known and available from reagent suppliers. Examples of such compounds include pyrene-1-carboxylic acid, pyrene-1-acetic acid, pyrene-1-propanoic acid, pyrene-1-butyric acid, anthracene-9-carboxylic acid, anthracene-9-acetic acid, etc. Or another part of compound (i), for example B １ However, -(OCH ２ CH ２ ) １－２ -OC(=O)-(CH ２ ) １－２ -, - (CH ２ ) １－４ -C(=O)NH-(CH ２ ) １－４ - or - (CH ２ ) １－７ Compounds of the form -C(=O)O-Ph- can be produced by known methods using compounds available from reagent suppliers. Examples of such methods include those described later in <Intermediate Compounds and Their Synthesis Methods> or the methods in the Examples (e.g., Example 4).

[0041] The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the raw materials to some extent, but examples include aromatic hydrocarbons such as benzene, toluene, or xylene; halogenated aliphatic hydrocarbons such as methylene chloride, chloroform, or 1,2-dichloroethane; ethers such as tetrahydrofuran, 1,2-dimethoxyethane, or 1,4-dioxane; nitriles such as acetonitrile or propionitrile; amides such as N,N-dimethylformamide, N,N-dimethylacetamide, or N-methylpyrrolidone; sulfoxides such as dimethyl sulfoxide; or any mixed solvents thereof. Preferably, halogenated aliphatic hydrocarbons such as methylene chloride, chloroform, or 1,2-dichloroethane, or ethers such as tetrahydrofuran, 1,2-dimethoxyethane, or 1,4-dioxane are used. The amount of solvent used is not particularly limited, but is usually 1 to 50 times the mass of compound (i), preferably 5 to 20 times.

[0042] The chlorinating agent used in the step of forming the acid chloride of compound (i) is not particularly limited, but examples include thionyl chloride, oxalyl chloride, and phosphorus trichloride. The amount of chlorinating agent used is usually 0.9 to 10 times the mole, preferably 1.1 to 3 times the mole, of compound (i). In addition, a catalyst such as N,N-dimethylformamide (DMF) may be added together with the chlorinating agent in the step of forming the acid chloride. The amount of catalyst used is usually less than 1 time the mole, preferably 0.01 to 0.9 times the mole, of compound (i).

[0043] The reaction temperature is not particularly limited, but for example, it is in the range from -20°C to the reflux temperature of the reaction mixture, preferably -20°C to 50°C, more preferably -10°C to 30°C, and most preferably room temperature (about 25 ± 5°C). The reaction time varies depending on the reaction temperature, etc., but is usually from 1 minute to 48 hours, preferably from 0.5 hours to 24 hours. The reaction pressure is set as appropriate as needed and may be pressurized, depressurized, or atmospheric pressure, but atmospheric pressure is preferred. The reaction atmosphere can be an atmosphere selected as appropriate as needed, but is preferably an air atmosphere or an inert gas atmosphere such as nitrogen or argon.

[0044] Next, the acid chloride of the formed compound (i) is reacted with compound (ii) in the presence of a base without isolating it from the reaction system to obtain compound (Ia). Compound (ii) is known and can be obtained from reagent suppliers. An example of such compound is N-(4-hydroxyphenyl)maleimide. Alternatively, compound (ii) can be produced by known methods using a compound available from a reagent supplier. The amount of compound (ii) used is usually 0.9 to 5 moles, preferably 1.1 to 3 moles, of compound (i) per mole.

[0045] Examples of bases used include alkali metal acetates such as sodium acetate or potassium acetate; alkali metal carbonates such as sodium carbonate, potassium carbonate, or cesium carbonate; or organic bases such as triethylamine or diisopropylethylamine, with potassium carbonate, cesium carbonate, triethylamine, or diisopropylethylamine being preferred. The amount of base used is usually 0.9 to 10 times the mole of compound (ii) per mole, and preferably 1 to 5 times the mole.

[0046] The reaction temperature is not particularly limited, but for example, it is in the range from -20°C to the reflux temperature of the reaction mixture, preferably -20°C to 50°C, more preferably -10°C to 30°C, and most preferably room temperature (about 25 ± 5°C). The reaction time varies depending on the reaction temperature, etc., but is usually from 1 minute to 48 hours, preferably from 0.5 hours to 24 hours. The reaction pressure is set as appropriate as needed and may be pressurized, depressurized, or atmospheric pressure, but atmospheric pressure is preferred. The reaction atmosphere can be an atmosphere selected as appropriate as needed, but is preferably an air atmosphere or an inert gas atmosphere such as nitrogen or argon.

[0047] If the compound represented by the obtained formula (Ia) contains protecting groups, it can be subjected to a deprotection step as needed. If the compound represented by general formula (Ia) has at least two different protecting groups, one protecting group can be selectively removed by selecting deprotection conditions. The deprotection conditions can be carried out as appropriate, according to methods commonly used in organic synthesis chemistry or in accordance with the examples herein.

[0048] In each manufacturing method, the obtained compounds may be isolated and purified by known means, or they may be directly immobilized onto nanosheet materials. Isolation and purification can be carried out using conventional procedures such as filtration, extraction, crystallization, and various types of column chromatography.

[0049] <Intermediate compound and method of synthesis thereof> For example, intermediate compound (ia), which is part of compound (i) in scheme 1 above: (In the formula, Z is as described above, preferably a 1-pyrenyl group, B ２ is, -(OCH ２ CH ２ ) １－２ -OC(=O)-(CH ２ ) １－２ -, - (CH ２ ) １－４ -C(=O)NH-(CH ２ ) １－４ - or - (CH ２ ) １－７-C(=O)O-Ph-) can be produced by known methods using compounds available from reagent suppliers. For example, B ２ However, -(OCH ２ CH ２ ) ２ -OC(=O)-(CH ２ ) ２ - The intermediate compound can be produced according to the method shown in Scheme 2 below.

[0050]

[0051] In the formula, Hal is a halogen atom, preferably a chlorine atom or a bromine atom.

[0052] In the reaction of Scheme 2, an intermediate is obtained in which a crosslinking portion is constructed in pyrenol to introduce the functional portion (maleimide structure) of the linker compound of the present invention.

[0053] In the first step, compound (ia-1-1) is obtained by reacting pyrenol with an alkyl halide compound under basic conditions (Williamson synthesis). Then, in the second step, compound (ia-1-1) is reacted with succinic anhydride in the presence of a base to obtain intermediate (ia-1), which is a carboxylic acid ester derivative.

[0054] Examples of bases used in the first step include inorganic bases such as sodium carbonate, potassium carbonate, lithium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, and potassium phosphate; aliphatic amines such as trimethylamine, triethylamine, tripropylamine, triisopropylamine, tributylamine, diisopropylethylamine, and N-methylmorpholine; aromatic amines such as pyridine, quinoline, colidine, and dimethylaminopyridine; and hydrides such as sodium hydride and potassium hydride. The use of inorganic bases such as potassium carbonate is preferred. The amount of base used is usually 1 to 50 times the molar amount of the starting material (pyrenol), preferably 1 to 10 times, and more preferably 1 to 5 times.

[0055] The solvent used in the first step can be any solvent that does not react with the raw materials, and examples include aprotic polar solvents (N-methyl-2-pyrrolidone (NMP), dimethylformamide (DMF), dimethyl sulfoxide, etc.), ethers (diethyl ether, diisopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, tetrahydrofuran (THF), dioxane, etc.), aliphatic hydrocarbons (pentane, hexane, heptane, petroleum ether, etc.), aromatic hydrocarbons (benzene, toluene, xylene, mesitylene, chlorobenzene, dichlorobenzene, nitrobenzene, tetralin, etc.), halogenated hydrocarbons (chloroform, dichloromethane, carbon tetrachloride, dichloroethane, etc.), lower fatty acid esters (methyl acetate, ethyl acetate, butyl acetate, methyl propionate, etc.), and nitriles (acetonitrile, propionitrile, butyronitrile, etc.). These solvents can be appropriately selected considering reactivity, etc., and may be used alone or in mixtures of two or more. The use of aprotic polar solvents such as NMP and DMF is preferred.

[0056] The reaction temperature for the first stage is not particularly limited, but is, for example, in the range from -20°C to the reflux temperature of the reaction mixture, preferably 20°C to 90°C, and more preferably about 80°C. The reaction time can be arbitrarily selected between 3 hours and 24 hours. After the reaction is complete, the product can be separated and purified by existing methods such as recrystallization, distillation, and silica gel column chromatography.

[0057] Examples of bases used in the second step include aliphatic amines such as triethylamine, diisopropylethylamine, and N-methylmorpholine, and aromatic amines such as pyridine, quinoline, colidine, and dimethylaminopyridine (DMAP), with the use of aromatic amines such as DMAP being preferred. The amount of base used is usually 1 to 50 times the molar amount of compound (ia-1-1), preferably 1 to 10 times the molar amount, and more preferably 1 to 5 times the molar amount.

[0058] The solvent used in the second step can be any solvent that does not react with the raw materials, and examples of such solvents are the same as those used in the first step. For the solvent used in the second step, the use of ethers such as THF is preferred.

[0059] The reaction temperature for the second stage is not particularly limited, but is, for example, in the range from -20°C to the reflux temperature of the reaction mixture, preferably 20°C to 90°C, and more preferably about 50°C. The reaction time can be arbitrarily selected between 3 and 24 hours. After the reaction is complete, the product can be separated and purified by existing methods such as recrystallization, distillation, and silica gel column chromatography.

[0060] <Linker Composition> The linker composition of the present invention is characterized by comprising the linker compound described above and a specific solvent, and being in the form of a solution. Any solvent that can dissolve the linker compound may be used in the linker composition of the present invention. Monoether and / or monoester solvents of ethylene glycol or propylene glycol are preferred, and examples include monomethyl ether or monobutyl ether of ethylene glycol or propylene glycol, or their acetate esters. Specifically, examples include ethylene glycol monomethyl ether (2-methoxyethanol or methyl cellosolve: MCS), ethylene glycol monobutyl ether (butyl cellosolve: BCS), propylene glycol monomethyl ether (PEGME), or propylene glycol methyl ether acetate (PGMEA), and preferably ethylene glycol monobutyl ether, propylene glycol monomethyl ether, or propylene glycol methyl ether acetate. These solvents can be appropriately selected considering the solubility of the linker compound, and may be used alone or in mixtures of two or more.

[0061] <Preparation of Linker Composition> The linker composition of the present invention can be prepared by mixing a linker compound and a specific solvent in any way. One embodiment of the linker composition of the present invention contains the linker compound at a concentration of 0.001 to 10% (w / w). The amount (concentration) of the linker compound used may be a concentrated concentration for storage, or a diluted concentration for use as a linker composition. In the latter case, the concentration is usually 0.001 to 10% (w / w), preferably 0.005 to 5% (w / w), more preferably 0.01 to 1% (w / w), and even more preferably 0.01 to 0.5% (w / w).

[0062] The linker composition of the present invention may further contain any additives, in addition to the linker compound and specific solvent described above, as long as they do not impair the purpose of the present invention.

[0063] <Manufacturing of GFET Biosensors> Biosensors comprising the linker / nanosheet material composite of the present invention exist in various types depending on the selection of the nanosheet material. Examples of nanosheet materials used in the present invention include graphene, graphene oxide, reduced graphene oxide, carbon nanotubes, fullerenes, and MoS2 (transition metal dichalcogenide). Here, we will describe in detail a GFET biosensor using graphene as the nanocarbon material.

[0064] GFET biosensors are manufactured through a series of steps: (i) graphene synthesis, (ii) transfer of graphene to a support substrate, (iii) sensor (GFET) conversion, and (iv) probe application (bio-GFET conversion).

[0065] (i) Synthesis of graphene Graphene is synthesized by chemical vapor deposition (CVD). What is CVD? CVD is a method of synthesizing two-dimensional materials by reacting raw materials and a substrate under heating (600°C to 1,200°C), and it is a method widely used worldwide. When manufacturing graphene, copper substrates and methane are generally used.

[0066] (ii) Transfer of graphene to a support substrate In the process of transferring graphene to a support substrate, it is common to protect the synthesized graphene with a polymer film prior to the transfer. This polymer film makes it possible to protect the graphene, which is extremely thin and easily torn. Subsequently, the copper is dissolved by immersing the substrate in an acid solution or the like, creating a composite state of the polymer film and graphene. After washing, it is transferred to the support substrate, and finally the polymer protective film is removed to obtain graphene transferred onto the support substrate. A silicon wafer is commonly used as the support substrate.

[0067] (iii) Sensor (GFET) Formation <Linker / Nanosheet Material Composite> In the sensor (GFET) formation process of (iii), a linker compound is immobilized on graphene transferred onto a support substrate. The linker compound of formula (I) of the present invention is used in this process and can be easily immobilized on nanosheet materials such as graphene to form a linker / nanosheet material composite. Specifically, a linker / nanosheet material composite is formed by dropping a solution of the linker compound or the above-mentioned linker agent composition onto a two-dimensional material transferred onto a specific support.

[0068] Any solvent that can be used with the above-mentioned linker agent composition may be used as the solvent for dissolving the linker compound. Preferably, ethylene glycol monomethyl ether (2-methoxyethanol or methyl cellosolve: MCS), ethylene glycol monobutyl ether (butyl cellosolve: BCS), propylene glycol monomethyl ether (PEGME), or propylene glycol methyl ether acetate (PGMEA) are used. Furthermore, when diluting the linker compound solution or the above-mentioned linker agent composition during immobilization, water, methanol, ethanol, isopropanol, dimethyl sulfoxide, dimethylformamide, etc. may be used as the diluent. From the viewpoint of immobilization efficiency, the concentration of the linker compound in the linker compound solution or linker agent composition during immobilization is preferably as high as possible, and the saturation solubility in the solvent used is ideal, but 1 μM or higher is particularly preferred. Normally, immobilization of the linker compound solution or linker composition onto the nanosheet material occurs immediately after dropping it onto the nanosheet material. However, to immobilize more of the linker compound onto the nanosheet material, it is preferable to leave it for 10 minutes to 6 hours, preferably 20 minutes to 3 hours, and more preferably 30 minutes to 2 hours. Heating or stirring is not necessarily required during this time, but may be done if necessary.

[0069] The solvent may be removed from the obtained linker / nanosheet material composite and the process may proceed immediately to step (iv) probe application (bio-GFET formation), or, after removing the solvent from the obtained linker / nanosheet material composite, the surface may be washed and dried, and if necessary, a polymer compound for surface protection may be applied, and the linker / nanosheet material composite may be preserved in its composite state and proceed to step (iv) after some time has passed.

[0070] (iv) Probe application (bio-GFET conversion) In the probe application (bio-GFET conversion) step of (iv), a probe compound solution is dropped onto the linker / nanosheet material composite obtained in (iii), and the probe is applied to the linker / nanosheet material composite by a nucleophilic substitution reaction (e.g., en-thiol reaction) between the functional portion of the linker compound and the probe compound. The probe compound can be obtained from a reagent supplier, or can be manufactured using a compound available from a reagent supplier in accordance with known methods, and can be selected from such compounds according to the purpose. A general physiological salt solution or ionic liquid is used as the solvent for the probe compound solution. Examples of physiological salt solutions include physiological saline, Ringer's solution, Locke's solution, phosphate buffer solution, Tyroht's solution, Hanks' solution, Earl's solution, Hepes's solution, etc. Among these, phosphate buffer solution is preferred. From the viewpoint of immobilization efficiency, the concentration of the probe compound solution is preferably as high as possible, and the saturation solubility in the solvent used is ideal, but a concentration of 100 μM or higher is particularly preferred. Normally, the nucleophilic substitution reaction described above proceeds immediately after dropping the probe compound solution onto the linker / nanosheet material composite, but in order to allow more reaction between the probe compound and the linker compound to proceed, it is good to leave it for 10 minutes to 6 hours, preferably 20 minutes to 3 hours, and more preferably 30 minutes to 2 hours. Heating or stirring is not necessarily required at this time, but may be done if necessary.

[0071] The present invention will be further described in detail below with reference to examples, but the present invention is not limited to these examples. The abbreviations of the compounds used and the methods for measuring each of their physical properties are as follows.

[0072] (Organic solvents / Commercial reagents) MCS: Methyl cellosolve (ethylene glycol monomethyl ether) THF: Tetrahydrofuran DMF: N,N-dimethylformamide TEA: Triethylamine DMAP: Dimethylaminopyridine D-PBS: Dulbecco's phosphate-buffered saline

[0073] ( １(H-NMR measurement) Apparatus: Fourier transform superconducting nuclear magnetic resonance spectrometer (FT-NMR) "AVANCE III" (BRUKER) 500 MHz. Solvent: Deuterated dimethyl sulfoxide (DMSO-d ６ (Standard substance: tetramethylsilane)

[0074] Example 1: Synthesis of a phenyl group-containing maleimide compound (PM-1) PM-1 was synthesized according to the route shown below.

[0075] 1-Pyrene butyric acid (0.66 g, 2.3 mmol) and THF (6 g) were added to a 50 mL flask and, while stirring, oxalyl chloride (0.58 g, 4.6 mmol) and a catalytic amount of DMF (0.1 g) were added, and the mixture was stirred at room temperature until the generation of bubbles stopped. The mixture was concentrated in an evaporator, and the residue was dissolved in THF (3 g) (Solution A). N-(4-hydroxyphenyl)maleimide (0.46 g, 2.4 mmol), THF (10 g), and TEA (0.34 g, 3.5 mmol) were added to another 50 mL flask and stirred at room temperature. Solution A was slowly added to this mixture and stirred at room temperature. After the reaction was complete, ethyl acetate (30 g) and pure water (10 g) were added, and liquid-liquid washing was performed. When the obtained organic layer was dried under reduced pressure, crystals precipitated, which were filtered off and dried to obtain PM-1 (0.7 g, 1.52 mmol, yield 66.3%, yellow crystals).

[0076] 1 H-NMR(500MHz)in DMSO-d6:δ(ppm)=8.44(d, 1H), 8.30-8.05(m, 7H), 8.01(d, 1H), 7.38-7.35(m, 2H), 7.26-7.22(m, 4H), 3.46(t, 2H), 2.79(t, 2H), 2.19-2.16(m, 2H)

[0077] Example 2: Synthesis of a phenyl group-containing maleimide compound (PM-2) PM-2 was synthesized according to the route shown below.

[0078] 4-oxo-4-(2-(pyrene-1-ioxy)ethoxy)butyric acid (0.72 g, 2.0 mmol) and THF (6 g) were added to a 50 mL flask. While stirring, oxalyl chloride (0.51 g, 4.0 mmol) and a catalytic amount of DMF (0.1 g) were added, and the mixture was stirred at room temperature until the formation of bubbles stopped. The mixture was concentrated in an evaporator, and the residue was dissolved in THF (3 g) (Solution A). N-(4-hydroxyphenyl)maleimide (0.38 g, 2.04 mmol), THF (10 g), and TEA (0.30 g, 3.0 mmol) were added to another 50 mL flask and stirred at room temperature. Solution A was slowly added to this mixture and stirred at room temperature. After the reaction was complete, ethyl acetate (30 g) and pure water (10 g) were added, and the mixture was separated and washed. When the obtained organic layer was dried under reduced pressure, crystals precipitated, which were then filtered and dried to obtain PM-2 (0.6 g, 1.14 mmol, yield 57.2%, pale orange crystals).

[0079] 1 H-NMR(500MHz)in DMSO-d6:δ(ppm)=8.37(d, 1H), 8.35-7.97(m,7H), 7.81(d, 1H), 7.33-7.31(m, 2H), 7.20-7.18(m, 4H), 4.65-4.60(m, 4H), 2.90(t, 2H), 2.79(t, 2H)

[0080] Example 3: Synthesis of a phenyl group-containing maleimide compound (PM-3) PM-3 was synthesized according to the route shown below.

[0081] 3-(4-(pyrene-1-yl)butanamide)propanoic acid (0.72 g, 2.0 mmol) and THF (6 g) were added to a 50 mL flask and, while stirring, oxalyl chloride (0.51 g, 4.0 mmol) and a catalytic amount of DMF (0.1 g) were added, and the mixture was stirred at room temperature until the generation of bubbles stopped. The mixture was concentrated in an evaporator, and the residue was dissolved in THF (3 g) (Solution A). N-(4-hydroxyphenyl)maleimide (0.38 g, 2.04 mmol), THF (10 g), and TEA (0.30 g, 3.0 mmol) were added to another 50 mL flask and stirred at room temperature. Solution A was slowly added to this mixture and stirred at room temperature. After the reaction was complete, ethyl acetate (30 g) and pure water (10 g) were added, and the mixture was separated and washed. When the obtained organic layer was dried under reduced pressure, crystals precipitated, which were then filtered and dried to obtain PM-3 (0.3 g, 0.58 mmol, yield 29.2%, light brown crystals).

[0082] 1 H-NMR(500MHz)in DMSO-d6:δ(ppm)=8.43(d, 1H), 8.30-8.01(m, 9H), 7.39-7.20(m,6H),3.96(t, 2H), 3.50(t, 2H), 2.96(t, 2H),2.72(t, 2H) 1,77-1.73(m, 2H)

[0083] Example 4: Synthesis of a phenyl group-containing maleimide compound (PM-4) PM-4 was synthesized according to the following steps.

[0084] 1-hydroxypyrene (3.05 g, 14 mmol), 2-(chloroethoxy)ethanol (3.47 g, 28 mmol), potassium carbonate (4.84 g, 35 mmol), and DMF (30 g) were added to a 100 mL four-necked flask and heated and stirred at 80°C. After the reaction, 60 g of pure water was added, and the precipitated solid was filtered off. The cake was washed with pure water (30 g) and methanol (30 g) in that order, and the resulting solid was filtered off and dried to obtain PM-4-1 (3.5 g, 11.4 mmol, yield 81.7%, gray solid).

[0085] 1H-NMR(500MHz)in DMSO-d6:δ(ppm)=8.41(d, 1H), 8.40-7.97(m, 7H), 7.76(d, 1H),4.69(s,1H), 4.48(t, 2H), 3.96(t, 2H), 3.66-3.54(m, 4H)

[0086]

[0087] PM-4-1 (4.29 g, 14 mmol) and THF (40 g) were added to a 100 mL four-necked flask and dissolved. The reaction solution was heated to 50°C, succinic anhydride (1.68 g, 16.8 mmol) and DMAP (0.86 g, 7 mmol) were added, and the mixture was stirred. After the reaction was complete, aqueous hydrochloric acid (80 g) and ethyl acetate (80 g) were added and the mixture was washed by liquid-liquid extraction. When the resulting organic layer was dried under reduced pressure, crystals precipitated, which were filtered off and dried to obtain PM-4-2 (5.0 g, 12.3 mmol, yield: 87.9%, gray crystals).

[0088] 1 H-NMR(500MHz)in DMSO-d6:δ(ppm)=12.15(s,1H),8.09(d, 1H), 7.89-7.60(m, 7H), 7.42(d, 1H), 4.17(t, 2H), 3.91(t, 2H), 3.68(t, 2H), 3.49(t, 2H)

[0089]

[0090] In a 50 mL flask, PM-4-2 (0.61 g, 1.5 mmol) and methylene chloride (6 g) were added and stirred. Then, oxalyl chloride (0.38 g, 3.0 mmol) and a catalytic amount of DMF (0.1 g) were added and stirred at room temperature until the generation of bubbles stopped. The mixture was concentrated in an evaporator, and the residue was dissolved in methylene chloride (3 g) (Solution A). In another 50 mL flask, N-(4-hydroxyphenyl)maleimide (0.29 g, 1.5 mmol), methylene chloride (3 g), and TEA (0.23 g, 2.3 mmol) were added and stirred at room temperature. Solution A was slowly added to this mixture and stirred at room temperature. After the reaction was complete, methylene chloride (12 g) and pure water (12 g) were added and liquid-liquid washing was performed. When the resulting organic layer was dried under reduced pressure, crystals precipitated, which were then filtered and dried to obtain PM-4 (0.7 g, 1.3 mmol, yield 84.3%, light brown crystals).

[0091] 1 H-NMR(500MHz)in DMSO-d6:δ(ppm)=8.39(d, 1H), 8.24-7.96(m, 7H), 7.76(d, 1H), 7.34(d, 1H), 7.22-7.16(m, 6H)4.48(t, 2H), 4.27(t, 2H), 3.99(t, 2H), 3.82(t, 2H), 2.84(t, 2H), 2.72(t, 2H)

[0092] Evaluation Example 1: Immobilization Characteristic Evaluation Using FETs A 39 μL mixture of D-PBS, pure water, and MCS in a 1:4:5 ratio was added to the surface of a GFET (Graphenea, mGFET-4D) as a gate liquid, and connected to a semiconductor parameter analyzer (KEYSIGHT, B1500A). The source-drain voltage was fixed at 20 mV, and the FET transfer characteristics of gate voltage-drain current were measured. Subsequently, 1 μL of MCS solution of PM-1 adjusted to 4 mM was added to the gate liquid, and the FET transfer characteristics after 30 minutes were measured and compared with the initial transfer characteristics. When molecules are immobilized on the graphene surface in liquid, the FET transfer characteristics change. The transfer characteristics when PM-1 is added are shown in Figure 1. In addition, the same evaluation was performed for PM-2, PM-3, and PM-4 as with PM-1, and it was confirmed that the transfer characteristics changed. The results are shown in Figures 2 to 4.

[0093] The results of Evaluation Example 1 demonstrate that linker molecules can be immobilized on graphene elements by using a specific maleimide compound of the present invention.

Claims

1. The following formula (I): [wherein, A is a single bond or C １－１０ alkylene group (C ２ For two or more alkylene groups, at least one -CH ２ - unit may be replaced by an atomic group selected from the group consisting of an amide bond, an ester bond and an ether bond), B is a single bond, an amide bond, an ester bond, an ether bond or C １－１０ alkylene group (C ２ For two or more alkylene groups, at least one -CH ２ - unit may be replaced by an atomic group selected from the group consisting of a phenylene group which may be substituted, an amide bond, an ester bond and an ether bond), Z is a condensed polycyclic hydrocarbon group containing three or more benzene rings, or a condensed heterocyclic group containing one or more benzene rings and one or more hetero atoms selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom, R １ ~R ６ are each independently a group selected from the group consisting of a hydrogen atom, a C １－１０ alkyl group and a phenyl group] (however, 1-pyreneacetic acid (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl ester is excluded).

2. The compound according to claim 1, wherein A is a single bond.

3. A is one -CH ２ - C where the unit is replaced by an ester bond or amide bond ２－１０ The compound according to claim 1, which is an alkylene group.

4. The compound according to claim 1, wherein B is a single bond.

5. B is one -CH ２ - C where the unit is replaced by an ester bond or amide bond ２－１０ The compound according to claim 1, which is an alkylene group.

6. B has at least two -CH ２ - A C atom in which each unit is independently replaced by an atomic group selected from the group consisting of a phenylene group, an amide bond, an ester bond, and an ether bond, which may be substituted. ４－１０ The compound according to claim 1, which is an alkylene group.

7. The compound according to claim 1, wherein Z is an anthryl group, a tetracenyl group, a pentacenyl group, a pyrenyl group, a perilenyl group, a coronenyl group, or a phenoxadinyl group.

8. The compound according to claim 1, wherein Z is a 1-pyrenyl group.

9. R １ ~R ６ The compound according to claim 1, wherein is a hydrogen atom.

10. Formulas 1-14 below: A compound selected from the group consisting of structures represented by the following:

11. A linker composition characterized by comprising a compound according to any one of claims 1 to 10 and at least one solvent selected from the group consisting of ethylene glycol monomethyl ether, propylene glycol monomethyl ether, propylene glycol methyl ether acetate, and ethylene glycol monobutyl ether, and being in the form of a solution.

12. A linker / graphene nanosheet composite obtained by immobilizing a compound according to any one of claims 1 to 10 onto a graphene nanosheet.

13. A GFET biosensor comprising the linker / graphene nanosheet composite described in claim 12.

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