Graphene linker compound and field effect transistor biosensor

Abstract

The purpose of the present invention is to provide: a linker agent which contains a specific solvent and a linker compound that enables surface modification of a two-dimensional material, and which has excellent storage stability; a novel linker compound; and a GFET biosensor which is provided with the linker compound.　The present invention provides: a linker agent which is characterized by containing a compound of formula (1) (wherein Z, X, A, Y, and B are as described in the claims and the description) and at least one solvent that is 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 which is also characterized by being a solution; a novel linker compound; and a GFET biosensor which is provided with the linker compound.

Description

Graphene linker compounds and field-effect transistor biosensors

[0001] The present invention relates to a linker agent with excellent storage stability, which comprises a linker compound that enables surface modification of two-dimensional materials such as graphene and a specific solvent, as well as a novel linker compound and a graphene FET biosensor equipped with the linker compound.

[0002] Methods and systems for detecting target substances such as proteins, viruses, and bacteria with high sensitivity and speed are important in medical diagnosis, environmental monitoring, bioresearch, etc., and various detection methods and means are being investigated. Many target substances have specific properties, such as surface shape, protein, and charge, and selective detection methods that utilize these specific properties have been proposed. Proposed detection means include optical means and electrical means using field-effect transistors (hereinafter abbreviated as FETs), and electrical means using FETs in particular are expected to enable high sensitivity and provide simple systems.

[0003] Graphene, a typical two-dimensional material, is composed 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 ideal for sensor applications. The development of graphene field effect transistors (GFETs) using graphene has been actively pursued in recent years.

[0004] Japanese Patent Application Laid-Open No. 2023-125621

[0005] Immobilization of biomolecules on the surface of two-dimensional materials, such as graphene, is crucial in the functionalization process of biosensors. To develop more sensitive and reliable biosensors, new linker compounds with high immobilization efficiency on two-dimensional materials are needed. Linker compounds are typically used as linker agents dissolved in a solvent during immobilization. However, because linker compounds contain crosslinking groups, long-term storage in solution as linker agents can induce decomposition or side reactions. Therefore, the development of linker agents with excellent storage stability is needed.

[0006] The present invention aims to provide a linker agent with excellent storage stability, which comprises a linker compound that enables surface modification of two-dimensional materials and a specific solvent, as well as a novel linker compound and a GFET biosensor equipped with the linker compound.

[0007] The present inventors have conducted extensive research to solve the above problems and have come up with the present invention. That is, the present invention relates to the following: 1. A compound represented by the following formula (1): [In the formula, Z represents a polycyclic aromatic ring or a polycyclic heteroaromatic ring; X and Y each independently represent a single bond, an alkylene group having 1 to 5 carbon atoms which may contain an ether bond, a phenylene group, a biphenyldiyl group, or a naphthylene group, and any hydrogen atom on the aromatic ring of the group may be substituted with another substituent; A represents a phenylene group, a biphenyldiyl group, a naphthylene group, an ether bond, an ester bond, an amide bond, a urea bond, or a urethane bond; and B represents a group represented by any of the following formulae (I) to (III): and (x and y are both single bonds, and x represents a group selected from the group consisting of 1 to 10 carbon atoms ...

[0008] 2. The linker agent according to 1., wherein Z is selected from the group consisting of anthracene, tetracene, pentacene, benzopyrene, chrysene, pyrene, perylene, triphenylene, corannulene, coronene, opalene, indole, isoindole, benzimidazole, purine, benzotriazole, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, pteridine, chromene, isochromene, acridine, xanthene, carbazole, and benzo[c]cinnoline. 3. The linker agent according to 1. or 2., wherein Z is pyrene.

[0009] 4. The following formula (1'): [In the formula, Z represents a polycyclic aromatic ring or a polycyclic heteroaromatic ring; X and Y each independently represent a single bond, an alkylene group having 1 to 5 carbon atoms which may contain an ether bond, a phenylene group, a biphenyldiyl group, or a naphthylene group, and any hydrogen atom on the aromatic ring of the group may be substituted with another substituent; A represents a phenylene group, a biphenyldiyl group, a naphthylene group, an ether bond, an ester bond, an amide bond, a urea bond, or a urethane bond; and B represents a group represented by any of the following formulae (I) to (III): 4. A compound of the following formula (1'): [In the formula, Z represents a polycyclic aromatic ring or a polycyclic heteroaromatic ring; X and Y each independently represent a single bond, an alkylene group having 1 to 5 carbon atoms which may contain an ether bond, a phenylene group, a biphenyldiyl group, or a naphthylene group, and any hydrogen atom on the aromatic ring of the group may be substituted with another substituent; A represents a phenylene group, a biphenyldiyl group, a naphthylene group, an ether bond, an ester bond, an amide bond, a urea bond, or a urethane bond; and B represents a group represented by any of the following formulae (I) to (III): wherein X and Y are both single bonds and B is a group of formula (II) or (III), and A may be an alkylene group having 1 to 10 carbon atoms.

[0010] 6. The compound according to 4. or 5., wherein Z is selected from the group consisting of anthracene, tetracene, pentacene, benzopyrene, chrysene, pyrene, perylene, triphenylene, corannulene, coronene, opalene, indole, isoindole, benzimidazole, purine, benzotriazole, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, pteridine, chromene, isochromene, acridine, xanthene, carbazole, and benzo[c]cinnoline. 7. The compound according to 4. or 5., wherein Z is pyrene.

[0011] 8. The following formula: A compound represented by the formula:

[0012] 9. A linker / nanosheet material composite obtained by immobilizing the compound according to any one of items 4 to 8 on a nanosheet material. 10. A GFET biosensor comprising the linker / nanosheet material composite according to item 9.

[0013] The compound of the present invention has high immobilization efficiency to nanosheet-shaped materials and is suitable as a linker compound for use in GFET biosensors. Furthermore, by using a specific solvent, the linker agent of the present invention has excellent storage stability in a solution state and can be stored for a long period of time.

[0014] In Example 1, 1 μL of AE-1 MCS solution adjusted to 4 mM was added to the gate liquid, and the FET transfer characteristics were measured after 30 minutes and compared with the initial transfer characteristics. In Example 2, 1 μL of AE-2 MCS solution adjusted to 4 mM was added to the gate liquid, and the FET transfer characteristics were measured after 30 minutes and compared with the initial transfer characteristics. In Example 3, 1 μL of AE-3 MCS solution adjusted to 4 mM was added to the gate liquid, and the FET transfer characteristics were measured after 30 minutes and compared with the initial transfer characteristics. In Example 4, 1 μL of AE-4 MCS solution adjusted to 4 mM was added to the gate liquid, and the FET transfer characteristics were measured after 30 minutes and compared with the initial transfer characteristics. In Example 5, 1 μL of AE-5 MCS solution adjusted to 4 mM was added to the gate liquid, and the FET transfer characteristics were measured after 30 minutes and compared with the initial transfer characteristics. In Example 6, 1 μL of a PEGME solution of AE-2 adjusted to 4 mM was added to the gate liquid, and the FET transfer characteristics were measured after 30 minutes, and compared with the initial transfer characteristics. In Example 7, 1 μL of a PGMEA solution of AE-2 adjusted to 4 mM was added to the gate liquid, and the FET transfer characteristics were measured after 30 minutes, and compared with the initial transfer characteristics. In Example 8, 1 μL of a BCS solution of AE-2 adjusted to 4 mM was added to the gate liquid, and the FET transfer characteristics were measured after 30 minutes, and compared with the initial transfer characteristics. In Comparative Example 1, 1 μL of only MCS was added without using a graphene linker compound, and the FET transfer characteristics were measured after 30 minutes, and compared with the initial transfer characteristics.

[0015] The linker agent of the present invention, a novel linker compound, a linker / nanosheet material composite using the linker compound, and a GFET sensor including the composite are described in detail below. Note that the nanosheet material referred to here refers to a nanocarbon material and a three-dimensional material using a nanocarbon material. Specific examples include graphene, graphene oxide, reduced graphene oxide, carbon nanotubes, and MoS2 (transition metal dichalcogenide), with graphene being preferred.

[0016] <Linker Compound> The linker agent of the present invention contains a linker compound and a specific solvent. The linker compound used in the linker agent of the present invention is represented by the following formula (1).

[0017] In the formula, Z represents a polycyclic aromatic ring or a polycyclic heteroaromatic ring.Specific examples include anthracene, tetracene, pentacene, benzopyrene, chrysene, pyrene, perylene, triphenylene, corannulene, coronene, opalene, indole, isoindole, benzimidazole, purine, benzotriazole, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, pteridine, chromene, isochromene, acridine, xanthene, carbazole, and benzo[c]cinnoline.Polycyclic aromatic rings such as anthracene, tetracene, pentacene, pyrene, perylene, or coronene are preferred.Among these, from the viewpoint of ease of synthesis and effect expression, anthracene or pyrene is more preferred, and pyrene is particularly preferred.

[0018] In the formula, X and Y each independently represent a single bond, an alkylene group having 1 to 5 carbon atoms which may contain an ether bond, a phenylene group, a biphenyldiyl group, or a naphthylene group, and any hydrogen atom on the aromatic ring of the group may be substituted with another substituent. An alkylene group having 1 to 5 carbon atoms which may contain an ether bond or a phenylene group is preferred.

[0019] An “alkylene group having 1 to 5 carbon atoms which may contain an ether bond” means an “alkylene group having 1 to 5 carbon atoms” or an “alkylene group having 1 to 5 carbon atoms and containing an ether bond”. In this specification, unless otherwise limited, “an alkylene group having 1 to 5 carbon atoms” includes a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 2,2-dimethyl-trimethylene group, and a 1-ethyl-trimethylene group, and an alkylene group having 1 to 3 carbon atoms such as a methylene group, an ethylene group, a propylene group, or a trimethylene group is preferred. The "alkylene group having 1 to 5 carbon atoms and containing an ether bond" is a group containing an ether bond (-O-) at the terminal of the alkylene group, or a group in which the alkylene group is interrupted by an ether bond (-O-), such as -OCH ２ -, -CH ２ O-, -OCH ２ CH ２ -, -CH ２ CH ２ O-, -OCH ２ CH ２ CH ２ -, -CH ２ CH ２ CH ２ O- or -CH ２ CH ２ OCH ２ CH ２ -, etc., -OCH ２ CH ２ - or -CH ２ CH ２ O- is preferred.

[0020] In this specification, unless otherwise limited, the term "phenylene group" refers to a 1,4-phenylene group, a 1,3-phenylene group, or a 1,2-phenylene group, the term "biphenyldiyl group" includes a 1,1'-biphenyl-4,4'-diyl group, and the like, and the term "naphthylene group" includes a 1,5-naphthylene group, a 2,6-naphthylene group, and the like. Any hydrogen atom on these aromatic rings may be replaced with another substituent, for example, at least one substituent selected from the group consisting of an alkyl group having 1 to 10 carbon atoms and a phenyl group.

[0021] In the formula, A represents a phenylene group, a biphenyldiyl group, a naphthylene group, an ether bond, an ester bond, an amide bond, a urea bond, or a urethane bond, with an ester bond or an amide bond being preferred.

[0022] In this specification, unless otherwise limited, an "amide bond" means -NHC(=O)- or -C(=O)NH-, an "ester bond" means -OC(=O)- or -C(=O)O-, a "urea bond" means -NHC(=O)NH-, and a "urethane bond" means -NHC(=O)O-.

[0023] B represents an active structural portion that reacts with an amino group and is selected from the following formulae (I) to (III).

[0024] Furthermore, the linker compound of the present invention may be a compound represented by the above formula (1) in which when X and Y are both single bonds, A is an alkylene group having 1 to 10 carbon atoms. Such a compound may be a compound represented by the following formula (2): [In the formula, Z represents a polycyclic aromatic ring or a polycyclic heteroaromatic ring, A represents an alkylene group having 1 to 10 carbon atoms, and B represents a group represented by the following formula (II) or (III): Some of these compounds (for example, the compound of formula (AE-6) given in the examples below) are known.

[0025] Therefore, the novel linker compound provided by the present invention has the following formula (1'): [In the formula, Z represents a polycyclic aromatic ring or a polycyclic heteroaromatic ring; X and Y each independently represent a single bond, an alkylene group having 1 to 5 carbon atoms which may contain an ether bond, a phenylene group, a biphenyldiyl group, or a naphthylene group, and any hydrogen atom on the aromatic ring of the group may be substituted with another substituent; A represents a phenylene group, a biphenyldiyl group, a naphthylene group, an ether bond, an ester bond, an amide bond, a urea bond, or a urethane bond; and B represents a group represented by any of the following formulae (I) to (III): represents a group selected from the group consisting of:

[0026] More specifically, the following compounds are preferably used:

[0027] The novel linker compound provided by the present invention may also be a compound represented by the above formula (1') in which when X and Y are both single bonds, A is an alkylene group having 1 to 10 carbon atoms (however, 1-pyreneacetic acid succinimidyl ester and 1-pyrenebutyric acid succinimidyl ester are excluded). Furthermore, the novel linker compound provided by the present invention may also be a compound represented by the above formula (1') in which when X and Y are both single bonds and B is a group of formula (II) or (III), A is an alkylene group having 1 to 10 carbon atoms. Such a compound may be a compound represented by the following formula (2'): [In the formula, Z represents a polycyclic aromatic ring or a polycyclic heteroaromatic ring, A represents an alkylene group having 1 to 10 carbon atoms, and B represents a group represented by the following formula (II) or (III): represents a group

[0028] More specifically, the following compounds are preferably used:

[0029] <Method for Synthesizing Linker Compound> A general method for synthesizing the linker compound of the present invention will be explained using as an example one of the preferred methods for synthesizing AE-2 shown in the following scheme.

[0030] In the first and second stage reactions, an active ester structure is introduced into pyrenol to introduce the terminal structure (active structure that reacts with an amino group) of the linker compound of the present invention.

[0031] In the first step, pyrenol is reacted with an alkyl halide compound under basic conditions (Williamson synthesis) to obtain intermediate AE-2-2, which is then reacted with succinic anhydride in the presence of dimethylaminopyridine (DMAP) to obtain precursor AE-2-1, a carboxylic acid ester derivative.

[0032] The base used here may be, for example, an inorganic base such as sodium carbonate, potassium carbonate, lithium carbonate, cesium carbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, or potassium phosphate; an amine such as trimethylamine, triethylamine, tripropylamine, triisopropylamine, tributylamine, diisopropylethylamine, pyridine, quinoline, or collidine; or a base such as sodium hydride or potassium hydride.

[0033] The solvent used here can be any solvent that does not react with the raw materials, and examples thereof 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 taking into consideration reactivity and the like, and can be used alone or in combination of two or more.

[0034] The reaction temperature can be selected arbitrarily within the range of the properties of the solvent used, such as the boiling point, but is preferably 90° C. The reaction time can be selected arbitrarily between 3 and 24 hours. The product can be separated and purified by conventional techniques such as recrystallization, distillation, and silica gel column chromatography.

[0035]

[0036] In the third step, the terminal carboxylic acid moiety of AE-2-1 is substituted with N-hydroxysuccinimide (NHS) by a condensation reaction to synthesize the target AE-2.

[0037] The condensation method used here may, for example, be a method using a carbodiimide condensing agent such as N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), or N,N'-diisopropylcarbodiimide (DIPC), or a method of converting to a carboxylic acid chloride using oxalyl chloride, thionyl chloride, phosphorus trichloride, phosphorus pentachloride, etc. This activates the terminal carboxylic acid moiety of AE-2-1, allowing the condensation reaction to occur efficiently.

[0038] The solvent used here can be any solvent that does not react with the raw materials, and examples thereof include aprotic polar solvents (N-methyl-2-pyrrolidone, dimethylformamide, dimethyl sulfoxide, etc.), ethers (diethyl ether, diisopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, tetrahydrofuran, 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 taking into consideration reactivity and the like, and can be used alone or in combination of two or more.

[0039] The reaction temperature can be selected arbitrarily within the range of the properties of the solvent used, such as the boiling point, but is preferably 23°C. The reaction time can be selected arbitrarily between 2 and 24 hours. The product can be separated and purified by conventional techniques such as recrystallization, distillation, and silica gel column chromatography.

[0040] In the above-described method for synthesizing AE-2, the construction of the pyrene skeleton, the spacer, and the activated ester structure is first carried out via the formation of an ether bond. However, these constructions can also be carried out via the formation of an ester bond or an amide bond, and these can be easily achieved by a person skilled in the art by using known organic synthesis techniques and selecting appropriate reaction compounds.

[0041] AE-7, another linker compound of the present invention, can be synthesized by subjecting the terminal carboxylic acid moiety of 1-pyrenebutyric acid to a condensation reaction with 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, as in the third step of the synthesis of AE-2, as shown in the following scheme.

[0042]

[0043] <Linker Agent> The linker agent of the present invention comprises a linker compound and a specific solvent. The specific solvent used in the linker agent of the present invention is a monoether and / or monoester solvent of ethylene glycol or propylene glycol, such as the monomethyl ether or monobutyl ether of ethylene glycol or propylene glycol, or an acetate ester thereof. Specific examples include ethylene glycol monomethyl ether (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. It has been found that the use of such a specific solvent allows the linker compound of the present invention to have excellent storage stability in the linker agent (i.e., in a solution state) and to be stored for a long period of time.

[0044] <Production of GFET Biosensor> There are various types of biosensors equipped with the linker / nanosheet material composite of the present invention 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, fullerene, MoS2 (transition metal dichalcogenide), etc. Here, a detailed description will be given using a GFET biosensor in which graphene is used as the nanocarbon material.

[0045] A GFET biosensor is manufactured through the following steps: (i) graphene synthesis, (ii) transfer of graphene to a support substrate, (iii) formation of a sensor (GFET), and (iv) attachment of a probe (formation of a bio-GFET).

[0046] <(i) Synthesis of Graphene> Graphene is synthesized by chemical vapor deposition (CVD). CVD is a method of synthesizing two-dimensional materials by reacting raw materials with a substrate under heating (600°C to 1,200°C), and is a method that is widely used worldwide. When producing graphene, a copper substrate and methane are generally used.

[0047] <(ii) Transfer of graphene to a support substrate> In the process of transferring graphene to a support substrate, a polymer film is generally used to protect the synthesized graphene prior to transfer. This polymer film makes it possible to protect the extremely thin and easily torn graphene. The substrate is then immersed in an acid solution or the like to dissolve the copper, resulting in a polymer film and graphene composite. After washing, the substrate is transferred to a support substrate, and finally, the polymer protective film is removed to obtain graphene transferred onto the support substrate. A silicon wafer is generally used as the support substrate.

[0048] <(iii) Formation of a Sensor (GFET)> <Linker / Nanosheet Material Composite> In the (iii) formation of a sensor (GFET), a linker compound is immobilized on graphene transferred onto a support substrate. The linker compound 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, the linker / nanosheet material composite is formed by dropping a solution of the linker compound onto a two-dimensional material transferred onto a specific support. The solvent used to dissolve the linker compound is at least one selected from water, methanol, ethanol, isopropanol, dimethyl sulfoxide, dimethylformamide, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, propylene glycol monomethyl ether, and propylene glycol methyl ether acetate. Among these, a mixed solvent of ethylene glycol monomethyl ether and water, or at least one solvent selected from the group consisting of ethylene glycol monobutyl ether, propylene glycol monomethyl ether, and propylene glycol methyl ether acetate, is preferred. From the viewpoint of immobilization efficiency, the concentration of the linker compound solution is preferably higher, with saturated solubility in the solvent being ideal, and 100 μM or higher is particularly preferred. Typically, immobilization to the nanosheet material proceeds immediately after the linker compound solution is dropped onto the nanosheet material. However, to immobilize as much of the linker compound as possible onto the nanosheet material, it is advisable to leave the solution 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, but may be performed as needed. The solvent may be removed from the resulting linker / nanosheet material composite, and the process may proceed immediately to step (iv) of probe application (bio-GFET formation). Alternatively, the solvent may be removed from the resulting linker / nanosheet material composite, the surface may be washed and dried, and then, if necessary, a polymer compound may be applied for surface protection. The linker / nanosheet material composite may then be stored in this state, and the process may proceed to step (iv) after a certain time has elapsed.

[0049] (iv) Probe Application (Bio-GFET Formation) In the probe application (bio-GFET formation) step (iv), a probe solution is added dropwise to the linker / nanosheet material composite obtained in (iii), and a nucleophilic substitution reaction of the probe compound occurs, with the structure at the end of the linker compound serving as a leaving group, thereby applying the probe to the linker / nanosheet material composite. Solvent B is typically a physiological salt solution or an ionic liquid. Examples of physiological salt solutions include physiological saline, Ringer's solution, Locke's solution, phosphate buffer solution, Tyrote's solution, Hank's solution, Earle's solution, and Hepes solution. Among these, phosphate buffer solution is preferred. From the viewpoint of immobilization efficiency, the higher the concentration of the probe compound solution, the better. While saturation solubility in the solvent used is ideal, a concentration of 100 μM or higher is particularly preferred. Usually, the above-mentioned nucleophilic substitution reaction proceeds immediately after the probe compound solution is dropped onto the linker / nanosheet material composite, but to allow the reaction between the probe compound and the linker compound to proceed more rapidly, it is advisable to leave the mixture for 10 minutes to 6 hours, preferably 20 minutes to 3 hours, and more preferably 30 minutes to 2 hours. In this case, heating or stirring is not necessarily required, but may be performed as needed.

[0050] The present invention will be described in more 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 the various physical properties are as follows:

[0051] (Organic solvents) MCS: ethylene glycol monomethyl ether THF: tetrahydrofuran D-PBS: Dulbecco's phosphate buffer solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) PEGME: propylene glycol monomethyl ether PGMEA: propylene glycol methyl ether acetate BCS: ethylene glycol mono-normal butyl ether NMP: N-methyl-2-pyrrolidone

[0052] (General Reagents) NHS: N-hydroxysuccinimide EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride DMAP: dimethylaminopyridine

[0053] (Pyrene compounds) AE-1 to AE-6: Compounds represented by the following formulas (AE-1) to (AE-6), respectively:

[0054] [Synthesis of Pyrene Compounds] AE-1 to AE-5 are novel compounds not previously disclosed in the literature, and their synthesis methods are described in detail below. The products described in Monomer Synthesis Examples 1 to 5 below are １ The product was identified by H-NMR analysis (analysis conditions are as follows): Apparatus: Fourier transform superconducting nuclear magnetic resonance spectrometer (FT-NMR) "AVANCE III" (manufactured by BRUKER) 500 MHz; Solvent: Deuterated dimethyl sulfoxide (DMSO-d ６ , standard substance: tetramethylsilane)

[0055] (Synthesis Example 1: Synthesis of AE-1) AE-1 was synthesized according to the route shown below.

[0056] 4-Carboxyphenyl 1-pyrenebutanoic acid (0.68 g, 1.7 mmol), NHS (0.38 g, 3.3 mmol), and THF (13 g) were added to a 50 mL four-neck flask and dissolved. EDC (0.77 g, 4.0 mmol) was slowly added thereto and stirred at room temperature. After completion of the reaction, ethyl acetate (25 g) and purified water (25 g) were added and washed by separation. The obtained organic layer was dried under reduced pressure, and crystals precipitated. These were filtered and dried to obtain AE-1 (0.56 g, 1.1 mmol, yield: 66.4%, light gray crystals). １ H-NMR (500MHz) in DMSO-d ６ : δ (ppm) = 8.44 (d, 1H), 8.30-8.23 (m, 4H), 8.15-8.13 (m, 4H), 8.07 (t, 1H), 8.01 (d, 1H), 7.42 (d, 2H), 3.47 (t, 2H), 2.90 (s, 4H), 2.83 (t, 2H), 2.22-2.16 (m, 2H)

[0057] (Synthesis Example 2: Synthesis of AE-2) AE-2 was synthesized according to the route shown below.

[0058] 2-(1-pyrenyloxy)ethanol (0.78 g, 3 mmmol) and THF (8 g) were added to a 50 mL four-neck flask and dissolved. The reaction solution was heated to 50°C, and succinic anhydride (0.36 g, 3.6 mmmol) and DMAP (0.18 g, 1.5 mmmol) were added and stirred. After completion of the reaction, 2 N aqueous hydrochloric acid solution (15 g) and ethyl acetate (15 g) were added for separation and washing. The resulting organic layer was dried under reduced pressure, and crystals precipitated. These were filtered and dried to obtain AE-2-1 (0.80 g, 2.2 mmol, yield: 73.6%, white crystals). １ H-NMR (500MHz) in DMSO-d ６ : δ (ppm) = 12.26 (s, 1H), 8.39 (d, 1H), 8.26-8.13 (m, 4H), 8.09-7.98 (m, 3 H), 7.77 (d, 1H), 4.61-4.57 (m, 4H), 2.63-2.61 (m, 2H), 2.56-2.53 (m, 2H)

[0059]

[0060] AE-2-1 (0.68 g, 1.9 mmol), NHS (0.43 g, 3.8 mmol), and THF (7 g) were added to a 50 mL four-neck flask and dissolved. EDC (0.87 g, 4.5 mmol) was slowly added thereto and stirred at room temperature. After completion of the reaction, ethyl acetate (12 g) and pure water (12 g) were added and washed by separation. When the obtained organic layer was dried under reduced pressure, crystals precipitated. These were filtered and dried to obtain AE-2 (0.54 g, 1.2 mmol, yield: 61.9%, white crystals). １ H-NMR (500MHz) in DMSO-d ６ : δ (ppm) = 8.37 (d, 1H), 8.27-7.98 (m, 7H), 7.78 (d, 1H), 4.61-4.59 (m, 4H), 2.98 (t, 2H), 2.79-2.77 (m, 6H)

[0061] (Synthesis Example 3: Synthesis of AE-3) AE-3 was synthesized according to the route shown below.

[0062] N-[1-oxo-4-(1-pyrenyl)butyl]-β-alanine (0.53 g, 1.5 mmol), NHS (0.34 g, 3.0 mmol), and THF (6 g) were added to a 50 mL four-neck flask and dissolved. EDC (0.69 g, 3.6 mmol) was slowly added thereto and stirred at room temperature. After completion of the reaction, ethyl acetate (10 g) and purified water (10 g) were added and washed by separation. When the obtained organic layer was dried under reduced pressure, crystals precipitated. These were filtered and dried to obtain AE-3 (0.4 g, 0.9 mmol, yield: 58.5%, white crystals). １ H-NMR (500MHz) in DMSO-d ６ : δ (ppm) = 8.39 (d, 1H), 8.26 (t, 2H), 8.23-8.21 (m, 2H), 8.14-8.13 (m, 2H) 8.08-8.06 (m, 2H), 7.9 4 (1H), 3.40-3.38 (m, 2H) 3.32 (t, 2H), 2.88 (t, 2H), 2.80 (s, 4H), 2.26 (t, 2H), 2.03-1.99 (m, 2H)

[0063] (Synthesis Example 4: Synthesis of AE-4)

[0064] AE-2-1 (0.82 g, 2.3 mmol), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (0.41 g, 2.5 mmol), and THF (13 g) were added to a 50 mL four-neck flask and dissolved. EDC (0.52 g, 2.7 mmol) was slowly added thereto and stirred at room temperature. After completion of the reaction, ethyl acetate (30 g) and pure water (30 g) were added and the mixture was separated and washed. The resulting organic layer was dried under reduced pressure, and crystals precipitated. These were filtered and dried to obtain AE-4 (0.60 g, 1.2 mmol, yield: 51.9%, brown crystals). １ H-NMR (500MHz) in DMSO-d ６ : δ (ppm) = 8.33 (d, 1H), 8.24-7.94 (m, 11H), 8.71 (d, 1H), 4.67-4.60 (m, 4H) 3.17 (t, 2H), 2.89 (t, 2H)

[0065] (Synthesis Example 5: Synthesis of AE-5)

[0066] 4-(anthracen-9-ylmethoxy)-4-oxobutyric acid (0.61 g, 2.0 mmol), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (0.36 g, 2.2 mmol), and THF (10 g) were added to a 50 mL four-neck flask and dissolved. EDC (0.46 g, 2.4 mmol) was slowly added thereto and stirred at room temperature. After completion of the reaction, ethyl acetate (18 g) and purified water (18 g) were added and the mixture was separated and washed. The resulting organic layer was dried under reduced pressure to precipitate crystals, which were filtered and dried to obtain AE-5 (0.50 g, 1.1 mmol, yield: 55.1%, orange crystals). １ H-NMR (500MHz) in DMSO-d ６ : δ (ppm) = 8.71 (s, 1H), 8.38-8.33 (m, 4H), 8.23-8.19 (m, 1H), 8.13 (d, 2H), 8 .06-8.03 (m, 1H), 7.63-7.53 (m, 4H), 6.19 (s, 2H), 3.14 (t, 2H), 2.79 (t, 2H)

[0067] [Evaluation of Immobilization Characteristics Using FET] A 39 μL gating liquid, a 1:4:5 mixture of D-PBS, pure water, and MCS, was added to the device surface of a GFET (mGFET-4D, manufactured by Graphenea), and the device was connected to a semiconductor parameter analyzer (B1500A, manufactured by KEYSIGHT). The source-drain voltage was fixed at 20 mV, and the FET transfer characteristics (gate voltage-drain current) were measured. Subsequently, 1 μL of a 4 mM solution of AE-1 to AE-5 was added to the gating liquid. The FET transfer characteristics were measured after 30 minutes and compared with the initial transfer characteristics (Examples 1 to 8). The MCS in the gating liquid was replaced with a specific solvent as appropriate. The FET transfer characteristics changed when each linker compound was immobilized on the graphene surface in the liquid. Table 1 shows the correspondence between the graphene linker compounds and the specific solvents in the gating liquid, and Figures 1 to 8 show the comparison results of the transfer characteristics for each example. In Comparative Example 1, no graphene linker compound was used, and 1 μL of MCS alone was added. The FET transfer characteristics were measured after 30 minutes and compared with the initial transfer characteristics. The comparison results of the transfer characteristics in the comparative example are shown in FIG.

[0068]

[0069] The results of Examples 1 to 8 demonstrate that the use of the pyrene compound of the present invention enables the immobilization of a linker compound onto a graphene element.

[0070] <Storage Stability of Linker Agent> As shown in Table 2 below, a linker compound was dissolved in a specific solvent to a concentration of 50 μM, and 10 mL of each linker agent was prepared. Each prepared linker agent was left at room temperature for 6 hours, and the relative area value of the linker compound was measured using a liquid chromatograph manufactured by Shimadzu Corporation. Regarding the effect of the specific solvent on storage stability, a value of 95 area% or more was rated as good, and a value of 95 area% or less was rated as poor.

[0071]

[0072] From the results of Examples 9 to 14, it was found that by using a specific solvent, the linker compound of the present invention has excellent storage stability in the linker agent (i.e., in a solution state) and can be stored for a long period of time.

[0073] (Pyrene Compounds) AE-7 to AE-8: Compounds represented by the following formulas (AE-7) to (AE-8)

[0074] AE-7 and AE-8 are novel compounds not previously disclosed in the literature, and their synthesis methods are described in detail below. (Synthesis Example 6: Synthesis of AE-7) AE-7 was synthesized according to the route shown below.

[0075] 1-Pyrenebutyric acid (1.15 g, 4.0 mmol), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (0.72 g, 4.4 mmol), and THF (18 g) were added to a 50 mL four-neck flask and dissolved. EDC (0.92 g, 4.8 mmol) was slowly added thereto and stirred at room temperature. After completion of the reaction, ethyl acetate (30 g) and purified water (30 g) were added and washed by separation. The obtained organic layer was dried under reduced pressure, and crystals precipitated. These were filtered and dried to obtain AE-7 (1.00 g, 2.3 mmol, yield: 57.7%, white crystals). １H-NMR (500MHz) in DMSO-d ６ : δ (ppm) = 8.46 (d, 2H), 8.37-8.02 (m, 12H), 3.52 (t, 2H), 3.07 (t, 2H), 2.26-2.23 (m, 2H)

[0076] (Synthesis Example 7: Synthesis of AE-8) AE-8 was synthesized according to the route shown below.

[0077] 1-Pyrenebutyric acid (1.4 g, 5.0 mmol), triethylamine (0.7 g, 7.5 mmol), and THF (15 g) were added to a 50 mL four-neck flask and dissolved. BOP reagent (2.4 g, 6.0 mmol) was slowly added thereto and stirred at room temperature. After the reaction was completed, pure water (42 g) was added, and crystals precipitated. These were filtered and dried to obtain AE-8 (1.38 g, 3.4 mmol, yield: 67.9%, white crystals). １ H-NMR (500MHz) in DMSO-d ６ : δ (ppm) = 8.45 (d, 2H), 8.31-8.02 (m, 9H), 7.87-7.81 (m, 1H), 7.65-7.60 (m, 1H), 7.53-7.50 (m, 1H), 3.52 (t, 2H), 3.17 (t, 2H), 2.30-2.24 (m, 2H)

[0078] [Evaluation of reaction rate with amine compound] To evaluate the immobilization efficiency of the probe by the linker compound, the proportion of amine adducts formed when the linker compound was mixed with the amine compound in solution was evaluated using a liquid chromatograph manufactured by Shimadzu Corporation. Samples were prepared as shown in Table 3, and aliquots of the solution were sampled 5 minutes, 35 minutes, and 65 minutes after the mixing operation, and the proportion of the adducts formed was analyzed. The experimental results for each sample are shown in Tables 4 to 6.

[0079]

[0080]

[0081]

[0082]

[0083] Examples 15 and 16 show that by using the pyrene compound of the present invention, the reaction efficiency with the amine compound is improved compared to Comparative Example 5, making it possible to prepare device elements in a shorter time.

Claims

1. The following formula (1): [In the formula, Z represents a polycyclic aromatic ring or a polycyclic heteroaromatic ring; X and Y each independently represent a single bond, an alkylene group having 1 to 5 carbon atoms which may contain an ether bond, a phenylene group, a biphenyldiyl group, or a naphthylene group, and any hydrogen atom on the aromatic ring of the group may be substituted with another substituent; A represents a phenylene group, a biphenyldiyl group, a naphthylene group, an ether bond, an ester bond, an amide bond, a urea bond, or a urethane bond; and B represents a group represented by any of the following formulae (I) to (III): and (x and y are both single bonds, and x represents a group selected from the group consisting of 1 to 10 carbon atoms ...

2. The linker agent of claim 1, wherein Z is selected from the group consisting of anthracene, tetracene, pentacene, benzopyrene, chrysene, pyrene, perylene, triphenylene, corannulene, coronene, opalene, indole, isoindole, benzimidazole, purine, benzotriazole, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, pteridine, chromene, isochromene, acridine, xanthene, carbazole, and benzo[c]cinnoline.

3. The linker agent of claim 1, wherein Z is pyrene.

4. The following formula (1'): [In the formula, Z represents a polycyclic aromatic ring or a polycyclic heteroaromatic ring; X and Y each independently represent a single bond, an alkylene group having 1 to 5 carbon atoms which may contain an ether bond, a phenylene group, a biphenyldiyl group, or a naphthylene group, and any hydrogen atom on the aromatic ring of the group may be substituted with another substituent; A represents a phenylene group, a biphenyldiyl group, a naphthylene group, an ether bond, an ester bond, an amide bond, a urea bond, or a urethane bond; and B represents a group represented by any of the following formulae (I) to (III): represents a group selected from the group consisting of: (excluding 1-pyreneacetic acid succinimidyl ester and 1-pyrenebutyric acid succinimidyl ester).

5. The following formula (1'): [In the formula, Z represents a polycyclic aromatic ring or a polycyclic heteroaromatic ring; X and Y each independently represent a single bond, an alkylene group having 1 to 5 carbon atoms which may contain an ether bond, a phenylene group, a biphenyldiyl group, or a naphthylene group, and any hydrogen atom on the aromatic ring of the group may be substituted with another substituent; A represents a phenylene group, a biphenyldiyl group, a naphthylene group, an ether bond, an ester bond, an amide bond, a urea bond, or a urethane bond; and B represents a group represented by any of the following formulae (I) to (III): wherein X and Y are both single bonds and B is a group of formula (II) or (III), and A may be an alkylene group having 1 to 10 carbon atoms.

6. The compound of claim 4 or 5, wherein Z is selected from the group consisting of anthracene, tetracene, pentacene, benzopyrene, chrysene, pyrene, perylene, triphenylene, corannulene, coronene, opalene, indole, isoindole, benzimidazole, purine, benzotriazole, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, pteridine, chromene, isochromene, acridine, xanthene, carbazole, and benzo[c]cinnoline.

7. The compound of claim 4 or 5, wherein Z is pyrene.

8. The following formula: A compound represented by the formula:

9. A linker / nanosheet-shaped material complex obtained by immobilizing the compound according to claim 4 or 5 on a nanosheet-shaped material.

10. A GFET biosensor comprising the linker / nanosheet material composite of claim 9.

Images