JPWO2018062542A1 - Electron mediator modified enzyme and enzyme electrode, spectroscopic analysis kit and enzyme test paper using the same - Google Patents

Abstract

An electron mediator modifying enzyme represented by the following structural formula (I) and having an oxidoreductase E, a phenazine derivative, and a linker site L that links the oxidoreductase E and the phenazine derivative is disclosed. In Structural Formula (I), Y represents a linear or branched alkyl group which may have a substituent having 1 to 5 carbon atoms, and R1 to R8 are independently a hydrogen atom or a substituent. Represents an alkyl group or an alkoxy group which may have a hydroxyl group, a halogen atom, a nitro group or an amino group which may have a substituent, at least one of which is a linker site L. In addition, an enzyme electrode, a spectroscopic analysis kit, and an enzyme test paper using the electron mediator-modified enzyme are disclosed.

Description

  The present invention relates to a novel electron mediator-modified enzyme that is excellent in electron transfer efficiency and enables highly sensitive and highly accurate measurement of biological materials, an enzyme electrode using the electron mediator-modified enzyme, and a spectroscopic analysis kit And enzyme test paper.

  An electrochemical biosensor is a device that combines the high substrate selectivity of an enzyme reaction by combining an oxidoreductase reaction with an electrode reaction, as well as electrochemical measurement characteristics that enable simple and highly sensitive measurement. It is. In order to increase the sensitivity of an electrochemical biosensor, it is important to improve the electron transfer rate between the oxidoreductase and the electrode. For this purpose, an organic compound or metal complex having a low molecular weight that mediates electron transfer between an oxidoreductase (coenzyme) and an electrode, which is called an electron mediator, may be used.

  For example, Patent Document 1 includes an insulating substrate, an electrode system having a working electrode and a counter electrode disposed on the substrate, and a reagent layer disposed on the electrode system, and the reagent layer includes Aspergillus A glucose sensor including an oryzae type FAD-GDH, a ruthenium compound, and PMS (phenazine methosulfate) is disclosed.

  Patent Document 2 discloses glucose oxidoreductase, a coenzyme selected from flavin nucleoside and nicotinamide nucleotide, 9- (dimethylamino) benzophenoxazine-7-ium chloride, N- (9H -Benzophenoxazine-9-ylidene) -N-methylmethanaminium chloride, 8-dimethylamino-2,3-benzophenoxazine hemi (zinc chloride) salt, 7-dimethylamino-1,2-benzophenoxy At least one electroactive organic molecule singly selected from sazine or 8-dimethylamino-2,3-benzophenoxazine and at least one coordination complex singly selected from each osmium or ruthenium complex And a reagent for detecting a specimen comprising a transmitter substance comprising Strips constructed electrochemical detection apparatus is disclosed in.

Japanese Patent No. 5585740 Japanese Patent No. 5453314

  However, the electrochemical glucose sensor described in Patent Documents 1 and 2 uses an electroactive organic molecule such as phenazine methosulfate or benzophenoxazine in combination with an expensive transition metal complex such as ruthenium as an electron mediator. Therefore, it is expensive and it is necessary to use an excessive amount of electron mediator in order to ensure the efficiency of electron transfer from the oxidoreductase.

  The present invention has been made in view of such circumstances, and uses an electron mediator-modifying enzyme that is inexpensive, excellent in electron transfer efficiency, enables highly sensitive and highly accurate measurement of biologically relevant substances, and the electron mediator-modifying enzyme. It is an object of the present invention to provide an enzyme electrode, a spectroscopic analysis kit, and an enzyme test strip.

A first aspect of the present invention that meets the above object is represented by the following structural formula (I):
Oxidoreductase E,
A phenazine derivative;
The present invention solves the above problems by providing an electron mediator modifying enzyme having a linker site L that links the oxidoreductase E and the phenazine derivative.

In structural formula (I),
X represents an anion,
Y represents a linear or branched alkyl group which may have a substituent having 1 to 5 carbon atoms,
R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are each independently a hydrogen atom, an optionally substituted alkyl group or alkoxy group, a hydroxyl group, Represents a halogen atom, a nitro group or an amino group which may have a substituent, and at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is Linker site L.

  The electron mediator modifying enzyme according to the first aspect of the present invention may be represented by the following structural formula (II).

  In Structural Formula (II), Y represents a methyl group or an ethyl group.

  The electron mediator modifying enzyme according to the first aspect of the present invention may be represented by the following structural formula (III).

  In the structural formula (III), Y represents a methyl group or an ethyl group.

  In the electron mediator modifying enzyme according to the first aspect of the present invention, the oxidoreductase E may be an oxidoreductase having FAD or FMN as a coenzyme.

  In the electron mediator modifying enzyme according to the first aspect of the present invention, the oxidoreductase E may be an oxidoreductase having NAD or NADP as a coenzyme.

  In the electron mediator modifying enzyme according to the first aspect of the present invention, the oxidoreductase E may be an oxidoreductase having PQQ as a coenzyme.

  In the electron mediator-modifying enzyme according to the first aspect of the present invention, the oxidoreductase E having FAD or FMN as a coenzyme may be glucose, and the oxidoreductase E is FAD. It may be a dependent glucose dehydrogenase.

  In the electron mediator-modifying enzyme according to the first aspect of the present invention, the oxidoreductase E uses any one of lactic acid, fructosyl amino acid, cholesterol and 1,5-anhydroglucitol as a substrate. Also good.

  In the electron mediator modifying enzyme according to the first aspect of the present invention, the oxidoreductase E comprises lactate oxidase (LOx), fructosyl amino acid oxidase (FAOx), cholesterol oxidase (ChOx), and pyranose oxidase (PyOx). ).

The second aspect of the present invention comprises a substrate, a working electrode, and a counter electrode,
The problem is solved by providing an enzyme electrode having at least one kind of the electron mediator modifying enzyme according to the first aspect of the present invention immobilized on the surface of the working electrode.

  According to a third aspect of the present invention, there is provided a spectroscopic analysis kit comprising at least one electron mediator-modifying enzyme according to the first aspect of the present invention and a redox indicator. It is.

  According to a fourth aspect of the present invention, there is provided an enzyme test paper comprising at least one electron mediator-modifying enzyme according to the first aspect of the present invention and a redox indicator. .

  The electron mediator modifying enzyme provided by the present invention has a structure in which a phenazine derivative that functions as an electron mediator is bound to an oxidoreductase through a linker site. As a result, since the phenazine derivative is always placed in the vicinity of the oxidoreductase, the transfer of electrons between the oxidoreductase and the phenazine derivative is efficient without using an expensive transition metal complex such as a ruthenium complex. To be done. Therefore, according to the present invention, an electron mediator-modifying enzyme that is inexpensive, excellent in electron transfer efficiency, and capable of measuring a biologically relevant substance with high sensitivity and high precision, an enzyme electrode using the electron mediator-modifying enzyme, and spectroscopy Analytical kits and enzyme test strips are provided.

4 is a graph showing the relationship between the glucose concentration and current of the glucose-responsive enzyme electrode prepared in Example 2. 6 is a graph showing a CA measurement result of a glucose responsive enzyme electrode prepared in Example 3. 6 is a graph showing the relationship between the glucose concentration and current of the glucose-responsive enzyme electrode prepared in Example 3. It is a graph which shows the relationship between the glucose concentration of the glucose responsive enzyme electrode produced in Example 6, and an electric current.

  Subsequently, an embodiment of the present invention will be described to provide an understanding of the present invention.

[Electron Mediator Modification Enzyme]
The electron mediator modifying enzyme (hereinafter sometimes abbreviated as “electron mediator modifying enzyme”) according to the first embodiment of the present invention is represented by the following structural formula (I), oxidoreductase E, It has a phenazine derivative, and a linker site L that connects the oxidoreductase E and the phenazine derivative.

In the structural formula (I), X represents an anion, Y represents a linear or branched alkyl group which may have a substituent having 1 to 5 carbon atoms, and R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ each independently have a hydrogen atom, an alkyl or alkoxy group which may have a substituent, a hydroxyl group, a halogen atom, a nitro group or a substituent. Represents an amino group, and at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is a linker moiety L.

Anion X
As the anion X which is a counter ion of the phenazine derivative, any organic or inorganic anion can be used without particular limitation as long as it does not affect the electron transfer characteristics and the like. Specific examples of the anion X include halide ions such as chloride ions and bromide ions, acetate ions, propionate ions, lactate ions, citrate ions, tartrate ions, methanesulfonate ions, ethanesulfonate ions, and trifluoromethane. Organic acid ions such as alkyl sulfonate ions such as sulfonic acid, benzene sulfonate ions, aryl sulfonate ions such as p-toluene sulfonate ions, nitrate ions, sulfate ions, methyl sulfate ions, ethyl sulfate ions, phosphate ions, etc. Inorganic acid ions and the like. Specific examples of preferable anions include methyl sulfate ions and ethyl sulfate ions.

Alkyl group Y
The alkyl group Y on the nitrogen atom of the phenazine ring is any of straight-chain or branched-chain alkyl groups having 1 to 5 carbon atoms. Specific examples of preferable alkyl group Y include a methyl group and an ethyl group.

Substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸
The substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ on the phenazine ring are each independently a hydrogen atom or an alkyl group which may have a substituent. Or it is an amino group which may have an alkoxy group, a hydroxyl group, a halogen atom, a nitro group, or a substituent. Adjacent substituents may be bonded to each other to form a saturated or unsaturated ring. The alkyl group and alkoxy group may be a linear or branched alkyl group or alkoxy group having any carbon number as long as the enzyme activity and electron transfer properties are not affected. The substituents on the alkyl group and the alkoxy group may be arbitrary as long as they do not affect the enzyme activity and the electron transfer property, but the oxidoreductase E and the phenazine derivative are linked as the linker site L. What fulfills the function is formed by reaction of a functional group such as a hydroxyl group, amino group, carboxyl group or thiol group on oxidoreductase E with a reactive functional group such as a carboxyl group, isocyanate group, amino group or thiol group. It may have a functional group such as an ester group, an amide group, a urethane group, a urea group, or a disulfide group. The functional group may be a reactive functional group or may have a function such as a chromophore or an electron transport function.

Linker site L
At least ^{one of} R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ^7, and R ⁸ is a linker site that links the oxidoreductase E and a phenazine derivative that acts as an electron mediator (L ). As the linker site L, any atomic group can be used without limitation as long as it does not affect the enzyme activity and the electron transfer characteristics. Specific examples of the linker site L include an oxygen atom, a nitrogen atom, a sulfur atom, an ester, an amide, a urethane, a urea, a cycloalkylene group, an aryl group, a heteroaryl group, or the like between CC bonds or side chains. And an alkylene group which may have a branch may be mentioned.

  A preferred example of the electron mediator-modifying enzyme is one represented by the following structural formula (II) or (III).

  In the structural formulas (II) and (III), X is a methyl sulfate ion or an ethyl sulfate ion, and Y is a methyl group or an ethyl group.

Oxidoreductase E
In the electron mediator modifying enzyme, the oxidoreductase E used is a dehydrogenase, oxidase, or reductase of any origin having a desired substrate specificity depending on the purpose of use, the biological substance to be measured, etc. Oxygenase, hydrogentransferase, etc. can be appropriately selected and used. Specific examples of the oxidoreductase include the following.
(1) FAD or FMN as a coenzyme: FAD-dependent glucose dehydrogenase, etc. (2) NAD or NADP as a coenzyme: alcohol dehydrogenase, etc. (3) PQQ as a coenzyme: Bacterial glucose dehydrogenase and the like (4) Others: fructosyl amino acid oxidase (FAOx), pyranose oxidase (PyOx), lactate oxidase (LOx), cholesterol oxidase (ChOx). Among them, FAOx is an oxidase that uses glycated amino acids produced by hydrolysis of glycated proteins such as glycated hemoglobin and glycated albumin. By using this enzyme, glycated protein that is an index of blood glucose control in the long term of diabetes is used. Measurement can be performed. PyOx is an enzyme that is used in an enzyme analysis method for 1,5-anhydroglucitol, which is an indicator of blood glucose control in the short and medium stages of diabetes. It is obvious that PyOx has a wide substrate specificity, and in order to detect the presence or absence of the activity of this enzyme, if the activity of glucose of the enzyme can be confirmed, 1,5-anhydroglucitol exhibits the same activity. is there.

  The synthesis of the electron mediator modifying enzyme is performed by reacting a reactive functional group of a substituent on the phenazine derivative that becomes the linker site L after the reaction with a functional group on oxidoreductase E. In order to avoid denaturation and inactivation of oxidoreductase E, the synthesis is preferably carried out by a reaction that proceeds under mild conditions in an aqueous solvent. For example, in the formation of an amide by the reaction of a carboxylic acid and an amine, which will be described later, N-hydroxysuccinimide ester (NHS ester) may be used as the carboxylic acid active ester.

  Specific examples of the combination of the reactive functional group on the phenazine derivative that becomes the linker site L after the reaction and the functional group on the oxidoreductase E include (1) carboxylic acid and amine: amide, and (2) carboxylic acid and hydroxyl group. : Ester, (3) isocyanate and amine: urea, (4) isocyanate and hydroxyl group: urethane, (5) thiol group: disulfide, and the like. Alternatively, chemical modification is required, but the phenazine derivative and oxidoreductase E are combined by azide and alkyne Huisgen cyclization (so-called “click chemistry”), or by the formation of thiosuccinimide by reaction of maleimide and thiol group. You may let them.

[Enzyme electrode]
The enzyme electrode according to the second embodiment of the present invention (hereinafter sometimes abbreviated as “enzyme electrode”) has at least an insulating base material, a working electrode, and a counter electrode. The electron mediator modifying enzyme according to the first embodiment of the present invention is immobilized on the surface of the electrode. The enzyme electrode may have a reference electrode as necessary. The enzyme electrode can read out the electron transfer accompanying the enzyme reaction catalyzed by the electron mediator-modifying enzyme as a change in current value, and can be used for quantifying the substrate concentration in the sample.

  The working electrode and the counter electrode may be of any material and shape. For example, a carbon electrode may be used as the working electrode and the counter electrode, or a metal electrode such as platinum, gold, silver, nickel, or palladium may be used. The reference electrode is not particularly limited, and a common electrode in electrochemical experiments can be used without particular limitation. Specific examples thereof include a saturated calomel electrode and a silver-silver chloride electrode. .

  Examples of the method for forming the electrode on the insulating base material include a photolithographic method, a printing method such as screen printing, gravure printing, flexographic printing, and the like. As the material for the insulating substrate, any known material can be used without particular limitation, and specific examples thereof include silicon, glass, glass epoxy, ceramic, polyethylene terephthalate (PET), polystyrene, polymethacrylate. , Polypropylene, acrylic resin, polyvinyl chloride, polyethylene, polypropylene, polyester, and polyimide.

  For fixing the electron mediator modifying enzyme to the working electrode surface, any known method can be used without particular limitation. Specific examples thereof include (1) hydrogen bonding, intermolecular force, hydrophobic interaction and the like. (2) A highly reactive functional group is introduced on the surface of the working electrode by utilizing a reaction between a surface functional group and a cross-linking molecule, etc. , React this with the functional group of the electron mediator modifying enzyme, and fix the electron mediator modifying enzyme to the surface of the working electrode via the formed covalent bond, (3) Crosslinking method that crosslinks and insolubilizes, (4) Inclusion method that encapsulates electron mediator modification enzyme in matrix such as polymer, etc. It is also possible to use Te Align. From the viewpoint of stability and the like, the covalent bonding method (2) is preferable, and the inclusion method (4) is also preferable in that a printing method such as an inkjet method can be applied.

  The spectroscopic analysis kit according to the third embodiment of the present invention (hereinafter sometimes abbreviated as “spectroscopic analysis kit”) is modified by the electron mediator according to the first embodiment of the present invention. Used to read out the electron transfer associated with the oxidation-reduction reaction of a substrate catalyzed by an electron mediator-modifying enzyme in solution as a change in the color of the oxidation-reduction indicator (absorbance at a specific wavelength). It is done. The spectroscopic analysis kit may contain the electron mediator-modifying enzyme and the redox indicator in a state of being accommodated in the same container, or may be included in a state of being individually accommodated in a separate container. As the container, any container that can stably store the electron mediator-modifying enzyme and the redox indicator can be used. Specific examples of the container include glass or synthetic resin bottles, vials, and ampoules. The electron mediator-modifying enzyme and the redox indicator may be contained in a container in the form of crystals, powder, or the like, or may be contained in a state dissolved or dispersed in a suitable solvent or dispersion medium.

  As the redox indicator, one that can exchange electrons with the electron mediator can be appropriately selected and used according to the redox potential of the electron mediator used for the electron mediator modifying enzyme.

  The spectroscopic analysis kit may contain a solvent, a dispersion medium, a diluting solution, and the like together with an instruction manual in a state of being packaged in a packaging container as necessary.

  The enzyme test paper according to the fourth embodiment of the present invention (hereinafter may be abbreviated as “enzyme test paper”), the electron mediator-modifying enzyme according to the first embodiment of the invention, and the redox It contains an indicator in an impregnated state on a sheet-like base material such as paper, and is catalyzed by an electron mediator-modifying enzyme, which oxidizes the electron transfer accompanying the redox reaction of the substrate dropped on the enzyme test paper. It is used to read out as a change in the color of the reduction indicator (absorbance at a specific wavelength).

  The electron mediator-modifying enzyme and redox indicator used for the enzyme test paper are the same as those in the above-described spectroscopic analysis kit, and thus detailed description thereof is omitted. As a base material used for enzyme test paper, it can be held in an impregnated state with an electron mediator-modifying enzyme and a redox indicator, has water absorption, and is used as a test paper, such as a sheet of paper or nonwoven fabric The shape can be used without particular limitation.

Next, examples carried out for confirming the effects of the present invention will be described.
Example 1: Preparation of PES-modified FADGDH 1- (3-carboxylpropane) oxy-5 with respect to 8.5 µL of a mold-derived FAD-dependent glucose dehydrogenase (FADGDH) aqueous solution (36.2 mg / mL, distilled water) -Add 5 or 8 μL of 50 mM aqueous solution of N-hydroxysuccinimide ester (arPES) of ethylphenazinium ethanesulfonate (arPES (final concentration 2.5 or 4.0 mM), and 40 μL of 50 mM TAPS buffer (pH = 8.3) Then, distilled water was added to make 100 μL. Moreover, this mixed reaction solution was shaken at 1200C for 2 hours at 25 ° C. After the reaction, in order to remove unreacted arPES, an ultrafiltration column (Amicon (registered trademark) ultra 30k, Merck), 20 mM P.A. P. B. The buffer was exchanged using (pH = 7.0).

  The enzyme activity of the PES-modified FADGDH (structural formula (II)) prepared as described above was measured using the MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) system, PMS ( It was measured by a phenazine methosulfate) / DCIP (2,6-dichloroindophenol) system or a DCIP system. In the MTT system, 1 mM MTT, 0.04% Triton, 20 mM P.I. P. B. In (pH = 7.0), the increase in absorbance at 570 nm was measured when 100 mM glucose was added. In the PMS / DCIP system, 0.6 mM PMS, 0.06 mM DCIP, 20 mM P.I. P. B. The decrease in absorbance at 600 nm when 100 mM glucose was added was measured in (potassium phosphate buffer, pH = 7.0).

  The modification reaction was carried out at an enzyme: arPES mixing ratio of 1:50 or 1:80 when the enzyme was modified with PES. A study was also conducted when the enzyme and arPES mixed solution during the reaction was diluted three times. In the modification reaction, aggregation was observed without dilution, but no aggregation was visually observed in the reaction solution in which the enzyme / arPES mixed solution was diluted 3 times. However, agglomeration was observed in the process of exchanging with distilled water and concentrating. The amount of enzyme recovered was greater in the diluted reaction solution. Table 1 shows the results of enzyme activity measurement (1 mM MTT or 0.6 mM PMS / 0.06 mM DCIP, glucose concentration 100 mM) of each modified enzyme prepared.

  Regarding the MTT activity, the enzyme prepared at a mixing ratio of 1:50 was 14.3 U / mg without dilution and 10.9 U / mg with the diluted reaction solution, and the PES modification efficiency decreased by dilution. It was thought that there was. On the other hand, when the mixing ratio was 1:80, it was 15.7 U / mg without dilution and 17.7 U / mg with the diluted reaction solution, and no decrease in PES modification efficiency was observed even after dilution. In order to examine the effect of freezing and thawing of arPES used in the modification reaction, the difference in modification efficiency between using condensate and arPES after condensing and freezing and thawing was evaluated by MTT activity. As a result, when arPES after freezing and thawing was used, the MTT activity was 11.8 U / mg, a decrease in activity of about 80% was observed. Based on this result, it was decided to use arPES without freezing and thawing in subsequent experiments.

Example 2: Preparation of enzyme electrode using PES-modified FADGDH (1)
Carbon printed electrode; P. B. (PH = 7.0), trehalose, arPES, mold-derived FADGDH, and ketjen black were mixed so that the final concentrations were 0.5%, 5 mM, 5.16 mg / mL, and 1.2%, respectively. This was designated as PES-modified enzyme ink. The prepared enzyme ink was applied on a carbon electrode at 160 nL / mm ² (160 nL × 1). After applying the PES-modified enzyme ink on the working electrode of the electrode chip, it was dried at room temperature and low humidity (McDry: 1% relative humidity) for 2 hours. The dried electrode tip was exposed to glutaraldehyde vapor at 25 ° C. for 1 hour and used as a PES-modified enzyme electrode tip. In addition, a PES non-modified enzyme electrode chip was produced using a PES non-modified enzyme ink prepared using the same procedure except that mold-derived FADGDH without PES modification was used. Each electrode chip was stored until measurement under low humidity (McDry: 1% relative humidity).

  20 mM P.I. P. B. A calibration curve for a PES-modified enzyme electrode chip was prepared using a glucose solution prepared at 0 to 600 mg / dL in (pH = 7.0). In addition, using a FADGDH that is not PES-modified using the same concentration of glucose solution and the same concentration of glucose solution containing 0.36 mM 1-methoxyPES (mPES), a calibration curve for a PES-unmodified enzyme electrode chip Made. The chronoamperometry (CA) method was used for the measurement, and the potential was applied 5 seconds after the sample was added (waiting time 5 seconds). The applied potential is +100 mV vs. Ag / AgCl, potential application time was 45 seconds.

  The measurement results are shown in FIG. The PES-modified enzyme electrode chip showed a substrate concentration-dependent response up to 600 mg / dL without adding mPES to the glucose solution. On the other hand, in the PES non-modified enzyme electrode chip, when mPES is not added to the glucose solution, a glucose concentration-dependent current response is not observed, and when mPES is added to the glucose solution, the current response is high at a high substrate concentration. Saturated. When the PES modified enzyme electrode chip was used, a glucose concentration-dependent current response could be confirmed over a wider range of glucose concentrations than when a PES non-modified enzyme electrode chip was used and mPES was added to the glucose solution.

Example 3: Preparation of enzyme electrode using PES-modified FADGDH (2)
10 mM P.I. P. B. (PH = 7.0) trehalose, arPES, mold-derived FADGDH so that the final concentration is 0.5%, 0.5, 2.5 or 5 mM, 5.0 mg / mL, 1.2%, respectively. Ketjen black was mixed and used as a PES-modified enzyme ink. The prepared three types of PES-modified enzyme inks were applied in layers by 200 nL twice on the circular carbon electrode of the DEP chip. After drying, 200 nL of 10% BSA solution was further applied and dried at room temperature and low humidity (McDry: 1% relative humidity) for 2 hours. Each dried chip was exposed to glutaraldehyde vapor at 25 ° C. for 1 hour. Each produced electrode chip was stored until measurement under low humidity (McDry: 1% relative humidity).

  The chip thus produced was connected to a potentiostat, immersed in a batch cell, and subjected to CA measurement at an applied potential of +100 mV (vs. Ag / AgCl) and a stirring speed of 250 rpm. The measurement solution was 100 mM P.I. P. B. (PH = 7.0), and the glucose solution prepared with the same buffer solution was added sequentially so as to be 5, 10, 25, 50, 100, 300, 600 mg / dL. After the calibration curve was prepared, continuous measurement was continued for 20 hours using the same buffer and the same conditions.

  Three types of glucose-responsive electrode tips were prepared by preparing different concentrations of PES-modified enzyme ink, and their current responses were compared. In the CA measurement, all glucose-responsive electrode tips showed a substrate concentration-dependent response (see FIG. 2), but the observed response current value was higher as the amount of arPES mixed was higher. The calibration curve prepared from the CA measurement showed no significant difference in linearity between the arPES concentration of 5 mM and 2.5 mM, but the lower glucose concentration when the arPES concentration was 0.5 mM. The response was saturated (see FIG. 3).

Example 4: Preparation of PES-modified LOx and PES-modified ChOx To 300 μg of lactic acid oxidase (LOx) derived from lactic acid bacteria, 20 μL of 50 mM TAPS buffer (pH = 8.3) and 5 μL of 50 mM arPES aqueous solution were added, and 120 μL was added by adding distilled water. . The mixed reaction solution was shaken at 1200C for 2 hours (1200 rpm). After the reaction, in order to remove unreacted arPES, an ultrafiltration column (Amicon (registered trademark) ultra 30k, Merck), 20 mM P.A. P. B. The buffer was exchanged using (pH = 7.0). Similarly, PES modification was performed on cholesterol oxidase (ChOx) derived from Streptomyces.

  The enzyme activities of the PES-modified LOx and PES-modified ChOx prepared as described above were measured by the MTT system and the PMS / MTT system. In the MTT system, 1 mM MTT, 0.04% Triton, 20 mM P.I. P. B. (PH = 7.0), in the PMS / MTT system, 0.6 mM PMS, 1 mM MTT, 0.04% Triton, 20 mM P.O. P. B. In (pH = 7.0), the increase in absorbance at 570 nm was measured when 1 mM lactic acid or 100 μM cholesterol was added. The results are shown in Table 2.

  When no electron mediator was added, no MTT coloration was observed in LOx or ChOx. In contrast, by PES modification of these enzymes, the MTT system to which no electron mediator was added showed a color development of about 10% of the PMS / MTT system to which the electron mediator was added. From the above results, it was shown that by using PES-modified LOx and PES-modified ChOx, each of lactic acid or cholesterol can be measured without adding another electron mediator.

Example 5: Preparation of PES-modified PyOx 50 mg TAPS buffer solution (pH = 8.3) 40 μL and 50 mM arPES aqueous solution 5 μL were added to 1 mg of Coralus genus pyranose oxidase (PyOx), and distilled water was added to make 100 μL. The mixed reaction solution was shaken at 1200C for 2 hours (1200 rpm). After the reaction, in order to remove unreacted arPES, an ultrafiltration column (Amicon (registered trademark) ultra 30k, Merck), 20 mM P.A. P. B. The buffer was exchanged using (pH = 7.0).

  The enzyme activity of the PES-modified PyOx prepared as described above was measured by the MTT system and the PMS / MTT system. In the MTT system, 1 mM MTT, 0.04% Triton, 20 mM P.I. P. B. (PH = 7.0), in the PMS / MTT system, 0.6 mM PMS, 1 mM MTT, 0.04% Triton, 20 mM P.O. P. B. In (pH = 7.0), the increase in absorbance at 570 nm was measured when 1 mM lactic acid or 100 μM cholesterol was added. The results are shown in Table 3.

  When the electron mediator was not added, the activity of the MTT system was improved 15 times or more by the PES modification, and the color development was about 7% as compared with the PMS / MTT system to which the electron mediator was added. PyOx is an enzyme that is used in an enzyme analysis method for 1,5-anhydroglucitol, which is an index of blood glucose control in the short and medium stages of diabetes. It is obvious that PyOx has a wide substrate specificity, and in order to detect the presence or absence of the activity of this enzyme, if the activity of glucose of the enzyme can be confirmed, 1,5-anhydroglucitol exhibits the same activity. is there. Therefore, these results show that 1,5-anhydroglucitol can be measured because MTT color development was observed using PES-modified PyOx without adding other mediators.

Example 6: Preparation of enzyme electrode using PES-modified PyOx 5 mM P.O. P. B. (PH = 7.0) trehalose, PES modified PyOx, BSA, ketjen black so that the final concentrations are 0.25%, 2.5 mg / mL, 1.2%, and 0.6%, respectively. This was mixed to obtain a PES-modified enzyme ink. The prepared PES-modified enzyme ink was applied in a stack of 200 nL twice on the circular carbon electrode of the DEP chip. The film was dried at room temperature and low humidity (McDry: 1% relative humidity) for 2 hours. The dried chips were exposed to glutaraldehyde vapor for 1 hour at 25 ° C. The produced enzyme electrode chip was stored until measurement under low humidity (McDry: 1% relative humidity).

  The chip thus fabricated was connected to a potentiostat, immersed in a batch cell, and subjected to CA measurement at an applied potential of +50 mV (vs. Ag / AgCl) and a stirring speed of 250 rpm. The measurement solution was 100 mM P.I. P. B. (PH = 7.0), and the glucose solution prepared with the same buffer solution was added sequentially so as to be 5, 10, 25, 50, 100, 300, 600 mg / dL.

  CA measurement showed a glucose concentration-dependent response (see FIG. 4), indicating that glucose using PES-modified PyOx can be measured. PyOx is an enzyme that is used in an enzyme analysis method for 1,5-anhydroglucitol, which is an index of blood glucose control in the short and medium stages of diabetes. PyOx has a wide substrate specificity, and in order to detect the presence or absence of the activity of this enzyme, if it is possible to measure glucose of this enzyme, 1,5-anhydroglucitol can be measured as well. Is self-explanatory. Therefore, these results indicate that 1,5-anhydroglucitol can be measured using PES-modified PyOx without adding other mediators.

The fructosyl amino acid oxidase (FAOX) was condensed with dH ₂ O, and exchange concentrated dH ₂ O using ultrafiltration column (Amicon (registered trademark) ultra 30k, Merck). To about 600 μg of enzyme, 2.5 μL of 50 mM ar-PES and 40 μL of 50 mM TAPS (pH 8.3) buffer solution were added, mixed up to 100 μL with dH ₂ O, and then shaken at 25 ° C. for 2 hours. (1200 rpm). In order to remove unreacted arPES after the reaction, the buffer solution was exchanged using an ultrafiltration column (Amicon (registered trademark) ultra 30k, Merck) and dH ₂ O. The activity of the PES modifying enzyme prepared as described above was measured by MTT or PMS / MTT system. 1 mM MTT, 0 or 0.6 mM PMS, 0.04% Triton, 20 mM P.I. P. B. In (pH 7.0), the increase in absorbance at 570 nm when the substrate was added was measured. The substrate concentration at the time of measurement was 1 mM fructosyl valine.

  Enzyme activity measurement using MTT was performed on PES-modified FAOx. As a result of measuring the enzyme activity of the prepared PES-modified FAOx using MTT, the MTT activity was 0.31 U / mg. The MTT activity when PMS was used as a mediator was 25 U / mg. On the other hand, since the MTT activity of unmodified FAOD was 0.12 U / mg and the activity in the PMS / MTT system was 23 U / mg, the activity using MTT as a coloring reagent by modifying FAOx with arPES was 3 It doubled. FAOx is an oxidase that uses glycated amino acids produced by hydrolyzing glycated proteins such as glycated hemoglobin and glycated albumin as substrates. By using this enzyme, glycated proteins, which are indicators of blood glucose control over the medium to long term, are measured. be able to. Therefore, these results show that glycated proteins can be measured without adding other electron mediators by using PES-modified FAOx.

  It should be noted that the present invention can be variously modified and modified without departing from the broad spirit and scope of the present invention. Further, the above-described embodiment is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is shown not by the embodiments but by the claims. Various modifications within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.

  This application is based on Japanese Patent Application No. 2006-192730 filed on Sep. 30, 2016, and includes the specification, claims, drawings, and abstract. The entire disclosure in the above Japanese patent application is incorporated herein by reference.

Claims (13)

It is represented by the following structural formula (I),
Oxidoreductase E,
A phenazine derivative;
An electron mediator-modifying enzyme comprising a linker site L that links the oxidoreductase E and the phenazine derivative.
In structural formula (I),
X represents an anion,
Y represents a linear or branched alkyl group which may have a substituent having 1 to 5 carbon atoms,
R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are each independently a hydrogen atom, an optionally substituted alkyl group or alkoxy group, a hydroxyl group, Represents a halogen atom, a nitro group or an amino group which may have a substituent, and at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is Linker site L. The electron mediator-modifying enzyme according to claim 1, which is represented by the following structural formula (II).
In structural formula (II),
Y represents a methyl group or an ethyl group. The electron mediator-modifying enzyme according to claim 1, which is represented by the following structural formula (III).
In structural formula (III),
Y represents a methyl group or an ethyl group.   The electron mediator-modifying enzyme according to any one of claims 1 to 3, wherein the oxidoreductase E is an oxidoreductase having FAD or FMN as a coenzyme.   The electron mediator-modifying enzyme according to any one of claims 1 to 3, wherein the oxidoreductase E is an oxidoreductase having NAD or NADP as a coenzyme.   The electron mediator-modifying enzyme according to any one of claims 1 to 3, wherein the oxidoreductase E is an oxidoreductase having PQQ as a coenzyme.   The electron mediator-modifying enzyme according to claim 4, wherein the oxidoreductase E uses glucose as a substrate.   The electron mediator-modifying enzyme according to claim 7, wherein the oxidoreductase E is a FAD-dependent glucose dehydrogenase.   The electron mediator-modifying enzyme according to claim 4, wherein the oxidoreductase E uses any one of lactic acid, fructosyl amino acid, cholesterol and 1,5-anhydroglucitol as a substrate.   The electron mediator modification according to claim 4, wherein the oxidoreductase E is any one of lactate oxidase (LOx), fructosyl amino acid oxidase (FAOx), cholesterol oxidase (ChOx), and pyranose oxidase (PyOx). enzyme. A substrate, a working electrode, and a counter electrode;
The enzyme electrode by which the electron mediator modification enzyme of any one of Claims 1-10 is fix | immobilized on the surface of the said working electrode.   The spectroscopic analysis kit containing at least 1 sort (s) of the electron mediator modification enzyme of any one of Claims 1-10, and a redox indicator.   The enzyme test paper containing at least 1 sort (s) of the electron mediator modification enzyme of any one of Claims 1-10, and a redox indicator.