JPH0690754A - Enzyme modified through spacer with mediator and sensor involving the same modified enzyme - Google Patents

Abstract

PURPOSE:To prepare a modified dehydrogenase useful, e.g. for a sensitive sensor capable of using in the body, for an electrode system and for an electrochemical system by covalently bonding mediator through a spacer to the enzyme. CONSTITUTION:A mediator such as ferrocene is covalently bonded through a spacer such as H2NNHC(=O)-(CH2)4-(C=O)-NHNH2 to dehydrogenase. The mediator can modify a carbon chain of the enzyme and/or the enzyme itself.

Description

Detailed Description of the Invention

[0001]

[Industrial application] Measurement of chemical substances is carried out in a wide range of fields including medical treatment, industrial processes, environment and the like. Various chemical substances exist in these fields, and in order to measure various chemical substances,
A variety of physical and chemical devices are used, and there is a strong demand for a method and a device that can directly and quickly measure these. However, chemical substances do not exist alone, and usually many kinds of substances are mixed. In order to select a specific chemical substance from such a system and measure it, a material and a reagent that identify these substances are necessary. For example, an enzyme that is a biocatalyst selectively promotes a reaction in a living body. The enzyme has a molecular recognition ability as well as a catalytic function. Therefore, by using this enzyme, a specific chemical substance can be identified and the concentration of the chemical substance can be measured.

INDUSTRIAL APPLICABILITY The present invention is not only useful as a measurement sensor used in various fields, but also in the field of clinical examination, it is possible to quickly and easily quantify a sample liquid for a specific component in various trace biological samples. It relates to an enzyme sensor that can be used.

[0003]

2. Description of the Related Art The usefulness of an enzyme sensor utilizing the high substrate selectivity of an enzyme has been recognized in the measurement of various compounds, and it has already been put to practical use particularly in the quantitative analysis of glucose and the like. The enzyme sensor currently in practical use is a hydrogen ion produced by immobilizing an enzyme having a high substrate specificity for a substance to be measured on a substrate, and the enzyme that controls the catalytic action of the target substance in the sample. The amount or the amount of oxygen consumed is quantified by electrochemically detecting with a hydrogen ion electrode or an oxygen electrode, respectively.
Therefore, the enzymes that can be used in this type of enzyme sensor are limited to oxidases that consume oxygen or generate hydrogen ions by the reaction, so-called dehydrogenases.

For example, a sensor can be constructed by entrapping and immobilizing dehydrogenase in a membrane and mounting it on a diaphragm oxygen electrode. Since the substrate is dehydrogenated by the action of this dehydrogenation, the concentration of the substrate can be measured from the change in the current value with the oxygen electrode that is in close contact with the enzyme membrane. Also, the substrate concentration can be obtained in the same manner by measuring this with a hydrogen ion electrode instead of this oxygen electrode.

These electrode reactions are electrochemical (redox)
Based on the dynamic mechanism. Depending on the type of substrate, there are systems in which the enzyme and substrate do not react easily, and there are cases where mediators and coenzymes are required. Research is progressing on various systems. For example, a method has been proposed in which an artificial mediator is applied to the surface of the electrode in a thin film form, and the surface of the electrode is coated with a semipermeable membrane (EP78636B1). In addition, a poorly water-soluble mediator may be contained in the electrode material (Agric. Biol.
Chem., 52 , 1557 (1988)), in the case of a highly water-soluble mediator, after including the mediator in the electrode in advance,
By forming a mixture of an ionic polymer and an enzyme as a thin film on the electrode surface, a method of preventing the mediator from eluting in the electrolytic solution (Agric. Biol. Chem., 52 ,
3187 (1988)) has been proposed.

However, in any case, the preparation of the electrode is complicated, and the enzyme is used so as to form a thin film on the surface of the electrode after containing the mediator.

[0007]

As described above, the conventional enzyme-modified electrode is complicated in its preparation method and requires a difficult work to mass-produce a quality-controlled product as a measuring instrument, and moreover, it is repeated many times. When used for a long time, there are various problems such as short life of the electrode itself and limitation of usable enzymes. Therefore, a method of coating the surface of the electrode step by step with a mediator thin film layer, an enzyme thin film layer and a semipermeable membrane layer has been conventionally used, but it has a problem that the preparation of the electrode is complicated.

Therefore, instead of these complicated methods, a complex formed by previously covalently binding a mediator to an enzyme via a spacer is prepared in advance, and this is used as a solution component, and the coexisting substrate component acts. We have established a sensor system that can measure the substrate concentration by electrochemically detecting the change in the substrate concentration during the reaction with the sample solution in the electrode system that combines the electrode, the counter electrode and the reference electrode.

In addition to portability, the above-mentioned measurement system is simple,
Since there is a problem in immediacy, we conducted research to make the above electrodes compact by using our printing technology. That is, an enzyme-modified electrode having a complex in which a mediator is covalently bound to an enzyme via a spacer as an electrode component is used as a working electrode, and a reaction with a sample solution is performed in an electrode system that is combined with a counter electrode and a reference electrode as described above. At the same time, we established a sensor that can measure the substrate concentration by electrochemically detecting the current value or potential value of the change in substrate concentration. Here, the enzyme-modified electrode has a uniform composition by adding the above-mentioned complex (mediator-spacer-enzyme) to a conductor used in a normal electrode such as graphite and carbon paste, and an appropriate vehicle, This uniform composition is used as an electrode base material (for example, a carbon electrode) on the surface of a base material that does not show conductivity by patterned screen printing (coating).
The manufacturing method was adopted as a simple method. Furthermore, by studying various enzymes, the method for producing an enzyme-modified electrode was established, and various studies were conducted for the purpose of providing a sensor with excellent performance incorporating the enzyme-modified electrode. completed.

[0010]

The first aspect of the present invention is to provide a space.
It is a modified enzyme obtained by covalently binding a mediator and dehydrogenase through a ser. Examples of the dehydrogenase used in the present invention include fructose devitrogenase. Examples of such dehydrogenase include alcohol dehydrogenase, aldehyde dehydrogenase, glucose devidogenase, glutamate devidogenase, cholesterol dehydrogenase, sorbitol dehydrogenase, and glycerol dehydrogenase.

Examples of mediators include ferrocene, ferrocene derivatives, p-benzoquinone, potassium ferricyanide, phenazine methosulfate, 2,
6-dichlorophenol indophenol, PQQ, F
Examples thereof include AD, NAD and NADP, with ferrocene and ferrocene derivatives being particularly preferred. Examples of the ferrocene derivative include 1,1′-dimethylferrocene, ferroceneacetic acid, hydroxyethylferrocene, and 1,1′-
Examples thereof include bis (hydroxymethyl) ferrocene, ferrocene monocarboxylic acid, ferrocene 1,1′-dicarboxylic acid, chloroferrocene, and methyltrimethylaminoferrocene.

As a combination of the enzyme and the mediator,
A combination of fructose dehydrogenase and ferrocene or a ferrocene derivative is particularly preferable. The modified enzyme of the present invention is characterized in that the enzyme is covalently bound to the mediator via a spacer. Examples of the spacer include isoprene, butadiene, acetylene, their derivatives and their respective copolymers.

However, as the spacer, a dihydrazito compound represented by the following formula is used because of the necessity of chemical modification of the enzyme. Particularly preferred is n = 4 adipyl dihydrazito.

[0014]

[Chemical 2]

The second aspect of the present invention is a sensor comprising a solution component containing the modifying enzyme of the present invention and an electrode system comprising a working electrode, a counter electrode and a reference electrode.
Is a sensor including an electrode system including an enzyme-modified electrode having the above-described modified enzyme of the present invention as an electrode component, a counter electrode, and a reference electrode. The third sensor of the present invention is preferably a sensor whose electrode system is made of a material mainly composed of carbon formed by screen printing on an insulating substrate.

The present invention will be described in more detail with reference to fructose dehydrogenase (hereinafter referred to as "FDH") which is a preferable combination of an enzyme, a mediator and a spacer, ferrocene or a ferrocene derivative and a hydrazide as a spacer. In this combination, the following two points are important. 1. Chemical modification of FDH with ferrocene or a ferrocene derivative.

2. Environmental pH control using a fructose sensor electrode. FDH catalyzes the following reactions. :

[0018]

[Chemical 3]

On the other hand, mediators such as ferrocene are
It replaces the acceptor in the above formula and acts as a redox reagent as follows.

[0020]

[Chemical 4]

When used as a biosensor, a redox reagent (hydrogen peroxide or other mediator) is
The reaction between the FADH site of the enzyme and the electrode is reciprocated, and electrons are transferred from the enzyme to the electrode to such an extent that a sufficient current can be measured. In addition, ferrocene has the following advantages over hydrogen peroxide. 1) Although the oxygen concentration in the solution is variable,
The ferrocene concentration can be easily controlled. 2) Ferrocene can be measured even at a lower electrode potential than hydrogen peroxide. Therefore, a biosensor using ferrocene is less affected by interference from a compound such as ascorbic acid that is reduced at a similar potential during measurement, as compared with the case where hydrogen peroxide is used.

Ferrocene can be immobilized on the electrode carbon by adsorption. Also, since it has low solubility in water, it does not dissolve so quickly from the electrode, but some loss is unavoidable. Further, there may be an approach of contacting dehydrogenase with the immobilized ferrocene. However, if ferrocene can bind to the carbon chain of dehydrogenase (this carbon chain is an alkyl chain branched from the enzyme, sugar chain, side chain of amino acid, etc.), the enzyme activity will be the activity of the protein (enzyme). The effect is less than when ferrocene is bound near the site and interferes with the enzymatic reaction. If ferrocene is attached to the carbon chain of the enzyme, it will be responsible for the reaction between the FADH reaction site of the enzyme and the electrode. Furthermore, if a sufficient (or excess) amount of ferrocene molecule can bind (modify) to the enzyme and the carbon chain of the enzyme, it will not adversely affect its catalytic ability and also the reaction between the enzyme's FADH site and the electrode. Without going back and forth, from the FADH reaction site of the enzyme itself to several ferrocene molecules or electrodes bound to various different sites of the enzyme,
Electronic delivery will be possible directly. So to speak, "conductive enzyme" could be produced. The process leading to the production of the mediator modifying enzyme and the modifying enzyme itself are the main points of the present invention.

Furthermore, when a complex formed by covalently binding a mediator to FDH via a spacer in advance in an actual measurement system is reacted with a substrate (fructose), the complex is a "conductive enzyme". From this, it was observed that the conductivity of the solution system greatly changed depending on the substrate concentration. In particular, FDH is a PQQ type enzyme, and since the enzyme reaction does not directly depend on coenzyme (NADH), it was a particularly preferable enzyme for obtaining the above phenomenon. Using this system as it is, carbon paste is further added, and an electrode pattern is printed on a certain substrate (plastic or the like) by a screen printing method. This chip was used as a disposable biosensor attachment. Furthermore, the machine body (reader) and the attachment were constructed as a set as a sensor system.

[0024]

EXAMPLES The present invention will be described in more detail below with reference to examples and comparative examples, but the scope of the present invention is not limited to these examples.

[0025]

Example 1 Preparation of ferrocene-FDH modified only on the carbon chain of the enzyme (FIG. 1 (A)) (1) The carbon chain of FDH was oxidized with sodium periodate. FDH (100 mg) was dissolved in 0.1 M pH 6.0 phosphate buffer (1 ml) and mixed with 20 mM sodium periodate (previously dissolved in the above buffer). Oxidation is 4
Proceed for 1 hour at ℃, followed by ethylene glycol (100
μl) was added to stop the reaction. Then, further 4 ℃, 30
After that, the reaction mixture was dialyzed for 48 hours or longer while the pH 6.0, 0.1 M phosphate buffer was changed 4 times. (2) Cross-linking reagent: Binding of adipyl dihydrazide to the oxidized carbon chain.

Adipyl dihydrazide (100 mg dry powder)
Was dissolved in a dialyzed solution of oxidized FDH, and the solution was allowed to react overnight at room temperature in a dark place. The reaction mixture was dialyzed with four changes of pH 6.0, 0.1 M phosphate buffer to remove excess adipyl dihydrazide. (3) Reaction of unreacted hydrazide group bonded to carbon chain of enzyme with ferrocene carboxaldehyde.

Ferrocene carboxaldehyde (concentration 1
mg / ml, pH 6.0, 0.1 M phosphate buffer 2 ml) was mixed with an adipyl hydrazide solution (2 ml) and reacted overnight at 4 ° C. in the dark. Sodium cyanoborohydride was mixed to a final concentration of 10 mM, reacted for 1 hour, and
The pH 6.0, 0.1 M phosphate buffer was changed 4 times and the reaction mixture was dialyzed to remove excess ferrocene carboxaldehyde.

[0028]

Example 2 Ferrocene-modified on both the carbon chain of the enzyme and the enzyme itself
Preparation of FDH (Fig. 1 (B)) The following two methods were tried in order to prepare a two-step modified enzyme.
(Method A, Method B). Method A Method A is based on Y. Degani and A. Heller (J. Physical Chemistr
The method by Y, 91 , 1285-1289 (1987) was referred to. In this method, by reacting ferrocenecarboxylic acid with the free amino group of the protein itself by the carbodiimide method,
Ferrocene is bound to the FDH itself. In order to obtain a two-step modification enzyme (that is, to attach ferrocene to both the protein body and the carbon chain of the protein), adipyl dihydrazide is first attached to a part of the carbon chain in the same manner as in Example 1. Then, the ferrocenecarboxylic acid is reacted with both the amino group of the protein body and the free hydrazide group of the protein carbon chain. (1) The carbon chain of FDH was oxidized with sodium periodate in the same manner as in Example 1 (1). (2) In the same manner as in Example 1 (2), the adipyl dihydrazide was reacted with oxidized FDH. (3) Both the amino groups of the protein body and the protein carbon chain hydrazide group were reacted with ferrocenecarboxylic acid. Urea was mixed into the reaction mixture to unwind the protein and expose the amino groups so that the reaction proceeded. Urea (810mg), water-soluble carbodiimide (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride; 100m
g) and ferrocenecarboxylic acid (80 mg) at pH 7.3, 0.15M
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (hereinafter referred to as "HEPES") buffer solution (4 ml)
Sonicated in and re-adjusted to pH 7.2-7.3. Ferrocenecarboxylic acid was saturated at this pH. Add adipyl hydrazide solution (2 ml) and adjust pH if necessary
Was confirmed and adjusted, and the reaction product was left overnight at 0 ° C. in the dark.
The excess amount of reagent was removed by dialysis exchange with a pH 6.0, 0.1 M phosphate buffer solution four times. Method B In method B, the aldehyde group (-CHO) of the oxidized carbon chain of the protein side chain and the free carboxyl group (-COOH) of the protein body
Both were reacted with adipyl dihydrazide. The ferrocene aldehyde was then reacted with the bound hydrazide. (1) A portion of the FDH carbon chain was oxidized with sodium periodate in the same manner as in Example (1). (2) Oxidized FDH was reacted with adipyldihydrazide in the presence of carbodiimide. 0.5M adipyl dihydrazide, adjusted to pH 5 (2ml), 9mg sodium chloride, oxidized enzyme solution (2ml) and 1M water-soluble carbodiimide (1m
The pH was adjusted to 5 by mixing all of 1) and 3M urea. The reaction proceeds by leaving it at room temperature in the dark for 4 hours: the pH is adjusted to 5 every 30 minutes. After 4 hours, the reaction solution was dialyzed and exchanged four times with 0.1 M phosphate buffer (pH 6.0). (3) Reaction with ferrocene carboxaldehyde FDH hydrazide solution (2.5 ml) was mixed with 0.4 mg / ml ferrocene carboxaldehyde (2.5 ml) and reacted at 4 ° C in the dark for 4 hours. Add sodium cyanoborohydride to a final concentration of 0.1 mM, and adjust the pH to 6.0, 0.1.
The reaction solution was dialyzed 4 times with M phosphate buffer.

Also, it was confirmed that there is no great difference between the two methods, judging from the electrochemical response of ferrocene-FDH finally obtained by methods A and B.

[0030]

Example 3 Ferrocene-FDH (Fig. 1) modified only on the enzyme itself.
(C)) As already mentioned in the section “Means for solving the problem”, in order to compare with the difference due to the above modification site (Examples 1 and 2), ferrocene in which the mediator is modified only in the enzyme itself. -FDH was also prepared. The manufacturing method is Example 2
According to the method A, the ferrocene can be bound to FDH itself by reacting the ferrocenecarboxylic acid with the free amino group of the protein itself by the carbodiimide method.

The amino group of the protein body was reacted with ferrocenecarboxylic acid. Urea was mixed into the reaction mixture to unwind the protein and expose the amino groups so that the reaction proceeded. Urea (810mg), water-soluble carbodiimide (100mg)
And ferrocenecarboxylic acid (80mg) at pH 7.3, 0.15M HEP
Sonicate in ES buffer (4 ml), pH 7.2 to
Retuned to 7.3. Ferrocenecarboxylic acid was saturated at this pH. Add adipyl hydrazide solution (2 ml),
The pH was checked and adjusted as needed and the reaction was left overnight in the dark at 0 ° C. The excess amount of reagent was removed by dialysis exchange with a pH 6.0, 0.1 M phosphate buffer solution four times.

[Comparative Example 1] System in which a mediator and a substrate are mixed without modifying the enzyme In order to clarify the significance of Examples 1 to 3, instead of the ferrocene modifying enzyme used in these systems, modification was performed. No FDH was used. Further, ferrocene was mixed as a mediator and subjected to the following electrochemical evaluation.

For the purpose of making the conditions such as FDH and ferrocene concentrations of each measurement system as uniform as possible, the reagent formulation described in Example 1 was referred to and prepared as follows. Conditions FDH (unmodified) 100 mg Ferrocene 1 mg Phosphate buffer (pH 6.0, 0.1 M) 30 ml Catalase (see Example 4) 3 mg Fructose (presence or absence) 10 mM or 0 mM Response in this measurement system (Comparative Example 1) Was observed to be extremely low. The reason for this may be that the ferrocene molecules that are mediators are uniformly present in the solution, and the number of ferrocene molecules that contribute to FDH and the redox reaction is considerably small. On the other hand, in each system of Examples 1 to 3, since the ferrocene molecule is localized on the surface of FDH or in the periphery thereof, the ferrocene molecule contributes to the redox reaction considerably efficiently as compared with the present measurement system. It is probable that these differences were recognized in the above.

[0034]

Example 4 Evaluation of Modified FDH Gold working electrode (diameter 1 mm), platinum counter electrode, Ag / AgCl
The ferrocene-modified FDH was evaluated by cyclic voltammetry with a reference electrode. Aluminum oxide before use
The working electrode was polished with (0.075 μm). Hokuto Denshi Potentiostat / Galvanostat model HAB-151 and Graphtec WX1200 recorder were used.

The modified enzyme solution was used without dilution according to Examples 1-3. Catalase at a final concentration of 0.1 mg / ml was added to avoid interference with hydrogen peroxide. First, the cyclic voltammogram of the enzyme was measured in the absence of fructose. Then, a fructose solution was added so that the final concentration was 10 mM, and the cyclic voltammogram was measured again under exactly the same conditions. Further, in order to eliminate the influence of oxygen, bubbling with nitrogen was sufficiently performed.

The cyclic voltammograms of the ferrocene-modified FDH obtained in Examples 1, 2 (method A) and 3 were measured, and the conductivity was improved by adding the substrate (fructose). It was seen.

[0037]

[Example 5] Measuring system using electrodes by screen printing method Dai Nippon Printing Co., Ltd. has developed a clinical test material typified by JP-A-3-28199 "Test piece for testing body fluid components". There is. Others include JP-A-60-178356.
No. "Body fluid test body", JP-A-60-238763 "Body fluid test body", JP-A-61-247967 "Glucose detection test body and its manufacturing method", JP-A-61-284661 "Glucose detection body""Inspectionbody" and JP-A-62-263468 "Glucose detection ink composition and inspection body". Some of these inventions make detailed reference to a method for producing a urine test paper by a screen printing method using a composition containing a plurality of kinds of enzymes as an ink. As its application, a new ink formulation as shown in Table 1 was developed by mixing a conductive component instead of removing a component such as a color change indicator that is not required in the following process. As other printing means, gravure printing, coating with a dispenser, or die coating can be used.

[0038]

[Table 1]

The screen printing method is an existing technology. The screen is Tetron, 120 mesh, 150 μm (film thickness).
Was prepared (Mino Group Co., Ltd.) and printing was carried out under normal conditions (see JP-A-61-284661). Immediately after printing, it was dried (60 ° C., 5 minutes), and then this printed material was cut and subjected to electrochemical measurement as an electrode (chip). The print patterns are shown in FIG. 2 (pattern A, pattern B).

Pattern A is a one-electrode chip formed of ink A (containing a modifying enzyme) from FIG. 2, and pattern B is a pattern of ink A (containing a modifying enzyme) as a working electrode.
This is a two-electrode chip using ink B (without enzyme) as a counter electrode. First, the measurement was performed with a one-electrode system. That is, although the measurement system is the three-electrode system described above, one electrode tip was loaded instead of the gold working electrode, and the current values shown in Table 2 were obtained. Furthermore, the same measurement was carried out using a two-electrode chip having a working electrode and a counter electrode. For the sample solution, three levels of fructose aqueous solution, 0 mM, 5 mM, and 10 mM were used as measurement points. printing,
The size of the electrode pattern after drying is 3 mm wide, 30 mm long,
It is 100 μm thick and is about 20 mm from the side where the
Was immersed in the sample for measurement.

[0041]

[Table 2]

In Table 2, the ferrocene-modified FDH obtained in Example 2 (method A) was used as the modifying enzyme. Also,
The measured current value is the working electrode when the potential value is +450 mV,
It is a current value flowing between the opposing electrodes. Moreover, since the amount of enzyme contained in the printing ink can be easily controlled, the dynamic range and sensitivity of the sensor can be freely selected. It has been confirmed that a portable fructose sensor can be produced with a two-electrode system (Pattern B). As a practical model, a system in which the disposable tip (electrode) is loaded on the tip of a pen type (digital display) I was able to devise. FIG. 3 shows a practical pen-type model.

[0043]

INDUSTRIAL APPLICABILITY In the present invention, it becomes possible to physically and firmly immobilize the enzyme and the mediator by covalently bonding them. This not only makes it possible to react the enzyme with the ordinary sensor, electrode system, and electrochemical system extremely efficiently, but also can prevent the leakage of the mediator to the outside of the reaction system. That is, the conventional sensor is prohibited from being used in the body because the mediator (often highly toxic) elutes, but the present invention enables the use in the body, and as described above. Since the enzyme surface is provided with conductivity, it can be easily used in an environment where oxygen does not exist, and is extremely useful in various fields. Furthermore, the present method, which allows the enzyme surface to be sufficiently modified with a mediator, exhibits higher sensitivity than conventional sensors.

From the above, an electrode system prepared by a screen printing method containing a solution system containing an enzyme-mediator complex and a substrate does not require (1) the original "mediator" to be used in combination, and (2) leakage of the mediator. So there is no such thing as in viv
can be used in (3) high sensitivity of response,
(4) The reaction is reversible, and a stable response can be obtained.
(5) Since unstable enzymes are chemically modified, they are resistant to denaturing factors such as solvents. (6) Various fine and complicated printing by devising printing plates such as screen plates. The effect that the pattern can be designed is recognized.

[Brief description of drawings]

FIG. 1 is a schematic diagram of ferrocene-FDH.

FIG. 2 is a diagram showing a print pattern of an electrode chip.

FIG. 3 is a diagram showing a pen-type practical model.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12Q 1/32 6807-4B 1/54 6807-4B

Claims (8)

[Claims] 1. A modified enzyme obtained by covalently binding a mediator and a dehydrogenase via a spacer. 2. The dehydrogenase is alcohol dehydrogenase, glutamate dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase,
The modified enzyme according to claim 1, which is selected from the group consisting of glucose dehydrogenase, fructose dehydrogenase, sorbitol dehydrogenase and glycerol dehydrogenase. 3. The modified enzyme according to claim 1, wherein the spacer is a dihydrazide compound represented by the following formula. [Chemical 1] 4. The modified enzyme according to claim 1, wherein the mediator is ferrocene or a ferrocene derivative, a nicotinamide derivative, a flavin derivative, a quinone or a derivative thereof. 5. A sensor comprising an electrode system comprising a working component, a counter electrode and a reference electrode, and a solution component containing the modifying enzyme according to claim 1. 6. A sensor comprising an electrode system comprising an enzyme-modified electrode containing the modified enzyme according to any one of claims 1 to 4 as an electrode component, a counter electrode and a reference electrode. 7. A sensor comprising an electrode system comprising a modified enzyme-immobilized electrode using the modified enzyme according to any one of claims 1 to 4 as an electrode component, a counter electrode and a reference electrode. 8. The sensor according to claim 6, wherein the electrode system is made of a carbon-based material formed by screen printing on an insulating substrate.