JPH05240828A - Electrode and measuring method using the same - Google Patents

Abstract

PURPOSE:To make it possible to perform accurate measurement without the effect of the obstructing material for the measurement by enlarging the enzyme activity on the far surface side than that of the surface side nearer to the working electrode of a water absorbing layer covering the working electrode. CONSTITUTION:For example, two platinum lines and one silver line are arranged on an acrylic plate 1. A working electrode 2, a counter electrode 3, a silver or silver chloride reference electrode 4 are formed. As the water absorbing body, scientific analyzing filter paper is used. The aqueous solution of glucose oxidase and bovine serum albumin is developed on the surface of one side and dried in air. Thus, an enzyme-fixed water-sucking body 5 is formed. Then, the sucking body 5 covers the surface of the electrodes 2 and 4. Then, the enzyme-fixed water-fixed sucking body 5 is mounted on a measuring cell so that the enzyme fixing surface faces outside, and a pushing plate 6 is mounted. Thus, the difference in diffusing speeds in arrival on the electrode surface between the hydrogen peroxide and the measurement obstructing material is generated. Therefore, the measurement, wherein the effect of the measurement obstructing material is suppressed at the lower value, can be achieved by processing the measured data in the time region after the starting of response to hydrogen peroxide to the time before the response to the measurement obstructing material.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrochemical measurement electrode provided with two electrodes of a working electrode and a counter electrode, or a three-electrode system of a working electrode, a counter electrode and a reference electrode, and a water absorbing body having an oxidoreductase. Regarding the measurement method used.

[0002]

2. Description of the Related Art In recent years, the number of analysis items and the number of analysis samples have been increasing with the progress of analysis technology. Under such circumstances, the development of a device that can process an analytical operation more easily and more rapidly continues. Among them, the method to which the electrochemical measurement is applied observes the electron transfer generated quantitatively from the target substance to be measured and can be measured relatively easily.

By applying this advantage, in addition to conventional measurement methods such as polarography, it is applied in a wide range by combining with liquid chromatography, flow injection analysis and the like. The features of electrochemical detection are as follows: (1) A chemical quantity that is generally difficult to measure is converted directly into an easy-to-measure quantity of electricity and measured easily. (2) Since the electrochemical reaction that proceeds according to Faraday's law is observed, a minute change in the substance can be captured from the current value or the amount of electricity, and highly sensitive measurement can be performed. (3) The reaction process can be immediately known, and rapid detection / quantification is possible. Etc., and has been applied to the measurement of various substances by methods such as potentiometry, amperometry, polarography, coulometry, impedance measurement, and cyclic voltammetry.

Further, the measuring method in which these detection methods are combined with a conversion element of biological origin, which is excellent in selective reactivity to various chemical substances, is a method which enables easy and selective measurement. In addition, research and development of conversion elements derived from living organisms used for it begins with enzymes and microorganisms,
The types of such as antibodies are increasing.

Among these conversion elements derived from organisms, enzymes that react with a substrate to consume oxygen or produce hydrogen peroxide have excellent response speed and measurement sensitivity, and are easily electrochemically detected. Widely used in. A typical example is hydrogen peroxide-producing oxidoreductase, which is used to quantify substances such as glucose by detecting oxygen consumed by the enzymatic reaction with the target substance to be measured and hydrogen peroxide produced. There is.

On the other hand, a problem in the electrochemical measurement is low selectivity in the electrochemical detector portion such as an electrode. This is because the final stage of detection depends only on the current value, and therefore it is not possible to distinguish between the current generated by the reaction of the measurement target substance and the current generated by the interfering substance of the measurement.

Conventionally, in electrochemical measurement, a measuring method such as cyclic voltammetry or pulse voltammetry for improving the selectivity has been used, but the measuring method is complicated as compared with the amperometric method, and the measurement target substance is also used. Sufficient results were not obtained in terms of the ratio of selective permeation between the and interfering substances and shortening of the measurement time. Target substance (substrate of enzyme) in amperometry
As a coping method in the case where the interfering substance and the interfering substance coexist in the sample, a selective permeable membrane is used in the oxygen electrode for detecting the oxygen concentration. In the oxygen electrode, the difference in molecular size is used, and a polymer membrane with minute holes that allow only oxygen to permeate and other substances do not permeate is attached to the oxygen electrode.

However, a porous polymer membrane generally used for an oxygen electrode has a slow permeation rate of hydrogen peroxide having a molecular weight larger than that of oxygen, and is fixed when made of a material such as fluororesin. Since it is difficult to form an enzyme membrane directly on the surface, it cannot be applied to a hydrogen peroxide electrode. On the other hand, when hydrogen peroxide is detected by the amperometry method, since the redox potential of the decomposition reaction of hydrogen peroxide on the electrode surface is 0.68 V (vs. standard hydrogen electrode, 25 ° C), Must be maintained at a potential of about 0.46 V or higher with respect to a commonly used silver / silver chloride reference electrode.

However, taking ascorbic acid, which is a typical substance that interferes with measurement, as the standard oxidation-reduction potential of ascorbic acid is 0.34 V (pH 7.0), the oxidation current of hydrogen peroxide is detected. If this is done, the oxidation current value of ascorbic acid will also be added, and hydrogen peroxide alone cannot be accurately measured. Therefore, in an amperometric biosensor in which a hydrogen peroxide electrode and an immobilized enzyme are combined, it is necessary to suppress the response of the interfering substance to hydrogen peroxide generated by the enzymatic reaction to a low level.

Therefore, conventionally, there has been proposed a measurement cell in which a measurement cell in which various hydrogen peroxide selective permeation membranes such as an acetyl cellulose membrane are formed in advance so as to cover the electrode surface is combined with an immobilized enzyme. For example, JP-A-52
No. 55691 exemplifies an electrode equipped with a hydrogen peroxide selective permeable membrane made of acetyl cellulose.
However, because acetyl cellulose is insoluble in water,
It must be dissolved in a volatile organic solvent and then developed to form a film. In this manufacturing method, since the organic solvent easily evaporates,
The concentration of acetyl cellulose was liable to change, and it was difficult to prepare a membrane having a constant thickness to obtain a membrane having reproducible selective permeability. In addition, the acetyl cellulose membrane must have a thickness of several tens of μm or less to obtain a sufficient response speed. Therefore, it requires skill to attach the acetyl cellulose membrane together with the enzyme-immobilized membrane to the measurement cell or the electrode. It was not possible to perform the analysis operation quickly and efficiently.

As described above, the electrode using the hydrogen peroxide selective permeable membrane which has been conventionally used has an insufficient selectivity for hydrogen peroxide produced by an enzymatic reaction or an insufficient response speed, It was not at a practical level.

[0012]

DISCLOSURE OF THE INVENTION The present invention solves the above problems and provides an electrochemical measurement electrode capable of performing accurate measurement without being affected by a measurement interfering substance, and a measurement method using the same.

[0013]

DISCLOSURE OF THE INVENTION The present invention provides an electric system having an electrode system including at least a working electrode and a counter electrode, and a water-absorbing layer formed by adsorbing or immobilizing a redox enzyme which catalyzes a reaction for producing hydrogen peroxide. An electrochemical measurement electrode, which is a chemical measurement electrode, in which the water-absorbent layer covers the working electrode, and the enzyme activity of the surface side far from the working electrode is greater than the enzyme activity of the surface side of the water-absorbent layer near the working electrode. Is.

Further, according to the present invention, the water-absorbent layer on which the oxidoreductase is adsorbed or immobilized is formed by applying the oxidoreductase-containing liquid on one surface of the water-absorbent layer, and the coated surface is the side far from the working electrode. An electrochemical measurement electrode mounted on the working electrode. Furthermore, the present invention is a measurement method of supplying a sample containing a substrate of oxidoreductase from the side far from the working electrode of the water absorbent layer of the electrochemical measurement electrode, and detecting hydrogen peroxide derived from the substrate, Detection of the hydrogen peroxide after the hydrogen peroxide permeates the water-absorber layer and reaches the working electrode, and the measurement-interfering substance having electrode activity in the sample permeates the water-absorber layer and reaches the working electrode. Disclosed is a measuring method for quantifying a substrate based on an output value before causing an error in the value. However, in the present invention, before the measurement-interfering substance permeates the water-absorbent layer and reaches the working electrode includes a case where the influence of the measurement-interfering substance gives a substantially negligible output value.

[0015]

[Function] Generally, in the electrochemical measurement, in order to detect hydrogen peroxide produced by a conversion element derived from a living organism such as an enzyme, the electrode configuration is simple and suitable for highly sensitive measurement. The amperometry that was used is often used. However, in this case, if a reducing substance that produces an electrolysis current at the same potential as hydrogen peroxide produced by an enzymatic reaction coexists in the sample to be measured, an error is given to the measured value. Such measurement-interfering substances include ascorbic acid, reduced glutathione, urea, uric acid, and many other substances that are generally present in samples such as foods and fermentation liquors. Therefore, in order to perform highly accurate measurement using the immobilized enzyme body and electrochemical measurement, some measures against these measurement interfering substances are required.

An example will be described below in which glucose oxidase is mainly immobilized on a water-absorbent layer and mounted on a hydrogen peroxide electrode, and glucose is measured from a sample containing glucose and ascorbic acid which are measurement-interfering substances. .. Glucose produces hydrogen peroxide by the reaction of glucose oxidase. The permeation rate of this hydrogen peroxide in the water absorber is higher than the permeation rate of the measurement-interfering substance. The present invention utilizes this phenomenon to perform accurate measurement.
In other words, glucose produces hydrogen peroxide by glucose oxidase, but the output current value before the produced hydrogen peroxide reaches the electrode and before the interfering substance reaches the electrode and at a level where its influence can be ignored is By performing the measurement, accurate measurement can be performed without the influence of the measurement interfering substance.

This is possible because the molecular weight of hydrogen peroxide is as small as about 34 so that it can be quickly permeated through the water-absorbing body, while the molecular weight of the measurement-interfering substance is large, so It is speculated that it is slow to penetrate. For example, in an amperometric detection method for hydrogen peroxide, ascorbic acid, which is a typical substance that interferes with measurement, has a molecular weight of about 176, and uric acid has a molecular weight of about 168, but hydrogen peroxide has a molecular weight of about 168. Since it is about 34, it is assumed that the transmission rate is high. However, probably because the molecular weight of glucose is about 180, the permeation rate is not much different from that of the interfering substance. If glucose is allowed to pass through the water absorbent layer,
The effects of interfering substances cannot be excluded.

Therefore, the glucose oxidase activity is fixed on the surface (sample supply surface) of the water absorbent layer far from the working electrode so that hydrogen peroxide is generated from glucose immediately after the sample is introduced to the water absorbent layer. Preferably. On the other hand, when the oxidoreductase is immobilized or adsorbed in the water absorbent layer, when the enzyme is uniformly immobilized or adsorbed,
It takes time for glucose to permeate to the location where the enzyme is immobilized in the water-absorbent layer, and until the hydrogen peroxide is generated and reaches the working electrode, measurement-interfering substances also reach the working electrode in the water-absorbing layer. Will pass through, and an accurate measured value proportional to the glucose concentration cannot be obtained.

That is, when the enzyme is uniformly and uniformly immobilized on the entire water-absorbing material, the production of hydrogen peroxide is rate-controlled by the permeation rate of the substrate, and the response shows no significant difference from the measurement-interfering substance. In order to immobilize the oxidoreductase on one surface of the water absorbent body with high activity, it is achieved by applying the water absorbent body on one surface of the water absorbent body instead of dipping it in an enzyme solution or impregnating a dilute enzyme solution. it can.

The water absorbing body may be of any type as long as it is impregnated with or permeates with water. For example, paper such as filter paper for analysis,
Cellulose derivatives such as nitrocellulose, collagen,
Cloths such as gauze or non-woven fabrics, filters made of water-insoluble material, and the like can be used. Cellulose or a cellulose derivative is particularly preferable. To immobilize so that the amount of enzyme activity localized near one surface of the water absorber is greater than the amount of enzyme activity localized near the other surface, apply from one surface of one side of the water absorber. Since the enzyme solution thus prepared needs to be retained on the surface of the application side to a certain extent, a water absorber having a specific drainage time (drainage time) is preferable. The freezing time is the time during which one end of a test piece cut into a square with a width of about 10 cm is immersed in distilled water at 20 ° C., water is absorbed and rises, and passes through a height of 10 cm. A water absorbent of 20 seconds or more is preferable. The freezing time is more preferably 40 to 300 seconds.

In the present invention, the oxidoreductase is immobilized on one surface of the water-absorbing body so that the activity of the oxidoreductase is unevenly distributed, and the water-absorbing body is attached to the measurement cell so that the surface thereof contacts the sample to be measured.

This causes a difference in the diffusion rate between hydrogen peroxide and other measurement-interfering substances before reaching the measurement electrode surface. Then, by processing the measurement data in the time range after the start of the response to hydrogen peroxide and before the response of the measurement-interfering substance, the measurement with the influence of the measurement-interfering substance suppressed can be performed.

In order to obtain the above-mentioned time range, one of the methods is to record the current when the water absorber is attached to the electrode and to make hydrogen peroxide act, and use the measurement data in the time range where the maximum velocity is obtained. It will be a guide. The setting of the time range is determined in that the signal / noise ratio becomes the best when the time is extended from a short time to a long time because the speed of current change differs depending on the water absorber used.

Examples of the electrode system include a two-electrode system composed of a working electrode and a counter electrode, or a three-electrode system composed of a working electrode, a reference electrode and a counter electrode. The electrode can be formed by, for example, embedding a conductive substance in the bottom surface of the measurement cell or depositing a metal on the inner wall surface, a solution plating method, an electroless plating method, a printing method, or the like. The enzyme-immobilized water absorbent according to the present invention can be used in combination with a hydrogen peroxide electrode based on platinum or the like. It is also possible to combine with not only platinum but also commonly used electrodes such as gold and carbon.

When a reference electrode is provided, a silver / silver chloride reference electrode, a saturated calomel reference electrode or the like can be used. By connecting the electrochemical measurement cell equipped with the enzyme-immobilized water absorbent disclosed in the present invention to a suitable detection device and a detection circuit, a hydrogen peroxide measurement device is configured, and an enzyme, a microorganism, an antigen, an antibody, or By combining these immobilization elements,
A biosensor can be constructed. More specifically, enzymes such as glucose oxidase, alcohol oxidase, lactate oxidase, galactose oxidase, and uricase that generate hydrogen peroxide can be used.

Examples of the method of immobilizing or adsorbing the enzyme include entrapping immobilization method, covalent bond immobilization method, or a method of simply applying to a water-absorbing body to adsorb. Immobilize enzyme with other proteins, if necessary, with polyhydric aldehydes such as glutaraldehyde and glyoxal (covalent bond)
Preferably. Examples of such a protein include albumin, gelatin, globulin, collagen, casein, etc., whereby the effect of stabilizing the enzyme can be obtained.

An insulating material such as acrylic, fluororesin, vinyl chloride resin, or glass can be used as the material of the cell in which the electrodes are provided, but a conductive material can also be used if it is insulated from the electrode system. It is also possible to have a two-layered water-absorber layer and attach an enzyme-free water-absorber layer to the electrode side, but if the layer is too thick, the measurement time will be long.

[0028]

EXAMPLES The contents of the present invention will be described in more detail with reference to the following examples, but of course the present invention is not limited thereto.

Example 1 will be described with reference to FIG. A three-electrode system consisting of a platinum working electrode (2), a silver / silver chloride reference electrode (4), and a counter electrode (3) connected to a commercially available potentiostat device (HECS1100 type: manufactured by Fuso Seisakusho, not shown) Then, the electrochemical measurement cell of the present invention was constructed and measured.

(1) Method for preparing a measuring cell for electrochemical measurement: An acrylic plate (1) having a size of 30 mm × 30 mm and a thickness of 3 mm, and two platinum wires having a diameter of 2 mm (2) and (3), and a silver wire having a diameter of 2 mm. One piece was arranged in a substantially straight line so that the end surface was the same as the acrylic plate surface, embedded, and sealed with an epoxy resin. The end surface of the silver wire was electrolyzed in a 0.1 M hydrochloric acid aqueous solution at a potential of 0.250 V against a saturated calomel reference electrode for 30 minutes to deposit silver chloride, thereby forming a silver / silver chloride reference electrode (4).

(2) Method for preparing enzyme-immobilized water-absorbing material As a water-absorbing material impregnated with or permeated with water, commercially available filter paper for chemical analysis (5A filter paper, manufactured by Advantech Toyo Co., Ltd., filtration time 60 seconds) was used. The filter paper was cut into 20 mm x 20 mm and 0.5 containing 0.2% by weight of glutaraldehyde.
Weight% glucose oxidase (Type II: Sigma) and 0.5 weight% bovine serum albumin (Fra
(action V: manufactured by Sigma) on one side surface
0 μl was developed and air-dried to form an enzyme-immobilized water absorbent.
The developing method was carried out by fixing the filter paper and spreading the aqueous solution on the end face immediately after spreading with a glass rod.

(3) Measuring method The enzyme-immobilized water-absorbing body (5) was attached so that the electrode surface of the measuring cell was covered and the enzyme-immobilized surface was on the outside, and 1 from the top.
An acrylic holding plate (6) having an opening of 5 mm × 15 mm was attached and screwed. This cell can be obtained by applying a potential of 0.6 V to a silver / silver chloride reference electrode to a platinum working electrode and immersing it in a 0.5 mM glucose solution dissolved in a 100 mM phosphate buffer containing 50 mM potassium chloride. The electrolytic current value was recorded with a recorder for 180 seconds immediately after the immersion. Similarly, the electrolytic current value obtained by immersion in a 0.5 mM hydrogen peroxide solution dissolved in 100 mM phosphate buffer containing 50 mM potassium chloride was recorded by a recorder for 180 seconds immediately after immersion. Similarly, 50
The electrolytic current value obtained by immersion in a 0.5 mM ascorbic acid aqueous solution dissolved in 100 mM phosphate buffer containing mM potassium chloride was recorded by a recorder for 180 seconds immediately after immersion. Similarly, the electrolytic current value obtained by immersing in a 0.5 mM uric acid aqueous solution dissolved in 100 mM phosphate buffer containing 50 mM potassium chloride was 18
It was recorded with a recorder for 0 seconds.

(4) Results When the changing state of the obtained electrolytic current value is corrected to the current value increasing rate, the result is as shown in FIG. The electrolytic current for glucose (indicated by a broken line and reference numeral 2) can be observed 6 seconds after the immersion, and reached a maximum rate of 1.85 nA / second 27 seconds later.
Electrolytic current for hydrogen peroxide (shown by solid line and symbol 1)
Can be observed 3 seconds after immersion, and 7.19nA after 19 seconds
Reached the maximum speed of / sec. The electrolysis current for ascorbic acid (indicated by the dotted line and reference numeral 3) can be observed 15 seconds after the immersion, and reached a maximum rate of 1.2 nA / second after 48 seconds. The electrolytic current for uric acid (indicated by the alternate long and short dash line and symbol 4) can be observed 19 seconds after the immersion and 0.7 seconds after 69 seconds.
The maximum speed of nA / sec was reached. This result shows that the response speed to glucose is faster than that of ascorbic acid or uric acid.

At 15 seconds, the electrolysis current due to glucose is sufficient, there is no effect of uric acid, and there is almost no effect of ascorbic acid. At 25 seconds, the electrolysis current due to glucose is sufficient, and the influence of uric acid and ascorbic acid is extremely small compared to the rate of increase of electrolysis current due to glucose. Therefore, 15
If measured from 2 seconds to 25 seconds, there is no influence of interfering substances or it can be almost ignored. Furthermore, after immersion of the electrolysis current value 15
FIG. 3 shows the calibration curve of the average change rate (second measured data for 15 seconds to 25 seconds is linearly approximated and the gradient is expressed as a speed) and the concentration from second to 25 seconds. Assuming that the response to glucose is 100%, the response to other substances is hydrogen peroxide 454% and ascorbic acid 8.6.
%, And uric acid was 0%.

Comparative Example 1 (1) Method for preparing measurement cell for electrochemical measurement The same cell as in Example 1 was prepared and used.

(2) Method for preparing enzyme-immobilized body The same commercially available filter paper as in Example 1 was used. However, Example 1
After being dipped in the enzyme solution used in 1. and dried in air, the enzyme was uniformly immobilized on the entire water-absorbing body. (3) Measurement method The same measurement as in Example 1 was performed.

(4) Results When the state of change of the obtained electrolytic current value was corrected to the current value increasing rate, the result was as shown in FIG. The electrolysis current for glucose (dashed line, symbol 2) can be observed 13 seconds after immersion, and
A maximum rate of 1.68 nA / sec was reached after 4 seconds. The electrolytic current for hydrogen peroxide can be observed 9 seconds after immersion, and
A maximum rate of 6.85 nA / sec was reached after 7 seconds. Electrolysis current for ascorbic acid (dotted line, code 3) is 1 after immersion
It was observed after 8 seconds and reached the maximum speed of 1.0 nA / sec after 52 seconds. The electrolytic current for uric acid was observed 20 seconds after the immersion, and reached the maximum rate of 0.7 nA / second after 85 seconds. It can be seen from FIG. 4 that there is little time difference between the start of glucose output and the start of ascorbic acid, and the effect of ascorbic acid cannot be eliminated.

Furthermore, 15 seconds to 2 after immersion in the electrolytic current value
The calibration curve of the average rate of change and the concentration during 5 seconds is as shown in FIG. When the response to glucose was set to 100%, the response to other substances was hydrogen peroxide 522%, ascorbic acid 83%, and uric acid 33%. As compared with Comparative Example 1, it is clear that Example 1 has a better output value selection ratio and can accurately measure glucose.

[0039]

Industrial Applicability According to the present invention, it becomes possible to construct an immobilized enzyme body capable of performing accurate measurement while suppressing the influence of a substance which interferes with the measurement.

[Brief description of drawings]

FIG. 1 is an example of a configuration diagram of an electrochemical measurement cell used in Example 1.

FIG. 2 is the glucose obtained in Example 1,
It is an electrolytic current value increase rate curve of a hydrogen peroxide solution, an ascorbic acid, and a uric acid solution. In the figure, a solid line (reference numeral 1) shows a response to hydrogen peroxide, a broken line (reference numeral 2) shows glucose, a dotted line (reference numeral 3) shows ascorbic acid, and a chain line (reference numeral 4) shows a response to uric acid. The vertical axis represents the rate of increase in electrolytic current value (nA / sec),
The horizontal axis represents the elapsed time (seconds) after the electrode was immersed.

FIG. 3 shows glucose obtained in Example 1,
After immersion in hydrogen peroxide solution, ascorbic acid, uric acid solution 1
It is a calibration curve of each substance obtained from the average change speed and concentration of the electrolysis current value from 5 seconds to 25 seconds. In the figure, a solid line (reference numeral 1) shows a response to hydrogen peroxide, a broken line (reference numeral 2) shows glucose, a dotted line (reference numeral 3) shows ascorbic acid, and a chain line (reference numeral 4) shows a response to uric acid. The vertical axis represents the average rate of increase of the electrolysis current value (nA / sec), and the horizontal axis represents the concentration of each substance (mM).
Is shown.

FIG. 4 shows glucose obtained in Comparative Example 1,
It is an electrolytic current value increase rate curve of a hydrogen peroxide solution, an ascorbic acid, and a uric acid solution. In the figure, the solid line shows hydrogen peroxide, the broken line shows glucose, the dotted line shows ascorbic acid, and the one-dot chain line shows the response to uric acid. The vertical axis represents the increasing rate of the electrolysis current value (n
(A / sec), the horizontal axis is the elapsed time (seconds) after dipping the electrode
Is shown.

FIG. 5 shows glucose obtained in Comparative Example 1,
After immersion in hydrogen peroxide solution, ascorbic acid, uric acid solution 1
It is a calibration curve of each substance obtained from the average change speed and concentration of the electrolysis current value from 5 seconds to 25 seconds. In the figure, the solid line shows hydrogen peroxide, the broken line shows glucose, the dotted line shows ascorbic acid, and the one-dot chain line shows the response to uric acid. The vertical axis represents the average rate of change in electrolytic current value (nA / sec), and the horizontal axis represents the concentration (mM) of each substance.

[Explanation of symbols]

 1 Cell bottom 2 Working electrode 3 Counter electrode 4 Silver / silver chloride reference electrode 5 Redox enzyme-immobilized water absorber 6 Acrylic holding plate

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location 7235-2J G01N 27/46 338

Claims (2)

[Claims] 1. An electrochemical measurement electrode having an electrode system including at least a working electrode and a counter electrode, and a water absorbent layer formed by adsorbing or immobilizing an oxidoreductase which catalyzes a reaction for producing hydrogen peroxide. An electrochemical measurement electrode in which the body layer covers the working electrode, and the enzyme activity on the surface side far from the working electrode is greater than the enzyme activity on the surface side near the working electrode of the water absorbing body layer. 2. A method for measuring, wherein a sample containing a substrate of oxidoreductase is supplied from a surface side of a water absorbent layer of the electrochemical measurement electrode according to claim 1 which is remote from the working electrode, and hydrogen peroxide derived from the substrate is detected. After the hydrogen peroxide permeates the water-absorber layer and reaches the working electrode, and the measurement-interfering substance having an electrode activity in the sample permeates the water-absorber layer and reaches the working electrode, the peroxidation occurs. A measurement method that quantifies the substrate based on the output value before the error in the detected hydrogen value is generated.