JP2007003265A - Electrode structure and enzyme sensor including it for measuring phosphoric acid in body fluids - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means capable of simply measuring phosphoric acid in body fluids without requiring optical quantification. <P>SOLUTION: The electrode structure of this enzyme sensor for measuring phosphoric acid in body fluids is equipped with: a hydrogen peroxide electrode; an enzyme immobilizing membrane applied to the hydrogen peroxide electrode and having an enzyme, which catalyzes a reaction consuming phosphoric acid to form hydrogen peroxide, immobilized thereon; and a porous film arranged outside of the enzyme immobilizing membrane to coat the enzyme immobilizing membrane. The enzyme sensor for measuring phosphoric acid in body fluids includes a reference electrode, a means for applying voltage across the hydrogen peroxide electrode and the reference electrode, and a sensor main body for housing the electrode structure and the reference electrode and the electrolyte housed in the sensor main body. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an enzyme sensor for measuring phosphate in body fluid and an electrode structure included therein.

  Phosphoric acid is an essential substance for carbohydrate metabolism in the living body as various phosphate esters and as a high energy phosphate compound for energy metabolism. In addition, it is important as a constituent of nucleic acids, phospholipids, proteins, nucleotides, and the like, and is a kind of constituent element indispensable for a living body. Phosphoric acid in serum varies depending on mechanisms such as absorption from the intestinal tract, mobilization from bone, translocation inside and outside cells, and excretion from the kidney, and is a useful measurement substance for clinical diagnosis. In particular, when renal function is impaired, it is a measurement item that needs to be monitored and managed regularly.

  As a method for measuring phosphoric acid in body fluids, a molybdate is added to a specimen to produce molybdic acid, which is reduced with a reducing agent to form molybdenum blue, and this blue is optically quantified. Method, and phosphorylases such as sucrose phosphorylase and nucleoside phosphorylase that catalyze the reaction that consumes phosphoric acid and pyruvate oxidase are used to carry out the enzymatic reaction that consumes phosphoric acid in the sample, and the reaction product is optical In general, a method collectively called an enzymatic method is used.

  On the other hand, a phosphoric acid sensor that measures phosphoric acid in river water using a method of electrically measuring the hydrogen peroxide production reaction by pyruvate oxidase immobilized on a membrane using a hydrogen peroxide electrode is also known. (Non-patent document 1, Patent document 1).

Hideaki Nakamura et al., Talanta 50 (1999) 799-807 Japanese Patent Publication No. 8-20401

  Conventional methods for measuring phosphoric acid in body fluids are methods for optically quantifying products of reactions involving phosphoric acid. Since optical quantification requires an apparatus including an optical system, the apparatus becomes complicated and expensive, and whole blood is colored, and thus whole blood cannot be used as a specimen.

  The objective of this invention is providing the means for measuring the phosphoric acid in a bodily fluid which does not require optical fixed_quantity | quantitative_assay and can measure the phosphoric acid in a bodily fluid simply.

  If phosphoric acid in a body fluid can be electrically measured, an optical system is not required, and automation of the measurement is easy. However, there is no known method for electrically measuring phosphoric acid in body fluids. On the other hand, as described in Non-Patent Document 1 and Patent Document 1, a method for measuring phosphoric acid in river water by measuring a hydrogen peroxide generation reaction by pyruvate oxidase with a hydrogen peroxide electrode is known. However, the range of phosphate concentrations contained in river water and body fluids such as blood and urine is greatly different, and body fluids contain various interfering substances that affect the measurement using hydrogen peroxide electrodes. Therefore, it is impossible to measure phosphoric acid in body fluids using a known phosphoric acid sensor.

  As a result of earnest research, the present inventor has surprisingly found that phosphate in whole blood can be quantified by arranging a porous membrane outside the enzyme-immobilized membrane on which pyruvate oxidase is immobilized. The headline and the present invention were completed.

  That is, the present invention relates to a hydrogen peroxide electrode, an enzyme-immobilized membrane that covers the hydrogen peroxide electrode and immobilizes an enzyme that catalyzes a reaction that consumes phosphoric acid to generate hydrogen peroxide, and the enzyme Provided is an electrode structure for an enzyme sensor for measuring a phosphoric acid concentration in a body fluid, comprising a porous membrane disposed outside the immobilized membrane and covering the immobilized enzyme membrane. The present invention also provides an electrode structure, a reference electrode, a means for applying a voltage between the hydrogen peroxide electrode and the reference electrode, a sensor body that houses the electrode structure and the reference electrode, Provided is an enzyme sensor for measuring a phosphoric acid concentration in a body fluid, including an electrolyte solution housed in a center body. Furthermore, the present invention provides a measurement of a phosphate concentration in a body fluid, comprising measuring the phosphate concentration in a body fluid separated from a living body by detecting the phosphate concentration as a current change using an enzyme sensor. Provide a method.

  The enzyme sensor of the present invention makes it possible for the first time to electrically measure phosphoric acid in body fluids. According to the enzyme sensor of the present invention, it is possible to measure phosphoric acid in whole blood without performing a pretreatment such as dilution or blood cell separation, and since an optical system is not required, a measuring device can be used. It can be simple and inexpensive, and since the electrical value is measured, the measurement can be easily automated.

  The enzyme sensor for measuring a phosphoric acid concentration in a body fluid of the present invention is characterized by an electrode structure of a hydrogen peroxide electrode. As described above, the electrode structure of the present invention includes a hydrogen peroxide electrode and an enzyme immobilization in which the hydrogen peroxide electrode is coated and an enzyme that catalyzes a reaction that consumes phosphoric acid to generate hydrogen peroxide is immobilized. And a porous membrane disposed outside the enzyme-immobilized membrane and covering the enzyme-immobilized membrane. An example of this structure is schematically shown in FIG.

An electrode structure 10 schematically shown in FIG. 1 is configured using the bottom of a glass tube 12, and a hydrogen peroxide electrode 14 made of platinum, for example, is disposed through the bottom of the glass tube 12. Has been. The tip 14a of the electrode 14 is exposed from the bottom of the glass tube 12, and this part functions as a hydrogen peroxide electrode. The lower part of the hydrogen peroxide electrode 14 is fixed by glass 16 which has been solidified after being melted. An enzyme-immobilized film 18 on which an enzyme that catalyzes a reaction that generates phosphoric acid by consuming phosphoric acid covers the tip portion 14a of the hydrogen peroxide electrode. Further, the porous membrane 20 is disposed outside the enzyme-immobilized membrane 18 and covers the enzyme-immobilized membrane 18. The enzyme-immobilized membrane 18 and the porous membrane 20 are fixed to the bottom of the glass tube 12 by an O-ring 22. FIG. 1 schematically shows a simple example. In short, the hydrogen peroxide electrode only needs to be covered in this order by the enzyme-immobilized membrane and the porous membrane. Moreover, since FIG. 1 is a schematic diagram, the ratio of the size of each member is not accurately described.

  The hydrogen peroxide electrode itself is well known and platinum is often used, but other metals can also be used.

  The enzyme to be immobilized on the enzyme-immobilized membrane is not particularly limited as long as it is an enzyme that catalyzes a reaction that consumes phosphoric acid to generate hydrogen peroxide. Pyruvate oxidase can be mentioned as a preferred example. . The enzyme-immobilized membrane is preferably a porous semipermeable membrane that allows liquid to pass through but does not allow a polymer such as an enzyme to be immobilized to pass through. Such a semipermeable membrane can be composed of a porous polymer membrane. Examples of polymers constituting the polymer membrane include celluloses such as cellulose acetate, vinyl chloride resin, polyvinyl alcohol, Azide- Unit Pendant Water-soluble Photopolymer (AWP), polycarbonate resin, nylon, polypropylene, polyethylene, Teflon (registered trademark) and the like can be mentioned. Methods for immobilizing enzymes on these porous membranes are known per se, and enzymes can be easily immobilized on semipermeable membranes by known methods. The preferred methods are also specifically described in the following examples. The thickness of the enzyme-immobilized membrane is not particularly limited, but is usually about 1 μm to 30 μm.

As a material constituting the porous membrane that is disposed outside the enzyme-immobilized membrane and covers the enzyme-immobilized membrane, a material that can form a porous membrane and has a required mechanical strength is used. It does not specifically limit, A polycarbonate, cellulose mixed ester, polytetrafluoroethylene, glass fiber etc. can be illustrated. The pore size of the porous membrane is preferably smaller than the size of blood cells so that clogging by blood cells does not occur. From the viewpoint of improving measurement reliability and expanding the measurement range, the pore size (diameter) is 0.01 μm to 1 μm are preferable, and 0.1 μm to 0.6 μm are more preferable. “Porosity” may be in the form of a sponge, but from the viewpoint of increasing the reliability and reproducibility of measurement, a porous membrane having a large number of cylindrical through holes is preferable. In this case, the density of the through holes is preferably about 10 ⁷ to 10 ⁹ pieces / cm ² , more preferably about 10 ⁸ to 10 ⁹ pieces / cm ² from the viewpoint of further improving the reliability and reproducibility of the measurement. . The thickness of the porous film is not particularly limited, but is usually about 3 μm to 50 μm, preferably about 5 μm to 15 μm. A film in which a cylindrical through hole having a pore diameter in the above-mentioned range is formed in the above density on a polycarbonate film is commercially available from Sterlitech, Inc. under the trade name “Nuclepore” (registered trademark). As the porous membrane in, one to five of this commercially available Nuclepore (registered trademark) can be laminated and used.

  The reason why phosphoric acid in body fluids such as whole blood can be measured with a hydrogen peroxide electrode by covering the enzyme-immobilized membrane with a porous membrane is not necessarily clear, but the substrate solution is retained by the porous membrane. At the same time, the diffusion of phosphate in the sample is limited, the enzyme does not come into contact with a reduced amount of phosphate, and the porous membrane prevents contact of interfering substances such as blood cells with the enzyme membrane. It is speculated that even phosphoric acid in whole blood can be measured by a hydrogen peroxide electrode as a result of being restricted or diffusion thereof.

  The enzyme sensor for measuring phosphoric acid in a body fluid according to the present invention is characterized in that it comprises the above-described hydrogen peroxide electrode structure, and the other configuration may be the same as a known phosphoric acid sensor. That is, the enzyme sensor for measuring phosphoric acid of the present invention comprises the electrode structure of the present invention, a reference electrode, means for applying a voltage between the hydrogen peroxide electrode and the reference electrode, the electrode structure, A sensor main body for storing the reference electrode; and an electrolyte solution stored in the center main body. An example of this structure is schematically shown in FIG.

In FIG. 2, 10 is the above-described electrode structure of the present invention, and 24 is a reference electrode. The electrode structure 10 and the reference electrode 24 are accommodated in the sensor body 26. An electrolyte solution 28 is accommodated in the sensor body 26 (the liquid level is indicated by a broken line), and the hydrogen peroxide electrode 14 and the reference electrode 24 are immersed in the electrolyte solution 28. Further, a positive voltage is applied to the hydrogen peroxide electrode 14 with respect to the reference electrode 24 by the battery 30. As the reference electrode, an Ag / AgCl electrode is often used. Further, as the electrolyte solution, a buffer solution containing sodium chloride or the like can be used. In addition, when a substrate necessary for an enzyme reaction and the enzyme require supply of a coenzyme, the coenzyme can be added to the electrolyte solution. For example, when the enzyme to be immobilized is pyruvate oxidase, pyruvate as a substrate and FAD and thiamine pyrophosphate (thiamine diphosphate, TPP) as coenzymes are required, but these are added to the electrolyte solution. I can keep it.

In use, the porous membrane of the electrode structure 10 is brought into contact with the specimen. When the enzyme sensor is incorporated in the automation device, the specimen flows in the direction indicated by the arrow in FIG. A part of the specimen reaches the enzyme-immobilized membrane through the porous membrane of the electrode structure. On the other hand, the substrate and necessary coenzyme contained in the electrolyte solution also reach the enzyme-immobilized membrane, and an enzyme reaction occurs here due to the catalytic action of the enzyme. The enzymatic reaction consumes phosphoric acid in the sample and produces hydrogen peroxide. For example, when the enzyme is pyruvate oxidase, the following reaction occurs.
Pyruvate + H ₂ O + O ₂ + Phosphate → Acetyl phosphate + CO ₂ + H ₂ O ₂
In this way, phosphoric acid in the specimen is consumed, and equimolar hydrogen peroxide is generated. Accordingly, the amount of hydrogen peroxide produced at this time varies depending on the phosphoric acid concentration in the specimen, and the amount of hydrogen peroxide produced increases as the phosphoric acid concentration in the specimen increases. Therefore, by quantifying the generated hydrogen peroxide using a hydrogen peroxide electrode, the phosphoric acid concentration in the specimen can be measured as an electrical value. The generated hydrogen peroxide is decomposed on the hydrogen peroxide electrode as follows, and an electric current is generated at this time.
^{_{H 2 O 2 → 2H + +}} O 2 + 2e -
Hydrogen peroxide can be quantified by measuring this current flowing through the hydrogen peroxide electrode. This is because, for example, the potential at which hydrogen peroxide is oxidized is applied to the hydrogen peroxide electrode, which is the working electrode, with respect to the potential of the reference electrode, and the amount of current corresponding to the hydrogen peroxide concentration is This can be done by measuring with an ammeter connected to the electrode and the reference electrode. If a calibration curve is created using a plurality of standard samples containing phosphoric acid at a known concentration in advance, the phosphoric acid concentration in the unknown sample can be known from the measured current value.

  The body fluid used for the phosphoric acid measurement using the phosphoric acid measurement enzyme sensor of the present invention is not particularly limited, and examples thereof include whole blood, serum, plasma, and urine. Among these, it is a great advantage of the present invention that whole blood and urine can be subjected to measurement without any pretreatment such as dilution or blood cell separation.

  The enzyme substrate and coenzyme can be included in the electrolyte solution as described above, but a solution containing the substrate and coenzyme can also be separately introduced into the enzyme-immobilized membrane. FIG. 3 schematically shows an example of a measurement system equipped with an enzyme sensor having a supply path for supplying a liquid containing a substrate and a coenzyme. In FIG. 3, the sampling nozzle is moved to a flow path position connected to the reagent tank 40 holding the liquid containing the substrate and the coenzyme, and the liquid containing the substrate and the coenzyme is transferred to the phosphate sensor by the peristaltic pump (liquid feed pump) 38. Introduce to the department. Next, the sampling nozzle is moved to a position where the measurement sample can be sucked, and the measurement sample is sucked by the peristaltic pump 38 and introduced into the phosphate sensor unit 32. In FIG. 3, 34 is a three-way valve, 40 is a substrate solution (including a coenzyme), 42 is a specimen, 44 is a current-voltage converter, 46 is an A / D converter, 48 is a computer, 50 is a printer, 54 Is a waste tank.

  It is preferable to supply a sufficient amount of the substrate and necessary coenzyme. In particular, when pyruvate oxidase is used as an enzyme, pyruvate as its substrate is also contained in body fluids such as blood, so that the measured value does not substantially change depending on the concentration of pyruvate in the body fluid. It is preferable to supply a sufficiently large amount of pyruvic acid compared to the amount of pyruvic acid in the body fluid. When pyruvate oxidase is used as an enzyme and the substrate solution is separately supplied via a supply channel as shown in FIG. 3, the pyruvate concentration in the substrate solution is preferably about 1 to 10 mmol / L, and the TPP concentration is preferably About 0.1 to 2 mmol / L, and the FAD concentration is preferably about 0.1 to 100 μmol / L.

  The body fluid contains various interfering substances such as ascorbic acid and uric acid that affect the measurement with the hydrogen peroxide electrode. In the enzyme sensor of the present invention described above, it is possible to measure the phosphoric acid concentration in the body fluid even when these interfering substances coexist, but the present invention further eliminates the influence of the interfering substances. An enzyme sensor capable of obtaining an accurate measurement value (hereinafter referred to as “second enzyme sensor” for convenience) is also provided. The second enzyme sensor includes a second hydrogen peroxide electrode structure in addition to the constituent elements of the enzyme sensor of the present invention described above (hereinafter sometimes referred to as “first enzyme sensor” for convenience). To do. The second electrode structure is obtained by removing the immobilized enzyme membrane from the above-described electrode structure of the present invention (hereinafter sometimes referred to as “first electrode structure” for convenience). Similarly to the first electrode structure, this second electrode structure is also applied with a voltage between the reference electrode and immersed in the above-described electrolyte solution. The reference electrode of the second electrode structure can be shared with the reference electrode of the first electrode structure, or may be provided separately. The second electrode structure is also accommodated in the sensor body described above, and the porous film is disposed adjacent to the porous film of the first electrode structure and simultaneously contacts the specimen.

  At the time of use, as in the case of the first enzyme sensor, the first and second electrode structures are brought into contact with the specimen, the value of the current flowing through the hydrogen peroxide electrode of each electrode structure is measured, and the difference between them is taken. . A calibration curve is created using the difference between the current values as a true current value, and the phosphate concentration in the unknown sample is measured.

  In the second enzyme sensor, since the second electrode structure does not have an immobilized enzyme film, an enzyme reaction that consumes phosphoric acid in the sample to generate hydrogen peroxide does not occur. Therefore, ideally, no current should flow through the hydrogen peroxide electrode of the second electrode structure, but if it does, it is due to interfering substances other than phosphoric acid, such as ascorbic acid or uric acid. Is. In the second enzyme sensor, the current value caused by the interfering substance can be separately measured as described above, and the true current value caused by the phosphoric acid in the sample can be measured by subtracting the current value. This eliminates the influence of interfering substances and enables more accurate measurement.

  The second electrode structure may be accommodated in the same sensor body of the enzyme sensor as the enzyme sensor including the first electrode structure, but an enzyme sensor including the second electrode structure alone is separately provided. It may be configured so that the current value resulting from the interfering substance is measured. That is, the present invention also provides an enzyme sensor for measuring a phosphate concentration in a body fluid, comprising the enzyme sensor of the present invention and an enzyme-free sensor having a structure obtained by removing the enzyme-fixed membrane from the enzyme sensor of the present invention. To do.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Production of Electrode Structure A hydrogen peroxide electrode structure having the structure shown in FIG. 1 was produced as follows. That is, the bottom of the glass tube was heated and melted with a burner, and a platinum rod having a diameter of 0.3 mm was penetrated. Upon cooling to room temperature, a basic structure was obtained in which the tip of the platinum rod was exposed from the bottom of the glass tube, and the platinum rod was fixed with molten and solidified glass (16 in FIG. 1). Next, an enzyme-immobilized membrane (manufacturing method will be described later) and a porous polycarbonate membrane (“Nuclepore” (registered trademark) manufactured by Sterlitech, USA, pore diameter 0.2 μm, pore density 3 × 10 ⁸ / cm ² , thickness 10 μm) The stacked ones were fixed to the side portion of the glass tube with an O-ring so that the enzyme-immobilized membrane was on the side in contact with the platinum electrode, thereby obtaining the electrode structure of the present invention.

  The enzyme-immobilized membrane used was prepared as follows. First, a 2% cellulose acetate aqueous solution was prepared. 500 μL of the solution was taken and spread over a 7 × 10 cm area on the substrate. After the development, the substrate was immersed in a hexane tank within 10 seconds to obtain a cellulose acetate film. 30 μL of 160 U / 150 μL MES buffer + 2% GA (glutaraldehyde) of biruvate oxidase was spread on the cellulose acetate membrane (5 cm × 8 cm) and allowed to stand at 4 ° C. for 1 hour to obtain an enzyme-immobilized membrane. The obtained enzyme-immobilized membrane was washed repeatedly with MES buffer, and then loaded with the above Nuclepore (registered trademark) membrane and dried overnight at 4 ° C. Provided.

2. Production of enzyme sensor Using the electrode structure produced in 1, an enzyme sensor having the structure shown in FIG. 2 was produced. Note that an Ag / AgCl electrode was used as the reference electrode, and +0.6 V was applied to the hydrogen peroxide electrode with respect to the reference electrode. As the electrolyte solution, NaCl and 0.2630g / 100mL, the Na ₂ HPO ₄ to prepare an aqueous solution containing 0.4216g / 100mL, the NaH ₂ PO ₄ at a concentration of 0.1836g / 100mL, which was approximately 0.3mL filled in the sensor main body Then, the platinum electrode and the reference electrode were immersed in the electrolyte solution.

Measurement of phosphoric acid Measuring device The enzyme sensor produced in Example 1 was incorporated into an automated device. The automation apparatus has the configuration shown in FIG. In FIG. 4, the number of enzyme sensors is 32. 34 is a three-way valve, 36 is a metering syringe, 38 is a liquid feed pump, 40 is a substrate solution (including coenzyme), 42 is a specimen, 44 is a current-voltage converter, 46 is an A / D converter, 48 is a computer, 50 is a printer, 52 is a waste liquid pump, and 54 is a waste liquid tank.

At the time of measurement, the specimen 20 was aspirated by the liquid feed pump 38 and the nozzle connected thereto, and transferred to the enzyme sensor 32. In addition, the substrate solution 40 was aspirated with the quantitative syringe 36. While measuring the signal output of the enzyme sensor, the substrate solution was introduced and mixed in the vicinity of the enzyme-immobilized membrane of the enzyme sensor. The concentration of the hydrogen peroxide solution generated here was automatically measured based on the value of the current flowing through the hydrogen peroxide electrode. The substrate solution was a MOPS buffer solution (0.2 M, pH 7.0) containing 1 mmol / L pyruvic acid, 10 μM FAD, and 200 μM TPP.

2. Calibration curve Fig. 5 shows calibration curves prepared using various concentrations of phosphoric acid aqueous solutions (standard samples) as specimens. FIG. 5 shows that the current value increases as the phosphoric acid concentration increases, and the phosphoric acid concentration in the specimen can be measured by the enzyme sensor of the present invention. Here, the rhombus mark represents a calibration curve when the substrate solution is introduced into the enzyme sensor part in the future with the sample in advance, and the circle mark and the triangle mark are respectively measured by introducing the sample after introducing the substrate solution 1 The second calibration curve and the second calibration curve are shown.

3. Measurement using actual specimen Table 1 shows the results obtained when measurement was performed using serum and whole blood as specimen specimens. The results shown in Table 1 are the results of simultaneous reproducibility for these specimens. Whole blood and serum samples 1 and 2 are samples taken from different donors. In Table 1, n is the number of measurements, SD is the standard deviation, and CV% is the coefficient of variation.

  From these results, it can be seen that according to the enzyme sensor of the present invention, the phosphoric acid concentration in the body fluid can be accurately measured.

It is a figure which shows typically an example of the electrode structure of this invention. It is a figure which shows typically an example of the enzyme sensor of this invention. It is a figure which shows typically an example of the measurement system which comprises the supply path of a substrate supply liquid. It is a block chart figure which shows the structure of the automatic measuring apparatus incorporating the enzyme sensor of this invention used for the measurement in an Example. In the Example of this invention, it is a figure which shows the calibration curve which measured the phosphoric acid concentration in a standard sample.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Electrode structure 12 Glass tube 14 Hydrogen peroxide electrode 16 Solidified glass 18 Enzyme fixed membrane 20 Porous membrane 22 O-ring 24 Reference electrode 26 Sensor main body 28 Electrolyte solution 30 Battery 32 Enzyme sensor 34 Three-way valve 36 Metering syringe 38 Liquid feeding Pump 40 Reagent liquid 42 Test sample 44 Current-voltage converter 46 A / D converter 48 Computer 50 Printer 52 Waste liquid pump 54 Waste liquid tank

Claims (14)

  A hydrogen peroxide electrode, an enzyme-immobilized membrane that coats the hydrogen peroxide electrode and immobilizes an enzyme that catalyzes a reaction that consumes phosphoric acid to generate hydrogen peroxide, and an outer side of the enzyme-immobilized membrane. An electrode structure of an enzyme sensor for measuring a phosphoric acid concentration in a body fluid, comprising a porous membrane disposed and covering the enzyme-immobilized membrane.   2. The electrode structure according to claim 1, wherein the enzyme is a pyruvate oxidase enzyme.   The electrode structure according to claim 1 or 2, wherein the pore diameter of the porous membrane is a size that blood cells cannot pass through.   The electrode structure according to claim 3, wherein the pore diameter of the porous membrane is 0.01 μm to 1 μm.   The electrode structure according to claim 4, wherein the porous membrane has a pore diameter of 0.1 μm to 0.6 μm.   6. The electrode structure according to claim 4, wherein the porous film is a flat film having a cylindrical through-hole opened. The electrode structure according to claim 6, wherein the density of the through holes is 10 ⁷ to 10 ⁹ holes / cm ² . The electrode structure according to claim 7, wherein the density of the through holes is 10 ⁸ to 10 ⁹ holes / cm ² .   The electrode structure according to any one of claims 1 to 7, a reference electrode, means for applying a voltage between the hydrogen peroxide electrode and the reference electrode, and the electrode structure and the reference electrode. An enzyme sensor for measuring a phosphoric acid concentration in a body fluid, comprising: a sensor body to be housed; and an electrolyte solution housed in the center body.   The enzyme sensor according to claim 9, wherein the internal solution contains the coenzyme when the enzyme substrate and the enzyme require supply of the coenzyme.   A second electrode structure having a structure in which the enzyme-immobilized membrane is removed from the electrode structure according to any one of claims 1 to 8, further including in the sensor body, the second electrode structure The enzyme sensor according to claim 9 or 10, further comprising means for applying a voltage between the second hydrogen peroxide electrode contained in the electrode and the reference electrode or the second reference electrode.   An enzyme sensor for measuring a phosphoric acid concentration in a body fluid, comprising the enzyme sensor according to claim 9 or 10, and an enzyme-free sensor having a structure obtained by removing the enzyme-immobilized membrane from the enzyme sensor according to claim 9 or 10.   The enzyme sensor according to any one of claims 9 to 12, wherein the body fluid is whole blood or urine. A bodily fluid comprising measuring the phosphoric acid concentration by detecting the phosphoric acid concentration in a bodily fluid separated from a living body as a current change using the enzyme sensor according to any one of claims 9 to 13. Of measuring the concentration of phosphoric acid in water.

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