JP3937283B2 - Concentration measuring electrode and concentration measuring sensor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an electrode for measuring the concentration of superoxide ion (O ₂ ⁻ ), which is active oxygen in a living body, and a sensor for measuring the concentration of superoxide ion using this concentration measuring electrode, and more particularly, in vivo in vivo. (in vivo) can be higher on the concentration measuring electrode and the density measurement sensors capable superoxide ion micronized applied.
[0002]
[Prior art]
Reactive oxygen species have an important role in vivo in connection with synthesis of bioactive substances, bactericidal action, aging development, and the like. This reactive oxygen species is generated in vivo by oxidation of xanthine and hypoxanthine into uric acid by xanthine oxidase (XOD), reduction of oxygen by hemoglobin, and the like.
Measurement of the concentration of superoxide ions, one of the active oxygen species, in vivo is important for identifying various diseases. Measurement of light absorption at 550 nm of cytochrome C (divalent) produced by reduction reaction of cytochrome C (trivalent, Fe ³⁺ ) with superoxide ion (formula (1)) in vitro (in vitro, in vitro) Accordingly, attempts have been made to quantify the superoxide ions and to measure the concentration of SOD using the same. The concentration measurement of SOD utilizes the fact that when SOD is active, the reaction of formula (1) does not proceed and cytochrome C (divalent) is not generated. However, there is a problem that the reaction is slow and the operation is complicated.
cyt c (trivalent) + O ₂ ⁻ → cyt c (divalent) + O ₂ ▲ 1 ▼
[0003]
In addition, NBT (nitroblue tetrazolium) reduction, TNM (tetranitromethane) reduction, adrenaline oxidation, Cypridina luciferin analog (MCLA, Cypridina luciferin analog, 2-methyl-6- [p-methoxyphenol] -3,7, dihydroimidazo -[1,2-a] pyrazine-3-one), or O ₂ to CLA (2-methyl-6-phenyl-3,7, dihydroimidazo- [1,2-a] pyrazine-3-one)) ^- by using the light emission or the like of the adduct concentration measurement of superoxide ions are attempted, but both are spectroscopic techniques in vitro.
[0004]
Attempts have also been made to detect superoxide ions in electrochemical blood. Cooper et al. Modified the surface of gold or platinum with N-acetylcysteine and produced an enzyme electrode on which a protein such as cytochrome C was bound with S-Au, and reduced cytochrome C (trivalent) with superoxide ions. The potential of the enzyme electrode is maintained so that the cytochrome C (divalent) can be oxidized (formula (2)), and the concentration of superoxide ions is measured from the oxidation current obtained thereby. [J. Electroanal. Chem., 347, 267-275 (1993)]. Furthermore, this method can indirectly measure the SOD concentration.
cyt c (divalent) → cyt c (trivalent) + e ▲ 2 ▼
[0005]
[Problems to be solved by the invention]
However, since the reaction rate of the formula (1) is slow (about 10 ⁵ M ^-1 s ^-1), cytochrome C and O ₂ ^- The method using a reaction between is not practical, and the reaction of the formula (1) is not specific to superoxide ions, since in other reduced species in vivo is reduced flows superoxide ions unique current or more current, the current and superoxide ions generated exactly correspond to such Ito There is a problem.
[0006]
In the superoxide ion measurement method using SOD, hydrogen peroxide produced by the reduction reaction (decomposition by SOD) of the superoxide ion to be measured is dissolved in the measurement solution as shown in formula (3) by dissolving SOD in the measurement solution. Keep the electrode potential at a level at which hydrogen oxide can be oxidatively decomposed (0.32 V or more), decompose it by the reaction shown in Formula (4), measure the oxidation current generated at this time, and determine the superoxide ion concentration from the measured value. [CJMcNeil et al., Free Rad. Res. Comms., 17, 399-406 (1992)].
2O ₂ ⁻ + 2H ⁺ → H ₂ O ₂ + O _{2 (} 3)
H ₂ O ₂ → O ₂ + 2H ⁺ + 2e (4)
[0007]
Since the reaction rate of formula (3) is fast (10 ⁷ to 10 ⁵ M ⁻¹ s ^{−1 at} pH 4 to ⁷ ), the correspondence between the current and the concentration is good, but it is indirect by measuring the concentration of hydrogen peroxide. Since it becomes concentration measurement, for example, when hydrogen peroxide is produced in other in vivo sites and stably present in the blood, the concentration increases by the amount of hydrogen peroxide that does not originate from superoxide ions, There is a disadvantage that it becomes an inaccurate concentration measuring method.
The present invention enables electrochemical detection that can be used for superoxide ion concentration detection and superoxide ion decomposition, and high-speed detection and high-precision detection that is not affected by coexisting substances such as hydrogen peroxide. and to provide a superoxide ion concentration measurement electrodes and sensors to.
[0008]
[Means for Solving the Problems]
The present invention relates to a conductive member, a gold base layer provided on the surface of the conductive member, the surface of which is modified into a thin film with an organic compound containing a thiol group, and a superoxide formed in a thin film on the surface of the gold base layer Ri comprising the ion-degrading enzyme (SOD), an electrochemical electrode, characterized in that the density measurement of superoxide ions by measuring the redox of the copper ions in the enzyme Motochu, and the electrode, Super For concentration measurement, measuring the concentration of superoxide ions by immersing in a solution containing oxide ions and measuring the current resulting from the redox reaction of copper ions between superoxide ion-degrading enzyme and superoxide ions It is a sensor. Instead of the conductive member, a base material obtained by modifying a gold surface with an organic compound containing a thiol group into a thin film may be used.
In the present invention, gold may be used as the conductive member instead of forming the gold base layer.
[0009]
The present invention will be described in detail below.
The present invention intends to measure the concentration of superoxide ions by utilizing the specificity of the electrode having SOD for superoxide ions. The electrode and sensor of the present invention can be applied in vivo. Further, in the present invention, the SOD concentration is measured by utilizing the bond due to the interaction between the hydrocarbon compound containing a thiol group and SOD.
A part of the surface of the SOD has a reverse truncated conical hole (the cross section is a downward trapezoidal shape) having an upper edge (entrance) side of 200 to 300 nm and a bottom (back) of about 40 nm. The reaction site of SOD is located at the bottom of this hole, and cationic groups are concentrated on the surface of the hole. Therefore, for example, only anions out of the components in blood can selectively move through the pores, and only small anions such as superoxide ions can reach the reaction site. In particular, it is considered that a disproportionation reaction occurs). The decomposition products oxygen and hydrogen peroxide are released from the reaction site. Therefore, only the current derived from O ₂ ⁻ can be detected to perform accurate concentration measurement.
[0010]
Degradation machinery to oxygen and hydrogen peroxide of superoxide ions by SOD, of the Cu-Zn-based ion contained therein is considered an oxidation-reduction pair of Cu ⁺ -Cu ²⁺ (0.15V) contributes [D. Klung et al., J. Biol. Chem., 247, 605-609 (1972); HJ Forman et al., Arch. Biochem. Biophys., 158, 396-400 (1973)].
In other words, Cu ⁺ of SOD reacts with O ₂ ⁻ to oxidize itself to Cu ²⁺ to reduce O ₂ ⁻ to H ₂ O ₂ (formula (5)). And the oxidized Cu ²⁺ further O ₂ ^- O ₂ as the reaction to itself is reduced to Cu ⁺ and ^{- -} if there O ₂ is oxidized to O ₂ (Equation ▲ 6 ▼). The state of the oxidation-reduction reaction (mediator reaction) by this Cu ⁺ -Cu ²⁺ pair is shown in FIG.
Cu ⁺ + 2H ⁺ + O ₂ ⁻ → Cu ₂ ⁺ + H ₂ O _{2 (} 5)
^{_{Cu 2+ + O 2 - → Cu}} + + O 2 ▲ 6 ▼
[0011]
Moreover, it becomes as follows when it sees for every electrode.
When an electrode having SOD is maintained in a potential range where Cu ²⁺ in the SOD is stable and O ₂ ⁻ is unstable (for example, −0.2 to +0.3 V), O ₂ ⁻ reaching the anode is expressed by the formula (6). Cu ²⁺ becomes oxidized by oxygen in accordance with ▼ becomes Cu ^+. The formed Cu ⁺ gives electrons taken from O ₂ ⁻ to the anode and becomes Cu ²⁺ again. This is shown in FIG.
On the other hand, when the electrode having SOD is maintained in a potential range where Cu ⁺ in the SOD is stable, O ₂ ⁻ is unstable, and hydrogen peroxide is stable (for example, −0.2 to −0.3 V), O ₂ reaching the cathode. ^- is reduced and becomes Cu ⁺ hydrogen peroxide according to equation ▲ 5 ▼ becomes Cu ^2+. The generated Cu ²⁺ replenishes Cu ⁺ again by taking the electron from the cathode in order to compensate for the electrons transferred to O ₂ ⁻ . This situation is shown in FIG.
[0012]
Since the oxidation-reduction current generated at this time is proportional to the total amount of superoxide ions consumed by this oxidation-reduction current, if the relationship between the current flowing in advance and the concentration of superoxide ions is determined, the superoxide ion concentration is determined from the current value. The concentration can be measured.
In the electrode in which the SOD is formed in a thin film shape on the surface of the conductive member of the present invention, Cu ⁺ and Cu ²⁺ contributing to the oxidation-reduction reaction are present in deep portions in the pores of the SOD and react with superoxide ions. Since it has the property of not reacting with hydrogen peroxide, even if hydrogen peroxide is stably present in the living body separately from this redox system, this hydrogen peroxide will contact with the Cu ⁺ and Cu ^2+. The electric current resulting from this does not flow, and it becomes possible to accurately measure the superoxide ion concentration.
[0014]
As the conductive member of the electrode according to the present invention, it is preferable to use carbon, titanium, nickel, iron or an oxide thereof, and particularly in the case of an electrode for SOD measurement, the surface of the conductive member is coated with gold. it exists as a gold underlayer Te.
Gold is formed to be 10 to 100 g / m ² by a thermal decomposition method, a resin fixing method, a vapor deposition method, an electroplating method, an electroless plating method, or the like. In the case of superoxide ion measurement, the gold underlying layer surface formed on the conductive member surface Ru to form a thin layer of an organic compound having a thiol group. The thin layer can be easily formed by immersing a conductive member on which a gold underlayer is formed in water or an organic solvent (for example, methanol or acetone) in which a thiol component is dissolved, and taking out and drying. The thiol component that did not adhere to the gold base layer even after drying can be easily dissolved and removed by immersing it in a solution containing only an organic solvent. Note that gold may be used as the conductive member instead of forming the gold base layer.
The reason for forming such a gold underlayer and a thin layer of an organic compound having a thiol group is that a strong Au—S bond is formed between the gold underlayer and sulfur of the thiol group, and the parent of the organic compound. Ru der to produce a strong interaction also between the organic compound and SOD by oil.
[0015]
The following compound can be illustrated as an organic compound which has the said thiol group. Thiophenol (C ₆ H ₅ —SH), 4-aminothiophenol (p—H ₂ N—C ₆ H ₄ —SH), 4-mercaptopyridine (p—C ₅ H ₄ NSH), bis (4-pyridyl) ) disulfide _{(p-C 5 H 4 N} -S-S-C 5 H 4 N-p), methionine _{[CH 3 -S-CH 2 -CH 2} -CH (NH 2) COOH ], p- thiocresol ( _{p-HS-C 6 H 4} -CH 3), 2- mercaptopyrimidine _{(C 4 N 2 H 3 -SH} ), butanethiol _{(C 3 H 7 -SH),} 2- aminoethanethiol (HS-CH ₂ CH ₂ NH ₂ ), cystine [HOOC—CH (NH) —CH ₂ —S—S—CH ₂ —CH (NH) —COOH], cysteine [HS—CH ₂ —CH (COOH) —NH ₂ ], isocysteine _{[H 2 N-CH 2 -CH (} SH) -COOH ], N- A Chill cysteamine _{(HS-CH 2 -CH 2 -NH} -COCH 3), N- acetylcysteine _{[HS-CH 2 -CH (COOH)} -NH-COCH 3 ], cysteinylglycine [HS-CH ₂ -CH ( _{NH 2) -CO-NH-CH} 2 -COOH ], alpha-homocysteine _{[HS-CH 2 -CH 2 CH (} NH 2) -COOH ], beta-homocysteine _{[HS-CH 2 -CH (NH 2} ) -CH ₂ -COOH], alpha-methyl cysteine _{[HS-CH 2 -C (NH 2} ) (CH 3) -COOH ], 3-mercaptopropionic acid _{(HS-CH 2 -CH 2 -COOH} ), mercaptoacetic acid ( HS-CH ₂ -COOH).
[0016]
The conductive member formed with a thin layer of an organic compound having a thiol group is preferably washed with water, and then a thin layer of SOD is formed. The thin layer is formed, for example, by placing the thiol-modified conductive member in an aqueous solution of SOD or an EDC (water-soluble carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) solution for 1 hour to 1 day. The electrode for concentration measurement according to the present invention is completed by forming the thin layer by immersing it to the extent that it has not been fixed and sufficiently immersing it with a phosphate buffer (pH 7) to remove it.
When this concentration measurement electrode is actually used for concentration measurement, a counter electrode is necessary, and the counter electrode often enters the living body. Therefore, the electrode is made of a highly safe material (for example, platinum, titanium, and carbon). It is preferable.
[0017]
As the reference electrode (reference electrode) for controlling the potential, silver / silver chloride and mercury / mercuric chloride are usually used, but a solid reference electrode can also be used. It is also preferable to apply a pulse of potential for the purpose of improving the detection limit concentration when maintaining the potential within a preferable range. In the case of a microelectrode or a structure composed of a plurality of electrodes, it may be used that a constant diffusion current is observed with respect to the concentration. These contrivances can eliminate the influence of flow velocity.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
Next, an embodiment of a concentration measuring device and a concentration measuring sensor having a concentration measuring electrode according to the present invention will be described based on the attached drawings, but the present invention is not limited to this.
FIG. 4 is a schematic sectional view showing an embodiment of the superoxide ion concentration measuring apparatus of the present invention, and FIG. 5 is an enlarged sectional view of the concentration measuring electrode of FIG.
In FIG. 4, the concentration measuring apparatus 1 includes a sample chamber 2, a sample supply tube 3 to which a sample solution in which superoxide ions are dissolved, and a discharge tube from which the sample solution after concentration measurement is discharged. It consists of four. A concentration measuring electrode 6, a carbon counter electrode 7 and a reference electrode 8 are immersed in the sample solution 5 in the sample chamber 2.
[0019]
As shown in FIG. 5, the concentration measuring electrode 6 is bonded to a conductive member 9 made of carbon or titanium, a gold base layer 10 coated on the conductive member 9, and gold atoms of the gold base layer 10. It consists of an organic compound 11 having a thiol group and a superoxide ion degrading enzyme (SOD) 12 that interacts with the organic compound 11.
When the sample solution 5 in which superoxide ions are dissolved is supplied to the sample chamber 2 of the apparatus 1 having such a configuration through the sample supply tube 3, if the copper in the SOD 12 of the concentration measuring electrode 6 is Cu ⁺ , the above formula is used. (5) oxidizes itself to Cu ²⁺ and reduces superoxide ions to produce hydrogen peroxide. If the copper in the SOD 12 is Cu ²⁺ , the copper itself is reduced to Cu ⁺ by the above formula (6) and superoxide ions are oxidized to generate oxygen gas.
[0020]
When the current flowing at this time is measured using the counter electrode 7 and the reference electrode 8, the concentration of superoxide ions dissolved in the sample solution 5 supplied to the sample chamber 2 can be detected.
Further, the copper ions of SOD12 supported on the concentration measuring electrode 6 are difficult to come into contact with hydrogen peroxide, and even when hydrogen peroxide is present, the oxidation current caused by the hydrogen peroxide hardly flows. Different and accurate concentration measurement.
Unlike the superoxide ion exemplified here, when measuring the concentration of superoxide ion degrading enzyme, an electrode to which SOD12 is not bound is used in FIG.
[0021]
FIG. 6 is a schematic cross-sectional view showing an embodiment of the concentration measuring sensor of the present invention.
The concentration measuring sensor 21 has a hollow shape with a reduced diameter at the lower end of the cylindrical main body. The reduced diameter portion is filled with a working electrode 22, and a conductive wire 23 to the working electrode 22 is formed on the side wall of the sensor 21. It is arranged along. A counter electrode 24 and a reference electrode 25 are disposed above the working electrode 22 so as to be spaced apart from each other, and are led to the outside by lead wires 26 and 27 through a stopper 28 fitted to the base of the sensor body.
The sensor 21 having such a configuration is inserted into a main part of the human body, and the concentration of superoxide ions and / or SOD is measured according to the principle described with reference to FIGS.
[0022]
Next, examples and comparative examples of concentration measurement using the concentration measuring sensor according to the present invention will be described, but these do not limit the present invention.
Example 1
The tip of a gold wire with an electrode area of 0.8 mm ² was used as an electrode, and the side surface was sealed. The electrode was immersed in methanol containing 50 mM cysteine for 1 hour. The gold electrode was taken out of the methanol solution of cysteine and then immersed in an organic solvent containing only methanol to remove cysteine remaining on the surface to obtain a thiol-modified electrode. This thiol-modified electrode is immersed in an EDC solution in which SOD is dissolved for 1 hour, then taken out, immersed in a phosphate buffer solution (pH 7), washed thoroughly to remove SOD remaining on the surface, and used for concentration measurement. An electrode was obtained.
A carbon rod having a diameter of 0.5 mm was installed as a counter electrode, and an SCE (silver / silver chloride electrode) was installed as a reference electrode in close proximity to the concentration measuring electrode, thereby constituting a concentration measuring sensor as shown in FIG.
[0023]
The sensor was filled with a phosphate buffer (pH 7), and the relationship between the obtained current and potential was shown by a curve (a) in the graph of FIG.
From this curve (a), an oxidation reaction response of Cu ⁺ , which is the active site, to Cu ²⁺ is observed near +200 mV, and a reduction reaction response of Cu ²⁺ to Cu ⁺ is observed near 0 mV. I understand that Therefore, for example, by maintaining the electrode potential at 300 mV, the concentration of O ₂ ⁻ can be evaluated by measuring the oxidation current of Cu ⁺ resulting from the reaction between Cu ²⁺ and O ₂ ⁻ according to the formula (6). Similarly, if the electrode potential is maintained at −200 mV, for example, the concentration of O ₂ ⁻ can be evaluated by measuring the Cu ²⁺ reduction current generated by the reaction of Cu ⁺ and O ₂ ⁻ according to the formula (5).
[0024]
Comparative Example 1
A concentration measuring sensor was constructed in the same manner as in Example 1 except that the surface of the thiol-modified electrode was not coated with SOD.
This concentration measurement sensor was filled with a phosphate buffer (pH 7) in the same manner as in Example 1, and the relationship between current and potential was measured. The result is shown by a curve (b) in the graph of FIG.
As can be seen from the curve (b) in FIG. 7, the current peaks at 200 mV and 0 mV as in Example 1 were not observed.
[0025]
Example 2
The same concentration measuring sensor as in Example 1 coated with SOD was used, and the electrode potential was fixed at 0.3 V. A phosphate buffer solution (pH 7) to which xanthine and xanthine oxidase were added was supplied as a sample solution to the concentration measuring sensor at a rate of 6 ml / min. Xanthine is oxidized to uric acid by xanthine oxidase, and O ₂ ⁻ is generated as an intermediate species during the oxidation process.
When the steady-state current value observed at that time is plotted against the concentration of xanthine oxidase (that is, O ₂ ⁻ ) contained in the sample solution, it is as shown in FIG. 8, and a linear relationship was obtained.
[0026]
Comparative Example 2
The gold wire of Example 1 was coated with the same amount of N-acetylcysteine under the same conditions as in Example 1, and cytochrome was supported on the S-Au bond thereon to form an enzyme electrode.
When this electrode was used and the current was observed under the same conditions as in Example 2, the same behavior as in Example 1 was observed. However, when hydrogen peroxide was allowed to coexist, the current increased and the determination of O ₂ ⁻ was impossible.
[0027]
Example 3
The potential of the electrode of Example 1 was fixed at −0.2 V, and a substantially linear relationship was obtained between the steady current value and the xanthine oxidase concentration in the same manner as in Example 2.
[0033]
【The invention's effect】
The present invention provides a gold underlayer provided on the surface of a conductive member, the surface of which is modified into a thin film with an organic compound containing a thiol group, and a superoxide ion-degrading enzyme (SOD) formed in a thin film on the gold underlayer surface. Is a superoxide ion concentration measuring electrode.
The SOD formed on the conductive member of the concentration measuring electrode of the present invention selectively oxidizes only O ₂ ⁻ to generate oxygen gas or reduce it to generate hydrogen peroxide. Is inactive. Thus O ₂ ^- When immersing the electrode in a solution containing, without being influenced by the presence of hydrogen peroxide and oxygen, O ₂ ^- only flows oxidation or reduction current corresponding. By measuring this current value, the exact O ₂ ⁻ concentration in the solution can be detected. In addition, since it is stable in vivo and has a high reaction rate, it can be suitably used in vivo.
[0034]
Also the conductive member surface is made of carbon or titanium, an organic compound containing a thiol group to form a gold underlayer has been modified into a thin film, gold underlayer This conductive member and SOD through the S-Au bond tied firmly Te, O ₂ ^- to improve the accuracy of the measurement.
Similarly, the concentration measurement sensor constructed using this electrode is not affected by the presence or absence of hydrogen peroxide, and an oxidation or reduction current corresponding to only O ₂ ⁻ flows, and this current is measured. An accurate O ₂ ⁻ concentration can be detected.
Moreover, the use of gold underlayer organic compound modified into a thin film containing a thiol group in place of the conductive member in the sensor of the present invention may, precise O ₂ in the same manner in this case ^- can be detected become.
[0035]
Further, the concentration measuring electrode of the present invention comprises a conductive member and a hydrocarbon compound containing a thiol group formed in a thin film on the surface of the conductive member, and is immersed in a solution containing a superoxide ion degrading enzyme. A concentration measuring electrode characterized by measuring a current due to oxidation or reduction of the superoxide ion degrading enzyme to detect the concentration of the superoxide ion degrading enzyme, and a concentration measuring sensor using the electrode.
According to this electrode or sensor, SOD (superoxide ion degrading enzyme) can be easily measured instead of superoxide ions, and analysis costs and analysis time can be reduced. Furthermore, when an electrode system is inserted into the living body, SOD can be detected directly and on-time, and improvement in measurement accuracy can be expected.
[Brief description of the drawings]
FIG. 1 is an explanatory diagram showing an O ₂ ⁻ redox cycle using copper ions in SOD.
FIG. 2 is an explanatory view showing an oxidation state of O ₂ ⁻ on the anode side in FIG. 1;
FIG. 3 is an explanatory view showing a reduction state of O ₂ ⁻ on the cathode side in FIG. 1;
FIG. 4 is a schematic cross-sectional view showing an embodiment of the concentration measuring apparatus of the present invention.
FIG. 5 is an enlarged view of the concentration measurement electrode of FIG. 1;
FIG. 6 is a schematic cross-sectional view showing an embodiment of a concentration measuring sensor according to the present invention.
FIG. 7 is a graph showing the redox response of the concentration measurement electrode of Example 1 and a similar electrode without SOD modification.
[8] graph showing the relationship between the steady-state current value and xanthine oxidase concentration in Example 2.

Claims (6)

A conductive member, a gold underlayer provided on the surface of the conductive member, the surface of which is modified into a thin film with an organic compound containing a thiol group, and a superoxide ion-degrading enzyme formed in a thin film on the surface of the gold underlayer comprise Ri formed, superoxide ion concentration measurement electrodes, characterized in that the density measurement of superoxide ions by measuring the redox of the copper ions in the enzyme Motochu. An electrically conductive member made of gold, an underlayer whose surface is modified with an organic compound containing a thiol group into a thin film, and a superoxide ion-degrading enzyme formed into a thin film on the surface of the underlayer An electrode for measuring superoxide ion concentration, comprising measuring the concentration of superoxide ions by measuring the oxidation-reduction of copper ions in the enzyme. A conductive member, comprising a gold base layer provided on the surface of the conductive member, the surface of which is modified into a thin film with an organic compound containing a thiol group, and is immersed in a solution containing a superoxide ion degrading enzyme. An electrode for concentration measurement, wherein the concentration of the superoxide ion-decomposing enzyme is detected by measuring an electric current due to oxidation or reduction of copper ion in the oxide ion-decomposing enzyme. A gold conductive member, comprising a base layer provided on the surface of the conductive member, the surface of which is modified into a thin film with an organic compound containing a thiol group, immersed in a solution containing a superoxide ion degrading enzyme, An electrode for concentration measurement, characterized in that the concentration of the superoxide ion decomposing enzyme is detected by measuring a current due to oxidation or reduction of copper ions in the superoxide ion decomposing enzyme. A conductive member, and a concentration measuring electrode, a counter electrode, and a reference electrode comprising a superoxide ion-degrading enzyme formed into a thin film on the surface of the conductive member obtained by modifying an organic compound containing a thiol group on the gold surface into a thin film. The superoxide ion concentration is measured by immersing in a solution containing superoxide ions and measuring the current resulting from the oxidation-reduction reaction between copper ions and superoxide ions in the superoxide ion-degrading enzyme. Concentration sensor. Concentration measuring electrode, counter electrode and reference electrode comprising a gold conductive member and a superoxide ion-degrading enzyme formed into a thin film on the surface of the conductive member obtained by modifying an organic compound containing a thiol group on the surface into a thin film Is immersed in a solution containing superoxide ions, the current resulting from the oxidation-reduction reaction between copper ions and superoxide ions in the superoxide ion-degrading enzyme is measured, and the superoxide ion concentration is measured. Sensor for concentration measurement.

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