WO2010095630A1

WO2010095630A1 - Electrochemical quantification method for hydrogen peroxide

Info

Publication number: WO2010095630A1
Application number: PCT/JP2010/052313
Authority: WO
Inventors: 正嗣盛満
Original assignee: 学校法人同志社
Priority date: 2009-02-18
Filing date: 2010-02-17
Publication date: 2010-08-26
Also published as: JP4859003B2; JPWO2010095630A1

Abstract

Disclosed is an electrochemical quantification method for hydrogen peroxide, which does not generate oxygen in an electrochemical reaction of hydrogen peroxide on a sensing electrode, and does not induce any reaction by dissolved oxygen contained in a test sample solution, and in which the sensing electrode is chemically stable against the reaction of hydrogen peroxide and the material of the electrode itself does not undergo oxidation, the influence by an interfering component that is contained in the test sample solution and interferes with the electrochemical reaction of hydrogen peroxide directly or interferes with the current measurement that relies only on the electrochemical reaction of hydrogen peroxide is suppressed, the sensitivity can be maintained at a high level steadily for a long period, and any complicated calibration procedure for a sensor is not required. Specifically disclosed is an electrochemical quantification method for hydrogen peroxide, which is characterized by comprising the steps of: oxidizing a target substance (which has been delivered to a sensor) through an enzymatic reaction to generate hydrogen peroxide; and measuring a current generated in an electrochemical reaction of hydrogen peroxide, wherein the current to be measured is a current generated upon the reduction of hydrogen peroxide on a sensing electrode having, formed therein, a catalyst layer comprising amorphous iridium oxide.

Description

Electrochemical determination of hydrogen peroxide

The present invention relates to biological fluids such as urine, saliva, blood, food production fluids, decomposition fluids, extracts, cooked products, cooking products and their extracts, and target substances contained in pharmaceuticals by enzymatic reaction. It relates to an electrochemical quantification method for determining the concentration of hydrogen peroxide generated when oxidized, and the concentration of the target substance is determined by measuring the current flowing in the electrochemical reaction of hydrogen peroxide generated by the enzymatic reaction of the target substance. The present invention relates to an electrochemical determination method of hydrogen peroxide which can be used as a determination method.

As a technique for quantifying a specific target substance, an enzyme reaction of the target substance and an electrochemical reaction of hydrogen peroxide generated by the enzyme reaction are continuously generated, and the concentration of the target substance is determined from the current flowing in the electrochemical reaction. The method is widely used. For example, when the target substance is glucose, glucose contained in a biological fluid is oxidized by glucose oxidase to generate gluconic acid and hydrogen peroxide, and this hydrogen peroxide is electrochemically detected at a detection electrode made of platinum or the like. There is known a method for quantitatively determining glucose from an electric current when it is oxidized. In such a method, the principles of chronoamperometry and polarography are often used for current measurement, and methods and apparatuses based on this principle are disclosed in, for example, Patent Documents 1 to 6. In addition, recently, sensors and devices that measure the glucose concentration in human urine have been developed by applying such principles, and some of them are small and portable and are arranged in conjunction with Western-style toilets. For example, it is disclosed in Patent Documents 7 to 9.

On the other hand, in addition to glucose, there are target substances that generate hydrogen peroxide by enzymatic reaction. For example, when the target substance is cholesterol, free cholesterol is oxidized with cholesterol oxidase to produce hydrogen peroxide, which is then converted into 4-aminoantipyrine and N-ethyl-N- ( A reddish purple quinone dye is produced by oxidative condensation with 3-methylphenyl) -N-acetylethylenediamine, and cholesterol is quantified from the absorbance of visible light by the quinone dye. Similarly, uric acid and the like are target substances that are quantified using the generation of hydrogen peroxide by an enzymatic reaction and the absorbance of light absorption accompanying the chemical reaction. In addition to these, target substances that generate hydrogen peroxide by enzymatic reaction include, for example, glutamic acid, L-amino acid, D-amino acid, alcohol, bilirubin, amine, choline, xanthine, pyruvic acid, lactic acid, and the like.

Japanese Patent Laid-Open No. 1-153952 JP 2005-17183 A JP 2007-33459 A JP 2007-163224 A Special table 2007-500136 gazette Special table 2007-518984 gazette JP 2004-233294 A JP 2006-234458 A Japanese Patent No. 3687789 Japanese Patent Laid-Open No. 2004-233302 JP 2007-248342 A

As described above, when the target substance that generates hydrogen peroxide by enzymatic reaction is quantified, the quantification of hydrogen peroxide is performed by electrochemical analysis that measures the current of the electrochemical reaction or by reacting hydrogen peroxide with quinone. Spectral analysis is mainly used in which a substance that absorbs light, such as a dye, is generated and its absorbance is measured. Here, glucose contained in blood or urine is a typical target substance that uses electrochemical analysis, but target substances that can be used for electrochemical analysis are relatively limited. One of the reasons is that when oxidizing hydrogen peroxide at the sensing electrode, other coexisting substances also react, and other coexisting substances interfere with hydrogen peroxide oxidation. . However, if the spectroscopic analysis as described above is used for the determination of hydrogen peroxide, a substance that reacts with hydrogen peroxide is required as compared with the electrochemical analysis. Has a problem that a large-scale spectroscopic analyzer capable of measuring absorbance is required. In other words, using electrochemical analysis as described above requires a shorter measurement time and does not require a substance that reacts with hydrogen peroxide or a large spectroscopic analyzer. Despite the desirability of using electrochemical analysis, the target substance that generates hydrogen peroxide by enzymatic reaction due to the selectivity of other substances that coexist with hydrogen peroxide in electrochemical analysis. However, there is still a problem that many target substances are difficult to quantify by electrochemical analysis.

Furthermore, there is a problem to be solved in the electrochemical determination method of hydrogen peroxide for a target substance that has already been used for electrochemical analysis such as glucose. The problem will be described below by taking glucose as an example of the target substance.

First, the conventional electrochemical quantification method of hydrogen peroxide oxidizes glucose in a test solution to be quantified with glucose oxidase, and oxidizes the hydrogen peroxide generated at that time electrochemically. Thus, the concentration of glucose is determined from the flowing current. The sensor configuration that enables such quantification includes an enzyme membrane containing glucose oxidase, a permselective membrane for the purpose of permeating only hydrogen peroxide generated by glucose oxidation toward the detection electrode, and peroxidation. A detection electrode that electrochemically oxidizes hydrogen is common, and there are many examples in which a permselective membrane and an enzyme membrane are laminated in this order on the detection electrode. At this time, when hydrogen peroxide is electrochemically oxidized at the detection electrode, oxygen and protons (H ⁺ ) are generated as products. In other words, when oxygen gas is generated on the detection electrode and the surface of the detection electrode is covered with oxygen, the reactivity of the detection electrode to hydrogen peroxide decreases, or an enzyme membrane or a selectively permeable membrane formed on the detection electrode is removed. There has been a problem that the efficiency of the enzymatic reaction is reduced by expansion and the function of the permselective membrane to selectively permeate only hydrogen peroxide is lowered.

Secondly, in the conventional electrochemical determination method of hydrogen peroxide, noble metals such as platinum and other metals are mainly used as the sensing electrode material of the sensor described above. The electrochemical oxidation potential also causes the sensing electrode material itself to oxidize, which overlaps the oxidation current of hydrogen peroxide and interferes with accurate measurement of the current only for hydrogen peroxide oxidation. As a result of the oxidation of the sensing electrode material itself, the problem is that the catalytic properties for the oxidation of hydrogen peroxide are reduced and the sensitivity is lowered, and it is impossible to maintain a constant sensitivity for a long time or a long period of use. There was a problem.

Third, it is a case where a component other than glucose, which is the target substance, is contained in the test solution, and that component is electrochemically oxidized at the detection electrode in the same potential range as hydrogen peroxide. In some cases, there is a problem that due to the influence, the current for only the oxidation of hydrogen peroxide cannot be measured accurately. Examples of substances that interfere with the measurement of the oxidation current of only hydrogen peroxide include ascorbic acid and uric acid as examples when the target substance is glucose and the test solution is human urine.

Fourth, even when the test solution itself does not contain a substance that interferes with the measurement of the oxidation current of hydrogen peroxide alone, it is added to the carrier solution for transporting the test solution to the sensor, for example. The preservative component reacts at the sensing electrode, preventing accurate measurement of the oxidation current of hydrogen peroxide alone. On the other hand, in Patent Document 8, when a detection electrode made of iridium oxide or an oxide containing iridium oxide is used, a preservative component is obtained when electrochemically oxidizing hydrogen peroxide generated by the enzymatic reaction of glucose. A concentration measuring device that can suppress the interference of hydrogen peroxide by inhibiting the reaction of hydrogen peroxide is disclosed, but even if such a sensing electrode is used, the product is obtained by electrochemically oxidizing hydrogen peroxide. Since oxygen is generated as described above, there is a problem that it is difficult to solve the decrease in sensitivity due to oxygen covering the detection electrode as described above.

Fifth, Patent Document 8 also discloses a concentration measuring device using a sensing electrode made of amorphous iridium dioxide or an oxide containing amorphous iridium dioxide. When used for the detection electrode, it is shown that the sensitivity to glucose is higher than when platinum is used. However, the sensing electrode made of amorphous iridium dioxide or an oxide containing the same is electrochemically flowing with respect to a carrier solution containing hydrogen peroxide of the same concentration as when crystalline iridium dioxide is used. However, the increase in this current is due to the electric double layer charging current at the sensing electrode interface that occurs simultaneously with the electrochemical oxidation of hydrogen peroxide. With respect to, the increase in the current flowing at the sensing electrode when the concentration of hydrogen peroxide is increased is smaller when amorphous iridium dioxide is used at the sensing electrode than with crystalline iridium dioxide. In a method in which hydrogen peroxide is electrochemically oxidized using a sensing electrode made of iridium dioxide or an oxide containing the same, even if the concentration of hydrogen peroxide increases, peroxidation occurs. Small increase in current only for the oxidation of hydrogen, has a problem that high sensitivity can not be obtained. In addition, even when a sensing electrode made of amorphous iridium dioxide or an oxide containing the same is used, the current due to the oxidation of hydrogen peroxide is measured, so that the decrease in sensitivity due to oxygen covering the sensing electrode is solved. There was a problem that it was difficult.

Sixth, in the conventional method for electrochemical determination of hydrogen peroxide, as described above, the generation of oxygen by the electrochemical oxidation of hydrogen peroxide, the chemical change of the sensing electrode material, the peroxidation at the sensing electrode Due to the electrochemical reaction at the sensing electrode material itself or the sensing electrode, such as the presence of a component that interferes with the electrochemical oxidation of hydrogen, The selective permeation membrane allows only hydrogen peroxide to pass, and the current flowing at the sensing electrode does not depend only on the oxidation of hydrogen peroxide, or the amount of change in the current with respect to the concentration of hydrogen peroxide is small. In other words, the problem of low sensitivity to hydrogen peroxide arises, which makes it necessary to replace the sensor in a short period of time, or use a glucose or hydrogen peroxide standard solution with a precisely regulated concentration. The task of calibrating the current flowing through the sensing electrode there is a problem frequently needed.

In response to the above problems, the present invention does not generate oxygen by an electrochemical reaction of hydrogen peroxide at the detection electrode, and also causes a reaction due to dissolved oxygen contained in the test solution or carrier solution. The sensing electrode is chemically stable against the reaction of hydrogen peroxide, the sensing electrode material itself is not oxidized or reduced, and is contained in the test solution or carrier solution. The effects of interfering components that directly interfere with the electrochemical reaction or interfere with the measurement of currents that depend solely on the hydrogen peroxide electrochemical reaction can be suppressed, and high sensitivity can be maintained stably over the long term. It aims to provide an electrochemical method for the determination of hydrogen peroxide, which does not require frequent sensor calibration.

As a result of various studies to solve the above problems, the present inventor has solved the above problems by reducing hydrogen peroxide using a detection electrode on which a catalyst layer containing amorphous iridium oxide is formed. As a result, they have reached the present invention.

That is, the first invention of the present invention includes a step of oxidizing a target substance conveyed to a sensor by an enzymatic reaction to generate hydrogen peroxide, and a step of measuring a current generated by an electrochemical reaction of the hydrogen peroxide. A method for electrochemical determination of hydrogen peroxide, comprising measuring a current at which hydrogen peroxide is reduced at a sensing electrode on which a catalyst layer containing amorphous iridium oxide is formed. Electrochemical quantification method. Here, the target substance is a substance that generates hydrogen peroxide when oxidized by an enzymatic reaction. For example, glucose, cholesterol, uric acid, glutamic acid, L-amino acid, D-amino acid, alcohol, bilirubin, amine, choline, xanthine, Examples thereof include pyruvic acid and lactic acid, but are not limited thereto.

In the sensing electrode formed with a catalyst layer containing amorphous iridium oxide, it is not seen when noble metals such as platinum, gold, palladium, iridium and other metals or crystalline iridium oxide are used for the sensing electrode. High catalytic properties for reduction of hydrogen peroxide are developed. Therefore, hydrogen peroxide can be detected with high sensitivity. In addition, in the conventional electrochemical determination method of hydrogen peroxide, an electric current when oxidizing hydrogen peroxide is detected. In this case, 1 mol of H ₂ O ₂ is oxidized for 1 mol of H ₂ O ₂ . This produces O ₂ , 2 moles of H ⁺ and 2 moles of electrons. That is, oxygen gas is generated on the surface of the detection electrode, which causes the enzyme membrane and the selectively permeable membrane formed on the detection electrode to expand, and when the surface of the detection electrode is covered with O ₂ , In the present invention, there is a problem that the reactivity is lowered, the efficiency of the enzyme reaction is reduced due to the expansion of the enzyme membrane or the selectively permeable membrane, and the function of the selectively permeable membrane to selectively permeate only hydrogen peroxide is reduced. In order to measure the current generated by the reduction of hydrogen peroxide, it has the effect that oxygen is not generated by the electrochemical reaction of hydrogen peroxide.

In addition, when a catalyst layer containing amorphous iridium oxide is used as the sensing electrode, amorphous iridium oxide itself does not oxidize or reduce in a potential range where hydrogen peroxide reduction occurs, and it is extremely chemically stable. Therefore, there is no reduction of the metal's own oxide as in the case where a metal such as platinum, which is well-known as a sensing electrode material, is used for the sensing electrode, and it contains amorphous iridium oxide. Since the catalyst layer has a very low catalytic property for the reduction of oxygen, unlike the case where a metal such as platinum is used for the detection electrode, the catalyst layer has an effect that the oxygen dissolved in the test solution or the carrier solution is not reduced. That is, when a metal such as platinum is used for the sensing electrode, the current when the metal's own oxide is reduced and the current when the dissolved oxygen is reduced are in the same potential range as the current when hydrogen peroxide is reduced. Therefore, only the current due to the reduction of hydrogen peroxide cannot be separated and measured. For this reason, in the conventional electrochemical determination method of hydrogen peroxide, when the target substance is quantified from the current value generated by the electrochemical reaction of hydrogen peroxide generated by the enzymatic reaction of the target substance, the peroxide is It is not preferable to use the reduction current of hydrogen, and the hydrogen peroxide reduction reaction is not used in sensors and devices that are put into practical use. However, according to the present invention, in the potential region where the reduction of hydrogen peroxide occurs, no electric current is generated due to reduction of oxides derived from the sensing electrode material itself or reduction of dissolved oxygen. Not disturbed by the reduction of dissolved oxygen. This is a characteristic behavior that is possible only by the method of reducing hydrogen peroxide with a catalyst layer containing amorphous iridium oxide, which was found in the present invention.

Further, even when the carrier solution contains a preservative such as sodium azide, according to the present invention, the detection electrode in which the catalyst layer containing amorphous iridium oxide is used is used. Reduction of azide ions (N ₃ ⁻ ) does not occur in the potential range where hydrogen oxide is reduced. That is, in the electrochemical determination method of hydrogen peroxide according to the present invention, the detection electrode is not catalytic to the reduction of azide ions, so even when a preservative such as sodium azide is used, the reduction of hydrogen peroxide is performed. Has the effect of having no effect.

In the electrochemical quantification method of hydrogen peroxide of the present invention, first, biological fluids such as urine, saliva, blood, food production liquid, decomposition liquid, extract liquid, cooked product, cooking process product and its extract liquid, A test solution containing a target substance such as a solution containing a pharmaceutical is collected, mixed with a carrier solution, and conveyed to a sensor. In addition, even if the target substance is a solid, it is possible to prepare a solution containing the target substance. Therefore, the above-mentioned “test solution containing the target substance” must be a single target substance that is liquid. This does not mean that the target substance must be previously dissolved or mixed in the test solution, but the target substance itself may be directly dissolved or mixed in the carrier solution. Moreover, in the above, before mixing a test liquid with a carrier solution, you may perform the pretreatment which removes the component which interferes with fixed quantity of a target substance previously. For such pretreatment, various known methods can be applied. A buffer solution is usually used as the carrier solution. This is because the pH of the solution in which hydrogen peroxide is dissolved affects the reduction of hydrogen peroxide. As such a buffer solution, aqueous solutions having various known compositions can be used. For example, an aqueous solution in which equimolar amounts of potassium dihydrogen phosphate and disodium hydrogen phosphate are mixed becomes a buffer solution having a substantially neutral pH. In the present invention, it can be used as an example of a carrier solution used in the step of carrying a target substance or a test solution containing the target substance to a sensor. Furthermore, in the present invention, when a sensor having a reference electrode for controlling the potential of the detection electrode is used, components necessary for the reaction of the reference electrode are added to the carrier solution. For example, when a silver-silver chloride electrode is used as the reference electrode, since chloride ions are involved in the reaction that defines the potential of this electrode, for example, potassium chloride is added to the carrier solution. Such addition of potassium chloride ensures that the chloride ion concentration in the carrier solution is always kept constant, even when the test solution contains chloride ions, so that the potential of the silver-silver chloride electrode is always constant. It is effective to ensure that When an electrode other than the silver-silver chloride electrode is used as the reference electrode, components necessary for the reaction that defines the potential of the reference electrode are similarly added to the carrier solution.

Next, the target substance transported to the sensor by the carrier solution is oxidized by an enzyme reaction to generate hydrogen peroxide, which is further reduced by an electrochemical reaction at the detection electrode. These reactions are possible, for example, by configuring the following sensor. In the sensor, a detection electrode and a counter electrode, or a detection electrode, a counter electrode, and a reference electrode are formed on a substrate, and an enzyme film containing an enzyme that oxidizes hydrogen peroxide is formed on at least the detection electrode. In addition, at least between the enzyme film formed on the detection electrode and the detection electrode, the permeation of components that interfere with the accurate measurement of the reduction current of hydrogen peroxide at the detection electrode is suppressed, and only hydrogen peroxide is detected. Alternatively, a permselective membrane used for the purpose of reaching the above may be arranged. Various known materials and configurations used in sensors for the purpose of quantifying target substances can be used for enzyme membranes and permselective membranes. Furthermore, with such known materials and configurations, Even if the enzyme membrane is provided with the target substance oxidase, the enzyme membrane only needs to have a function that allows the carrier solution to penetrate and hydrogen peroxide generated by the enzyme reaction to reach the detection electrode. Further, the permselective membrane only needs to have a function of suppressing the passage of substances other than hydrogen peroxide so that only hydrogen peroxide generated by the enzyme membrane reaches the detection electrode. For example, when the target substance is glucose, a glucose oxidase supported on bovine serum albumin is used as the enzyme membrane, and a cross-linking agent such as glutaraldehyde or a buffer solution is added thereto. An enzyme membrane solution is used which is formed as an enzyme membrane by the drop method or the like. The permselective membrane for selectively permeating hydrogen peroxide to the detection electrode is made of, for example, cellulose acetate or a derivative thereof, an anion exchange resin containing perfluorosulfonic acid, or bovine serum albumin. A permselective membrane solution prepared by mixing in a deionized water together with a cross-linking agent and formed by the same method as the enzyme membrane is used. Further, the substrate may be subjected to a surface treatment such as silanization so that the enzyme membrane or the selectively permeable membrane does not easily peel from the substrate. Moreover, you may form the restriction | limiting permeation | transmission film | membrane which has a function which prevents and suppresses substances other than a target substance infiltrating in an enzyme membrane with a carrier solution on an enzyme membrane.

As the sensor substrate, use a material and shape that does not short-circuit the detection electrode and counter electrode, or the detection electrode, counter electrode, and reference electrode. For example, ceramics such as alumina and silicon nitride, glass, quartz, diamond Silicon, resin, or the like in which silicon oxide is formed can be used in a plate shape, a cylindrical shape, a rod shape, or the like, but is not particularly limited thereto.

In the sensor of the present invention, a detection electrode having a catalyst layer containing amorphous iridium oxide is used. For this detection electrode, a catalyst layer containing amorphous iridium oxide alone or a structure in which a conductive layer is formed on a substrate and a catalyst layer containing amorphous iridium oxide is formed thereon can be used. For example, a conductive layer made of titanium, platinum or the like is first formed on a substrate, a catalyst layer is formed on the conductive layer, and the conductive layer is controlled to the potential of the detection electrode or the voltage between the detection electrode and the counter electrode. Can be used as a lead. Further, for example, in the case of the catalyst layer alone, this may be detected by a means for preventing contact with the carrier solution on the part of the catalyst layer separated from the enzyme film, for example, by masking with an insulating substance. It can be used as a lead to a device that controls a potential or a voltage between a detection electrode and a counter electrode. The catalyst layer containing amorphous iridium oxide can be produced by various known methods such as thermal decomposition, physical vapor deposition, chemical vapor deposition, electrochemical oxidation, sol-gel, and electrodeposition. . At this time, amorphous iridium oxide is prepared in the form of particles or powder in advance and mixed alone or with other components, and then formed as a catalyst layer on the substrate or conductive layer by a known method. A precursor solution in which an iridium compound is dissolved is applied onto a substrate or a conductive layer as in a thermal decomposition method and heated, and an amorphous iridium oxide or a catalyst layer containing amorphous iridium oxide is directly applied to the substrate. Alternatively, it can be formed over a conductive layer.

Here, a method of forming a catalyst layer containing amorphous iridium oxide as a detection electrode by thermal decomposition using an alumina plate as a base will be further described as an example. Iridium acid hexahydrate chloride _{_{_{(H 2 IrCl 6 · 6H 2}}} O) was dissolved in 1-butanol as a 70 g / L in terms of metal iridium to prepare a precursor solution, which was coated on an alumina plate And then pyrolyzed by heating in an electric furnace. Here, if the temperature at the time of thermal decomposition is, for example, 340 to 380 ° C., a catalyst layer made of amorphous iridium dioxide is formed on the alumina plate. Moreover, when the temperature at the time of thermal decomposition is higher than 380 ° C. and lower than 440 ° C., a catalyst layer in which crystalline and amorphous iridium dioxide are mixed is formed on alumina. On the other hand, if the temperature at the time of thermal decomposition is, for example, 440 ° C. to 600 ° C., a catalyst layer made only of crystalline iridium dioxide is formed on alumina, and if pyrolysis occurs at a temperature higher than 600 ° C., crystalline iridium dioxide At the same time, a catalyst layer in which metal iridium is co-deposited may be formed, and a temperature at which a catalyst layer composed of crystalline iridium dioxide or a catalyst layer in which metal iridium is co-deposited with crystalline iridium dioxide is formed. Is unsuitable as a condition for producing the catalyst layer of the detection electrode used in the electrochemical determination method of hydrogen peroxide of the present invention. However, in the case of the above thermal decomposition method, the temperature at which the iridium dioxide becomes amorphous depends on the type of iridium compound used, the type of solvent used in the precursor solution, and the action that promotes or delays the thermal decomposition of the iridium compound. The temperature varies depending on whether or not the additive having the additive is present in the precursor solution and the concentration of the additive, so that the temperature forms a catalyst layer made of amorphous iridium oxide for achieving the present invention. It is an example regarding manufacture of the detection pole which performed.

In the above example, tantalum pentachloride (TaCl ₅ ) is dissolved in 1-butanol so that the molar ratio of iridium and tantalum is 80:20 together with iridium chloroiridate hexahydrate, and this is used as a precursor solution. When it is thermally decomposed after being coated on the alumina plate, a catalyst layer composed of iridium dioxide and tantalum pentoxide is formed on the alumina plate. For example, if the temperature at the time of thermal decomposition is 400 ° C., a catalyst layer made of crystalline, amorphous iridium dioxide and amorphous tantalum pentoxide is formed on an alumina plate. A catalyst layer made of high quality iridium dioxide and amorphous tantalum pentoxide is formed on the alumina plate. On the other hand, if the temperature at the time of thermal decomposition is 470 ° C., a catalyst layer composed of crystalline iridium dioxide and amorphous tantalum pentoxide is formed on alumina. It is unsuitable as a condition for preparing a detection electrode catalyst layer used in the quantitative method. However, even in the above case, the temperature at which the iridium dioxide becomes amorphous is such that the type of iridium compound or tantalum compound used, the type of solvent used in the precursor solution, and further the thermal decomposition of the iridium compound is accelerated or delayed. The temperature varies depending on whether or not an additive having an action is present in the precursor solution and the concentration of the additive, if any, so that the above temperature is applied to the catalyst layer containing amorphous iridium oxide for achieving the present invention. It is an example regarding preparation of the formed detection electrode.

The presence of amorphous iridium oxide in the catalyst layer formed on the substrate or the conductive layer can be known by a generally known X-ray diffraction method. For example, the diffraction peak of iridium dioxide is not seen in the X-ray diffraction image of the catalyst layer, or a broad diffraction line is seen in the vicinity of the 2θ value at which the diffraction peak of crystalline iridium dioxide should be generated. The presence of iridium oxide can be known. Moreover, when such a broad diffraction line and the diffraction peak of crystalline iridium dioxide overlap, it can be known that amorphous and crystalline iridium dioxide are mixed. In addition to the analysis by the X-ray diffraction method, the binding energy of each element of iridium and oxygen is measured by X-ray photoelectron spectroscopy (XPS), and the chemical state of each element is analyzed. Useful to know.

For the counter electrode formed on the sensor substrate, various known materials such as platinum and the like, metals used in sensors for electrochemical determination of hydrogen peroxide, and conductive ceramics can be used. . A counter electrode in which a catalyst layer containing amorphous iridium oxide is formed on the counter electrode can also be used. In the case of a sensor having a detection electrode, a counter electrode, and a reference electrode, for example, a silver-silver chloride electrode is used as the reference electrode. A silver-silver chloride electrode forms silver on a substrate or a conductive layer, and then electrochemically oxidizes silver in an aqueous solution containing chloride ions, or by carrying silver chloride on silver. Can be produced. However, the reference electrode is not limited to the silver-silver chloride electrode, and may be any electrode suitable for the purpose of controlling the potential of the detection electrode.

In the electrochemical determination method of hydrogen peroxide according to the present invention, an electrode on which a catalyst layer containing amorphous iridium oxide is formed as a detection electrode, and the potential of the detection electrode is controlled so that reduction of hydrogen peroxide occurs. Measure the detection pole current. For example, if the sensor has a sensing electrode, a counter electrode, and a reference electrode, these electrodes are connected to a generally known potentiogalvanostat or a device having a similar function, and the potential of the sensing electrode with respect to the reference electrode is controlled. The detection electrode causes a reduction reaction of hydrogen peroxide, and the counter electrode causes an oxidation reaction, and the current flowing through the detection electrode is measured. The potential of the detection electrode is controlled to a potential at which hydrogen peroxide is reduced with respect to the reference electrode. For example, 0.05 mol / L of potassium chloride is added to a carrier solution prepared by mixing 0.033 mol / L of potassium dihydrogen phosphate and disodium hydrogen phosphate with distilled water, and a silver-silver chloride electrode is added to the reference electrode. When hydrogen peroxide is used, reduction of hydrogen peroxide at the detection electrode on which the catalyst layer containing amorphous iridium oxide is formed occurs at a potential lower than 0.35 V with respect to the reference electrode. When the potential is controlled to be higher than this, hydrogen peroxide is oxidized, not reduced. In addition, the reduction of hydrogen peroxide is generally promoted by increasing the overvoltage necessary for the reaction as the potential becomes lower, and the current also increases. However, if the potential is too low, not only hydrogen peroxide but also the carrier is increased. Since a reduction reaction of other components contained in the solution occurs, it may be controlled within a range in which such a reaction does not occur. However, the potential of the detection electrode described above is shown as an example because it may change depending on the composition of the carrier solution, the presence or absence of components other than amorphous iridium oxide in the catalyst layer of the detection electrode, and the ratio thereof. It is.

In addition, when the sensor has a reference electrode and a counter electrode without a reference electrode, for example, these electrodes are connected to a generally known potentiogalvanostat or a device having a similar function, and between the detection electrode and the counter electrode, for example. The applied voltage is controlled to cause a reduction reaction of hydrogen peroxide at the detection electrode and an oxidation reaction at the counter electrode, and measure the current flowing through the detection electrode. In such a case, the relationship between the voltage applied to the detection electrode and the counter electrode and the potential of the detection electrode with respect to an appropriate reference electrode is clarified in advance, and the sensor is used without using the reference electrode without using the reference electrode. By controlling the voltage applied between the counter electrodes, the sensing electrode can be controlled to a potential at which hydrogen peroxide is reduced. That is, even when the sensor has a reference electrode and a counter electrode without a reference electrode, hydrogen peroxide is reduced at the detection electrode by controlling the potential of the detection electrode with the voltage applied between the detection electrode and the counter electrode. Can be made. In this case, as shown in the previous example, the voltage to be applied between the detection electrode and the counter electrode is such that hydrogen peroxide is reduced without oxidation of hydrogen peroxide at the detection electrode. The range is controlled so that the reduction reaction of other components contained in the carrier solution does not occur. The voltage applied between the detection electrode and the counter electrode is the overvoltage for the reaction occurring at the detection electrode, the overvoltage for the reaction occurring at the counter electrode, the ohmic loss of the detection electrode and the counter electrode, the ohmic loss in the carrier solution, the detection electrode and the counter electrode, respectively. It includes at least ohm loss in the connection between the sensing electrode and the device that controls the voltage of the counter electrode and measures the current flowing through the sensing electrode. Therefore, the range of the voltage applied between the detection electrode and the counter electrode is appropriately selected according to these ranges. In addition, the voltage range may change depending on the composition of the carrier solution, the presence or absence of components other than amorphous iridium oxide in the catalyst layer of the detection electrode, and the ratio thereof. Is done.

When measuring the current at the sensing electrode due to the reaction of hydrogen peroxide generated from the target substance carried to the sensor by the carrier solution, the current at the sensing electrode is determined by the method of feeding the carrier solution or the potential control method or sensing at the sensing electrode. Differences occur depending on the method of controlling the voltage applied between the pole and the counter electrode. As an example of such a method, a flow injection method can be used. For example, the carrier solution is flowed through the sensor at a constant flow rate in advance, and the potential of the detection electrode is set to a value that causes reduction of hydrogen peroxide, and then a test solution containing a certain amount of target substance is injected into the carrier solution. Then, the reduction current flowing through the detection electrode is measured. In this case, the current usually changes so as to give a peak with respect to time, and the concentration of hydrogen peroxide or the concentration of the target substance can be calculated from this peak current. In addition, when increasing the injection amount of the test solution containing the target substance into the carrier solution, continuously performing injection, or slowing the flow rate of the carrier solution, the current measured at the detection electrode is After showing a large value at first, it changes so as to attenuate with respect to time. This is normally a diffusion-controlled decay of hydrogen peroxide that reacts at the sensing electrode, and the current at a certain time after the current starts to flow depends on the concentration of the target substance and the target substance in the carrier solution. It depends on the concentration of hydrogen peroxide produced by the enzymatic reaction. In each of the above examples, a calibration curve indicating the relationship between the peak current measured at the detection electrode or the current at a certain time and the concentration of hydrogen peroxide or the target substance can be prepared in advance. Using this, the concentration of the target substance in an actual measurement object such as a biological fluid can be quantified. The potential of the detection electrode is always maintained at a constant potential, maintained at a constant potential after injecting a target substance or a test solution containing the target substance into the carrier solution, or maintained before injection. There is a method of measuring by changing to a lower potential than the measured potential. Although it is not preferable to change the potential of the detection electrode in a state where hydrogen peroxide is reduced, the target solution or the test solution containing the target material is not held in advance in the carrier solution, and is kept at a constant potential. In that state, the electric double layer formation current generated at the detection electrode is sufficiently attenuated to minimize and stabilize, and then the hydrogen peroxide generated from the target substance is reduced by holding it constant at a lower potential. Such two-stage potential control is effective for the purpose of suppressing the influence of the electric current for forming the electric double layer.

The second invention of the present invention uses a sensing electrode, a counter electrode, and a reference electrode, and the potential of the sensing electrode determined as a silver-silver chloride electrode of a saturated potassium chloride solution is + 0.35V to -0.6V. The electrochemical determination method of hydrogen peroxide is characterized in that the potential of the detection electrode is controlled so as to be in the range of Here, “the potential of the sensing electrode in which the reference electrode is defined as a silver-silver chloride electrode of saturated potassium chloride solution is in the range of +0.35 V to −0.6 V” means that the reference electrode used in the sensor is saturated with potassium chloride. It is not limited to silver-silver chloride electrodes using a solution. A silver-silver chloride electrode that is in contact with an aqueous solution of potassium chloride other than a saturated solution of potassium chloride, or an electrode other than a silver-silver chloride electrode can be used as a reference electrode, in which case the reference electrode and potassium chloride are used. What is necessary is just to correct | amend the difference of the electric potential of the silver-silver chloride electrode immersed in the saturated solution to the value of + 0.35V and -0.6V. That is, since the potential of the electrode reaction is a relative value determined with respect to the reference reaction potential, as described above, “the detection that is determined as a silver-silver chloride electrode of a saturated potassium chloride solution as a reference electrode”. The expression “electrode potential” does not mean that the reference electrode actually used is limited to a silver-silver chloride electrode in contact with a saturated potassium chloride solution.

By controlling the potential of the sensing electrode so that the potential of the sensing electrode determined as a silver-silver chloride electrode of a saturated potassium chloride solution as a reference electrode is in the range of +0.35 V to -0.6 V, hydrogen peroxide It has an effect of preventing oxidation and suppressing decomposition of the carrier solution. At this time, if the potential of the detection electrode becomes nobler than +0.35 V, hydrogen peroxide is not reduced but becomes an oxidation potential, which is not suitable, and if the potential of the detection electrode is lower than −0.6 V, This is not preferable because the potential difference between the detection electrode and the counter electrode is increased and the carrier solution is decomposed. The potential of the detection electrode is more preferably in the range of +0.2 V to −0.4 V with respect to the silver-silver chloride electrode of the saturated potassium chloride solution. In this range, a stable reduction current corresponding to the concentration of hydrogen peroxide can be obtained, and other components contained in the test solution can react to reduce the influence of hydrogen peroxide on the reaction. Have. For example, when the target substance is glucose in a biological fluid, components such as uric acid and ascorbic acid that are originally contained in the biological fluid may coexist in the carrier solution. These components are interfering components that interfere with accurate quantification of the target substance glucose, and in order to prevent such components from reaching the detection electrode, normally only hydrogen peroxide is used between the detection electrode and the enzyme membrane. A permselective membrane intended for permeation is disposed, or a restrictive permeation membrane intended to restrict permeation of components other than the target substance is disposed on the enzyme membrane. According to the present invention, even when the functions of the permselective membrane or the restricted permeation membrane are deteriorated, the excess due to the reduction reaction of the interfering component is detected in the potential range of the detection electrode that causes the reduction of hydrogen peroxide of the present invention. The effect of hydrogen oxide on the reduction current is effectively suppressed.

The third invention of the present invention uses a detection electrode and a counter electrode so that the potential of the detection electrode is in the range of +0.35 V to −0.6 V with respect to the silver-silver chloride electrode of the saturated potassium chloride solution. This is an electrochemical determination method of hydrogen peroxide characterized by controlling the voltage between the detection electrode and the counter electrode. Since the detection electrode and the counter electrode are used and the reference electrode is not used, the number of electrodes in the sensor is small, the configuration of the sensor is simplified, and the sensor can be made more compact. Even when the detection electrode and counter electrode are used, the potential of the detection electrode is applied between the detection electrode and the counter electrode so that the potential of the detection electrode is in the range of +0.35 V to -0.6 V with respect to the silver-silver chloride electrode of the saturated potassium chloride solution. By controlling the voltage, the oxidation of hydrogen peroxide is prevented and the decomposition of the carrier solution is suppressed. As a result of controlling the voltage between the detection electrode and the counter electrode, when the potential of the detection electrode becomes nobler than +0.35 V with respect to the silver-silver chloride electrode of the saturated potassium chloride solution, hydrogen peroxide is not reduced but oxidized. Therefore, it is unsuitable. Similarly, as a result of the voltage control, when the potential of the detection electrode based on the silver-silver chloride electrode of the saturated potassium chloride solution becomes lower than -0.6 V, the potential difference between the detection electrode and the counter electrode increases, This is not preferable because it causes decomposition of the carrier solution.

As described above, the relationship between the voltage applied between the detection electrode and the counter electrode and the potential of the detection electrode with respect to the silver-silver chloride electrode of the saturated potassium chloride solution is clarified in advance. It is possible to control the potential of the detection electrode by controlling the voltage applied between them. It is more preferable to apply a voltage between the detection electrode and the counter electrode so that the potential of the detection electrode is in the range of +0.2 V to -0.4 V with respect to the silver-silver chloride electrode of the saturated potassium chloride solution. is there. In this range, a stable reduction current corresponding to the concentration of hydrogen peroxide can be obtained, and other components contained in the test solution can react to reduce the influence of hydrogen peroxide on the reaction. Have. For example, when the target substance is glucose in a biological fluid, components such as uric acid and ascorbic acid that are originally contained in the biological fluid may coexist in the carrier solution. These components interfere with accurate quantification of the target substance glucose, and in order to prevent such components from reaching the detection electrode, normally only hydrogen peroxide is used between the detection electrode and the enzyme membrane. A permselective membrane intended for permeation is disposed, or a restrictive permeation membrane intended to restrict permeation of components other than the target substance is disposed on the enzyme membrane. According to the present invention, even when the functions of the selectively permeable membrane and the restricted permeable membrane are deteriorated, the detection electrode and the counter electrode are set so as to have the potential range of the detection electrode that causes reduction of hydrogen peroxide according to the present invention. By controlling the voltage between them, the effect of the reduction reaction of the interfering component on the reduction current of hydrogen peroxide is effectively suppressed.

According to a fourth aspect of the present invention, there is provided an electrochemical hydrogen peroxide characterized in that the catalyst layer uses a sensing electrode composed of amorphous iridium dioxide or amorphous and crystalline iridium dioxide. It is a quantitative method. A catalyst layer composed of amorphous iridium dioxide or amorphous and crystalline iridium dioxide can be produced by various known methods such as pyrolysis, physical vapor deposition, chemical vapor deposition, and electrolysis. Although possible, the catalyst layer containing amorphous iridium dioxide has a particularly high catalytic property for the reduction of hydrogen peroxide, and has an effect of improving the sensitivity to hydrogen peroxide. In addition, when the catalyst layer is formed with amorphous and crystalline iridium dioxide, crystalline iridium dioxide is inferior in catalytic properties for the reduction of hydrogen peroxide, but crystalline iridium dioxide detects amorphous iridium dioxide. By having an anchor effect that is strongly fixed to the electrode substrate or the conductive layer, the adhesion between the catalyst layer and the substrate or the conductive layer is improved.

According to a fifth aspect of the present invention, the catalyst layer is made of amorphous iridium dioxide or amorphous and crystalline iridium dioxide, and at least one metal selected from tantalum, titanium, niobium, zirconium, and tungsten. This is a method for electrochemical determination of hydrogen peroxide, characterized by using a sensing electrode composed of an oxide of the above. A sensing layer is a catalyst layer in which amorphous iridium dioxide or amorphous and crystalline iridium dioxide and an oxide of at least one metal selected from tantalum, titanium, niobium, zirconium, and tungsten are mixed. When used, the mixed metal oxide does not participate in the reduction reaction of hydrogen peroxide and interfering components, strongly adheres amorphous iridium dioxide and the substrate or conductive layer of the sensing electrode in the catalyst layer, and It plays a role as a binder for densifying the catalyst layer itself, and has the effect of suppressing the consumption of amorphous iridium dioxide and the peeling and dropping off of the catalyst layer.

According to a sixth aspect of the present invention, the catalyst layer is composed of amorphous iridium dioxide and amorphous tantalum pentoxide, or amorphous and crystalline iridium dioxide and amorphous tantalum pentoxide. This is a method for electrochemical determination of hydrogen peroxide, characterized by using a detection electrode. When a catalyst layer containing amorphous iridium dioxide and amorphous tantalum pentoxide is used as the sensing electrode, amorphous tantalum pentoxide is not involved in the reduction reaction of hydrogen peroxide and interfering components. First, in order to form a dense mixture in which amorphous iridium dioxide and amorphous tantalum pentoxide are mixed together without segregation in the catalyst layer, the catalyst layer is used as a substrate or a conductive layer of the detection electrode. In particular, it has an effect that it can more effectively suppress the consumption of amorphous iridium dioxide and the peeling and dropping off of the catalyst layer, and can reduce the amount and thickness of the catalyst layer. . In addition, when a catalyst layer in which amorphous and crystalline iridium dioxide and amorphous tantalum pentoxide are mixed is used as the sensing electrode, the anchor effect due to crystalline iridium dioxide and amorphous tantalum pentoxide are obtained. With the above effect, the action of bringing the catalyst layer into close contact with the base of the detection electrode or the conductive layer becomes stronger, and it is possible to more effectively suppress the consumption of iridium dioxide and the separation and dropping off of the catalyst layer, It has the effect | action that the quantity and thickness of a catalyst layer can be reduced further. In addition, tantalum pentoxide increases the dispersibility of iridium dioxide in the catalyst layer, promotes the amorphization of iridium dioxide, or forms iridium dioxide nanoparticles in the catalyst layer. Therefore, the sensitivity to reduction of hydrogen peroxide is improved, and the denseness of the catalyst layer is improved by a binder action as compared with the case of iridium dioxide alone.

The seventh invention of the present invention is an electrochemical quantification method of hydrogen peroxide characterized by using a counter electrode on which a catalyst layer containing amorphous iridium oxide is formed. By using amorphous iridium oxide for the counter electrode as well as the sensing electrode, the reaction of interfering components such as oxidation of azide ions that occurs when other materials such as platinum are used for the counter electrode is suppressed. Therefore, it has the effect of eliminating the factor that obstructs energization at the counter electrode, thereby preventing the problem that the current flowing to the detection electrode does not become a value proportional to the concentration of hydrogen peroxide due to the counter electrode. .

In addition, the present invention provides an electrochemical determination method for hydrogen peroxide, characterized in that the catalyst layer contains iridium dioxide in an amount of 40 mol% to 99 mol% and tantalum pentoxide in an amount of 60 mol% to 1 mol%. It is preferable to do. The catalyst layer in which iridium dioxide is in the range of 40 mol% to 99 mol% and tantalum pentoxide is in the range of 60 mol% to 1 mol% is highly sensitive to hydrogen peroxide, and the reaction of interfering components is effectively suppressed. There is no effect on the reduction of hydrogen peroxide, and the catalyst layer is in close contact with the substrate or conductive layer of the detection electrode, so that the consumption, separation and removal of amorphous iridium dioxide are more effectively suppressed. Have. If iridium dioxide is smaller than 40 mol% and tantalum pentoxide is larger than 60 mol%, the effect of amorphous iridium dioxide cannot be obtained sufficiently, and iridium dioxide is larger than 99 mol%. If tantalum pentoxide is smaller than 1 mol%, the effect of tantalum pentoxide cannot be sufficiently obtained, which is not preferable.

Further, the present invention is preferably an electrochemical quantification method for hydrogen peroxide characterized in that a preservative is contained in a carrier solution for transporting a target substance to a sensor. The carrier solution directly contacts the sensor and penetrates into the enzyme membrane that constitutes the sensor. Here, since it is virtually impossible to eliminate the germs and molds in the carrier solution mixed with the test solution containing the target substance, the germs and molds may be propagated using an enzyme or the like as food. As a result, the target substance is oxidized by the enzyme reaction in the enzyme membrane, and the efficiency of producing hydrogen peroxide is reduced, so that the current measured at the detection electrode is reduced, or the carrier solution is altered and the reference electrode is changed. Since the hydrogen peroxide cannot be kept constant, hydrogen peroxide cannot be accurately determined. Therefore, these problems can be suppressed by adding a preservative to the carrier solution. Here, for example, sodium azide is used as the preservative. Sodium azide is preferable because it provides antibacterial and antifungal properties required for a carrier solution even at a very low concentration and is inexpensive. In addition, according to the electrochemical determination method of hydrogen peroxide of the present invention, azide ions generated in the carrier solution by addition of sodium azide are preferable because neither oxidation nor reduction occurs at the detection electrode. is there. In addition, although sodium azide was shown as an example of an antiseptic | preservative, it is not specifically limited to this. Such preservatives are disclosed in, for example, Patent Document 10 and Patent Document 11.

As described above, according to the present invention, oxygen is not generated by the electrochemical reaction of hydrogen peroxide at the detection electrode, so the sensitivity is lowered even when continuously or highly quantifying hydrogen peroxide is quantified. In addition, since there is no effect on the sensitivity due to the previous measurement history in repeated measurement, hydrogen peroxide can be measured with high sensitivity, and high reproducibility and reliability of the results can be achieved even if repeated measurements are made. The effect that it can maintain is acquired.
In addition, according to the present invention, there is no reaction due to dissolved oxygen contained in the test solution or the carrier solution, there is no influence of the concentration change of dissolved oxygen, and dissolved oxygen is generated to maintain the dissolved oxygen concentration. Since there is no need to provide an auxiliary electrode on the sensor, the reliability of hydrogen peroxide determination is improved, and the sensor can be configured with the minimum number of electrodes, making the sensor structure simple and complicated to manufacture. In addition, the manufacturing cost can be reduced as compared with the case where the auxiliary electrode is used.
In addition, according to the present invention, the sensing electrode is chemically stable against the reaction of hydrogen peroxide, and the sensing electrode material itself is not oxidized or reduced. Since replacement is not necessary frequently and maintenance is easy even in long-term use, the burden on the user can be reduced, and the cost for maintenance can be reduced.
Further, according to the present invention, the interference that is contained in the test solution and directly interferes with the hydrogen peroxide electrochemical reaction or interferes with the measurement of the current that depends only on the hydrogen peroxide electrochemical reaction. Since the influence of the components is suppressed, the process of removing the disturbing components from the test solution in advance becomes unnecessary or simple, and the time and cost related to such a process are reduced, and the conventional inexpensive and low-cost process is used. Preservatives that are effective even at concentrations can be used as they are, and there is no need to change or improve the specifications of sensors and devices that perform electrochemical quantification of target substances, and target substances can be quantified with high sensitivity and stability. This makes it possible to develop sensors and devices that can be used.
In addition, according to the present invention, high sensitivity can be stably maintained over a long period of time, and the complicated calibration of the sensor is not necessary. Therefore, the burden on the user is reduced, and it is easier to use and more maintenance cost. It is possible to obtain an effect that it is possible to quantify a target substance having a low level.
In addition, according to the present invention, it is possible to apply to a target substance that has so far determined the concentration of the target substance by quantifying hydrogen peroxide other than electrochemical analysis such as spectroscopic analysis. This eliminates the need for a large-scale device such as spectroscopic analysis and greatly reduces the time required for the determination of hydrogen peroxide, making it easier and faster to generate hydrogen peroxide in an enzymatic reaction. The effect that the quantity of the substance can be obtained is obtained.

It is a relationship diagram of the reduction current density and concentration of hydrogen peroxide when the potential of the detection electrode is + 0.1V. It is a relationship diagram of the reduction current density and concentration of hydrogen peroxide when the potential of the detection electrode is 0V. It is the figure which compared the sensitivity of hydrogen peroxide. It is a figure which shows the reduction current density of hydrogen peroxide in the presence of ascorbic acid. It is a figure which shows the reduction current density of hydrogen peroxide in the presence of uric acid.

First, the results of quantifying hydrogen peroxide by changing the material of the catalyst layer will be described (Example 1, Example 2, Comparative Example 1 and Comparative Example 2).

[Example 1]
The titanium plate simulating the conductive layer was ultrasonically washed in acetone, further immersed in a 10 wt% oxalic acid solution at 90 ° C. for 60 minutes to etch the surface, washed with distilled water and dried. Next, a catalyst layer precursor solution was prepared by dissolving tantalum pentachloride and iridium (IV) chloride hexahydrate in a 1-butanol solution containing 6% concentrated hydrochloric acid. The molar ratio of iridium and tantalum in the precursor solution was 80:20, and the total concentration of iridium and tantalum was 70 g / L in terms of metal. This precursor solution was applied on a titanium plate, and then heated in an electric furnace at 360 ° C. for 20 minutes to thermally decompose the precursor solution. This application and thermal decomposition were repeated 5 times to form a catalyst layer on the titanium plate. As a result of analyzing the obtained catalyst layer with an X-ray diffractometer, no peak was observed in the 2θ value that produced a diffraction peak for crystalline iridium dioxide or crystalline tantalum pentoxide. Further, the XPS analysis results reveal the presence of iridium dioxide and tantalum pentoxide in the catalyst layer, and the obtained catalyst layer is made of a mixture of amorphous iridium dioxide and amorphous tantalum pentoxide. It was confirmed. A titanium electrode with the catalyst layer thus formed was used as a sensing electrode, a three-electrode type measuring cell was assembled using a platinum plate as a counter electrode and a silver-silver chloride electrode immersed in a saturated potassium chloride solution as a reference electrode.

Next, 0.033 mol / L of potassium dihydrogen phosphate and disodium hydrogen phosphate are mixed with distilled water to prepare a buffer solution having a pH of approximately neutral, and 0.05 mol / L of potassium chloride is added thereto. The obtained solution was used as a simulated solution of the carrier solution, and the detection electrode and the counter electrode were immersed in this solution. The carrier solution and the saturated potassium chloride solution of the reference electrode were connected by a salt bridge. Further, the contact area between the detection electrode and the carrier solution was regulated to be 1 cm × 1 cm.

Cyclic voltammograms were measured at a scanning speed of 5 mV / s in a carrier solution and in a solution in which hydrogen peroxide was added to the carrier solution to a hydrogen peroxide concentration of 1 to 3 mmol / L. First, in the cyclic voltammogram obtained with the carrier solution, only the electric current associated with charging of the electric double layer was observed, and there was no oxidation wave or reduction wave indicating oxidation reaction or reduction reaction caused by the catalyst layer. It was found that no oxidation or reduction of the catalyst layer occurred in the carrier solution. However, when the potential scan range of the cyclic voltammogram becomes lower than -0.6V, a reduction current due to decomposition of the carrier solution was observed, so that the reduction current of hydrogen peroxide at a lower potential than -0.6V. It has also been found that it is not preferable to measure. Next, in the solution in which hydrogen peroxide is added to the carrier solution, the current due to the reduction of hydrogen peroxide in the base potential range from +0.35 V to the cyclic voltammogram obtained with the carrier solution. An increase was seen. In addition, an increase in current due to oxidation of hydrogen peroxide was also observed in a potential region higher than + 0.35V. Therefore, with respect to the cyclic voltammograms obtained with the carrier solution and the carrier solution added with hydrogen peroxide, the reduction current density at the detection electrode potentials of +0.1 V and 0 V was read, and the solution was added with hydrogen peroxide. The reduction current density of hydrogen peroxide was subtracted from the reduction current density in the carrier solution to obtain the reduction current density of hydrogen peroxide.

As a result of organizing the relationship between the reduction current density of hydrogen peroxide at +0.1 V and the concentration of hydrogen peroxide, a proportional relationship was obtained as shown in FIG. As a result of arranging the relationship of hydrogen oxide concentration, a proportional relationship was obtained as shown in FIG. The reduction current density is a reduction current per contact area of the detection electrode with respect to the carrier solution. Such a measurement is performed on a plurality of sensing electrodes produced at the same time, the proportional relationship as shown in FIG. 1 or FIG. 2 is obtained, the slope of the straight line is calculated, the average value is calculated, The average value when the potential is +0.1 V and 0 V is shown in FIG. 3 as the sensitivity to hydrogen peroxide. From the comparison with Example 2 and Comparative Example 1 described later, the sensitivity of hydrogen peroxide in Example 1 in which the catalyst layer contains amorphous iridium dioxide and amorphous tantalum pentoxide is In comparison with Comparative Example 1, it was found that the potential of the detection electrode was 6.6 times higher when the potential was + 0.1V, and four times higher when 0V.

Furthermore, a hydrogen peroxide solution is added to the carrier solution to make the hydrogen peroxide concentration 3 mmol / L, and a hydrogen peroxide solution is added to the carrier solution to make the hydrogen peroxide concentration 3 mmol / L, and azide Cyclic voltammograms were measured at a scanning speed of 5 mV / s with a solution containing 0.05% sodium. As a result of comparing the cyclic voltammograms obtained with these two types of solutions, even if sodium azide was added, the cyclic voltammograms were the same, and even if sodium azide was present, the reduction current density of hydrogen peroxide was completely different. It turns out that there is no change.

[Example 2]
A catalyst layer was formed on a titanium plate simulating a conductive layer by the same method as in Example 1 except that the thermal decomposition temperature in Example 1 was changed from 360 ° C. to 400 ° C. As a result of analyzing the obtained catalyst layer with an X-ray diffractometer, no peak was observed in the 2θ value that produced a diffraction peak for crystalline tantalum pentoxide, but it was diffracted for crystalline iridium dioxide. A broad diffraction line overlapping with a weak diffraction peak was observed in the 2θ value that produced the peak. The XPS analysis also revealed the presence of iridium dioxide and tantalum pentoxide in the catalyst layer. The resulting catalyst layer was composed of amorphous and crystalline iridium dioxide and amorphous tantalum pentoxide. It was confirmed to consist of a mixture. The titanium plate on which the catalyst layer was formed as described above was used as a detection electrode, and measurement was performed under the same conditions using the measurement cell and carrier solution described in Example 1.

Cyclic voltammograms were measured at a scanning speed of 5 mV / s in a carrier solution and in a solution in which hydrogen peroxide was added to the carrier solution to a hydrogen peroxide concentration of 1 to 3 mmol / L. First, in the cyclic voltammogram obtained with the carrier solution, only the electric current associated with charging of the electric double layer was observed, and there was no oxidation wave or reduction wave indicating oxidation reaction or reduction reaction caused by the catalyst layer. It was found that no oxidation or reduction of the catalyst layer occurred in the carrier solution. Next, in the solution in which hydrogen peroxide is added to the carrier solution, the potential of the current due to the reduction of hydrogen peroxide in the base potential range from +0.31 V to the cyclic voltammogram obtained with the carrier solution. An increase was seen. Therefore, with respect to the cyclic voltammograms obtained with the carrier solution and the carrier solution added with hydrogen peroxide, the reduction current density at the detection electrode potentials of +0.1 V and 0 V was read, and the solution was added with hydrogen peroxide. The reduction current density of hydrogen peroxide was subtracted from the reduction current density in the carrier solution to obtain the reduction current density of hydrogen peroxide.

As a result of organizing the relationship between the reduction current density of hydrogen peroxide at +0.1 V and the concentration of hydrogen peroxide, a proportional relationship was obtained as shown in FIG. As a result of arranging the relationship of hydrogen oxide concentration, a proportional relationship was obtained as shown in FIG. Such a measurement is performed on a plurality of sensing electrodes produced at the same time, the proportional relationship as shown in FIG. 1 or FIG. 2 is obtained, the slope of the straight line is calculated, the average value is calculated, The average value when the potential is +0.1 V and 0 V is shown in FIG. 3 as the sensitivity to hydrogen peroxide. From the comparison with Comparative Example 1 described later, the sensitivity of hydrogen peroxide in Example 2 in which the catalyst layer contains amorphous and crystalline iridium dioxide and amorphous tantalum pentoxide is compared with Comparative Example 1. On the other hand, it was found that the potential of the detection electrode was 5.5 times higher when the potential was + 0.1V, and 3.6 times higher when the potential was 0V.

(Comparative Example 1)
A catalyst layer was formed on a titanium plate simulating a conductive layer by the same method as in Example 1 except that the thermal decomposition temperature in Example 1 was changed from 360 ° C. to 470 ° C. As a result of analyzing the obtained catalyst layer with an X-ray diffractometer, no peak was observed in the 2θ value that produced a diffraction peak for crystalline tantalum pentoxide, but it was diffracted for crystalline iridium dioxide. A sharp diffraction peak was observed in the 2θ value that produced the peak. In addition, the XPS analysis results reveal the presence of iridium dioxide and tantalum pentoxide in the catalyst layer, and the resulting catalyst layer is composed of a mixture of crystalline iridium dioxide and amorphous tantalum pentoxide. It was confirmed. The titanium plate on which the catalyst layer was formed as described above was used as a detection electrode, and measurement was performed under the same conditions using the measurement cell and carrier solution described in Example 1.

First, a cyclic voltammogram was measured at a scanning speed of 5 mV / s in a carrier solution and a solution in which hydrogen peroxide was added to the carrier solution to adjust the hydrogen peroxide concentration to 1 to 3 mmol / L. In the cyclic voltammogram obtained with the carrier solution, only the electric current associated with charging of the electric double layer was observed, and no oxidation wave or reduction wave indicating oxidation reaction or reduction reaction due to the catalyst layer was observed. It was found that no oxidation or reduction of the catalyst layer occurred in the solution. Next, in the solution obtained by adding hydrogen peroxide to the carrier solution, the reduction current increased in the base potential range from +0.12 V to the cyclic voltammogram obtained with the carrier solution. The increase was very small compared to Example 1 and Example 2. However, since the reduction current increased with increasing concentration of hydrogen peroxide, this increase in reduction current was considered to be reduction of hydrogen peroxide. Therefore, with respect to the cyclic voltammograms obtained with the carrier solution and the carrier solution added with hydrogen peroxide, the reduction current density at the detection electrode potentials of +0.1 V and 0 V was read, and the solution was added with hydrogen peroxide. The reduction current density of hydrogen peroxide was subtracted from the reduction current density in the carrier solution to obtain the reduction current density of hydrogen peroxide.

As a result of organizing the relationship between the reduction current density of hydrogen peroxide at +0.1 V and the concentration of hydrogen peroxide, a proportional relationship was obtained as shown in FIG. As a result of arranging the relationship of hydrogen oxide concentration, a proportional relationship was obtained as shown in FIG. Such a measurement is performed on a plurality of sensing poles produced at the same time, the proportional relationship as shown in FIG. 1 or 2 is obtained, the slope of the straight line is calculated, the average value is calculated, and the average value Is shown in FIG. 3 as the sensitivity of hydrogen peroxide. As described in Example 1 and Example 2, the reduction current density and sensitivity for hydrogen peroxide in Comparative Example 1 in which the catalyst layer contains crystalline iridium dioxide and amorphous tantalum pentoxide are It was very small compared to 1 and Example 2.

(Comparative Example 2)
A platinum thin film is formed on an alumina substrate by screen printing, ultrasonically washed in acetone, washed with distilled water, further immersed in a 0.5 mol / L sulfuric acid solution for 1 minute, and then washed again with distilled water. And dried. Using this as a detection electrode, a three-electrode measurement cell was assembled using a platinum plate as a counter electrode and a silver-silver chloride electrode immersed in a saturated potassium chloride solution as a reference electrode. Next, 0.033 mol / L of potassium dihydrogen phosphate and disodium hydrogen phosphate are mixed with distilled water to prepare a buffer solution having a pH of approximately neutral, and 0.05 mol / L of potassium chloride is added thereto. The obtained solution was used as a simulated solution of the carrier solution, and the detection electrode and the counter electrode were immersed in this solution. The carrier solution and the saturated potassium chloride solution of the reference electrode were connected by a salt bridge. Further, the contact area between the detection electrode and the carrier solution was regulated to be 1 cm × 1 cm.

As a result of measuring a cyclic voltammogram in the carrier solution at a scanning speed of 5 mV / s, an increase in reduction current from a potential of the detection electrode near +0.35 V and a reduction wave having a peak near +0.1 V were obtained. Next, the carrier solution was bubbled with nitrogen gas to sufficiently remove dissolved oxygen, and then the cyclic voltammogram was measured at a scanning speed of 5 mV / s. As a result, the reduction current observed in the cyclic voltammogram before removing the dissolved oxygen was measured. Although a reduction wave showing an increase and a peak was still seen, both the reduction current and the peak current of the reduction wave decreased compared to the cyclic voltammogram before the dissolved oxygen was removed. Such an increase in reduction current from around +0.35 V and a reduction current having a peak near +0.1 V are due to the reduction of dissolved oxygen contained in the carrier solution and the reduction of platinum oxide. Excluding it indicates that the reduction current decreased. In addition, the reduction current of dissolved oxygen was observed in a broad potential range lower than + 0.35V. In addition, the reduction current flowing at a potential lower than +0.35 V shown above is not stable every measurement, and depends on the dissolved oxygen in the carrier solution and the amount of platinum oxide formed on platinum. It was shown to change.

Thus, in Comparative Example 2 in which the material of the detection electrode is platinum, the reduction current in the carrier solution is not stable. Therefore, in the cyclic voltammogram measured with a solution obtained by adding hydrogen peroxide to the carrier solution, hydrogen peroxide The current for the reduction alone could not be separated, and the hydrogen peroxide could not be quantified.

Subsequently, the results of quantifying hydrogen peroxide using a carrier solution containing ascorbic acid (Example 3) and uric acid (Example 4), which are interfering substances, will be described.

[Example 3]
A titanium plate on which a catalyst layer was formed by the same method as in Example 1 was used as a detection electrode and a counter electrode, and a three-electrode measurement cell was assembled using the reference electrode described in Example 1. Next, the detection electrode and the counter electrode were immersed in the same carrier solution simulation solution as in Example 1. Further, as in Example 1, the carrier solution and the reference electrode were connected by a salt bridge. The contact area between the detection electrode and the carrier solution was regulated to 1 cm × 1 cm. Chronoamperometry was performed as follows under the condition that the carrier solution was stirred at a rotation speed of 600 rpm with a stir bar, and the reduction current of hydrogen peroxide was measured under the condition that the potential of the detection electrode was kept at −0.15V. .

First, after applying a potential with only the carrier solution, hydrogen peroxide solution was added at a time of about 110 s to measure the current at a hydrogen peroxide concentration of 1 mmol / L, and then the hydrogen peroxide solution at about 140 s. Was added and hydrogen peroxide concentration was 2 mmol / L, and the current was measured again. Then, hydrogen peroxide solution was further added for about 170 s to measure the current again at a hydrogen peroxide concentration of 3 mmol / L. As a result, as shown by waveform 1 in FIG. 4, a reduction current density proportional to the concentration of hydrogen peroxide was observed. Next, after applying a potential to the carrier solution to which 10 mmol / L ascorbic acid was added, hydrogen peroxide solution was added in about 110 seconds to measure the current with a hydrogen peroxide concentration of 1 mmol / L. As shown by waveform 2 in FIG. 4, the same value as the reduction current density was obtained when the hydrogen peroxide concentration in waveform 1 to which ascorbic acid was not added was 1 mmol / L. Next, after applying a potential to the carrier solution to which 10 mmol / L ascorbic acid was added, hydrogen peroxide solution was added in about 140 seconds to measure the current with a hydrogen peroxide concentration of 2 mmol / L. As shown by waveform 3 in FIG. 4, the same value as the reduction current density was obtained when the hydrogen peroxide concentration in waveform 1 to which ascorbic acid was not added was 2 mmol / L. Furthermore, after applying a potential to the carrier solution to which 10 mmol / L ascorbic acid was added, hydrogen peroxide solution was added in about 170 seconds to measure the current with a hydrogen peroxide concentration of 3 mmol / L. As in waveform 4, the same value as the reduction current density was obtained when the hydrogen peroxide concentration in waveform 1 to which ascorbic acid was not added was 3 mmol / L.

Thus, according to the present invention, hydrogen peroxide could be quantified without being affected by ascorbic acid that hinders detection of hydrogen peroxide.

[Example 4]
The titanium electrode on which the catalyst layer was formed by the same method as in Example 1 was used as a detection electrode, and the three-electrode measurement cell described in Example 1 was assembled. Next, the detection electrode and the counter electrode were immersed in the same carrier solution simulation solution as in Example 1. Further, as in Example 1, the carrier solution and the reference electrode were connected by a salt bridge. The contact area between the detection electrode and the carrier solution was regulated to 1 cm × 1 cm. Chronoamperometry was performed as follows under the condition that the carrier solution was stirred at a rotation speed of 600 rpm with a stir bar, and the reduction current of hydrogen peroxide was measured under the condition that the potential of the detection electrode was kept at −0.15V. .

First, after applying a potential with only the carrier solution, hydrogen peroxide solution was added at a time of about 110 s to measure the current at a hydrogen peroxide concentration of 1 mmol / L, and then the hydrogen peroxide solution at about 140 s. Then, the current was measured again at a hydrogen peroxide concentration of 2 mmol / L, and then the hydrogen peroxide solution was further added at a time of about 170 seconds to measure the current again at a hydrogen peroxide concentration of 3 mmol / L. As a result, as shown by the waveform 5 in FIG. 5, a reduction current density proportional to the concentration of hydrogen peroxide was observed. Next, after applying a potential to the carrier solution to which 1 mmol / L of uric acid has been added, hydrogen peroxide solution is added at a time of about 110 s to measure the current with a hydrogen peroxide concentration of 1 mmol / L, and then for about a further time. Hydrogen peroxide solution was added at 140 s and the current was measured again at a hydrogen peroxide concentration of 2 mmol / L. Then, hydrogen peroxide solution was added at about 170 s and the hydrogen peroxide concentration was set at 3 mmol / L and the current was turned on again. As a result of measurement, the result of waveform 6 in FIG. 5 was obtained, and the same result as that obtained when no uric acid was added (waveform 5) was obtained.

Thus, according to the present invention, hydrogen peroxide could be quantified without being affected by uric acid that interfered with the detection of hydrogen peroxide.

The present invention relates to target substances contained in biological fluids such as urine, saliva, blood, food production fluids, degradation fluids, extracts, cooked products, cooking products and extracts thereof, and pharmaceuticals by oxidase. The present invention can be applied to the concentration of hydrogen peroxide generated upon oxidation, the electrochemical determination method for determining the concentration of a target substance from the concentration of hydrogen peroxide, and a sensor or apparatus using the same. In addition, target substances contained in biological fluids such as urine, saliva, blood, food production fluids, decomposition fluids, extracts, cooked products, cooking products and their extracts, and pharmaceuticals are oxidized by oxidases. Instead of electrochemically reducing the hydrogen peroxide produced during the process, the hydrogen peroxide or the target substance is quantified by a method other than electrochemically reducing the hydrogen peroxide or the target substance. By reducing, it can be used as an electrochemical quantification method for determining the concentration of the target substance from the concentration of hydrogen peroxide or the concentration of hydrogen peroxide, or for a sensor or apparatus using the same. Applications include medical testing, food testing, industrial measurement, plant analysis, health management, etc. Available forms include portable, small, hospitals, individual houses, factories, laboratories, etc. It can be used for medium-sized or large-sized devices that are permanently installed.

Claims

A method for electrochemical determination of hydrogen peroxide, comprising: a step of oxidizing a target substance conveyed to a sensor by an enzymatic reaction to generate hydrogen peroxide; and a step of measuring a current generated by an electrochemical reaction of the hydrogen peroxide. Because
A method for electrochemical determination of hydrogen peroxide, comprising measuring a current at which the hydrogen peroxide is reduced at a sensing electrode on which a catalyst layer containing amorphous iridium oxide is formed.
Using the detection electrode, the counter electrode, and the reference electrode, the detection electrode is defined such that the reference electrode is defined as a silver-silver chloride electrode of a saturated potassium chloride solution. 2. The method for electrochemical determination of hydrogen peroxide according to claim 1, wherein the potential of the electrode is controlled.
Using a sensing electrode and a counter electrode, the potential between the sensing electrode and the counter electrode is such that the potential of the sensing electrode is in the range of +0.35 V to -0.6 V with respect to the silver-silver chloride electrode of the saturated potassium chloride solution. The method for electrochemical determination of hydrogen peroxide according to claim 1, wherein the voltage is controlled.
4. The hydrogen peroxide electricity according to claim 1, wherein the catalyst layer uses a sensing electrode composed of amorphous iridium dioxide or amorphous and crystalline iridium dioxide. Chemical quantification method.
A sensing electrode in which the catalyst layer is composed of amorphous iridium dioxide or amorphous and crystalline iridium dioxide, and an oxide of at least one metal selected from tantalum, titanium, niobium, zirconium, and tungsten. The method for electrochemical determination of hydrogen peroxide according to any one of claims 1 to 3, wherein:
The catalyst layer uses a sensing electrode composed of amorphous iridium dioxide and amorphous tantalum pentoxide, or amorphous and crystalline iridium dioxide and amorphous tantalum pentoxide. The method for electrochemical determination of hydrogen peroxide according to any one of claims 1 to 3.
The method for electrochemical determination of hydrogen peroxide according to any one of claims 1 to 6, wherein a counter electrode on which a catalyst layer containing amorphous iridium oxide is formed is used.