JP4654411B2 - Hydrogen peroxide concentration measurement method, enzyme activity measurement method and immunochemical measurement method - Google Patents

Description

  The present invention is capable of measuring the hydrogen peroxide concentration with high sensitivity, and in particular, is capable of measuring the activity of an oxidase that gives hydrogen peroxide as a product with high sensitivity, and is useful for immunochemical measurement methods. The present invention relates to a concentration measurement method, an enzyme activity measurement method, an immunochemical measurement method, and devices thereof.

A chemiluminescence method is currently used as a highly sensitive method for measuring hydrogen peroxide. This method utilizes the fact that light is emitted when hydrogen peroxide and luminol react in the presence of a catalyst such as an iron complex. However, this method has to carry out the reaction in an alkaline environment in order to increase the luminous efficiency, leaving problems in terms of handling and waste liquid treatment.
On the other hand, the electrochemical method (current measurement method) is simple and relatively sensitive, but the conventional detection lower limit concentration is usually ^{limited to} about 10 ⁻⁷ M.

In the analysis by measuring the current of hydrogen peroxide, in order to increase the sensitivity, platinum having a high catalytic activity for the electrode reaction of hydrogen peroxide is often used as an electrode material (see, for example, Patent Document 1). When other electrode materials are used, for example, a mediator such as ferrocene is used to increase current and increase sensitivity (for example, see Patent Document 2).
However, the conventional method has a problem that although the reduction current on the electrode has a positive correlation with the hydrogen peroxide concentration up to several mM, the detection lower limit concentration remains at about 1 μM (S / N = 3).
JP-A-8-338824 Japanese Patent Laid-Open No. 11-64271

  The present invention aims to solve the above-mentioned problems, and makes use of the simplicity of the electrochemical method to further increase the sensitivity. That is, it is a simple general-purpose electrochemical device that cannot be achieved conventionally. The purpose is to enable the highly sensitive measurement of hydrogen peroxide concentration, oxidase activity, and antigen concentration.

In view of the above problems, the following new invention is provided.
(1) An electrode made of a redox active species thin film having an oxidation potential lower than that of hydrogen peroxide formed on an electron conductor substrate is immersed in a hydrogen peroxide-containing sample solution for a certain period of time, and then an electrolyte solution. And the concentration of hydrogen peroxide in the sample is measured from the reduction current response obtained when the potential is swept or stepped from the open potential to the base potential. Method for measuring hydrogen concentration.
(2) A method for measuring the activity of oxidase by detecting the concentration of hydrogen peroxide, which is an oxidation-reduction active species that exhibits a lower oxidation potential than the reduction potential of hydrogen peroxide formed on an electron conductor substrate. Obtained by immersing an electrode consisting of a thin film in a hydrogen peroxide-containing sample solution for a certain period of time and then transferring it to an electrolyte solution, using this as a working electrode, and sweeping or stepping the potential from its open potential to a base potential A method for measuring enzyme activity, comprising measuring a concentration of hydrogen peroxide in a sample from a reduction current response, and measuring an activity of an oxidase using hydrogen peroxide as an enzyme reaction product from the concentration of hydrogen peroxide.
(3) An immunochemical measurement method for measuring an oxidase activity by detecting a hydrogen peroxide concentration and determining an antigen concentration using the oxidase as a labeling enzyme for an antibody molecule, which is formed on an electron conductor substrate. After immersing an electrode consisting of a redox active species thin film showing a base oxidation potential lower than the reduction potential of hydrogen peroxide for a certain period of time in a hydrogen peroxide-containing sample solution, it is transferred to an electrolyte solution, and this is used as a working electrode. The concentration of hydrogen peroxide in the sample is measured from the reduction current response obtained when the potential is swept or stepped from the open potential to the base potential, and the hydrogen peroxide is determined as the enzyme reaction product from the hydrogen peroxide concentration. The oxidase activity is measured, and the amount of antibody bound or unbound to the antigen after the antigen-antibody reaction is determined from the measured oxidoreductase activity, thereby determining the antigen concentration.疫化 science measurement method.
(4) When measuring the concentration of hydrogen peroxide, a substance having a catalytic action for promoting the reduction of hydrogen peroxide is included in the hydrogen peroxide-containing sample solution or the redox active species thin film. The hydrogen peroxide concentration measuring method, enzyme activity measuring method or immunochemical measuring method according to any one of (1) to (3) above.
(5) The substance having a catalytic action for promoting reduction of hydrogen peroxide is a metal-porphyrin compound such as hemin, an oligopeptide such as microperoxidase containing these, or an enzyme such as horseradish peroxidase. The hydrogen peroxide concentration measuring method, enzyme activity measuring method or immunochemical measuring method according to any one of the above (1) to (4).
(6) The reduced form of the redox active species includes ferrocene and derivatives thereof or a polymer compound containing the above structure, hydroquinone and derivatives thereof or a polymer compound containing these structures, or an osmium complex of polyvinylpyridine or this structure. The method for measuring the concentration of hydrogen peroxide, the method for measuring enzyme activity or the immunochemical assay according to any one of (1) to (5) above, wherein the polymer is a polymer.
(7) The redox active species thin film is formed on the electron conductor substrate by a self-organization method, a spin coating method, or a casting method, as described in any one of (1) to (6) above Hydrogen peroxide concentration measurement method, enzyme activity measurement method or immunochemical measurement method.

The present application also provides the following novel invention.
(8) A hydrogen peroxide-containing sample solution for immersing an electrode made of a thin film of redox active species having a base oxidation potential lower than the reduction potential of hydrogen peroxide formed on an electron conductor substrate for a certain period of time, and using this as an electrolyte Electrochemical measurement cell for measuring the concentration of hydrogen peroxide in a sample from the reduction current response obtained when the potential is swept or stepped from the open potential to the base potential from the open potential of the electrode as a working electrode after being transferred into the solution The electrochemical measurement cell has the working electrode, the reference electrode, and the counter electrode, and each electrode is connected to a potentiostat for regulating the potential, and the concentration measurement of hydrogen peroxide, apparatus.
(9) A hydrogen peroxide-containing sample solution for immersing an electrode made of a redox active species thin film having a base oxidation potential lower than the hydrogen peroxide reduction potential formed on the electron conductor substrate for a predetermined time, The solution contains an oxidase to be measured, a substance having a catalytic action for promoting reduction of hydrogen peroxide, and a substrate for the enzyme. The electrode is transferred into an electrolyte solution to be used as a working electrode, and the base is removed from the open potential of the electrode. An electrochemical measurement cell for measuring the concentration of hydrogen peroxide in the sample from the reduction current response obtained when the potential is swept or stepped to a different potential, the electrochemical measurement cell comprising the working electrode, the reference electrode, An enzyme activity measuring apparatus comprising a counter electrode and each electrode connected to a potentiostat for regulating a potential.
(10) A hydrogen peroxide-containing sample solution for immersing an electrode made of a redox active species thin film having a base oxidation potential lower than the hydrogen peroxide reduction potential formed on the electron conductor substrate for a predetermined time, The solution contains an oxidase to be measured, a substance having a catalytic action for promoting the reduction of hydrogen peroxide, and a substrate for the enzyme. The electrode is transferred into an electrolyte solution to form a working electrode, and the base potential is reduced from the open potential of the electrode. An electrochemical measurement cell for measuring the concentration of hydrogen peroxide in the sample from the reduction current response obtained when the potential is swept or stepped to the potential, the electrochemical measurement cell comprising the working electrode, the reference electrode, and the counter electrode Oxidase that has electrodes, each electrode is connected to a potentiostat for regulating the potential, and hydrogen peroxide is the enzyme reaction product from the concentration of hydrogen peroxide measured in the electrochemical cell After measuring the activity, an antigen-antibody reaction using an antibody with oxidase as a labeling enzyme is performed, and after the reaction, the amount of antibody bound to the antigen or unbound is determined from the oxidoreductase activity, and the antigen concentration is determined from the amount of antibody An immunochemical measurement device characterized by being obtained.

  According to the method and apparatus of the present invention, it is possible to measure a highly sensitive hydrogen peroxide concentration, oxidase activity, and antigen concentration, which has been impossible to achieve with a simple general-purpose electrochemical device. Have

In the method for measuring the concentration of hydrogen peroxide according to the present invention, an electrode comprising a thin film of a redox active species having a base oxidation potential lower than the reduction potential of hydrogen peroxide formed on an electron conductor substrate is placed in a sample solution containing hydrogen peroxide. After being immersed in the electrolyte solution for a certain period of time, the concentration of hydrogen peroxide in the sample is determined from the reduction current response obtained when the potential is swept or stepped from the open potential to the base potential using this as the working electrode. Measure.
That is, the present invention focuses on the following mediator reaction.
H ₂ O ₂ + Mred = 2H ₂ O + Mox (1)
Mox + 2e = Mred (2)
Here, Mred and Mox represent the reduced form and the oxidized form of the mediator, respectively. If the reaction rates of (1) and (2) are both high, a higher current than that obtained by direct electrolysis of hydrogen peroxide is applied, and high sensitivity in hydrogen peroxide measurement is achieved.

In the state where the mediator is immobilized on the electrode, first, only the reaction of (1) is caused to accumulate Mox on the electrode, and then the accumulated Mox is rapidly reduced by applying a negative potential to the electrode. By measuring the transient current response at that time or the amount of electricity integrated therewith, a large signal (transient current response) is obtained, and measurement of hydrogen peroxide at a significantly low concentration is possible.
This conceptual diagram is shown in FIG. Charges are accumulated in the left diagram of FIG. 1, and then electrochemically reduced in the right diagram. This makes it possible to measure the hydrogen peroxide concentration, oxidase activity, and antigen concentration with high sensitivity that could not be achieved in the past.

As a specific apparatus for measuring the concentration of hydrogen peroxide according to the present invention, an electrode composed of a thin film of redox active species having a base oxidation potential lower than the reduction potential of hydrogen peroxide formed on an electron conductor substrate. When a sample solution containing hydrogen peroxide for immersion for a certain period of time, a container therefor, and the treated electrode are transferred into an electrolyte solution to be used as a working electrode, and the potential is swept or stepped from the open potential of the electrode to a base potential The electrochemical measurement cell for measuring the concentration of hydrogen peroxide in the sample from the reduction current response obtained in (1).
The electrochemical measurement cell may be provided with the working electrode, the reference electrode, and the counter electrode, and each electrode may be configured by connecting a potentiostat for regulating the potential.

The activity of the oxidase using hydrogen peroxide as an enzyme reaction product can be measured from the concentration of hydrogen peroxide thus obtained. Further, from the measured oxidoreductase activity, the amount of antibody bound or unbound to the antigen after the antigen-antibody reaction can be determined, and thereby the antigen concentration can be determined.
In such an immunochemical measurement system using an oxidase as a labeling enzyme, such high sensitivity of enzyme activity leads to high sensitivity of immunochemical measurement, and can greatly contribute to the medical and environmental fields.

As a specific example of the enzyme activity measuring apparatus, the same apparatus as the hydrogen peroxide concentration measuring apparatus can be used. It should be understood that the following apparatus is an example, and that the present invention is not limited to this example apparatus.
A sample solution containing hydrogen peroxide for immersing an electrode made of a thin film of redox active species having an oxidation potential lower than that of hydrogen peroxide formed on an electron conductor substrate for a certain period of time and a container for holding the sample solution prepare.
The solution contains an oxidase to be measured, a catalyst that promotes reduction of hydrogen peroxide as necessary, and an enzyme substrate. In addition, the concentration of hydrogen peroxide in the sample is measured from the reduction current response obtained when the electrode is moved into the electrolyte solution and used as the working electrode, and the potential is swept or stepped from the open potential to the base potential. An electrochemical measurement cell is installed.
The working electrode, the reference electrode, and the counter electrode are arranged in the electrochemical measurement cell. Each electrode is connected to a potentiostat for regulating the potential.

Furthermore, the same immunochemical measuring apparatus as that of the enzyme activity measuring apparatus can be used. It should be understood that the following apparatus is an example, and that the present invention is not limited to this example apparatus.
This apparatus includes a hydrogen peroxide-containing sample solution for immersing an electrode made of a thin film of redox active species having an oxidation potential lower than that of hydrogen peroxide formed on an electron conductor substrate for a predetermined time.
The solution contains an oxidase to be measured, a catalyst that promotes reduction of hydrogen peroxide as necessary, and an enzyme substrate, and the electrode is transferred into an electrolyte solution to form a working electrode. Furthermore, an electrochemical measurement cell is provided for measuring the concentration of hydrogen peroxide in the sample from the reduction current response obtained when the potential is swept or stepped from the open potential to the base potential. In the electrochemical measurement cell, the working electrode, the reference electrode, and the counter electrode are arranged, and each electrode is connected to a potentiostat for regulating the potential.
The activity of oxidase using hydrogen peroxide as an enzyme reaction product is measured from the concentration of hydrogen peroxide measured in the electrochemical cell. Then, an antigen-antibody reaction using an antibody having an oxidase as a labeling enzyme is performed, and after the reaction, the amount of the antibody bound or unbound to the antigen is determined from the oxidoreductase activity, and the antigen concentration is determined from the amount of the antibody.

As described above, when measuring the concentration of hydrogen peroxide, a catalyst that promotes the reduction of hydrogen peroxide can be contained in the hydrogen peroxide-containing sample solution or in the thin film of redox active species. As this catalyst, metal-porphyrin compounds such as hemin, or oligopeptides such as microperoxidase containing these, ligand-binding heme proteins such as myoglobin and heme-substituted myoglobin, and enzymes such as horseradish peroxidase are effective. .
In addition, the reduced form of the redox active species includes ferrocene and its derivatives or polymer compounds containing the above structure, hydroquinone and its derivatives or polymer compounds containing these structures, or an osmium complex of polyvinylpyridine or this structure. Polymers can be used.
As a means for forming a redox active species thin film on the electron conductor substrate, a self-assembly method, a spin coating method, and a casting method can be used, which is effective.

  Hereinafter, the present invention will be described based on examples. In addition, the following description is for making an understanding of this invention easy, and is not restrict | limited to this. That is, modifications, embodiments, and other examples based on the technical idea of the present invention are included in the present invention.

Example 1
(Preparation of hydrogen peroxide measuring electrode and high sensitivity measuring method)
Gold disk electrode (diameter: 1.6 mm, manufactured by BAS, trade name: AUE gold electrode) is alumina-polished, ultrasonically cleaned, and electrochemically cleaned in sulfuric acid by an ordinary method to obtain 1 mM ferroceneundecanethiol in anhydrous hexane. A ferrocene self-assembled monolayer-coated electrode was fabricated by immersion for 2 hours. This redox potential was estimated to be +400 mV vs. Ag / AgCl (solution is 75 mM PB + 50 mM NaClO4, pH 7).

Add each concentration of hydrogen peroxide to the above aqueous solution (10 mL) to which 1 μM hemin has been added as a catalyst, insert the modified electrode (in the state of release potential), leave it for 15 hours, and then change the potential from the release potential to +100 step _{mV vs. Ag / AgCl, H 2} O 2 + 2Fc + 2H + = 2H 2 O + 2Fc + current re-reduced to Fc ⁺ produced in the reaction - was measured time curve (see Figure 2).
As the measurement system, a potentiostat (Hokuto Denko, trade name: HA-150) and a digital oscilloscope (IWATSU, trade name: DS-8812) were used.

When the amount of electricity was determined from the current-time curve and the relationship between the amount of hydrogen and the concentration of hydrogen peroxide was determined, as shown in FIG. 3, a marked increase in the amount of electricity was observed as the concentration increased to about 15 nM. The lower detection limit was estimated to be 0.5 nM (S / N = 3).
Compared with the general-purpose amperometry method shown below, this method has made it possible to measure the hydrogen peroxide concentration at a concentration of 1/1000 or less.

(Comparative Example 1)
An electrode similar to the above was prepared, and amperometric measurement was performed while adding a constant concentration of hydrogen peroxide at +100 mV vs. Ag / AgCl in the presence of hemin.
Although the reduction current on the electrode had a positive correlation with the hydrogen peroxide concentration up to several mM, the detection lower limit concentration remained at 1 μM (S / N = 3).

(Example 2)
An experiment similar to that in Example 1 was performed using a gold comb electrode (manufactured by NTT-AT, trade name: comb electrode Au). Similar results to Example 1 were obtained with an immersion time of 20 minutes by increasing the diffusion rate of hydrogen peroxide / hemin to the microelectrode.

(Example 3)
The same experiment as in Example 2 was performed using ₂ -mercaptohydrotoquinone (H ₂ Q) instead of ferrocene undecanethiol used in Example 2.
However, a voltage of −100 mV was applied to re-reduction of the quinone produced by the reaction of H ₂ O ₂ + H ₂ Q = 2H ₂ O + Q. As a result, the same result as in Example 2 was obtained.

Example 4
Add the appropriate amount of glucose oxidase to the buffer solution of Example 1 (10 mL) to which 1 μM hemin and 10 mM glucose have been added and let stand for 1 hour. Analyzed. As a result, even an enzyme with an activity of 500 nU / L could be measured. The sensitivity was increased by 1000 times compared to general-purpose amperometry and spectroscopy. An enzyme activity of 500 nU / L corresponds to 50 fg as the amount of enzyme in the reaction solution.

(Example 5)
After reacting with ANP using an anti-ANP antibody modified with glucose oxidase, removing the unreacted antibody by the ANP immobilization base, only the solution containing the reacted antibody was recovered, and this glucose oxidase activity was Measured by the same method as in No. 4, the relationship between enzyme activity and ANP concentration was examined. As a result, it was possible to measure ANP up to 0.1 ppt.

The present invention is particularly important in the field of biochemistry for the measurement of hydrogen peroxide concentration, especially its high sensitivity. For example, high-sensitivity measurement of hydrogen peroxide concentration enables high-sensitivity measurement of the activity of oxidase that gives hydrogen peroxide as a product, which often contributes to efficient screening of oxidase required for enzyme analysis. .
Furthermore, in an immunochemical measurement system using an oxidase as a labeling enzyme, increasing the sensitivity of the enzyme activity leads to higher sensitivity of the immunochemical measurement and greatly contributes to the medical and environmental fields.

It is a conceptual explanatory view of the hydrogen peroxide concentration measurement of the present invention. FIG. 4 is a graph showing the results of current-time response measured in Example 1. 3 is a graph showing the relationship between the hydrogen peroxide concentration measured in Example 1 and the measured electric quantity.

Claims (7)

  An electrode made of a thin film of a redox active species having an oxidation potential lower than that of hydrogen peroxide formed on an electronic conductor substrate is immersed in a hydrogen peroxide-containing sample solution containing an electrolyte for a certain period of time. A method for measuring the concentration of hydrogen peroxide, comprising measuring the concentration of hydrogen peroxide in a sample from a reduction current response obtained when the potential is swept or stepped from its open potential to a base potential as a working electrode. In measuring the concentration of hydrogen peroxide, according to claim 1, characterized in that the inclusion of substances having a catalytic action for promoting the reduction of hydrogen peroxide in the thin film of hydrogen peroxide-containing sample solution or redox active species The method for measuring the concentration of hydrogen peroxide as described in 1. The substance having a catalytic action for promoting reduction of hydrogen peroxide is a metal-porphyrin compound such as hemin, an oligopeptide such as microperoxidase containing these, or an enzyme such as horseradish peroxidase. 2. The method for measuring the concentration of hydrogen peroxide according to 2 . The reduced form of the redox active species is ferrocene and derivatives thereof or a polymer compound containing the above structure, hydroquinone and derivatives thereof or a polymer compound containing these structures, or an osmium complex of polyvinylpyridine or a polymer containing this structure. The method for measuring the concentration of hydrogen peroxide according to any one of claims 1 to 3 , wherein: The electron conductor substrate, self-organizing method, a spin coating method, the concentration determination of hydrogen peroxide according to any one of claims 1 to 4, characterized in that to form a redox active species of the thin film by casting Law. A method for measuring the activity of an oxidase by detecting a hydrogen peroxide concentration, wherein the concentration of hydrogen peroxide in a sample is measured by the method according to any one of claims 1 to 5 , and the peroxidation is performed. A method for measuring enzyme activity, comprising measuring the activity of an oxidase using hydrogen peroxide as an enzyme reaction product from the concentration of hydrogen. Measuring the activity of the oxidase by detecting the hydrogen peroxide concentration, an immunochemical assay to determine the antigen concentration oxidizing enzyme as a labeling enzyme for the antibody molecule, according to any of claims 1 to 5 The concentration of hydrogen peroxide in the sample is measured by this method, and the activity of oxidase using hydrogen peroxide as an enzyme reaction product is measured from the concentration of hydrogen peroxide. An immunochemical assay, wherein the amount of antibody for binding is determined from the measured oxidoreductase activity, thereby determining the antigen concentration.

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