JP2000321234A - Polyphenol sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily and rapidly measure polyphenol with high sensitivity by equipping an enzyme electrode containing an enzyme for decomposing hydrogen peroxide and an electron mediator and containing the enzyme for decomposing the hydrogen peroxide and the hydrogen peroxide in a reaction bath. SOLUTION: The polyphenol sensor is provided with an enzyme electrode, a reference electrode, and an outer electrode as counter electrodes, and the enzyme electrode simultaneously contains an enzyme for the hydrogen peroxide and an electron mediator. The enzyme and the electron mediator include peroxytase or the like and metallocene such as ferrocene, respectively, and the electron mediator accelerates the migration of electrons between the enzyme and the electrode. On measurement, an enzyme electrode is dipped into a buffer liquid that does not contain any samples, and a constant amount of hydrogen peroxide is added to the reaction bath. Then, after a measurement sample liquid containing polyphenol or that being diluted by the buffer liquid is added, and a steady current after a fixed amount of time passes is measured, thus easily measuring polyphenol concentration in a sample solution in approximately 4-7 minutes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polyphenol sensor for electrochemically quantifying polyphenol in a sample solution. The present invention also relates to a method for measuring polyphenol.

[0002]

2. Description of the Related Art Polyphenols are a general term for compounds having a plurality of hydroxyl groups on a benzene ring, and those corresponding to a wide range such as tannins, flavonoids, lignans, ligulins, and coumarins. In recent years, it has been revealed that polyphenols are superior to α-tocopherol and ascorbic acid in antioxidant action by radical scavenging action, such as preventing oxidation of low-density lipoprotein having an atherosclerosis accelerating action. Polyphenols are contained in many edible plants and medicinal plants, and with increasing health consciousness, the effects of red wine and tea rich in polyphenols are attracting attention.

[0003] Conventionally, as a method for measuring these polyphenols, Folin Ciocalteau has been used.
A spectrophotometric method of expressing the polyphenol content of a sample solution by the amount of gallic acid using a method (J. Biol. Chem., 73, 627 (1927)) is mainly used. However, in this method, the influence of the turbidity and coloring of the sample liquid may become a problem, and there is a disadvantage that the reaction time requires 2 to 3 hours.

[0004] As a method for separating and quantifying individual polyphenols, high performance liquid chromatography (HPLC) for detecting by UV (ultraviolet) absorption or the like is used (Chroma).
torgaphia, 34, 146 (1992), J. Chromatogr., 642, 175 (199
3), JP-A-10-287605). However, HPLC measurement devices are expensive and require specific equipment and locations. In this method, a suitable solvent differs depending on the target phenol, and a measurement time of one hour or more is required.

On the other hand, an electrochemical measurement method using electrodes has been proposed as a method that can perform measurement more easily and in a shorter time. The influence of turbidity and coloring of the sample solution poses a problem in the above-mentioned Folin-Tiocult method and the HPLC method, but these effects do not pose a problem in the electrochemical measurement method. The electrochemical measurement method has an advantage that it can be performed regardless of a measurement place because instruments are compact and easy to carry. The measuring instruments also have the advantage of being inexpensive as compared with the instruments used in the above-described measuring method. Further, the measurement time of the sample solution can be reduced to several minutes or several tens of minutes. That is, the electrochemical measurement method has the advantages of being simple, rapid, and highly sensitive.

As an electrochemical measurement method, measurement of phenol or polyphenol has been attempted (Anal. Che.
m., 53, 1695 (1981), J. Chromatogr., 360, 271 (1986)).
However, in order to directly measure the oxidation of phenol, it is necessary to apply a positive potential of 650 to 1100 mV vs. Ag / AgCl. At this potential, substances other than phenol contained in the measurement solution may be oxidized. And the concentration of phenols could not be determined accurately.

[0007] Therefore, when a phenol sensor in which one or two enzymes capable of oxidizing only a specific phenol are immobilized on an electrode is used to electrochemically reduce oxidized phenol produced by an enzymatic reaction, it flows. A method of measuring current has recently been used (Anal. Chim.
Acta., 311,245 (1995), Anal.Chim. Acta., 347, 51 (199
7)). However, the oxidized form of the phenol produced by the enzymatic reaction is easily polymerized, so that it is not reduced again and becomes an electrochemically inactive polymer, and the resulting current value does not accurately reflect the amount of the phenol. There was a problem.

[0008] As another electrochemical measurement method, a biosensor utilizing the polyphenol oxidase activity of plant tissue has been reported (Japanese Patent Publication No. 7-111415), but in this case, stability for a long period of several weeks. And reproducibility problems.

[0009]

SUMMARY OF THE INVENTION In view of the above circumstances, the present invention can measure polyphenol more easily and quickly,
Moreover, it is an object of the present invention to provide a polyphenol sensor capable of measuring with high sensitivity.

[0010]

Means for Solving the Problems The present inventors, in a method for measuring polyphenols, used a peroxidase (hereinafter, referred to as peroxidase).
An enzyme electrode containing an enzyme that decomposes hydrogen peroxide such as POD) and an electron mediator such as ferrocene (hereinafter also referred to as Fc) that promotes the electron transfer between the enzyme and the electrode is used. In order to solve the above-described problems, an enzyme and a polyphenol that decompose hydrogen peroxide are added to a reaction tank in the presence of hydrogen oxide, and the decrease in hydrogen peroxide concentration after a certain period of time is electrochemically measured. They have found that they can do this and have completed the present invention.

That is, the present invention has the following configuration. (1) A polyphenol sensor having an enzyme electrode containing an enzyme for decomposing hydrogen peroxide and an electron mediator, and further comprising an enzyme for decomposing hydrogen peroxide and hydrogen peroxide in a reaction tank. (2) The polyphenol sensor according to (1), wherein the enzyme that decomposes hydrogen peroxide is peroxidase. (3) The polyphenol sensor according to (1) or (2), wherein the electron mediator is ferrocene. (4) The polyphenol sensor according to any one of (1) to (3), wherein an enzyme for decomposing hydrogen peroxide at the enzyme electrode and an electron mediator are kneaded into the carbon paste. (5) The enzyme electrode is covered with a dialysis membrane (1)-
The polyphenol sensor according to any of (4). (6) Using an enzyme electrode containing an enzyme that decomposes hydrogen peroxide and an electron mediator, adding an enzyme and a sample solution that decompose hydrogen peroxide to a reaction vessel containing a certain concentration of hydrogen peroxide, and after a certain period of time A polyphenol sensor that detects polyphenols in a sample solution by electrochemically measuring the decrease in hydrogen peroxide concentration in the sample. (7) The polyphenol sensor according to (6), wherein the enzyme that decomposes hydrogen peroxide is peroxidase. (8) The polyphenol sensor according to (6) or (7), wherein the electron mediator is ferrocene. (9) The polyphenol sensor according to any one of (6) to (8), wherein an enzyme for decomposing hydrogen peroxide at the enzyme electrode and an electron mediator are kneaded into the carbon paste. (10) The enzyme electrode is covered with a dialysis membrane (6)-
The polyphenol sensor according to any one of (9). (11) Using an enzyme electrode containing an enzyme that decomposes hydrogen peroxide and an electron mediator, adding an enzyme and a sample solution that decompose hydrogen peroxide to a reaction vessel containing hydrogen peroxide at a certain concentration, and after a certain period of time A method for measuring polyphenol, characterized in that the concentration of polyphenol in a sample solution is quantified by electrochemically measuring the decrease in the concentration of hydrogen peroxide.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, an enzyme electrode used in the polyphenol sensor of the present invention will be described, and then the polyphenol sensor of the present invention will be described.

(1) Enzyme electrode The enzyme electrode used in the polyphenol sensor of the present invention is characterized by simultaneously containing an enzyme that decomposes hydrogen peroxide (hereinafter referred to as an enzyme) and an electron mediator. Examples of the enzyme include peroxidase and catalase. The electron mediator is a substance that promotes electron transfer between the enzyme and the electrode and has high reactivity with the electrode, and examples thereof include metallocenes such as ferrocene and ferrocene derivatives.

The enzyme electrode is for allowing an enzyme and an electron mediator to exist on the electrode surface, and the form is not particularly limited. For example, a solution containing an enzyme may be placed on an electrode, dried, and coated with a dialysis membrane. Although the molecular weight cut off of the dialysis membrane is not particularly limited,
About 0 is preferable.

The polyphenol sensor of the present invention using the enzyme electrode obtained as described above will be described. (2) Polyphenol Sensor The polyphenol sensor of the present invention has a reference electrode and a counter electrode in addition to the enzyme electrode serving as a working electrode. In order to measure the concentration of polyphenol in a sample solution using the enzyme electrode of the present invention, first, the enzyme electrode is immersed in a buffer containing no sample, and a certain amount of hydrogen peroxide is added. Subsequently, a measurement sample solution containing a polyphenol or a measurement sample solution diluted with a buffer solution is added, and the concentration of polyphenol in the sample solution can be measured by measuring a steady current after a certain period of time.

That is, when hydrogen peroxide is added to the reaction tank outside the enzyme electrode prepared as described above, the hydrogen peroxide passes through the dialysis membrane and comes into contact with the enzyme of the enzyme electrode. Hydrogen peroxide inside the dialysis membrane is reduced by an enzyme, and the resulting oxidized enzyme reacts with ferrocene to produce ferrocenium ions. When a potential capable of reducing ferrocenium ion to ferrocene is applied to the electrode, the ferrocenium ion is reduced again to ferrocene on the electrode surface, and a reduction current is observed. The current value in the steady state at this time is proportional to the concentration of hydrogen peroxide in the reaction tank.

Furthermore, when a measurement sample solution containing polyphenol is added to a reaction vessel together with a certain amount of enzyme, if the polyphenol acts as an electron donor, the oxidized enzyme produced by the reaction with hydrogen peroxide is immediately reduced. , Further reacting with hydrogen peroxide. As a result, the concentration of hydrogen peroxide in the reaction tank decreases in proportion to the amount of polyphenol, and the concentration of hydrogen peroxide passing through the dialysis membrane also decreases. as a result,
The reduction current value of ferrocenium ion decreases, and the amount of reduction reflects the concentration of polyphenol in the reactor. By this method, the amount of polyphenol contained in the sample can be measured indirectly.

The polyphenol sensor according to the present invention does not directly measure the oxidized form of polyphenol produced by the enzymatic reaction, and thus is not affected by the polymerization of the oxidized polyphenol. Further, since the sample component is separated from the electrode by the dialysis membrane, it is not affected by turbidity or coloring of the sample solution. Further, since the polyphenol contained in the sample solution can be measured in about 4 to 7 minutes, the measurement can be performed more easily and quickly than the conventional Folin-thiocult method or liquid chromatography method.

[0019]

The present invention will be specifically described below with reference to examples. An example of an enzyme electrode used in a polyphenol sensor and an example of a polyphenol measurement configuration having the enzyme electrode will be described with reference to the drawings.

(1) Peroxidase-ferrocene (P
OD-Fc) Preparation of enzyme electrode Ferrocene, horseradish peroxidase, liquid paraffin and graphite powder were mixed at a weight ratio of 4: 4: 20: 40 and kneaded sufficiently, and then commercially available carbon paste electrode (1) (BAS Made in the recess (3 mm inside diameter)
The surface was rubbed with paraffin paper to make it smooth, and the surface was further coated with a circular dialysis membrane (3) (product of Spectrum) having a diameter of 6 mm and a molecular weight cut off of 100, and further coated with a nylon net (4). PO stopped with ring (5)
A D-Fc enzyme electrode was prepared (FIG. 1).

(2) Cell for measuring polyphenol The POD-Fc enzyme electrode (6) prepared as described above,
Each of the reference electrode (7) and the counter electrode (8) was connected to a potentiostat (9), and immersed in a reaction tank containing a buffer solution to obtain a measurement cell (FIG. 2). A recorder (10) was connected to the potentiostat to record the change in the current value. Place the reactor on a magnetic stirrer (11)
And a stir bar (12) in the buffer of the reaction tank.
And the sample solution was stirred during the measurement of polyphenol.

In all the embodiments described below,
The following measurement conditions were used. Except for the measurement, the POD-Fc enzyme electrode was stored at 4 ° C. Applied potential: 0.1 V vs. Ag / AgCl buffer: 0.1 M phosphate buffer (pH 6.9) Stirring speed: 887 rpm Measurement temperature: room temperature

(Example 1) P produced as described above
Under the measurement conditions described above, the POD-Fc electrode was first immersed in a buffer containing no sample under the measurement conditions described above in the OD-Fc enzyme electrode (FIG. 1) and the measurement cell (FIG. 2) configured as described above.
A potential of 1 V was applied. The background current stabilized within 1 minute, after which a constant amount of hydrogen peroxide was added to the reactor. The concentration of hydrogen peroxide to be added is desirably 20 μM or less. After the current change becomes stable, (+)-
A sample solution containing catechin was added.

FIG. 3 shows current-time curves obtained by adding a certain amount of hydrogen peroxide to a buffer solution and then adding different concentrations of (+)-catechin. When the sample solution containing (+)-catechin was added, a reduction in reduction current was observed (increased in the positive direction).

In the absence of hydrogen peroxide, no change in current was observed even when a sample solution containing (+)-catechin was added. This decrease in current was due to the electrochemical change of (+)-catechin itself. It is not the result of the reaction, but the result of the decrease in the concentration of hydrogen peroxide in the dialysis membrane on the electrode due to the addition of (+)-catechin. Since the method of the present invention does not directly measure the oxidized polyphenol, it is not affected by the polymerization of the oxidized polyphenol. In addition, the method for measuring polyphenol of the present invention can measure polyphenol more easily and quickly.

Example 2 In order to quantify the concentration of (+)-catechin, a calibration curve was prepared using sample solutions containing different amounts of (+)-catechin. The measurement was performed in exactly the same manner as in Example 1 above. Figure 4 shows the steady-state current value and (+)
-Relationship between catechin concentrations is shown. FIG. 4 also shows an enlarged calibration curve of 20 μM or less. The experimental value is 3
The average value and the standard deviation are shown by error bars.
From the calibration curve shown in FIG. 4, (+)-catechin increased linearly in the concentration range up to 20 μM, and the detection limit was 0.3 μM.
It was shown to be. The time required to reach 90% of the steady-state current (hereinafter referred to as reaction time) was 160 seconds at a (+)-catechin concentration of 10 μM, 137 seconds at a concentration of 20 μM,
At 50 μM, it was 94 seconds.

Example 3 The detection characteristics of the POD-Fc enzyme electrode for seven kinds of polyphenols or phenol compounds other than (+)-catechin contained in wine and tea were examined. The measurement was performed in exactly the same manner as in Example 1 above. As a result, this POD-Fc enzyme electrode was as sensitive as (+)-catechin to (−)-epicatechin and caffeic acid. Meanwhile, tannic acid,
For gallic acid, lower detection sensitivity was obtained compared to the above three compounds. Vanillic acid, syringeic acid, p
-Almost no detectable for hydroxybenzoic acid. The reaction time was 5 minutes or less.

Example 4 One kind of commercially available white wine and 3
The concentration of polyphenols contained in different kinds of red wine was measured. The measurement was performed in the same manner as in Example 1 described above. One half of white wine and one tenth of red wine were added. These reaction times were less than 5 minutes. In order to convert the apparent concentration of polyphenol from the obtained steady-state current value, the current of (+)-catechin in Example 2 was used.
A concentration calibration curve was used. Table 1 shows the concentration of all polyphenols in the obtained wine.

[0029]

[Table 1]

Example 5 The polyphenol concentration in a commercially available tea beverage was measured. The measurement was performed in Example 1 described above.
The same was done. Nine different types of commercially available tea beverages, specifically, four types of green tea, one type of oolong tea, and four types of mixed tea, were used as measurement sample liquids. In the case of green tea or oolong tea, it was diluted and added to one fifth, and the mixed tea was added without diluting the stock solution. These reaction times were less than 5 minutes. Table 1 shows the values of the total polyphenol concentration obtained from the measurement of each tea beverage.

(Example 6) In order to examine the stability of the enzyme electrode, a POD-Fc enzyme electrode was prepared by the method described above.
Stored at 4 ° C. When the response of (+)-catechin was measured for two weeks after the preparation of the electrode, no change was observed in the measured current value within the measurement period, and good reproducibility was obtained.

(Comparative Example) The measurement results of the polyphenol concentration obtained by the electrochemical measurement method using the POD-Fc enzyme electrode of the present invention were compared with the results determined by a spectroscopic measurement method using a phenol reagent. Evaluation of polyphenol concentration by a spectroscopic measurement method followed the Folin-Ciocult method. A calibration curve of (+)-catechin absorbance / concentration was used to convert the apparent polyphenol concentration from the obtained absorbance.

Table 1 also shows the values of the total polyphenol concentration determined by the spectroscopic method using the wine and tea beverages used in Examples 4 and 5 as measurement samples. As is clear from the comparison in Table 1, the value of the polyphenol concentration obtained by the electrochemical measurement method using the POD-Fc enzyme electrode of the present invention almost coincided with the value obtained by the spectroscopic measurement method. This indicates that the electrochemical measurement method using the enzyme electrode of the present invention can be applied as a method for measuring the concentration of polyphenol in a test sample solution.

[0034]

As described above, the present invention provides a polyphenol sensor capable of measuring polyphenol contained in a measurement sample solution simply, quickly and with high sensitivity. Further, the method for measuring polyphenol of the present invention is an excellent measuring method which enables accurate measurement without being affected by easy polymerization of oxidized polyphenol.

[Brief description of the drawings]

FIG. 1 shows the PO used in the polyphenol sensor of the present invention.
It is a figure showing an example of a D-Fc enzyme electrode.

FIG. 2 is a view showing an embodiment of the polyphenol sensor of the present invention.

FIG. 3 shows (+) using the polyphenol sensor of the present invention.
-It is a figure which shows the result of a catechin measurement.

FIG. 4 shows (+) using the polyphenol sensor of the present invention.
-It is a figure which shows the calibration curve of a catechin density | concentration and a steady-state current.

[Explanation of symbols]

1. 1. Carbon paste electrode 2. ferrocene, peroxidase, liquid paraffin and graphite Dialysis membrane Nylon net 5. O-ring 6. Enzyme electrode 7. Reference electrode 8. Counter electrode 9. Potentiostat 10. Recorder 11. Magnetic stirrer 12. Stir bar

Continued on the front page. (72) Inventor Kenji Kano 6-3-504, Shichitakeshita Takashicho, Kita-ku, Kyoto-shi, Kyoto (72) Inventor Atsushi Ikeda 10-5 F-term (Kamitakahatacho, Sakyo-ku, Kyoto-shi, Kyoto-fu) Reference) 4B063 QA01 QQ16 QQ61 QR02 QR50 QR84 QS39 QX04

Claims (11)

[Claims] 1. A polyphenol sensor comprising an enzyme electrode containing an enzyme for decomposing hydrogen peroxide and an electron mediator, and a reaction vessel containing an enzyme for decomposing hydrogen peroxide and hydrogen peroxide. 2. The polyphenol sensor according to claim 1, wherein the enzyme that decomposes hydrogen peroxide is peroxidase. 3. The polyphenol sensor according to claim 1, wherein the electron mediator is ferrocene. 4. The polyphenol sensor according to claim 1, wherein the enzyme electrode is an enzyme that decomposes hydrogen peroxide and an electron mediator are kneaded into the carbon paste. 5. The polyphenol sensor according to claim 1, wherein the enzyme electrode is covered with a dialysis membrane. 6. An enzyme electrode containing an enzyme that decomposes hydrogen peroxide and an electron mediator, an enzyme that decomposes hydrogen peroxide and a sample solution are added to a reaction vessel containing hydrogen peroxide at a certain concentration, A polyphenol sensor that detects polyphenol in a sample solution by electrochemically measuring the decrease in hydrogen peroxide concentration after a lapse of time. 7. The polyphenol sensor according to claim 6, wherein the enzyme that decomposes hydrogen peroxide is peroxidase. 8. The polyphenol sensor according to claim 6, wherein the electron mediator is ferrocene. 9. The polyphenol sensor according to claim 6, wherein the enzyme electrode and the electron mediator, which decompose hydrogen peroxide, are kneaded into the carbon paste. 10. The polyphenol sensor according to claim 6, wherein the enzyme electrode is covered with a dialysis membrane. 11. Using an enzyme electrode containing an enzyme that decomposes hydrogen peroxide and an electron mediator, an enzyme and a sample solution that decompose hydrogen peroxide are added to a reaction vessel containing a certain concentration of hydrogen peroxide, A method for measuring polyphenols, characterized by quantifying the concentration of polyphenol in a sample solution by electrochemically measuring the decrease in hydrogen peroxide concentration after a lapse of time.