JP3093086B2 - Electric analysis method using battery cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electric analysis method,
The present invention relates to an electroanalysis method for quantifying the amount of a target sample substance (chemical species) by measuring a change in voltage, current, or amount of electricity using a battery cell.

[0002]

2. Description of the Related Art In a measurement using an electroanalytical method, the original mass of a sample is measured by measuring an electrical change when a reaction such as oxidation-reduction occurs on the sample material under a constant voltage or a constant current. A method based on electrolysis for quantification is generally known, and recently applied to a measurement method using a conductive porous electrode or the like.

[0003] In the case of an ion electrode, a method of immersing an electrode serving as a detection unit in a sample and electrically measuring an equilibrium potential between ions passing through the diaphragm and ions generated from the electrode film and the sample substance is generally widely used. Have been. In the method using a conductive porous electrode, a method is known in which a sample is directly electrolyzed and measured in a detection chamber in which a conductive porous body is impregnated with an electrolytic solution (JP-A-1-195358). I have. However, in the method of Japanese Patent Application Laid-Open No. 1-195358, electric energy must be externally applied, and a long time is required for stabilizing the measuring device.

[0004] In general, as a reaction mediated by a mediator, nicotinamide adenine dinucleotide (NAD) is reduced to nicotinamide adenine dinucleotide (NADH) in an enzyme reaction system using a dehydrogenase.
Glutamic acid measurement kit (manufactured by BMY) using a colorimetric method for quantifying glutamic acid using, for example, glutamate dehydrogenase
Are commercially available.

[0005]

However, in the above-mentioned ion electrode, the speed of ion movement in the diaphragm and the response speed until the equilibrium is reached are slow, and a long time is required for the measurement. There were problems such as necessity. In addition, when measuring by electroanalysis using electrolysis, apply a constant voltage to the electrodes or apply a constant current to the electrodes in order to perform the electrolysis of the target sample substance. It is necessary to keep
A non-equilibrium state is established between the two electrodes, and a so-called capacitive current is generated. Therefore, there is a large problem that a long stabilization time is required every time a voltage is applied before a target sample substance can be electrolyzed and measured. There is also a problem that the device itself becomes complicated, such as the need for a constant voltage generator or a constant current generator.

[0006] In the method of measurement using an enzymatic reaction mediated by a mediator, NADH produced by the enzymatic reaction is usually further reacted with diaphorase or the like.
Thereafter, a complicated operation such as measuring the absorbance was required. In addition, when these enzymes are used, the preservability of the enzymes to be used is poor, and refrigerated storage is necessary. Therefore, they cannot be applied to sensors intended to be used at ordinary temperature at ordinary temperature, and there is a problem in practicality. Further, when measuring the absorbance, there is a problem that an accurate measurement value cannot be obtained when a coloring substance is present in the sample.

The present invention solves the above-mentioned problems in the prior art, and does not require external electric energy such as voltage or current, and simply, quickly and quantitatively determines a sample substance to be measured. It is an object of the present invention to provide an electric analysis method capable of performing the following.

[0008]

Means for Solving the Problems In order to achieve the above object, the present inventors have conducted intensive studies and as a result, have found that the redox reaction between the components of the working electrode of the analytical electrode and the sample substance to be quantified. A potential difference was created between the working electrode and the counter electrode based on the difference in electrode chemical potential, and it was found that the amount of electricity generated by the potential difference was closely related to the quantification of the sample substance, and the components of both electrodes were selected. As a result, they found that the electrode reaction spontaneously and rapidly progressed, and completed the present invention.

That is, the present invention is selected from the group consisting of a working electrode which is a liquid containing a sample substance, hexacyanoferrate (III) ion, hexacyanoferrate (II) ion, permanganate ion or a mixture thereof. Using a battery cell composed of a counter electrode, which is a liquid containing a battery active material, and an ion-permeable diaphragm in contact with both electrodes, between the two electrodes without applying an external voltage between the two electrodes An electrical analysis method for a sample substance, characterized in that at least one or more of the generated voltage, current, and electric quantity are measured to quantify the sample substance.

Further, the present invention provides an N-type battery using the above-mentioned battery cell.
This is an electroanalysis method of glutamic acid in which glutamic acid is measured using ADH or a derivative thereof as an index. Further, the present invention, in the above battery cell, including an electrolyte in the working electrode,
This is a COD electrical analysis method for measuring chemical oxygen demand (COD) using potassium permanganate as an index using the above-mentioned battery cell using hexacyanoferrate (II) ion as a battery active material of a counter electrode.

Further, the present invention provides a battery using the above-mentioned battery cell in which an electrolyte is contained in a working electrode and hexacyanoferrate (II) ion and hexacyanoferrate (III) ion are used as a battery active material of a counter electrode. This is a COD electrical analysis method in which COD is measured using potassium manganate as an index. Further, the present invention provides the above battery cell, wherein the working electrode contains an electrolyte and / or 3,7 bis (dimethylamino) phenothiazine-5-ium chloride, and the counter electrode battery active material contains hexacyanoferrate (III) ion. An ascorbic acid electroanalytical method for measuring ascorbic acid using the above-mentioned battery cell using the above method.

A sample substance is a substance (chemical species) that generates a battery active material by reacting with a battery active material or a mediator. The sample substance discharges electric energy generated when the sample substance is discharged in a short time by a battery reaction. This is an electrical analysis method whose main purpose is to measure and quantify the amount. Therefore, it is based on a completely different principle from the conventional method of electrolyzing and quantifying a sample by applying electric energy from the outside.

The working electrode used in the present invention is an electrode containing the above-mentioned sample substance, and the working electrode can contain an electrode solution and / or a mediator. The electrode solution may be any electrolyte solution, for example, hydrochloric acid, sulfuric acid, potassium chloride solution or phosphate buffer solution. Examples of mediators include 5-methylphenazinium methyl sulfate (phenazine methosulfate), 1-methoxy-5-methylphenazinium methyl sulfate (methoxyphenazine methosulfate), and 9-dimethylaminobenzo [α] phenoxazine-7. -Ium chloride (Meldra blue) or 3,7 bis (dimethylamino) phenothiazine-5-ium chloride (methylene blue) and salts thereof can be used.

The counter electrode is an electrode containing a battery active material selected from the group consisting of hexacyanoferrate (III) ions, hexacyanoferrate (II) ions, permanganate ions and mixtures thereof. Further, as the ion, an ion obtained by dissolving a salt such as potassium hexacyanoferrate (III) or potassium hexacyanoferrate (II) in an electrode solution can be used. As the ion-permeable diaphragm in contact with both electrodes, any material can be used as long as the material contained in both electrodes can pass ions without directly mixing, for example, an ion-exchange membrane, An agar gel containing an electrolyte used as a salt bridge can be used.

The device used in the present invention does not require a voltage generator or the like for applying an external voltage between the electrodes.
The detector is not particularly limited as long as it has at least one of a voltmeter, an ammeter and a current integrator. In addition, a conductor having a high current collecting effect, such as carbon or metal, may be inserted into both electrodes to quickly perform a battery reaction when measuring the quantity of electricity or the like. For example, any commercially available carbon may be used, and carbon felt and the like can be used. Any metal may be used as long as it has high conductivity. For example, gold, platinum, silver, lead and the like can be used.

[0016]

The structure and operation of the present invention will be described. Each electrochemically active substance (electrochemically active species) dissolved in the solution exhibits a unique electrode potential. These substances generate a potential difference between the electrodes, and can be electrolyzed by applying a sufficient voltage from the outside. In this case, an oxidation reaction occurs on one electrode and a reduction reaction occurs on the other electrode.
This is the principle of electrolysis and can be considered as the conversion of electrical energy into chemical energy.

In general, the potential at which electrode oxidation occurs is often higher than the potential at which electrode reduction occurs. In this case, unless electrical energy is applied, such as by applying an external voltage, electrolysis occurs. Therefore, electrical analysis cannot be performed in this state. However, the inventors of the present invention have conducted intensive studies, and as a result, have been selected from the group consisting of hexacyanoferrate (III) ion, hexacyanoferrate (II) ion, permanganate ion, and a mixture thereof as a battery active material at the counter electrode. A material that reduces the electrode at a very noble potential is used as a battery active material for the working electrode, and reduced 5-methylphenazinium methyl sulfate, reduced 1-methoxy-5-methylphenazinium methyl sulfate, reduced 9- Dimethylaminobenzo [α] phenoxazine-7-ium chloride, reduced 3,7bis (dimethylamino) phenothiazine-5-ium chloride, hexacyanoferrate (II) ion or ascorbic acid By using a material that can be oxidized, the battery cell has a diaphragm through which ions can pass between both electrodes. Without applying electrical energy from chemical energy it has been found to be spontaneously and rapidly converted into electrical energy. That is, the inventors have invented an electric analysis method capable of quantifying a sample substance by measuring the chemical energy of the sample substance as electric energy generated by a battery reaction.

For example, when a phenazine methosulfate solution is used for the working electrode and a potassium hexacyanoferrate (III) solution is used for the counter electrode, both the mediator and the electrochemically active species contained in the solutions in both electrodes are oxidized, so that they are used as they are. No reaction occurs. However, a substance that reduces phenazine methosulfate at the working electrode, for example, NAD
When H or a derivative thereof is added and reduction is performed, the oxidation of the resulting reduced phenazine methosulfate occurs at a potential lower than that of the reduction of the hexacyanoferrate (III) ion at the counter electrode. A potential difference based on the electrochemical potential occurs between the (counter electrode) and, as a result, a battery reaction occurs, and if there is a sufficient solution of the counter electrode, the reduction continues until all of the reduced phenazine methosulfate becomes the oxidized form. By measuring the current flowing through the battery reaction, the amount of the reducing agent that reduced phenazine methosulfate can be determined. That is, NADH and its derivatives can be quantified.

FIG. 1 shows an example of a schematic diagram of an apparatus used in the present invention. The battery cell 1 has a working electrode 2 and a counter electrode 3, and
It is composed of an ion exchange membrane (Cation Exchange Membrane CMV manufactured by Asahi Glass Co., Ltd.) used as the diaphragm 4 and is connected to a current integrator 6 (NDCM-1 manufactured by Nissan Keisoku) through a platinum wire 5. A predetermined electrode solution is put into the working electrode 2 and the counter electrode 3, and a certain amount of the solution containing the sample substance to be measured is added to the working electrode 2 with a syringe or the like, and a battery reaction based on the above principle is caused to flow and the amount of electricity flowing at that time By measuring the measurement, the substance to be measured in the sample can be quantified.

[0020]

The present invention will be described below in more detail with reference to examples. However, the technical scope of the present invention is not limited by these examples. (Example 1) Selection of mediator The difference in the measurement time due to the difference in the mediator used when measuring NADH was measured using the apparatus in Fig. 1.

Potassium hexacyanoferrate (III), vitamin K ₃ , methylene blue, phenazisimethosulphate (all manufactured by Wako Pure Chemical Industries), methoxyphenazine methsulphate (manufactured by Dojindo Laboratories), Meldora Blue (made by Nacalai Tesque) was used. Each mediator (0.1M) was dissolved in 0.1M phosphate buffer (pH 7.0) to make 3 ml, and each solution was used as an electrode solution. The counter electrode is a 0.1 M potassium hexacyanoferrate (III) solution (0.1 M phosphate buffer pH 7.0, 3
NADH (10 ^-3 M)
Was used as a sample substance, and measurement was performed without applying an external voltage between both electrodes.

The measurement results are shown in Table 1. The results are shown in Table 1. Phenazine methosulfate, methoxyphenazine methosulfate,
The measurement time when Meldora Blue was used as a mediator was as short as about 30 seconds.

[0023]

[Table 1]

Example 2 Measurement of NADH Using the apparatus shown in FIG. 1, a 0.1 M phenazine methosulfate solution was used for the working electrode, and a 0.1 M potassium hexacyanoferrate (III) solution (each 0.1 M phosphate buffer) was used for the counter electrode. (Dissolved in pH 7.0 solution) 3 ml each was added, and NADH solutions of various concentrations (dissolved in 40 mM phosphate buffer pH 7.0) were measured. The measurement results for each 10 μL sample are shown in FIG. 2, and a good linear relationship was obtained between the concentration of NADH and the value measured by the current integrator. Regarding the repetition reproducibility, the coefficient of variation (hereinafter referred to as CV value) after repeating 10 times was within 2% for all samples, showing excellent reproducibility. In addition, carbon felt (Nippon Carbon
GF-20-5F) was inserted and the same measurement was performed. As a result, a good linear relationship was obtained between the concentration of NADH and the value measured by the current integrator. Furthermore, as a result of repeatedly measuring 10 μL of the 5 × 10 ⁻⁴ M NADH solution, the measurement was stably performed 200 times or more.
The CV value at that time was within 2%.

Example 3 Measurement of Glutamate Amount by Quantifying NADH Produced by Glutamate Dehydrogenase Glutamate dehydrogenase found by the present inventors and having excellent substrate specificity and stability (Japanese Patent Application No. 4-194245) ) Was used to determine the amount of glutamic acid according to the present invention. The reaction of this enzyme is as follows.

[0026]

Embedded image

That is, 10 μL of the glutamate dehydrogenase-containing solution described in Example 1 of Japanese Patent Application No. 4-194245 (4 U / ml 20 mM phosphate buffer pH 7.0 solution) and NAD ⁺ as a substrate were used ^.
Sodium glutamate solution of various concentrations containing (20 mM phosphate buffer pH 7.0 solution, NAD ⁺ contains 20 mg / ml) 990 μ
10 μL of a solution obtained by mixing L at 30 ° C. for 3 minutes was measured under the same conditions as in Example 2.

FIG. 3 shows the results. A good linear relationship was obtained between the measured value using NADH as an index and the glutamic acid concentration. Similarly, the glutamic acid content in commercially available soy sauce was measured and compared with the measured value of the amino acid analyzer. As a result, the measured value by the amino acid analyzer (JLC-300 manufactured by JEOL Ltd.) was 11.3 g / L, and the measured value by the present invention was This was in good agreement with 11.2 g / L. The same sample was measured using a glutamic acid measurement kit (manufactured by BMY). The result was 11.0 g / L.
Met. The measurement time was about one sample for the amino acid analyzer.
According to the present invention, the measurement was performed in about 50 seconds (approximately 4 minutes including the enzyme reaction time), whereas the glutamic acid measurement kit required 20 minutes in 1.5 hours, and rapid measurement was possible.

Further, in this example, the enzyme used could be immobilized in the working electrode by a commonly used immobilization method. Example 4 NA Generated by Glutamate Dehydrogenase
Measurement of Glutamate Amount by Quantification of DH Derivative Using 3-acetylpyridine NAD (manufactured by Sigma) as a coenzyme for glutamate dehydrogenase reaction, 50
The measurement was performed under the same conditions as in Example 3 except that the measurement was performed at 20 ° C. for 20 minutes. A good linear relationship was found between the measured value according to the present invention using 3-acetylpyridine NADH generated by the enzyme reaction as an index and the glutamic acid concentration. The relationship was obtained. The measurement time was as short as about 30 seconds.

Example 5 Chemical oxygen demand (CO
D) Measurement (1) Using the apparatus of FIG. 1, 3 ml of a 1N hydrochloric acid solution containing 0.1 M calcium chloride was used as a working electrode as an electrode solution, and 0.01 M hexacyanoferrate (II) as a battery active material was used as a counter electrode. 3 ml of a 1N hydrochloric acid solution containing potassium and 0.1 M potassium chloride was placed as an electrode solution, and the COD of each sample was measured.

As a sample, a wastewater from a factory in Handa City, Aichi Prefecture 2
Type (A, B), 2 types of sewage treated water (C, D) and glucose standard solution (described in JIS K8824) 1 mgO / L, 10 mgO
/ L was used. The measurement was performed as follows. 2 ml of each sample, 0.4 ml of 47% sulfuric acid, N / 40
0.2 ml of potassium permanganate was added in this order, mixed, and sealed. After heating for 5 minutes, the mixture was cooled to room temperature, and 10 μL of this mixed solution was added to the working electrode of the above-mentioned apparatus with a syringe to measure.

Table 2 shows the results of comparing the results with values measured by the method described in JISK0102 for the same sample.
Both measured values agreed well. Approximately 30 minutes
In a short period of time, a small amount of sample could be measured. When carbon felt (GF-20-5F manufactured by Nippon Carbon Co., Ltd.) was inserted into the working electrode and the counter electrode of this example, and the measurement was performed under the same conditions, the same measurement as described above was performed.

[0033]

[Table 2]

Example 6 Chemical oxygen demand (CO
D) Measurement (2) Using the apparatus shown in FIG. 1, 3 ml of a 1.5 N sulfuric acid solution was used as an electrode solution for the working electrode, and 0.02 M potassium hexacyanoferrate (II) and 0.02 M hexacyanoferron which were battery active materials were used as counter electrodes. (I
II) 3 ml of a 1.2N sulfuric acid solution containing potassium acid was added as an electrode solution, and the COD of each sample was measured. As samples, two types of wastewater from septic tanks (A, B), two types of wastewater from office (C, D) and glucose standard solution (JI
SK8824) 1 mgO / L and 10 mgO / L were used.

The measurement was performed as follows. 2 ml of various samples, 0.4 ml of 47% sulfuric acid, 1M
0.2 ml of silver nitrate solution and 0.2 ml of N / 40 potassium permanganate were added in this order, mixed, and sealed. After heating for 5 minutes, the mixture was cooled with water, and 10 μL of this mixed solution was added to the working electrode of the above-mentioned apparatus with a syringe and measured.

Table 3 shows the results of comparison of the same sample with values measured by the method described in JIS K0102, and the measured values of the two samples agreed well. The time required for the measurement was as short as about 30 seconds, and a small amount of the sample could be measured. In addition, carbon felt (GF-20-5F made by Nippon Carbon Co., Ltd.)
Was measured under the same conditions, and the same measurement as described above was performed.

[0037]

[Table 3]

Example 7 Measurement of Ascorbic Acid Using the apparatus shown in FIG. 1, 3 ml of a 10 mM methylene blue solution (dissolved in 0.4 M phosphate buffer, pH 5.0) was used for the working electrode, and 0.4 M potassium hexacyanoferrate (III) was used for the counter electrode. 3 ml of a solution (0.4 M phosphate buffer, pH 7.3) was added as an electrode solution, and 1 to 200 mg / 100
As a result of measurement of ascorbic acid solutions having various concentrations of ml, a good linear relationship was obtained between each concentration of the ascorbic acid solution and the measured value of electricity according to the present invention. The amount of the sample substance at that time was as small as 5 μL, and the measurement time was about 40 μL.
Seconds and the C
The V value was very stable at 2% or less.

Further, the same measurement was possible even when a 0.4 M phosphate buffer of pH 5.0 was used as the electrode solution of the working electrode. In addition, carbon felt (GF-20- made by Nippon Carbon Co., Ltd.)
Similar measurement results were obtained using the electrode into which 5F) was inserted. Example 8 Measurement of Ascorbic Acid in Various Commercial Foods Using the apparatus of FIG. 1, 3 ml of 0.4 M phosphate buffer at pH 5.0 was used as the working electrode as the electrode solution, and 0.1 M hexacyanoiron (III) was used as the counter electrode. 3 ml of potassium acid solution (0.4 M phosphate buffer pH
7.3) was used as an electrode solution, and ascorbic acid in various commercial foods was measured. In addition, the same sample was measured by indophenol titration method and HPLC method (high-performance liquid chromatography method) (published by the Japan Food Sanitation Association, Food Sanitation Inspection Guideline 1989, pages 361 to 363), and the results were compared. As shown in FIG. 4, well-matched measurement results were obtained.

[0040]

[Table 4]

[0041]

The present invention provides an electroanalytical method for quickly quantifying a sample substance without applying electric energy such as voltage from the outside, and analyzing food components such as glutamic acid and ascorbic acid and water quality analysis such as COD. It is extremely useful in industry.

[Brief description of the drawings]

FIG. 1 is a diagram showing an apparatus used to carry out the method of the present invention.

FIG. 2 is a graph showing the relationship between NADH concentration and measured values.

FIG. 3 is a graph showing a relationship between a glutamic acid concentration and a measured value.

[Explanation of symbols]

 Reference Signs List 1 battery cell 2 working electrode 3 counter electrode 4 diaphragm 5 platinum wire 6 current integrator

Continuation of the front page (56) References JP-A-2-213762 (JP, A) JP-A-59-124351 (JP, U) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27 / 416 G01N 27/28

Claims (7)

(57) [Claims] A working electrode which is a liquid containing a sample substance;
Hexacyanoferrate (III) ion, hexacyanoferrate (I
I) A battery cell comprising a counter electrode which is a liquid containing a battery active material selected from the group consisting of acid ions, permanganate ions or a mixture thereof, and an ion-permeable diaphragm in contact with both electrodes A voltage generated between the electrodes without applying an external voltage between the electrodes,
Measuring at least one of electric current or electric quantity,
An electrical analysis method for a sample substance, which comprises quantifying the sample substance. 2. The method according to claim 1, wherein the working electrode contains an electrode solution and / or a mediator. 3. The method according to claim 1, wherein the mediator is 5-methylphenazinium methyl sulfate, 1-methoxy-5-methyl-
Phenazinium methyl sulfate, 9-dimethylaminobenzo [α] phenoxazine-7-ium chloride, 3,7bis (dimethylamino) phenothiazine-5
3. The method according to claim 2, wherein the method comprises at least one of -ium chloride and salts thereof. 4. An electrical analysis method for glutamic acid, comprising using the battery cell according to claim 1 to measure glutamic acid using nicotinamide adenine dinucleotide or a derivative thereof as an index. 5. The method according to claim 1, wherein the working electrode contains an electrolyte, and hexacyanoferrate (II) ion is used as a battery active material of the counter electrode. An electrical analysis method for chemical oxygen demand that measures oxygen demand. 6. The method according to claim 5, wherein the electrolyte comprises hydrochloric acid and potassium chloride or sulfuric acid. 7. The method according to claim 1, wherein the working electrode contains an electrolyte and / or 3,7 bis (dimethylamino) phenothiazine-5-ium chloride, and hexacyanoferrate (III) ion is used as a counter electrode battery active material. A method for analyzing ascorbic acid, comprising measuring ascorbic acid using the battery cell according to 1.