JP3800350B2 - Biosensor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a biosensor, and more specifically, an immobilized physiologically active substance provided with a hydrogen peroxide selective permeable membrane on the surface of a hydrogen peroxide electrode and a physiologically active substance immobilized on the hydrogen peroxide selective permeable membrane. Membranes are provided, and these membranes are wetted with a buffer solution to cause the measurement target substance to react in the presence of a physiologically active substance, and an electric signal corresponding to the amount of hydrogen peroxide generated by the reaction is generated by hydrogen peroxide. The present invention relates to a biosensor that outputs from an electrode.
[0002]
[Prior art]
Conventionally, an immobilized physiologically active substance film (hereinafter simply referred to as an immobilized enzyme film) obtained by immobilizing a physiologically active substance like an immobilized enzyme film on a hydrogen peroxide electrode has been provided. A hydrogen peroxide selective permeable membrane is provided between the hydrogen oxide electrode, and these membranes are wetted with a buffer solution of about 50 mM / l, and in the presence of a physiologically active substance (for example, contained in a body fluid). There has been proposed a biosensor that causes a reaction of a substance to be measured) and outputs an electrical signal corresponding to the amount of hydrogen peroxide generated by the reaction from a hydrogen peroxide electrode. Since such a biosensor has high selectivity with respect to the measurement target substance, the concentration of an organic substance or the like having a complicated structure can be measured with high accuracy. In addition, the buffer solution is set to a concentration of about 50 mM / l in order to bring the measurement environment close to the biological conditions when, for example, a body fluid is the measurement target solution.
[0003]
In particular, when the solution to be measured is blood, if no hydrogen peroxide permselective membrane exists, noise derived from interfering substances in the blood (such as ascorbic acid) is superimposed on the detection current, resulting in a concentration Although the measurement accuracy is reduced, if a hydrogen peroxide selective permeable membrane is provided, only hydrogen peroxide will reach the surface of the hydrogen peroxide electrode, which may cause noise from interfering substances. In addition, highly accurate concentration measurement can be achieved.
[0004]
[Problems to be solved by the invention]
However, the hydrogen peroxide permselective membrane is generally produced by entanglement of a polymer like acetylcellulose, and has a partial charge bias. The biosensor also reverses the polarity of the bias voltage applied to the hydrogen peroxide electrode for a short time to remove the impurities formed on the surface of the hydrogen peroxide electrode during measurement and restore the activity of the hydrogen peroxide electrode. I try to let them.
[0005]
Therefore, the hydrogen peroxide permselective membrane provided in contact with the surface of the hydrogen peroxide electrode is disturbed by the inversion of polarity and the influence of OH radicals, and the stable state is disturbed. The membrane structure of the hydrogen peroxide permselective membrane produced by entanglement of the film gradually deteriorates. Further, since the concentration of the buffer solution is set as described above, the buffer capacity by the buffer solution cannot be increased so much, and the output signal from the hydrogen peroxide electrode is lowered in a low pH region. There is convenience.
[0006]
OBJECT OF THE INVENTION
The present invention has been made in view of the above-described problems, and can be stably maintained for a longer period of time than a hydrogen peroxide selective permeable membrane, and can prevent a decrease in output signal in a low pH region. It aims to provide a biosensor.
[0007]
[Means for Solving the Problems]
In the biosensor of claim 1, the concentration of the phosphate buffer solution for wetting the immobilized physiologically active substance membrane and the hydrogen peroxide selective permeable membrane is set to 200 mM / l or more and 300 mM / l or less .
[0008]
[Action]
In the biosensor according to claim 1, hydrogen peroxide selective permeation is performed on the surface of the hydrogen peroxide electrode where the polarity of the applied bias voltage is reversed with respect to the polarity at the time of the measurement operation to restore the activity of the electrode. Measured in the presence of a physiologically active substance in a state where an immobilized physiologically active substance film is formed by immobilizing a physiologically active substance on a hydrogen peroxide permselective membrane and these membranes are wetted with a phosphate buffer solution. A biosensor that causes a reaction of a target substance and outputs an electrical signal corresponding to the amount of hydrogen peroxide generated by the reaction from a hydrogen peroxide electrode, wherein the concentration of the phosphate buffer solution is 200 mM / l or more, and since the set below 300 mM / l, even if the polarity of the bias voltage applied to the hydrogen peroxide electrode is inverted, the effect of the polarity inversion is sufficiently absorbed in phosphate buffer solution layer, over Effects on hydrogen permselective membrane is greatly reduced. Therefore, the hydrogen peroxide permselective membrane can be stably maintained over a long period of time. Further, it has been found that by setting the concentration of the phosphate buffer solution to 200 mM / l or more and 300 mM / l or less, it is possible to prevent a decrease in output signal in a low pH region.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Embodiments of the present invention will be described below with reference to the accompanying drawings.
FIG. 1 is a diagram schematically showing one embodiment of the biosensor of the present invention.
This biosensor has a hydrogen peroxide permselective membrane 2 made of an acetylcellulose membrane, Nafion membrane, cellophane membrane, etc. on the surface of a hydrogen peroxide electrode 1 having at least a working electrode made of Pt or the like (not shown) and a counter electrode made of Ag or the like. And an immobilized enzyme membrane 3 so as to cover the hydrogen peroxide selective permeable membrane 2. In addition, an interfering substance separation membrane (not shown) that covers the immobilized enzyme membrane 3 is provided as necessary. When the measurement target solution is blood, the interfering substance separation membrane functions to separate blood cells or the like and allow components other than blood cells to reach the immobilized enzyme membrane 3. A phosphate buffer solution (disodium hydrogen phosphate / potassium dihydrogen phosphate or each hydrate) for wetting the hydrogen peroxide selective permeable membrane 2 and the immobilized enzyme membrane 3 is added. The phosphate concentration of this phosphate buffer solution is set to 300 mM / l.
[0011]
FIG. 2 is a diagram showing a state in which the output due to the leaking substance changes as the number of polarity inversions of the bias voltage for impurity removal increases. In the figure, a1 and a2 each employ a 50 mM / l phosphate buffer solution, and a3, a4, a5 and a6 each employ a 300 mM / l phosphate buffer solution.
As is apparent from FIG. 2, it is understood that the leakage of the reactant is suppressed by setting the phosphate concentration of the phosphate buffer solution to 300 mM / l, and the protective effect on the hydrogen peroxide permselective membrane is improved. .
[0012]
FIG. 3 shows the degree of deterioration of the hydrogen peroxide permselective membrane with the increase in the number of days when the biosensor is used normally (for example, when the polarity reversal of the bias voltage and the concentration measurement are alternately performed). It is a figure which shows a change. In FIG. 3, the broken line indicates a case where a 50 mM / l phosphate buffer solution is employed, and the solid line indicates a case where a 300 mM / l phosphate buffer solution is employed. Further, the deterioration degree indicates the output of the leaking substance with respect to the electrode output.
[0013]
In addition, the vertical axis | shaft of FIG. 3 has shown the output ratio by {(interfering substance + glucose solution) / glucose solution}.
As is clear from FIG. 3, it can be seen that the deterioration degree of the hydrogen peroxide selective permeable membrane can be suppressed by setting the phosphate concentration of the phosphate buffer solution to 300 mM / l. When the glucose concentration in the glucose solution is measured using a biosensor that employs an immobilized glucose oxidase membrane as the immobilized enzyme membrane 3, if the phosphate concentration of the phosphate buffer solution is set to 50 mM / l, The pH value decreases as the number of measurements increases, and the output signal value decreases accordingly. However, if the phosphate concentration of the phosphate buffer solution is set to 300 mM / l, the number of measurements increases. Even in the low pH range, almost no decrease in the output signal value was observed. This seems to be because the buffer capacity was improved by increasing the phosphate concentration of the phosphate buffer solution.
[0014]
Specifically, when the phosphate concentration of the phosphate buffer solution was set to 50 mM / l, 100 mM / l, 200 mM / l, and 300 mM / l, the measurement was repeated using a glucose solution with a known glucose concentration. The measurement results shown in FIGS. 4 to 7 were obtained. Each data shows data for two lots under each condition.
As is apparent from these measurement results, when a phosphate buffer solution having a phosphate concentration of 50 mM / l or 100 mM / l was used, the output decreased significantly as the number of measurements increased. When a phosphate buffer solution having a phosphate concentration of 200 mM / l or 300 mM / l was used, the output hardly decreased regardless of the increase in the number of measurements.
[0015]
In the above embodiment, only the case where a phosphate buffer solution is employed as the buffer solution has been described. , Glycine / hydrochloric acid, formic acid / sodium formate, citric acid / sodium citrate, acetic acid / sodium acetate, maleic acid / sodium hydroxide, tris / hydrochloric acid, diethanolamine / hydrochloric acid, boric acid / sodium hydroxide, sodium hydroxide / potassium chloride Etc.) can be adopted.
[0016]
【The invention's effect】
According to the first aspect of the present invention, even when the polarity of the bias voltage applied to the hydrogen peroxide electrode is reversed, the influence of the polarity reversal is sufficiently absorbed by the phosphate buffer solution layer, and the hydrogen peroxide selective permeable membrane The hydrogen peroxide permselective membrane can be stably maintained over a long period of time , and the concentration of the phosphate buffer solution is set to 200 mM / l or more and 300 mM / l or less. By doing this, there is a specific effect that it is possible to prevent the output signal from being lowered in the low pH region.
[Brief description of the drawings]
FIG. 1 is a diagram schematically showing a configuration of a biosensor to which a hydrogen peroxide selective permeable membrane protection method of the present invention is applied.
FIG. 2 is a diagram showing a state in which an output due to a leaking substance changes with an increase in the number of times of polarity inversion of a bias voltage for removing impurities.
FIG. 3 is a diagram showing a change in the degree of deterioration of a hydrogen peroxide selective permeable membrane with an increase in the number of days when a biosensor is used in a normal manner.
FIG. 4 is a diagram showing measurement results when measurement is repeated using a glucose solution having a known glucose concentration with the phosphate concentration of the phosphate buffer solution set to 50 mM / l.
FIG. 5 is a diagram showing measurement results when measurement is repeated using a glucose solution having a known glucose concentration with the phosphate concentration of the phosphate buffer solution set to 100 mM / l.
FIG. 6 is a diagram showing measurement results when measurement is repeated using a glucose solution with a known glucose concentration with the phosphate concentration of the phosphate buffer solution set to 200 mM / l.
FIG. 7 is a diagram showing measurement results when the measurement is repeated using a glucose solution with a known glucose concentration with the phosphate concentration of the phosphate buffer solution set to 300 mM / l.
[Explanation of symbols]
1 Hydrogen peroxide electrode 2 Hydrogen peroxide selective permeable membrane 3 Immobilized enzyme membrane

Claims (1)

A hydrogen peroxide selective permeation membrane (2) is provided on the surface of the hydrogen peroxide electrode (1) where the activity of the electrode is restored by reversing the polarity of the applied bias voltage with respect to the polarity during the measurement operation, An immobilized physiologically active substance membrane (3) formed by immobilizing a physiologically active substance on the hydrogen peroxide permselective membrane (2) is provided, and these membranes (2) and (3) are wetted with a phosphate buffer solution. to perform the reaction of the analyte in the presence of a physiologically active substance, an electric signal corresponding to the amount of hydrogen peroxide generated by reacting a biosensor output from the hydrogen peroxide electrode (1), phosphoric acid A biosensor characterized in that the concentration of the buffer solution is set to 200 mM / l or more and 300 mM / l or less .

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