JP2512112B2 - Measurement method using enzyme electrode - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for mixing hydrogen peroxide-producing oxidase into a buffer solution by using an enzyme electrode having a hydrogen peroxide-forming oxidase immobilized on a solid electrode directly or through a permselective membrane. By oxidizing the substance to be detected in the sample and detecting the hydrogen peroxide produced,
The present invention relates to a method for measuring the concentration of a substance to be detected in a sample.

A conventional measuring device using an immobilized enzyme acting electrode has excellent features such as simplicity, speed and substrate specificity, and is widely used in a wide range of fields such as clinical analysis, food analysis and environmental measurement.
Its application range is expanding. Among them, the device that performs amperometric measurement, that is, the device that captures the increase or decrease of the electrode active substance produced or consumed by the enzyme reaction as the change of the current output value from the working electrode to which a constant voltage is applied is used. Highly sensitive measurement can be easily performed.
Therefore, various electrodes, devices and methods used for such a measuring method have been developed.

Typical examples of amperometric measurement methods include a method of measuring oxygen consumed by an electrode reaction by an oxygen electrode and a method of measuring hydrogen peroxide generated by an electrode reaction by a hydrogen peroxide electrode. There is a way to do it. These two measurement methods are most commonly used for immobilized enzyme electrodes,
In terms of response speed and S / N ratio, a method combining an immobilized hydrogen peroxide-producing oxidase and a hydrogen peroxide electrode is excellent.

However, one drawback of the enzyme electrode is that the hydrogen peroxide electrode used is generally a solid electrode such as solid platinum, gold or carbon, so that sensitivity fluctuation due to contamination of the solid electrode surface is unavoidable. Is mentioned. The cause of this contamination is various compounds other than the substance to be detected in the sample, products other than hydrogen peroxide due to the enzymatic reaction, and the substance to be detected itself may cause contamination. Such contamination of the surface of the solid electrode is particularly noticeable and unavoidable in the electrode in which the enzyme is directly immobilized on the solid electrode.

Further, in general, in a hydrogen peroxide electrode, in order to prevent interference of the measurement by an oxidizable compound other than hydrogen peroxide, for example, ascorbic acid or uric acid, a permselective membrane that permeates hydrogen peroxide and does not permeate these obstacles. Are provided on the surface of the solid electrode. Therefore, if the molecular weight of the cause of contamination is extremely higher than that of hydrogen peroxide, the contamination is not caused only on the surface of the solid electrode. Contamination by such relatively large molecules occurs on the liquid contact surface of the enzyme electrode, that is, the surface of the immobilization membrane on which the enzyme is immobilized, and causes the trouble that the substance to be detected, which is the substrate, is difficult to permeate. become. At present, by devising a sample preparation method,
The above troubles are avoided by removing the cause of such a large molecular weight contamination.

On the other hand, compounds with a molecular weight similar to that of hydrogen peroxide, that is, relatively low molecular weight compounds, permeate the permselective membrane and adsorb on the surface of the solid electrode even though they do not undergo a chemical reaction on the electrode. There are things that fluctuate. Therefore, even when the permselective membrane is provided, contamination of the surface of the solid electrode still poses a problem.

For such sensitivity fluctuations due to contamination of the surface of the solid electrode, application of a reverse potential for the purpose of cleaning the surface of the solid electrode (JP-A-57-60255, JP-A-60-155959, JP-A-60-155959, and JP-A-60-155959). No. 62-156555) and a method of performing triangular wave potential scanning (Japanese Patent Laid-Open No. 63-122942). However,
Disadvantages of these methods are that, when sensitivity fluctuations gradually occur, the criteria for determining the time when the cleaning operation is performed must be determined, and it is necessary to provide a mechanism for changing the potential, and the device itself It is complicated and the cost of making the device is high.

In addition, some hydrogen peroxide-producing oxidases produce compounds that themselves can contaminate the surface of the solid electrode as a result of enzymatic reactions. For example, an alcohol oxidase that catalyzes the oxidation reaction of a lower alcohol produces an aldehyde corresponding to that alcohol as a product. The aldehyde itself or a carboxylic acid produced by oxidizing a part of the aldehyde on the electrode is adsorbed on the surface of the solid electrode and causes a decrease in measurement sensitivity.

A phenomenon similar to this phenomenon is also observed in the determination of lactic acid using lactate oxidase. In this case,
Sensitivity fluctuation may occur due to lactic acid itself which is a substrate and pyruvic acid produced by an electrode reaction. Up to now, no effective solution has been presented to such problems.

The object of the present invention is to carry out a measurement using an enzyme electrode,
Prevents low-molecular compounds that are included in the sample in advance or generated as a result of enzymatic reaction from contaminating the solid electrode surface of platinum, gold, carbon, etc., resulting in reduced sensitivity of the enzyme electrode or poor reproducibility. However, it is another object of the present invention to provide a measurement method using an enzyme electrode, which can perform measurement with high accuracy and good reproducibility.

Means for Solving the Problems The present invention is to oxidize a substance to be detected in a sample by mixing a sample in a buffer solution and using an enzyme electrode in which hydrogen peroxide-forming oxidase is directly immobilized on a solid electrode, A method for measuring a concentration of a substance to be detected in a sample by detecting hydrogen peroxide produced, wherein the buffer solution contains a compound represented by the general formula (1), R1, R2-CHCOOH ... (1) R1 is a substituent selected from the group consisting of an amino group and a hydroxyl group, and R2 is a substituent selected from the group consisting of a hydrogen atom and an alkyl group having 1 to 4 carbon atoms. This is a measurement method using an enzyme electrode.

Furthermore, the present invention uses a enzyme electrode in which a sample is mixed in a buffer solution and hydrogen peroxide-producing oxidase is immobilized on a solid electrode through a permselective membrane that selectively permeates hydrogen peroxide. A method for measuring the concentration of a substance to be detected in a sample by detecting the hydrogen peroxide produced by oxidizing the substance to be detected therein, comprising: adding a compound represented by the general formula (1) to the buffer solution. R1, R2-CHCOOH (1) R1 is a substituent selected from the group consisting of an amino group and a hydroxyl group, and R2 is a substituent selected from the group consisting of a hydrogen atom, a methyl group and an ethyl group. It is a measuring method using an enzyme electrode, which is characterized in that

Operation Generally, it is known that a clean solid electrode surface is easily polluted by various compounds, and particularly polluted by amines or carboxylic acids.
In the measurement using the enzyme electrode, the sample to be measured is a clinical sample, a food sample, or an environmental sample such as drainage,
It contains the pollutants mentioned above. Therefore, it is unavoidable that the surface of the solid electrode is contaminated by these contaminants.

The present inventor oxidizes a substance to be detected on the enzyme electrode by using an enzyme electrode on which hydrogen peroxide-forming oxidase is immobilized, and electrochemically detects the hydrogen peroxide produced, thereby detecting the substance to be detected. In the method of measuring the concentration, a method of causing various pollutants to act on the enzyme electrode and preventing the influence thereof was examined, and the present invention was completed.

In order to prevent the adsorption of contaminants on the surface of the solid electrode,
If a particular compound is used, it must reach the solid electrode surface. Therefore, the present inventor first, in part, a permselective membrane for removing an immobilized enzyme layer constituting the enzyme electrode or an interfering substance provided between the solid electrode surface and the immobilized enzyme layer. We searched for compounds that penetrated but did not undergo an electrode reaction by themselves, and still interacted with the solid electrode in some way.

The permselective membrane preferentially permeates hydrogen peroxide and limits the permeation of reducing substances such as ascorbic acid and uric acid coexisting in the sample. It is desired that only hydrogen peroxide of 34 is permeated, and no compound having a molecular weight higher than that is permeated. However, in reality, with a molecular weight of around 100 as the boundary,
A membrane which is less likely to permeate a compound having a higher molecular weight and is more likely to permeate a compound having a lower molecular weight is used as the selectively permeable membrane. As such a permselective membrane, a known acetylcellulose membrane, a silicone membrane, an acrylic membrane,
Further, a film utilizing a protein (Japanese Patent Laid-Open No. 63-182559)
No.) etc., but both films have similar characteristics.

Even when the enzyme layer is formed directly on the surface of the solid electrode without providing a permselective membrane, a substance having a smaller molecular weight than the compound serving as the substrate easily diffuses to the surface of the solid electrode and easily reaches the surface of the solid electrode. You can Therefore, the target compound is selected from compounds having a molecular weight of about 200 or less.

Specifically, based on a platinum electrode as a solid electrode,
Using bovine serum albumin as the permselective membrane, a glucose measuring electrode on which glucose oxidase was immobilized was used.
Place in 0 mM sodium phosphate buffer. Next, 1 mM acetic acid is added to the buffer solution as a pollutant, and an experiment is conducted to measure glucose. In such an experiment, within 15 minutes after adding acetic acid to the above-mentioned buffer, the electrode response to hydrogen peroxide decreased to about 70%, and a phenomenon corresponding to the case where the solid electrode surface was contaminated occurred. Various compounds were previously contained in the above-mentioned buffer solution, the above-mentioned experiment was carried out, and a compound which prevents the above-mentioned sensitivity deterioration was searched for.

As a result, when the compound according to the present invention, R1, R2-CHCOOH (1), that is, the compound having the structure of the general formula (1) is added, such sensitivity decrease is not observed, and further, these compounds are added. It was found that the sensitivity did not change even if acetic acid of 10 mM or more was added to the buffer containing about 10 mM.

The compound contained in the buffer solution has the structure of the general formula (1). In an enzyme electrode in which an enzyme is directly immobilized on a solid electrode, R1 is an amino group or a hydroxyl group, R2 is hydrogen or a carbon number of 1 It is selected from any of the alkyl groups from 1 to 4. In other words, as such a compound,
Glycolic acid and its derivatives, amino acids such as glycine and alanine, and their derivatives are preferably used.

Further, in the enzyme electrode in which the selective permeation membrane is provided on the solid electrode and the enzyme is immobilized on the solution side through the selective permeation membrane, R1 is either an amino group or a hydroxyl group, R2 is hydrogen, either a methyl group or an ethyl group. Is chosen from.

In an electrode having no such permselective membrane, a compound having a relatively large molecular weight can reach the surface of the solid electrode. However, if the length of the alkyl group exceeds the hexyl group, the water solubility decreases, and such a compound may be adsorbed as an oily contaminant on the surface of the solid electrode. Therefore, the addition of these compounds to the buffer solution causes a decrease in sensitivity, and it is difficult to use a compound having a large molecular weight. In order to obtain particularly good results, it is desired that the alkyl group has 4 or less carbon atoms.

In an electrode in which a permselective membrane is provided on a solid electrode and an enzyme is immobilized on the solution side through the permselective membrane, R1 is selected from an amino group or a hydroxyl group, R2 is hydrogen, a methyl group or an ethyl group. It is what is done. In such a case, R2 is limited in order to pass through the permselective membrane to some extent and interact with the surface of the solid electrode.

The reason why these compounds are effective is probably because they have a carboxyl group and an amino group or a hydroxyl group that enhance the interaction with the electrode surface, and contain a larger atomic group than acetic acid which is an obstacle. it is conceivable that.
In other words, the compound represented by the general formula (1) preliminarily adsorbs on the surface of the solid electrode, and therefore, it obstructs the adsorption of a compound such as acetic acid, but due to the steric hindrance of the side chain. It is considered that the coverage is low, and that the molecular weight of hydrogen peroxide is not enough to cover the electrode surface until the electrode reaction is inhibited.

Since these compounds have high water solubility, it is extremely easy to add them to the buffer used for the enzyme reaction. Generally, in a buffer solution, about 0.1 to 100 mM, more preferably 1
It is effective when added so that the concentration becomes about 10 mM.
If the concentration is too low, the effect is diminished, and if the concentration exceeds this range, the buffering ability of the buffer solution may be affected, or the added compound itself may precipitate due to temperature fluctuations, which is not preferable.

Further, these compounds do not particularly have an action of inhibiting an enzymatic reaction, and are also easy to use in that respect. However, in the case of using an amino acid oxidase among hydrogen peroxide-producing oxidases, for example, for L-amino acid oxidase, it is not possible to use the L-amino acid, and consideration should be given to using the D-amino acid. Alternatively, glycolic acid based compounds should be used. In general, amino acid compounds are cheaper and thus easier to use, but when they are used, various bactericides and bacteriostatic agents are used in combination because amino acid compounds are prone to decomposition due to microbial contamination. Is desirable. Further, the buffer containing the compound of the present invention,
The effect is exhibited both in the batch measuring system and the measuring system using the flow type measuring device.

Examples The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these. In addition,% represents weight%.

Example 1 (1) Pretreatment Method of Electrode A side surface of a platinum wire having a diameter of 2 mm was covered with heat-shrinkable Teflon (registered trademark), and one end of the platinum wire was smoothed with a file and No. 1500 emery paper. This platinum wire is the working electrode,
Using a 1 cm square platinum plate as a counter electrode and a saturated calomel electrode (hereinafter abbreviated as SCE) as a reference electrode, electrolytic oxidation treatment was performed in 0.1 M sulfuric acid at +1.4 V for 10 minutes. After that, the platinum wire was thoroughly washed with water, dried at 40 ° C. for 5 minutes, immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour, and then washed. An enzyme was immobilized on the aminosilane-treated platinum wire.

(2) Immobilization method of enzyme Alcohol oxidase (manufactured by Sigma, Pichiapastol
5 mg of bovine serum albumin (Fraction V, manufactured by Sigma) was added to 100 μl of a liquid enzyme preparation derived from is, and a 100 mM sodium phosphate buffer solution (pH 7.
1) of 5) was added under ice cooling. By this operation, the solution became cloudy and the enzyme and albumin were insolubilized. Add this solution to 4
Centrifugation was carried out at 20000 xg (g represents gravity velocity) at 10 ° C for 10 minutes. The supernatant was removed, and 100 mM sodium phosphate buffer (pH 7.5) was added to 100 μl to redissolve the precipitated protein. This solution was dialyzed against the same buffer used for reconstitution for 1 hour at 4 ° C. After dialysis,
It was used for the following immobilization operation. By the operation up to dialysis, saccharides and glycerol contained in the liquid enzyme preparation as a stabilizer and ammonium sulfate used for precipitating protein were removed.

Glutaraldehyde was added to the solution after dialysis so as to be 0.2%. 3 μl of this mixed solution was quickly put on an aminosilanized platinum wire prepared in advance, and dried and cured at 40 ° C. for 15 minutes. Then, 100 mM sodium phosphate buffer (pH
7.5) at room temperature.

(3) Measuring Method The flow type measuring device used in the measuring method according to the present invention is shown in FIG. The enzyme electrode prepared as described above was used as the working electrode 6, and the silver / silver chloride electrode 8 was used as the reference electrode, and these were attached to the measuring cell 5.
The internal volume of this measuring cell 5 was 40 μl. The working electrode 6 and the silver / silver chloride electrode 8 are arranged so as to face each other via a buffer solution line. As the counter electrode 7, the measuring cell 5
A stainless tube attached to the outlet side of was used.

In FIG. 1, the buffer solution contains 50 mM potassium chloride in order to stabilize the potential of the silver / silver chloride electrode 8 used as the reference electrode, and DL-le as a compound according to the present invention.
ucic acid ((CH ₃ ) ₂ CHCH ₂ CH (OH) COOH), that is, in the general formula (1), R1 = OH, R2 = (CH ₃ ) ₂ CHCH ₂ is added at 100 mM in 100 mM sodium phosphate buffer (pH 7). .5) and supplied from the buffer solution reservoir 1. This buffer solution was sent by a high performance liquid chromatograph pump 2 at a flow rate of 1.0 ml / min.

The sample was injected from a Rheodyne Model 7125 sample injection device 3 equipped with a sampling loop for 5 μl. In order to mix and dilute the injected sample with a buffer solution and to equilibrate the temperature, a diluting and mixing pipe 4 in which a Teflon tube having an inner diameter of 0.5 mm and a length of 1 m is coiled is installed.

The injection device 3 to the measuring cell 5 are arranged in a constant temperature bath 12 controlled at 37 ± 0.2 ° C. The waste liquid that has left the measurement cell 5 is discharged to the waste liquid reservoir 11. In addition, a voltage of +0.45 V is applied to the working electrode 6 with respect to the silver / silver chloride electrode 8 by the potentiostat 9 that regulates the potential of the working electrode 6 and detects the current. The generated current was voltage-converted, amplified, and output to the recorder 10, which recorded the output current value.

(4) Measurement method and results First, after the temperature of the constant temperature bath was stabilized, 5 μl of an aqueous ethanol solution having a concentration of 0.1 mM to 4 mM was injected, and the peak height from the baseline of the curve recorded by the recorder 10 was increased. Was measured. A calibration curve shown in FIG. 2 was prepared from the concentration of injected ethanol and the peak height (current value).
The obtained data was linearly approximated by the method of least squares, and the slope of the calibration curve was obtained.

Next, as a real sample, 5 μl of 250-fold diluted beer solution was diluted to 60
Inject once and make a calibration curve again. When this operation was repeated 5 times, the slope of the calibration curve hardly changed, and
It was constant as indicated by a circle in the figure. The change in the slope of the calibration curve at this time is shown by the line l1. In addition, DL-leucic
In a measurement system that does not contain acid, it has been separately confirmed that the sensitivity of this enzyme electrode does not fluctuate due to heat deactivation by injecting only the standard substance, and therefore the effect of DL-leucic acid is an electrode contaminant contained in beer, for example, It is considered to prevent contamination by organic acids such as acetic acid. Furthermore, even if the enzyme electrode was stored in this buffer, there was no difference in storage stability compared with the case where only the phosphate buffer was used.

Comparative Example 1 (1) Electrode Pretreatment Method The electrode was treated in the same manner as in Example 1.

(2) Immobilization method of enzyme Immobilization was performed in the same manner as in Example 1.

(3) Measuring device The same device as in Example 1 was used except that DL-leucic acid was removed from the buffer solution used for the measurement.

(4) Measurement Method and Results The same measurement as in Example 1 was performed to evaluate the variation in electrode sensitivity. The result is indicated by the ● mark in FIG.
The change in the slope of the calibration curve at this time is shown by the line l2. From this, it is understood that the added DL-leucic acid does not affect the electrode sensitivity and prevents the sensitivity fluctuation due to the actual sample injection.

Example 2 (1) Electrode pretreatment method An electrode was treated in the same manner as in Example 1.

(2) Immobilization method of enzyme Immobilization was performed in the same manner as in Example 1.

(3) Measuring device Instead of DL-leucic acid in the buffer used for measurement, 1
0 mM glycine, that is, R1 = N in the general formula (1)
The same apparatus as in Example 1 was used except that H ₂ and R2 = H were added.

(4) Measurement Method and Results The same measurement as in Example 1 was performed to evaluate the variation in electrode sensitivity. The result is indicated by a circle in FIG. Glycine added as shown in FIG.
Like leucic acid, it does not affect the electrode sensitivity,
Moreover, it can be seen that the sensitivity fluctuation due to the actual sample injection is prevented.

Example 3 (1) Pretreatment of electrode The same treatment as in Example 1 was performed.

(2) Immobilization method of enzyme Bovine serum albumin (manufactured by Sigma, Fractio) in 1 ml of distilled water
n V) 2 mg was melt | dissolved, and glutaraldehyde was added so that it might become 0.2% under ice cooling. 3 μl of this solution was applied on the platinum electrode for which pretreatment was completed, and the solution was allowed to stand at 4 ° C. for 30 minutes. Then, heat treatment was performed at 40 ° C. for 15 minutes to complete the crosslinking reaction and form a permselective membrane. Next, bovine serum albumin (Sigma, Fraction V) 5 mg and glucose oxidase (Sigma, Type II, Aspergillus niger derived) 5 mg were added to 100 mM sodium phosphate buffer (pH 7.0) 1
It was dissolved in ml, and glutaraldehyde was added to 0.2%. 3 μl of this solution was applied onto the electrode having the permselective membrane previously formed thereon, and heat treatment was carried out at 40 ° C. for 15 minutes to form an immobilized enzyme layer. Then, it preserve | saved in 100 mM sodium phosphate buffer (pH 6.0).

(3) Measuring device For the measurement, 50 mM potassium chloride and 5 mM alanine as a buffer solution, that is, R1 in the general formula (1)
= NH ₂ , R 2 = CH ₃ , and the same apparatus as in Example 1 was used, except that a 100 mM sodium phosphate buffer (pH 6.0) containing

(4) Measurement method and results After reaching the temperature equilibrium of the thermostat 12, record the response value of the 10 mM glucose standard solution,
Using a 20-fold diluted solution as a sample, 500 injections were performed per day. Next day, record the response value of the glucose standard solution,
The injection of the diuse diluent was repeated again. This measurement was repeated until the 10th day thereafter, and the relative sensitivity up to the 10th day when the response value to the standard solution on the 1st day was 100% is shown by a circle in FIG. As can be seen from FIG. 5, the sensitivity variation is within ± 2%, and the sensitivity is not substantially reduced.

Comparative Example 2 (1) Pretreatment of electrode The same treatment as in Example 3 was performed.

(2) Immobilization method of enzyme Immobilization was performed in the same manner as in Example 3.

(3) Measurement device Measurement was performed using the same device as in Example 3 except that alanine was removed from the buffer solution used for measurement.

(4) Measurement Method and Results Similar to Example 3, repeated measurement was carried out, and the relative sensitivity up to the 10th day when the response value to the standard solution on the 1st day was 100% is shown by a ● mark in FIG. Indicated by. As can be seen from FIG. 5, when the buffer solution containing no alanine was used, the sensitivity was reduced by about 15% in 10 days. Further, the sensitivity was recovered by repeating the triangular wave potential scanning 100 times in the range of -0.1 V to +0.7 V with respect to the silver / silver chloride electrode 8 on the immobilized enzyme working electrode with reduced sensitivity. confirmed. Therefore, it can be seen that the effect of alanine does not contribute to the stabilization of the enzyme but prevents the contamination of the solid electrode.

In the above-mentioned Examples, by adding each compound represented by the general formula (1) to a commonly used buffer solution for such measurement, it is possible to prevent a decrease in measurement sensitivity. This does not require any special improvement in the hardware of the measuring device, and can be carried out extremely easily as compared with the potential scanning method and the like.

EFFECTS OF THE INVENTION As described above, according to the present invention, it is possible to easily obtain a highly accurate and stable measurement result when performing measurement using an immobilized enzyme working electrode.

[Brief description of drawings]

FIG. 1 is a system diagram showing a flow-type measuring apparatus in one embodiment of the present invention, FIG. 2 is a graph showing a calibration curve in Embodiment 1 of the present invention, and FIG. 3 is a result of Example 1 and Comparative Example 1. 4 is a graph showing the result of Example 2, and FIG.
The figure is a graph showing the results of Example 3 and Comparative Example 2. 1 ... buffer reservoir, 2 ... pump, 3 ... injection device, 4 ... dilution mixing pipe, 5 ... measuring cell, 6 ...
Immobilized enzyme working electrode, 7 ... Counter electrode, 8 ... Reference electrode, 9
…… Potentiostat, 10 …… Recorder, 11 …… Waste liquid reservoir, 12 …… Constant temperature bath

Claims (2)

(57) [Claims] 1. A method in which a sample is mixed in a buffer solution and a hydrogen peroxide producing oxidase is directly immobilized on a solid electrode and an enzyme electrode is used to oxidize a substance to be detected in the sample to produce hydrogen peroxide. A method for measuring the concentration of a substance to be detected in a sample by detecting, wherein the buffer solution contains a compound represented by the general formula (1), and R1, R2-CHCOOH (1) R1 is an amino acid. An enzyme, wherein R2 is a substituent selected from the group consisting of a group and a hydroxyl group, and R2 is a substituent selected from the group consisting of a hydrogen atom and an alkyl group having 1 to 4 carbon atoms. Measuring method using electrodes. 2. A sample is prepared by mixing a sample in a buffer solution and using an enzyme electrode in which hydrogen peroxide-producing oxidase is immobilized on a solid electrode through a selective permeation membrane that selectively permeates hydrogen peroxide. A method for measuring the concentration of a substance to be detected in a sample by detecting the hydrogen peroxide produced by oxidizing the substance to be detected therein, comprising: adding a compound represented by the general formula (1) to the buffer solution. R1, R2-CHCOOH (1) R1 is a substituent selected from the group consisting of an amino group and a hydroxyl group, and R2 is a substituent selected from the group consisting of a hydrogen atom, a methyl group and an ethyl group. A measuring method using an enzyme electrode characterized by being present.