JPH05123185A - Determination by immobilized enzyme - Google Patents

Abstract

PURPOSE:To enable the continuous, accurate and easy determination of a number of specimens in high precision while preventing the contamination of electrode by adding an alcohol to an aqueous medium for determination in the electrochemical determination of the concentration a substance using an immobilized enzyme. CONSTITUTION:A specimen is added to an aqueous medium containing a >=3C alcohol (preferably 3-12C alcohol such as isopropyl alcohol and butyl alcohol). The specimen in the aqueous medium is brought into contact with an immobilized enzyme to effect the enzymatic reaction and the variation in the concentration of the electrode active substance increasing or decreasing by the reaction is measured by an electrical means.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring method using an immobilized enzyme and, more particularly, to a method for measuring an accurate concentration by preventing sensitivity fluctuation due to electrode contamination.

[0002]

2. Description of the Related Art Immobilized enzymes are widely applied in the fields of clinical examination, fermentation production, analytical chemistry and the like. In particular, the method of electrochemically measuring the substance concentration using an immobilized enzyme is widely used because of the high selectivity of the enzyme reaction, the rapidity of measurement, and the simplification of the device configuration.

Many of these electrochemical biosensors use a solid electrode, such as platinum, gold or carbon, as a conductive substrate. However, as a drawback of the solid electrode, there is a problem that the electrode reaction rate and hence the sensor sensitivity are changed due to contamination of the electrode surface or oxidation reaction. A large amount of impurities are contained in test samples such as general foods and fermented liquids. As the impurities, many compounds such as proteins, lipids and mucilage polysaccharides can be exemplified, and their attachment to the surface of the electrode causes the decrease in the sensitivity of the biosensor.

Further, if the calibration curve is measured again after measuring the analyte containing a large amount of impurities, and the analyte is subsequently measured, the impurities deviate from the electrode surface during the calibration curve measurement and the sensitivity decreases. Although it was back to some extent, the measured value suddenly shifts because impurities adhere immediately when measuring the subject. Therefore, accurate measurement cannot be performed even if the calibration curve is measured again.

Many proposals have hitherto been made to avoid problems caused by these impurities. For example,
A method has been disclosed in which a surfactant is included in the measurement when the test object comes into contact with the electrode (Japanese Patent Laid-Open No. 61-25496).
issue). This is because the surfactant prevents adsorption of proteins to the electrode and thus prevents fluctuations in the sensitivity of the electrode. Is it because impurities include not only proteins but also low-molecular substances? However, sufficient effects have not been obtained with surfactants.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to solve the above problems, prevent electrode contamination that cannot be completely removed with a surfactant or the like, and provide a highly accurate measuring method.

[0007]

Means for Solving the Problems The present invention provides a measuring method in which a sample in an aqueous medium is brought into contact with an immobilized enzyme to electrochemically detect a change in the concentration of an electrode active substance which increases or decreases due to an enzymatic reaction. Is a measuring method using an immobilized enzyme, wherein the alcohol contains 3 or more carbon atoms.

The present invention also has 3 to 10 carbon atoms in the aqueous medium.
The said measuring method which contains 0.001 to 0.1 volume% of 12 alcohols is disclosed.

[0009]

[Function] Most biosensors of the type in which an enzyme is immobilized near the surface of a conductive substrate and electrochemically detect changes in the electrode active substance that increase and decrease due to the enzyme reaction use solid electrodes such as platinum, gold, and carbon. ing. The contamination phenomenon on the electrode surface, which is a drawback of these conductive substrates or solid electrodes, is roughly classified into two types.

First, a high molecular compound such as protein is adsorbed on the surface to form an adsorbed layer, which interferes with the diffusion of a substance from a solution, resulting in a change in sensitivity. Second
In the case of relatively low molecular weight lipids, amines, and aromatic compounds are adsorbed on the electrode surface, or electrochemical reactions occur on the electrode surface, insoluble compounds are produced and the same pollution occurs.

The former proteins and the like can often be removed quite effectively by bringing anionic, cationic and nonionic surfactants into contact with the electrode surface. However, general foods, fermented liquors, etc. often contain the latter low-molecular contaminants, which cannot be removed by surfactants.

The present inventors, however, have found that adding an alcohol having 3 or more carbon atoms to an aqueous medium for measurement such as a buffer solution allows stable and accurate measurement. In the present invention, the substance to be measured in the sample reaches the detector in the state of coexisting with an alcohol having 3 or more carbon atoms. However, when the measurement is performed in this way, a low-molecular contaminant such as a low-molecular contaminant that interferes with the measurement is obtained. Even if the impurities are included, it is not affected by this and a large number of samples can be continuously measured with high accuracy.

Here, the alcohol having 3 or more carbon atoms means
Examples include n-propyl alcohol, isopropyl alcohol, butyl alcohol, sec-butyl alcohol, isobutyl alcohol, t-butyl alcohol, pentyl alcohol, cyclohexanol, ethylene glycol, glycerin, octyl alcohol and dodecanol. Of course, an alcohol having a higher molecular weight can be used, but an alcohol having a too high molecular weight may have a problem in adding water in an aqueous medium due to a decrease in water solubility. Therefore, the number of carbon atoms is preferably 12 or less.

Methanol and ethanol are not suitable for the purpose of the present invention because they have high hydrophilicity and adversely affect enzyme molecules. In addition, for example, alcohol oxidase generally oxidizes n-propyl alcohol, so when using immobilized alcohol oxidase for the purpose of measuring ethanol and the like, isopropyl alcohol or butyl having 4 or more carbon atoms is used for the purpose of preventing electrode contamination. It is essential to use alcohol etc.

Although not particularly limited, the alcohol having 3 or more carbon atoms is preferably added to an aqueous medium such as a buffer in a concentration range of 0.001% by volume to 0.1% by volume.
If it is less than 0.001%, a sufficient effect may not always be obtained, while if it exceeds 0.1%, an inhibition phenomenon may be observed depending on the type of enzyme. The reason why the addition of these alcohols prevents electrode contamination and improves the accuracy is not necessarily clear, but it is presumed that these alcohols, which have a certain degree of hydrophobicity, wash away the compounds adsorbed on the electrode surface. It

The measuring method of the present invention is effective whether the method of contacting the electrode with the sample is a batch type or a flow type. However, the flow method is preferable because the electrode surface can be more effectively washed with a buffer solution containing an alcohol having 3 or more carbon atoms while the sample is not injected. The alcohol may be added in advance to the buffer solution, or in the case of the flow type, the purpose may be achieved by sending the alcohol-free buffer solution and the alcohol by a separate pump to combine them.

The measuring method of the present invention is particularly effective for samples containing low molecular weight impurities such as low molecular weight amines, low molecular weight lipids and aromatic compounds such as fermented liquor, samples in the process of producing sake and beer.
Electrochemical detection is carried out at oxygen electrodes and hydrogen peroxide electrodes by amperometric measurement of decrease or increase of oxygen and hydrogen peroxide, which are electrode active substances, or when detecting electrode active substances by coulometric detection. Including etc.

The aqueous medium is not particularly limited, and an ordinary buffer solution is used. The enzyme may be formed into a film in the vicinity of the working electrode, or may be formed into an immobilized column. The enzymes include glucose oxidase, alcohol oxidase,
Examples thereof include oxidases such as lactate oxidase and galactose oxidase, and examples of dehydrogenases include lactate dehydrogenase and glutamate dehydrogenase. Besides, it can be applied to enzymes such as oxygenase system.

[0019]

The present invention will be described in more detail with reference to the following examples, but it goes without saying that the present invention is not limited thereto.

Example 1 An experiment was carried out using a glucose oxidase-immobilized electrode. [Measuring Device] For the measurement according to the present invention, the flow type measuring device shown in FIG. 1 was used. First, the buffer solution is continuously sent from the buffer solution bottle (1) by the solution sending pump (2), and the sample injected from the autosampler (3) is sent to the constant temperature bath (5). The measurement unit (4) in the thermostat has a working electrode having glucose oxidase immobilized, a counter electrode, and a silver / silver chloride reference electrode. Glucose oxidase immobilized on the working electrode oxidizes glucose in the sample. The generated hydrogen peroxide is electrolyzed on the platinum electrode surface, and the current value at that time is converted into a voltage by the potentiostat (7).
This voltage value is A / D in the single board computer (8).
After conversion, it is sent to the personal computer (10) via the RS-232C cable (9). A glucose concentration is calculated by a personal computer from a calibration curve prepared in advance using a standard solution. When one measurement is completed, a signal is sent to the autosampler control signal line (11) to inject the next sample. Note that (12) in FIG. 1 is a pump control signal, (6)
Indicates waste liquid. The flow path from the autosampler to the electrode is made of a fluororesin tube having an inner diameter of 0.5 mm and a length of 1.5 m.

[Measurement Method] Commercially available soy sauce, which is a sample containing a large amount of impurities that easily contaminate the electrodes, was diluted 10 times to prepare a sample solution. As a buffer solution to be sent to the apparatus, a solution containing 100 mM sodium phosphate, 50 mM potassium chloride, 1 mM sodium azide was adjusted to pH 7.0, and further 0.0
The one to which 1% isopropyl alcohol was added was used.

5 μl of each sample was injected at 180 second intervals. The soy sauce diluted solution was repeatedly measured 12 times, after which the calibration curve was measured again and then 12 times continuously, and a total of 24 times were measured to examine the stability of the glucose concentration measurement value.

[Results] The results are shown in FIG. As can be seen from this figure, the values measured 24 times were extremely stable, and the glucose concentration could always be measured accurately.

Comparative Example 1 The same measurement as in Example 1 was carried out except that a buffer solution containing no isopropyl alcohol was used. 5 μl of each sample was injected at 180 second intervals. Repeatedly measure 12 times for the diluted soy sauce solution, re-measure the calibration curve, then continue 1
The measurement was performed twice, and a total of 24 times was measured to examine the stability of the glucose concentration measurement value.

FIG. 4 shows the measured glucose concentration of the sample. As can be seen from this figure, the measured values of the first and thirteenth injections immediately after the measurement of the calibration curve are high, and there is a phenomenon that the value suddenly deviates from the next measurement, and the measured value of 24 times compared with Example 1. Is unstable. 3 and 4, in Example 1, the addition of isopropyl alcohol did not cause an abnormal measurement value to appear, and the improvement in measurement accuracy was clearly recognized.

As shown in Table 1, the coefficient of variation (CV
%), The effect of adding isopropyl alcohol is clear.

[0027]

[Table 1]

Example 2 Next, an experiment was carried out using a combination of an alcohol oxidase-immobilized column and a hydrogen peroxide electrode.

[Measuring Device] For the measurement, a flow type measuring device shown in FIG. 2 was used. First, the buffer solution is continuously sent from the buffer solution bottle (1) by the solution sending pump (2), and the sample containing ethanol injected from the autosampler (3) is sent to the constant temperature bath (5). Ethanol in the sample is oxidized by alcohol oxidase immobilized on the granular carrier packed in the column (13) in the thermostat, and at the same time hydrogen peroxide is produced. The generated hydrogen peroxide is measured
Detected by (4), the change of current value at that time is converted into voltage by potentiostat (7). The measurement unit has a hydrogen peroxide electrode (working electrode), a counter electrode, and a silver / silver chloride reference electrode.

Voltage signal is a single board computer
After being A / D converted at (8), it is sent to the personal computer (10) via the RS-232C cable (9). The ethanol concentration is calculated by applying it to a calibration curve measured in advance using a personal computer. In addition, (1 of FIG.
1) shows a sampler control signal, (12) shows a pump control signal, and (6) shows waste liquid.

[Measurement Method] As in Example 1, 1 of soy sauce was used.
A 0-fold dilution was used as the sample. As a buffer solution to be sent to the apparatus, a solution containing 100 mM sodium phosphate, 50 mM potassium chloride, 1 mM sodium azide has a pH of 7.
It was set to 0 and isopropyl alcohol was added so that the concentration would be 0.01%.

5 μl of each sample was injected at 180-second intervals. The soy sauce diluted solution was repeatedly measured 12 times, the calibration curve was measured again, and then the measurement was repeated 12 times.
The stability of the measured value of ethanol concentration was examined by measuring 4 times. As a result, the coefficient of variation of 24 measurements (CV%)
Became 1.1 (Table 2).

Comparative Example 2 The same measurement as in Example 2 was carried out except that a buffer solution containing no isopropyl alcohol was used. 5 μl of each sample was injected at 180 second intervals. Repeatedly measure 12 times for the diluted soy sauce solution, re-measure the calibration curve, then continue 1
The measurement was performed twice, and a total of 24 times were measured to examine the stability of the measured value of ethanol concentration.

As a result, the coefficient of variation (CV
%) Was 1.6 (Table 2).

Comparative Example 3 A surfactant (trade name: Triton X-100) containing polyethylene glycol and mono-p-octylphenyl ether in place of isopropyl alcohol was used.
The same measurement as in Example 2 was carried out except that a buffer solution containing 10% was used. 5 μl of each sample was injected at 180 second intervals. The soy sauce diluted solution was repeatedly measured 12 times, the calibration curve was measured again, and then 12 times were continuously measured. A total of 24 times were measured to examine the stability of the measured ethanol concentration value.

As a result, the coefficient of variation (CV
%) Became 1.5 (Table 2).

[0037]

[Table 2]

From Table 2, it can be seen that according to the measuring method of Example 2, the ethanol concentration can be measured more stably than in Comparative Examples 2 and 3.

Example 3 As in Example 2, experiments were carried out using a combination of an alcohol oxidase-immobilized column and a hydrogen peroxide electrode. [Measuring Device] For the measurement, the same flow-type measuring device as that of Example 2 shown in FIG. 2 was used.

[Measurement Method] As in Example 2, 1 of soy sauce was used.
A 0-fold dilution was used as the sample. As a buffer solution to be sent to the apparatus, a solution containing 100 mM sodium phosphate, 50 mM potassium chloride, 1 mM sodium azide has a pH of 7.
It was set to 0 and isopropyl alcohol was further added to 0.5% to use.

5 μl of each sample was injected at 180 second intervals. The soy sauce diluted solution was repeatedly measured 12 times, the calibration curve was measured again, and then the measurement was repeated 12 times.
The stability of the measured value of ethanol concentration was examined by measuring 4 times. As a result, the coefficient of variation of 24 measurements (CV%)
Became 1.1. However, the average of the detected current values was about 87% of that of Example 2. It is considered that this is because the enzyme reaction was suppressed by the high concentration of isopropyl alcohol.

Comparative Example 4 An experiment was carried out using the glucose oxidase-immobilized electrode as in Example 1. [Measurement Device] As in Example 1, the flow-type measurement device shown in FIG. 1 was used.

[Measurement Method] Similar to Example 1, commercially available soy sauce was diluted 10 times to prepare a sample solution. As a buffer solution to be sent to the device, a solution containing 100 mM sodium phosphate, 50 mM potassium chloride, 1 mM sodium azide is added to pH.
It was adjusted to 7.0 and 0.1% methanol was added.

5 μl of each sample was injected at 180-second intervals. The soy sauce diluted solution was repeatedly measured 12 times, after which the calibration curve was measured again and then 12 times continuously, and a total of 24 times were measured to examine the stability of the glucose concentration measurement value.

[Results] As in Comparative Example 1, a change in sensitivity was recognized. Further, the average of the detected current values was about 95% as compared with Example 1, and the output was small.

[0046]

According to the measuring method of the present invention, since the measurement is carried out in such a manner that the analyte in the sample and the alcohol having 3 or more carbon atoms coexist, even if the sample contains impurities that interfere with the measurement. It was possible to remove the influence of low-molecular contaminants and to measure a large number of samples continuously and accurately.

[Brief description of drawings]

1 is a measuring device used in Example 1. FIG.

2 is a measuring device used in Example 2. FIG.

FIG. 3 shows the results of Example 1.

FIG. 4 shows the results of Comparative Example 1.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Buffer bottle 2 Liquid feed pump 3 Autosampler 4 Measuring part 5 Constant temperature bath 6 Waste liquid 7 Potentiostat 8 Single board computer 9 RS232C cable 10 Personal computer 11 Sampler control signal line 12 Pump control signal line 13 Alcohol oxidase fixed column

Claims (2)

[Claims] 1. A measuring method in which a sample in an aqueous medium is brought into contact with an immobilized enzyme to electrochemically detect a change in the concentration of an electrode active substance that increases or decreases due to an enzymatic reaction, and an alcohol having 3 or more carbon atoms is added to the aqueous medium. A measuring method using an immobilized enzyme, which is characterized in that it is included. 2. The measuring method according to claim 1, wherein the aqueous medium contains 0.001 to 0.1% by volume of an alcohol having 3 to 12 carbon atoms.