JPS613048A - Measurement using biosensor - Google Patents

Abstract

PURPOSE:To measure a specimen with high sensitivity and high accuracy by removing the interaction between an electrode for detecting the quantity of a substance to be measured and an electrode for detecting the quantity of a measurement obstructing substance, by providing both electrodes in a mutually separated state. CONSTITUTION:The electrode 8 for detecting the quantity of a substance to be measured is constituted by providing a platinium electrode on which glucoseoxidase is imobilized, as an working electrode 8a and a Ag-AgCl electrode as a counter electrode 8b. As the acting electrode 7a of an electrode 7 for detecting the quantity of a measurement obstructing substance, a platinum electrode is provided and a Ag-AgCl electrode is provided as a counter electrode 7b. The electrodes 8, 7 are separated through a liquid passing part (l) so as to generate no interaction. Then, a buffer solution 1 is sent and a specimen is introduced from an injection port 6 and both of them are sent to the electrodes 7, 8 at a constant flow speed. The quantity of the substance to be measured is calculated by subtracting the current IA corresponding to the quantity of the obstructing substance at the electrode 7 from the current IB corresponding to the sum of the quantities of the substance to be measured and the obstructing substance at the electrode 8. Because the electrodes for respectively detecting the substance to be measured and the obstructing electrode are provided, highly accurate measurement is enabled.

Description

[Detailed description of the invention] [Bujo j (affirmation/?r total)] The present invention relates to a measurement method using a biosensor.

[Background technology]

Biosensor has selectivity of the substance to be measured! 14 degrees.

Due to its excellent responsiveness, it has recently been used for clinical testing.

It is becoming widespread, including in the food industry.

This trend is thought to become even stronger with the improvement of immobilization technology for physiologically active substances such as enzymes.

Electrode-type biosensors are electrochemical devices in which physiologically active substances such as enzymes are immobilized, and the amount of substances consumed or produced is measured electrochemically by the reaction between the immobilized enzymes and the target substance. It is detected by converting it into an electrical quantity such as voltage or current using a device. Electrochemical devices include various types of electrodes such as oxygen electrodes and hydrogen peroxide electrodes, and biosensors generally consist of such electrodes provided with a membrane of a physiologically active substance.

Such a biosensor has a drawback in that it reacts (electrode reaction) with reducing substances (measurement interfering substances) such as ascorbic acid present in the subject, in addition to the target substance. Methods to overcome this drawback include attaching a porous membrane to the biosensor that allows the analyte to pass through but not the interfering substances, electrolytically oxidizing the interfering substances, or adding interfering substances to the column. Although there are methods such as adsorption, sensitivity and responsiveness have to be sacrificed because electrolytic oxidation of interfering substances takes time and columns become clogged.

Therefore, as another method to eliminate the influence of interfering substances, we installed an electrode that reacts only to the interfering substance, and used an electrode for detecting the amount of the substance to be measured (for the target substance) (an electrode on which a physiologically active substance is immobilized and an electrode that reacts only to the interfering substance). There is a method of subtracting the fixed value obtained by the electrode for detecting the amount of interfering substances from the measured value (output) obtained by the 1-pole. However, this method requires electrolytic oxidation of interfering substances,
Since it is not adsorbed to the column, it does not have the disadvantage of sacrificing sensitivity or responsiveness, but it does have the disadvantage that measurement accuracy is not very good.

[Purpose of the invention]

This invention was made in view of these circumstances,
The purpose of the present invention is to provide a measurement method using a biosensor with excellent sensitivity and responsiveness, and high measurement accuracy.

[Disclosure of the invention]

The inventors have conducted repeated research in an attempt to achieve the objective by improving the method of using the electrode for detecting the amount of interfering substances. As a result, in the conventional method, the electrode for detecting the amount of the substance to be measured and the electrode for detecting the amount of the interfering substance were too close to each other, and interaction occurred between the two electrodes, resulting in noise that reduced measurement accuracy. Do you get it. Therefore, we thought that reducing this interaction would improve measurement accuracy, and thus completed this invention.

Therefore, the present invention uses an electrode for detecting the amount of a substance interfering with measurement in addition to an electrode for detecting the amount of the substance to be measured, and corrects the amount of electricity obtained by the former with the amount of electricity obtained from the latter. The gist of the present invention is a measurement method using a biosensor, which is characterized in that, when quantifying a substance to be measured, the two electrodes are separated from each other so that a sample solution phase is interposed between them.

The present invention will be explained in detail below.

FIG. 1 shows a measuring device used to carry out the measuring method according to the present invention. As shown in the figure, this measuring device
It is a flow type measurement device, and includes a solvent reservoir into which a solvent (buffer solution) 1 is placed. Passage through which the solvent l passes3. Each of them is provided with a receiver (waste liquid reservoir) 4 for the solvent 1 . The passage 3 has one end facing the solvent reservoir 2 and the other end facing the receiver 4. and,
In the middle part of the passage 3, there are pumps (metering pumps) 5. Sample injection port6. An interfering substance detection device 7 and a biosensor 8 are respectively arranged. The interfering substance detection device 7 is provided with a working electrode 7a and a counter electrode 7b as electrodes for detecting the amount of interfering substances to be measured.
A device 9a equipped with a power source and a current needle is connected to a and 7b. The biosensor 8 is provided with an electrode (working electrode) 8a to which a physiologically active substance is immobilized and its counter electrode 8b, and both electrodes (target substance detection electrodes) 8a and 8b are equipped with a device equipped with a power source and a current needle. 9b is connected. The interfering substance detection bag W7 and the biosensor 8 are separated via a passage (liquid passage) 3 to prevent interaction between their electrodes. Electrodes 7a, 7b and electrodes 8a, 8b are 3
It is preferable that the distance be 0 cm or more. Devices 9a, 9
A recording device IO such as a recorder is connected to b.

The measuring method according to this description is carried out using the measuring device, for example, in the following manner. First, the pump 5 passes the 18 medium 1 through the passage 3. And devices 9a, 9b
A constant voltage is applied between the electrodes 7a and Vb and between the electrodes 13a and 13b. Thereafter, a sample is injected through the sample injection port 6, changes in current are measured with the ammeters of the devices 9a and 9b, and the results are recorded with the recording device 10. The magnitude A of the current change measured by the device 9a is a value corresponding to the amount of the interfering substance, and the magnitude B of the current change measured by the device 9b is a value that corresponds to the magnitude of the current change corresponding to the amount of the interfering substance. It is the sum of the magnitude of the current change corresponding to the amount of material. Therefore, from the magnitude of current change B to the magnitude of current change A
The value obtained by subtracting the value accurately corresponds to the amount of the substance to be measured. In the measurement method according to the present invention, an electrode 8a and a counter electrode 8b on which a physiologically active substance is immobilized, and electrodes 7a and 7b for detecting an obstruction yt are separated through a sample solution phase so that no interaction occurs. Therefore, the magnitude of the current change A, B
can now be measured accurately, and the value obtained by subtracting the magnitude of current change Δ from the magnitude of current change B corresponds precisely to the amount of the substance to be measured. Therefore, measurement accuracy is high. Moreover, it is easy to operate, and since interfering substances are not electrolytically oxidized or adsorbed to the column, the sensitivity and response are excellent.

In the above measurement, an example was explained using a flow-type measuring device, but even a hatch-type measuring device has an electrode on which a physiologically active substance is immobilized, a counter electrode thereof, and an electrode for detecting the amount of interfering substances. If the two are separated via the sample solution phase so that no interaction occurs, measurement accuracy will be increased.

Next, the measurement methods of the Examples and Comparative Examples were carried out using the measuring apparatus shown in FIG. 1 to explain the Examples and Comparative Examples. However, the solvent is 30'C, pH 7.5,
A platinum electrode is used as the working electrode 7a of the interfering substance detection device 7, an Ag-AgC1 electrode is used as the counter electrode 7b, and a platinum electrode on which glucose okindase is immobilized as the working electrode 8a of the biosensor 8.

A Heg-AgC1 electrode was used as the counter electrode 8b, and the interfering substance detection device 7 and the biosensor 8 were connected directly or via a Teflon tube with an inner diameter of 0.5 mmφ. The length p of the Teflon tube is 0
It is decided to change it to ~2m, and i1! =Om was used as a comparative example, and when N = 0.3.0°8.1.1.5.2m was used as Examples 1 to 5.

While flowing the solvent 1 through the passage 3 at a constant flow rate, a voltage of +6V was applied between the electrodes 7a and 7b of the interfering substance detection device and the electrodes 8a and 8b of the biosensor, and the sample was injected. 10 μe of standard serum was injected through inlet 6.

The output current between electrodes 7a and 7b and between electrodes 3a and 3b was recorded by a recording device 10. Comparative example (ji = Om
) and the results of Example 3 (ff=1 m) are shown in FIG. 2(a) and FIG. 2(b), respectively. However, in the figure,
The solid line represents the output current generated by the biosensor 8, and the broken line represents the output current generated by the interfering substance detection device.

In Comparative Example and Example 3, (a) of FIG.

(b) Glucose in the standard serum was determined by determining the difference between the output current peak height of the solid line and the output current peak height of the broken line. Glucose was determined in the same manner as in Example 1 and 2.4°5.

From (a) and (b) in Figure 2, in the measurement results of the output current generated by the biosensor, in the comparative example, the output drops at the rise of the output current peak, causing noise, but in the example, the output drops and noise occurs. It can be seen that there is no noise at all, although the height of the output current peak corresponding to the measured substance is somewhat lower. Therefore, in Example 3, it was possible to quantify crucose more accurately than in the comparative example.

The same results as in Example 3 were obtained for Example 1.2.4.5, and it was possible to quantify crucose more accurately than in the comparative example.

The peak height h of the output current generated by the biosensor (see (b) in Figure 2) and the noise size n (see (a) in Figure 2) in Comparative Examples and Examples 1 to 5. The relationship with the length p of the Teflon tube is shown in FIG. However, in the figure, ○ represents the size of h, and △ represents the size of n.

From Figure 3, if the distance is 30cn or more, the sensitivity will hardly be sacrificed (h will not become too small).
It can be seen that the noise could be reduced.

〔Effect of the invention〕

The measurement method using the biosensor according to the present invention uses an electrode for detecting the amount of a substance interfering with measurement in addition to an electrode for detecting the amount of the substance to be measured, and the amount of electricity obtained with the former is used as the amount of electricity obtained with the latter. When quantifying the substance to be measured by correcting the amount,
Since both electrodes are separated from each other so that the sample solution phase is present between them, sensitivity and responsiveness are excellent, and measurement accuracy is high.

[Brief explanation of the drawing]

Fig. 1 is an explanatory diagram of the structure of the measuring device used in the first implementation of the measuring method according to the present invention, Fig. 2 (a) is a graph showing the output current measurement results in a comparative example, and Fig. 2 (b) is a graph showing the output current measurement results in a comparative example. is a graph showing the output current measurement results in Example 3,
Figure 3 is a graph showing the relationship between the peak height h of the output current generated by the biosensor, the noise size n, and the Teflon tube length p in Comparative Example and Examples 1 to 5. It is.

Claims (3)

[Claims] (1) In addition to the electrode for detecting the amount of the substance to be measured, an electrode for detecting the amount of the substance interfering with the measurement is also used, and the amount of electricity obtained with the former is corrected with the amount of electricity obtained with the latter. A measurement method using a biosensor, characterized in that, in performing quantitative determination, the two electrodes are separated from each other so that a sample solution phase is interposed between them. (2) A measurement method using the biosensor according to claim 1, wherein the distance between the electrode for detecting the amount of the substance to be measured and the electrode for detecting the amount of interfering substance is 30 cm or more. (3) A measurement method using the biosensor according to claim 1 or 2, which is a flow measurement method.