JPH0612356B2 - Substrate concentration measurement method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for detecting a specific component in a biological sample, which can be widely applied to the medical field, food engineering and the like.

Conventional technology With the advancement of medical technology, by measuring specific components in blood and urine, it has become possible to understand the effects of health check, disease state, and treatment. However, conventionally, there is a problem that it takes time and cost because the examination is performed in a clinical laboratory of a hospital with a large machine or a complicated method. Therefore,
There is a demand for a sensor that can more easily measure on the spot. As one of such attempts, a simple measuring instrument using light has been proposed. FIG. 5 shows the measuring chip. A reagent layer 9 carrying an enzyme and a coloring reagent is laminated on a support 8. When blood is dropped on the reagent layer 9, the reagent layer 9
In, the reaction proceeds and color develops depending on the substrate concentration. This is a method in which blood cells are removed after a certain period of time, light is applied from the direction of the arrow to examine the degree of color development, and the substrate concentration is detected. In the case of this sensor, there is an advantage that the measurement can be easily performed by dropping a small amount of blood. As a calibration method of this system, a standard solution of a substrate having a known concentration is used in the same manner as the sample solution, and a calibration curve is obtained from the degree of color development and the substrate concentration. Further, in order to calibrate easily, photometric measurement is performed using a colored calibration chip to obtain a calibration curve.

Problems to be Solved by the Invention In the calibration method of the conventional example, when a standard solution of a substrate having a known concentration is used, the standard solution of the substrate must be stored at a low temperature in a refrigerator and returned to room temperature at the time of measurement, which is troublesome. There was a drawback of this. Further, a colored calibration chip can be easily calibrated, but the degree of color development differs even at the same temperature due to the influence of temperature, so that measurement at a constant temperature is required.

Therefore, it is an object of the present invention to provide a method capable of easily performing temperature correction and highly accurate measurement.

Means for Solving the Problems The present invention uses an electrode system consisting of a measurement electrode, a counter electrode and a reference electrode provided on an insulating substrate, and reacts an enzyme with an oxidized electron acceptor and a sample solution, The concentration of the reduced electron acceptor produced is detected as an oxidation current in the electrode system to measure the substrate concentration, and a solution containing a known concentration of the oxidized and reduced electron acceptors is used as a calibration solution. , Dropping on the electrode system, measuring the oxidation current according to the concentration of the reduced electron acceptor, and comparing it with the oxidation current of the calibration solution measured at each temperature in advance, the temperature at the time of measurement was detected and obtained with the sample solution. When calculating the substrate concentration from the obtained values, temperature correction is applied.

Action Since a solution in which a fixed amount of the oxidized and reduced forms of the electron acceptor is dissolved is dropped on the electrode as a calibration liquid to cause an electrode reaction to measure an oxidation current, the temperature can be detected without using a thermistor. Further, the state of the electrode surface can be understood from the waveform of the oxidation current.

Example A glucose sensor will be described as an example of one of biosensors. Glucose oxidase was used as a redox enzyme, and potassium ferricyanide was used as an electron acceptor. FIG. 1 shows a schematic view of an embodiment of the glucose sensor. Platinum was embedded in an insulative substrate 1 made of vinyl chloride resin to form an electrode system consisting of a measurement electrode 2, a counter electrode 3 and a reference electrode 4 to form an electrode portion. A liquid-retaining layer 5 made of rayon paper, a superlayer 6 made of a polycarbonate porous film having a pore diameter of 1 μm, and a reaction layer 7 supporting a high concentration of potassium ferricyanide of glucose oxidase on a nonwoven fabric of pulp were provided so as to cover the electrode system. .

When blood is added to this reaction layer 7, glucose in blood is oxidized by glucose oxidase, and at the same time potassium ferricyanide is reduced to potassium ferrocyanide. Subsequently, the reacted blood has a blood cell component removed in the overlayer 6, and the blood (plasma) passed by the liquid retaining layer 5 is retained on the electrode. With respect to potassium ferrocyanide in the reacted plasma, the potential of the measurement electrode was set to 0 to +0.
Oxidation is performed by sweeping in a sawtooth shape at 0.1 V / sec between 1 V, at which time an oxidation current flows. This oxidation current corresponds to the concentration of the produced potassium ferrocyanide and further corresponds to the concentration of the substrate. Therefore, the concentration of glucose as the substrate can be detected by measuring the current value. The peak value of the oxidation current flowing at this time was measured as the response current.

In the case of the present glucose sensor, the measured current affects the rate of enzymatic reaction and the rate of diffusion of the produced potassium ferrocyanide to the electrode. When the reaction layer 7 is loaded with a high concentration of glucose oxidase and potassium ferricyanide to promptly carry out the enzymatic reaction, the influence of temperature on the enzymatic reaction can be reduced. Therefore, in order to investigate the effect of temperature on the diffusion rate, potassium ferricyanide and potassium ferrocyanide were each added to 0.0
A solution containing 2M was used as a calibration solution, and the oxidative current was measured at the same temperature as that of blood by dropping it on the electrode part at each temperature. As shown in FIG. A linear relationship was obtained. The response current at each temperature was obtained with good reproducibility if the area of the measuring electrode 2 was the same and the concentration of potassium ferrocyanide was the same. Therefore, even if the measurement temperature is unknown, the measurement temperature can be detected from the straight line A in FIG. Therefore,
When measuring blood, first, the response current i ₁ of the calibration liquid is measured. The response current of the calibration solution at 25 ° C. is i ₁ .
When the response current i obtained by actually dropping blood is multiplied by i ₂ / i ₁ , it can be converted into the response current of the blood at 25 ° C. The glucose concentration in blood as a sample can be calculated from the converted response current in the relational expression between the blood glucose concentration at 25 ° C. and the response current. When glucose standard solution 200mg / dl was measured at each temperature, the response current was 3rd
As shown in B of the figure, or when the temperature was corrected by the calibrating solution, the same values as in C were obtained, and the influence of temperature was eliminated and accurate measurement was possible. When the blood samples were also measured and temperature corrected, a very good correlation was obtained with the values analyzed in the hospital. As a result, even if the thermistor is not attached, the temperature can be corrected only by the calibration liquid, and the calibration liquid can be stored stably in the shielded container at room temperature for a long period of time, which is convenient for handling.

As for the waveform of the response current due to the calibration liquid, the position of the peak and the magnitude of the peak current are stable as shown by D in FIG. However, if an excessive current flows on the surface of the electrode and it is oxidized, or if proteins in the blood adhere to the surface of the electrode and become dirty, the peak position shifts as shown in E of FIG. 4, and the magnitude of the response current is small. became. Therefore, when the electrode was washed and the response current was measured again with the calibration solution, the original waveform was restored.
From this, it is possible to know not only the temperature but also the state of the electrode surface by measuring with the calibration liquid,
It was possible to eliminate the error caused by the contamination of the electrodes.

As an electron acceptor used as a calibration solution, potassium ferricyanide is suitable because it reacts stably.
-Benzoquinone, 2,6-dichlorophenolindophenol, methylene blue, phenazine methosulfate, β-naphthoquinone-4-potassium sulfonate and the like can also be used.

The sensor in the above-mentioned examples is not limited to glucose, but can be used in systems involving oxidoreductase such as alcohol sensor and cholesterol sensor.

EFFECTS OF THE INVENTION According to the present invention, temperature can be easily corrected with a calibration liquid,
Furthermore, the state of the electrode surface can be confirmed, and high-precision measurement becomes possible.

[Brief description of drawings]

FIG. 1 is a longitudinal sectional view of a glucose sensor according to an embodiment of the present invention, FIGS. 2 and 4 are diagrams showing a relationship between temperature and response current, and FIG. 3 is a diagram showing a relationship between current and voltage. FIG. 5 is a vertical sectional view of a conventional biosensor. 1 ... Substrate, 2 ... Measurement electrode, 3 ... Counter electrode, 4 ... Reference electrode, 5 ... Liquid retaining layer, 6 ... Overlayer, 7 ... Reaction layer.

Claims (1)

[Claims] 1. A concentration of a reduced electron acceptor produced by a reaction between an enzyme, an oxidized electron acceptor and a sample solution, using an electrode system comprising a measuring electrode, a counter electrode and a reference electrode provided on an insulating substrate. Is detected as an oxidation current, and the substrate concentration of the sample solution is measured, which is measured in the same manner as above using a solution containing a known concentration of the oxidized and reduced forms of the electron acceptor as a calibration solution. However, the substrate is characterized in that the temperature at the time of measurement is detected from the relational expression between the temperature measured in advance and the response of the calibration liquid, and the temperature is corrected when the substrate concentration is calculated from the value obtained in the sample liquid. How to measure concentration.