JP2517151B2 - Measuring method of substrate concentration by biosensor - Google Patents

Description

TECHNICAL FIELD The present invention relates to the determination of a specific component by electrochemically measuring a redox current corresponding to the specific component (substrate concentration) in a biological sample using a biosensor. It relates to the method of quantification.

2. Description of the Related Art Conventionally, a biosensor shown in FIG. 1 has been proposed as a sensor that can easily quantify a specific component in a biological sample such as blood without diluting or stirring the sample solution. This biosensor forms an electrode system composed of a counter electrode 2 and a measurement electrode 3 by printing a conductive carbon paste by screen printing on an insulating substrate 1 made of polyethylene terephthalate and heating and drying. Next, a counter electrode reaction part 2'which becomes an electrochemically acting part of each electrode and a measurement electrode reaction part 3'having a size of 1 mm ^2.
The insulating layer 4 is formed by applying an insulating paste so as to cover the electrode system while leaving the above, and performing heat curing treatment. After that, an enzyme reaction layer 5 composed of a hydrophilic polymer, an oxidoreductase and an electron acceptor is formed so as to cover the counter electrode reaction part 2'and the measurement electrode reaction part 3 '. When the sample solution is dropped onto the enzyme reaction layer 5 thus formed, the enzyme reaction layer 5
The redox enzyme and the electron acceptor contained in the solution dissolve in the sample solution, and the enzyme reaction proceeds with the substrate in the sample solution to reduce the electron acceptor. After completion of the reaction, the above electrode systems 2'and 3 '
When a voltage is applied to the substrate, the electron acceptor reduced by the enzymatic reaction is electrochemically oxidized, and the substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention In such a conventional biosensor, the substance to be measured and the enzyme are pre-reacted, and the measurement is started from the end point of the enzyme reaction. Therefore, it takes a long time to obtain the measurement result. There was a problem.

The present invention solves such a problem, and an object of the present invention is to provide a substrate concentration measuring method capable of accurately measuring in a short time.

Means for Solving the Problems In order to solve such problems, the measuring method of the present invention provides an electrode system composed of a measuring electrode and a counter electrode on an insulating substrate, and a redox enzyme on the surface of the electrode system. An enzyme reaction layer consisting of a hydrophilic polymer and an electron acceptor is provided, and the change in the concentration of the substance produced by the reaction between this enzyme, the electron acceptor and the sample solution is electrochemically detected by the electrode system to determine the substrate concentration. In the biosensor to be measured, a sample liquid is supplied onto the enzyme reaction layer while applying a constant DC voltage between the measurement electrode and the counter electrode,
It measures the flowing current.

Furthermore, the sample liquid is supplied onto the enzyme reaction layer while applying a constant DC voltage, then the application of the voltage is temporarily stopped after the supply of the sample liquid is detected, and the constant voltage is applied again after a certain period of time to flow. It measures the current.

Effect According to the present invention, the time when the supply of the sample liquid is started can be accurately detected, and further, the substrate concentration can be measured in a short time. In addition, since the reducing substance contained in the sample can be electrolytically oxidized and removed by once applying the voltage, the accuracy of the measurement can be improved.

Example An example of the substrate concentration measuring method of the present invention will be described below with reference to a glucose sensor.

Example 1 FIGS. 1 and 2 show the structure of a glucose sensor. FIG. 1 is a perspective view of the sensor, and FIG.
The drawing is a vertical sectional view taken along the line A-B in FIG. By printing a conductive carbon paste by screen printing on an insulating substrate 1 made of polyethylene terephthalate and heating and drying, a counter electrode 2 and a measurement electrode 3
To form an electrode system. Next, insulating paste printing to expose the 'size of the measuring electrode reaction portion 3 with 1 mm ^2' counter reaction unit 2 to be electrochemically acting portion of each of the electrodes (1 mm ^2), the heating The insulating layer 4 is formed by performing a curing process. A 0.5% by weight aqueous solution of carboxymethylcellulose (CMC), which is a kind of cellulosic hydrophilic polymer, is applied and dried so as to cover the surfaces of the counter electrode reaction part 2'and the measurement electrode reaction part 3 ', and 0.5% of CMC is further applied. A solution prepared by dissolving 10 mg of glucose oxidase (GOD) as an oxidase and 20 mg of potassium ferricyanide as an electron acceptor was added dropwise to 1 g of a weight% aqueous solution and dried at 40 ° C. for 10 minutes to form an enzyme reaction layer 5. When a glucose aqueous solution is dropped on the enzyme reaction layer 5 while applying a constant voltage of +0.5 V to the measurement electrode based on the counter electrode, an oxidation current flows. This current flows because potassium ferricyanide is reduced to potassium ferrocyanide when glucose in the sample is oxidized by glucose oxidase, and this potassium ferrocyanide is electrolytically oxidized on the electrode. A correlation was obtained between this oxidation current, for example, the peak current value and the glucose concentration, and good linearity was exhibited up to a glucose concentration of 500 mg / dl. The applied voltage was set to + 0.5V, but the reduced form of the electron acceptor was oxidized,
Moreover, any voltage may be used as long as it does not generate gas. Since the peak of the oxidation current can be obtained within 5 seconds at the latest, the measurement time required by the conventional biosensor for 1 to 2 minutes to complete the reaction could be greatly shortened. In addition to the peak current, the result of measuring the total amount of electricity flowing until the current reaches its maximum is also correlated with the glucose concentration, and the reproducibility of the total amount of electricity is good even if the method of supplying the sample to the sensor varies. It was

(Example 2) A sensor is formed in the same manner as in Example 1, and a constant voltage of +0.5 V is applied to the measurement electrode in the anode direction based on the counter electrode. An aqueous glucose solution was dropped, and after the rise of the oxidation current was detected, the voltage application was stopped, and after 10 seconds, a voltage of +0.5 V was applied again for 0.1 second to measure the maximum value of the oxidation current. Furthermore, after 50 seconds, that is, 1 minute after the start of measurement, a voltage of +0.5 V was applied in the same manner, and the current value after 5 seconds was measured. The maximum current value after 10 seconds and the maximum current value after 1 minute both correlate with glucose concentration. The response after 10 seconds is 700 mg / dl, and the response after 1 minute is linear up to a high concentration of 1000 mg / dl. was there.

The linearity was extended more than in the case of Example 1 because potassium ferricyanide was reacting while being dissolved, and after waiting 10 seconds, potassium ferricyanide corresponding to a high concentration of glucose could not be supplied at 5 seconds. It is considered that the higher concentration of potassium ferricyanide could be supplied. Further, for a sample having a high viscosity such as blood, the rising of the oxidation current was uneven, and the reproducibility was poor in the measuring method of Example 1. It is considered that this is because the dissolution rate of the oxygen reaction layer varied depending on the sample having high viscosity. However, by waiting the measurement for 10 seconds as in Example 2, the reproducibility could be improved considerably.

The data after 10 seconds may be directly converted to glucose concentration, but if it is detected as 500 mg / dl or more, the glucose sensor with the above configuration requires about 1 minute 30 seconds to complete the reaction, so extend the measurement time. When it is set, more accurate measurement becomes possible. In other words, when it is determined (measured) that the concentration is high, the measurement time can be extended and the voltage can be applied again to perform measurement to obtain a more accurate measured value. When a reducing substance such as an acid is contained, it is oxidized on the electrode like potassium ferrocyanide and causes an error.
It was possible to remove the influence on the measurement after 1 minute by applying a voltage once to oxidize after 10 seconds.

In this way, by applying the voltage before dropping the sample, it is possible to detect when the sample is supplied to the sensor, and the waiting time of 10 seconds can be accurate, so there is no need to indicate the start time. The accuracy of measurement is improved.

The biosensor of the present invention is not limited to the glucose sensor shown in the above examples, but can be used in systems involving oxidoreductase such as alcohol sensor and cholesterol sensor. As the oxidoreductase, glucose oxidase was used in the examples, but other enzymes such as alcohol oxidase, cholesterol oxidase,
Xanthine oxidase can also be used. Further, potassium ferricyanide used in the above-mentioned examples stably reacts as an electron acceptor, so that it is suitable for increasing the measurement accuracy. However, if P-benzoquinone is used, the reaction rate is large and thus it is suitable for speeding up. . Also, 2,6-dichlorophenolindophenol, methylene blue, phenazine methosulfate, potassium β-naphthoquinone 4-sulfonate, ferrocene and the like can be used as the electron acceptor.

EFFECTS OF THE INVENTION As is clear from the description of the above embodiments, according to the present invention, an enzyme system comprising an oxidoreductase, a hydrophilic polymer and an electron acceptor is printed by printing an electrode system on an insulating substrate. In a sensor in which a reaction phase is formed, a constant voltage is applied to a sensor in a dry state, and a current flowing when a sample liquid is supplied to the sensor is measured. The measurement time can be significantly shortened to 10 seconds. Furthermore, since the voltage is applied in a dry state, the time when the sample is supplied can be detected by the rise of the current, and the time can be accurately detected. Therefore, the accuracy of measurement can be improved and the operation can be simplified. In addition, after detecting the supply of the sample solution while applying the voltage to the dry sensor, stop the application of the voltage and apply the voltage again to measure the flowing current. It was possible to measure even a highly viscous sample in 10 seconds. Alternatively, based on the response value after 10 seconds,
By adjusting the measurement time, the accuracy of the measurement 1 minute after the sample is supplied can be improved, or the reducing substance existing in the sample and causing an error in the measurement can be electrolytically removed by applying a voltage after 10 seconds. It became possible.

[Brief description of drawings]

FIG. 1 is a perspective view of a biosensor used in one embodiment of the present invention, and FIG. 2 is a vertical sectional view taken along the line AB of the biosensor of FIG. 1 ... Substrate, 2 ... Counter electrode, 2 '... Counter electrode reaction part, 3 ...
Measuring electrode 3 '... Measuring electrode reaction part, 4 ... Insulating layer, 5 ...
Enzyme reaction layer.

Claims (2)

(57) [Claims] 1. An electrode system comprising a measuring electrode and a counter electrode is provided on an insulating substrate, and an enzyme reaction layer comprising an oxidoreductase, a hydrophilic polymer and an electron acceptor is provided on the surface of the electrode system.
In the biosensor for measuring the substrate concentration by electrochemically detecting the concentration change of the substance produced by the reaction of the enzyme, the electron acceptor and the sample liquid, between the measurement electrode and the counter electrode. A method for measuring a substrate concentration by a biosensor, in which a sample liquid is supplied onto the enzyme reaction layer while applying a constant DC voltage to the sample and the flowing current is measured. 2. An electrode system comprising a measuring electrode and a counter electrode is provided on an insulating substrate, and an enzyme reaction layer comprising a redox enzyme, a hydrophilic polymer and an electron acceptor is provided on the surface of the electrode system.
In the biosensor for measuring the substrate concentration by electrochemically detecting the concentration change of the substance produced by the reaction of the enzyme, the electron acceptor and the sample liquid, between the measurement electrode and the counter electrode. The sample solution is supplied onto the enzyme reaction layer while applying a constant direct current voltage to the enzyme reaction layer, and then the application of the voltage is stopped once the supply of the sample solution is detected, and a constant voltage is applied again after a certain time to flow the current. A method for measuring a substrate concentration by a measuring biosensor.