JP2727704B2 - Biosensor manufacturing method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a biosensor electrode test method capable of quickly and easily quantifying specific components in various trace biological samples without diluting a sample solution. Related to manufacturing method.

2. Description of the Related Art Conventionally, a biosensor as shown in FIG. 1 has been proposed as a method for easily quantifying a specific component in a biological sample such as blood without diluting or stirring a sample solution. In this biosensor, an electrode system 2 or 3 made of carbon or the like is formed on an insulating substrate 1 by a method such as screen printing, and an enzyme made of a hydrophilic polymer, an oxidoreductase, and an electron acceptor is formed on the electrode. The reaction layer 5 is formed. When the sample solution is dropped onto the enzyme reaction layer 5, the oxidoreductase and the electron acceptor 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 reduced electron acceptor is oxidized by applying a voltage to the electrode system, and the substrate concentration in the sample solution is determined from the oxidation current value obtained at this time. Since this biosensor is a disposable type, the area of each electrode system needs to be uniform.

Problems to be Solved by the Invention As a method for inspecting the area of the electrode, two-dimensional image processing by a camera can be considered, but the screen surface printing does not smooth the surface of the electrode, so the two-dimensional processing alone correlates with the response of the biosensor. Was not obtained. Therefore, JP 63
-144245 proposed a simple electrode inspection method.
In this method, an equimolar solution (electrode test solution) of an oxidized form and a reduced form of an electron acceptor is dropped on an electrode in advance, a voltage is applied to the electrode system to oxidize the reduced form of the electron acceptor, and the oxidation current is measured. Thus, a fairly good correlation with the response of the biosensor was obtained in the method of examining the electrode area (reaction area). Further, the disconnection of the lead, which is difficult to detect by the image processing, could be easily detected because the oxidation current did not flow.

However, in this method, a step of washing and drying the electrode is required in order to remove the electrode test solution after performing the electrode test. Furthermore, since a hydrophilic polymer layer and an oxidoreductase layer were formed on the electrode surface, they were adsorbed on the electrode surface, reducing the reaction area and causing a phenomenon that the correlation in the electrode test solution was not bad.

In addition, in the conventional production of biosensors, the enzyme reaction layer is prepared by dissolving the enzyme in water and then dropping it over time at a temperature at which the enzyme is not deactivated. There was a problem that the reaction rate also decreased due to a decrease in the dissolution rate.

Means for Solving the Problems In order to solve the above problems, the present invention provides an electrode system comprising at least a measurement electrode and a counter electrode on an insulating substrate, and a substance for reacting an enzyme, an electron acceptor, and a sample solution. In a biosensor in which a change in concentration is electrochemically detected by the electrode system and a substrate concentration in a sample solution is measured, an enzyme reaction layer comprising a redox enzyme, a hydrophilic polymer, and an electron acceptor is provided on the surface of the electrode system. Is a method in which a voltage is applied to the electrode system to inspect the electrodes when forming the electrodes. Furthermore, as described above, the present invention is a method for producing a biosensor in which an enzyme reaction layer is formed by heating and drying an oxidoreductase, a hydrophilic polymer, and an electron acceptor after inspecting an electrode.

According to the present invention, the electrode system can be inspected in the process of configuring a disposable type biosensor including the electrode system. Furthermore, since the inspection is performed after the hydrophilic polymer and the oxidoreductase are formed on the electrode surface, it is possible to inspect the reaction area at the time of response in the sensor. In addition, since the enzyme reaction layer was formed in a short time by heating when forming the enzyme reaction layer, the particle diameter of the electron acceptor was reduced, the dissolution rate was large, and the reaction could be completed quickly.

Embodiment 1 Hereinafter, an embodiment of the present invention will be described. A glucose sensor will be described as an example of a biosensor. 1 and 2 show a glucose sensor, and are a perspective view and a longitudinal sectional view of the sensor. A conductive carbon paste is printed on the insulating substrate 1 made of polyethylene terephthalate by screen printing,
By heating and drying, an electrode system including the counter electrode 2 and the measurement electrode 3 is formed. Next, the electrode system is partially covered, and 2 ', 3' (1 m
The insulating paste is printed in the same manner as described above and subjected to a heat treatment to form the insulating layer 4 so as to leave m ² ).

A 0.5% aqueous solution of carboxymethylcellulose (CMC), which is a kind of cellulosic hydrophilic polymer, is applied so as to cover the surface of the electrode system (2 ′, 3 ′).
A solution prepared by dissolving 20 mg of glucose oxidase (GOD) as an oxidoreductase, 33 mg of potassium ferricyanide as an oxidant of an electron acceptor, and 3 mg of potassium ferrocyanide as a reductant of an electron acceptor in 1 ml of a 0.5% aqueous solution was added dropwise. A constant voltage of +0.5 V is applied to the measurement electrode in the direction of the anode, and the current after 5 seconds is measured. This electric current flows as potassium ferrocyanide is oxidized to potassium ferricyanide, and since the concentration of potassium ferrocyanide is constant, it depends on the area of the electrode and the electrode can be inspected.

With this inspection method, inspection can be performed during the manufacturing process of the sensor and current does not flow if there is a broken wire of the lead which is difficult to find by image processing, and if the insulation is defective, excess current flows, so it is possible to easily detect defects in the printing process . Furthermore, the correlation between the response of this test method and the response of the sensor to glucose actually measured is better than the correlation between the response obtained with the electrode test solution containing only the oxidized form and the reduced form of the electron acceptor and the sensor response. Obtained. This is presumably because the inspection was performed in a state where CMC and GOD were present on the electrode, and thus the viscosity and the state of the electrode surface were more similar when measuring the sensor. Although the constant voltage is applied in the first embodiment, the voltage may be swept toward the anode to measure the peak current. Although the applied voltage is set to +0.5 V, it is preferable that the applied voltage is such that the reduced form of the electron acceptor is oxidized and does not involve generation of hydrogen. If the reduced form of the electron acceptor remains after the electrode inspection, a small amount of the reduced form is desirable because it gives a positive error in the actual measurement.

After confirming that the oxidation current was within 5% of the coefficient of variation (CV value) by electrode inspection, the components of the enzyme reaction layer were heated at 40 ° C. for 5 minutes to form an enzyme reaction layer.

10 μl of a glucose standard solution is dropped as a sample solution on the glucose sensor configured as described above, and after 1 minute, a constant voltage of +0.5 V is applied to the measurement electrode toward the anode with reference to the counter electrode, and the current is measured after 5 seconds. I do. Potassium ferricyanide is dissolved by the glucose standard solution, and is reduced to potassium ferrocyanide when glucose is oxidized in the enzyme reaction layer. Thus, by applying the above constant voltage, an oxidation current based on the concentration of the generated potassium ferrocyanide is obtained, and this current value corresponds to the concentration of glucose as a substrate. When the response current was measured, good linearity was obtained up to a high concentration of 600 mg / dl.

As in the conventional case, when the enzyme reaction layer was dried at 25 degrees, it took 25 minutes to dry it. When the response was examined for the biosensor of the enzyme reaction layer with and without the enzyme reaction layer heated, when the glucose concentration was 100 mg / dl, the reaction was completed in 30 seconds when heating was performed in about 30 minutes compared with the case where the reaction was not completed in about 1 minute. It took the reaction to finish. This is because, when heated, potassium ferricyanide particles are uniformly distributed in a fine state because drying is performed quickly, whereas when not heated, potassium ferricyanide grows into large crystals and the dissolution rate is reduced. It is considered that the reaction rate decreased. In addition, since the linearity does not change even when heated to 40 degrees, it is considered that heating for a short time does not affect the activity of the enzyme.

The biosensor of the present invention is not limited to the glucose sensor described in the above embodiment, but can be used for a system involving an oxidoreductase, such as an alcohol sensor or a cholesterol sensor. In the examples, glucose oxidase was used as the oxidoreductase, but other enzymes such as alcohol oxidase, cholesterol oxidase, and xanthine oxidase can be used. Also,
As the electron acceptor, potassium ferricyanide used in the above example is suitable because it reacts stably, but P-benzoquinone is suitable, for example, because of its high reaction speed. Also, 2.6-dichlorophenol indophenol, methylene blue, phenazine methosulfate, β
-Potassium naphthoquinone 4-sulfonate, ferrocene and the like and their respective reductants can be used.

Effect of the Invention As described above, the electrode inspection method of the present invention involves printing an electrode system on an insulating substrate and forming an enzyme reaction layer comprising a oxidoreductase, a hydrophilic polymer, and an electron acceptor. By applying a voltage after dropping the components of the enzyme reaction layer onto the substrate, it was possible to easily inspect the printing state of the electrode.
Further, the production method of the present invention can form a fine particle size of the electron acceptor by heating and quickly drying the components of the enzyme reaction layer after the inspection of the electrode, and can greatly contribute to the improvement of the reaction speed of the sensor. Was. This electrode inspection method is suitable for mass production because it can be performed during the production of the sensor.

[Brief description of the drawings]

FIG. 1 is a perspective view of a biosensor according to one embodiment of the present invention, and FIG. 2 is a longitudinal sectional view of the biosensor. 1 ... substrate, 2 ... counter electrode, 3 ... measurement electrode, 4 ... insulating layer, 5 ... enzyme reaction layer.

Claims (1)

(57) [Claims] An insulating substrate provided with an electrode system comprising at least a measurement electrode and a counter electrode, wherein the surface of the electrode system is composed of an oxidoreductase, a hydrophilic polymer, and an oxidized form and a reduced form of an electron acceptor. In a biosensor in which an enzyme reaction layer is provided and a substance concentration change during the reaction between the enzyme, the electron acceptor, and the sample solution is electrochemically detected by the electrode system to measure the substrate concentration of the sample solution, an enzyme reaction is applied to the electrode system. A method for producing a biosensor, comprising: dropping a component of a layer, inspecting an electrode system, and heating and drying the component of the enzyme reaction layer to form an enzyme reaction layer.