JP3370414B2 - Manufacturing method of biosensor - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a biosensor capable of easily and quickly quantifying a specific component in a sample.

[0002]

2. Description of the Related Art Conventionally, the following biosensor has been proposed as a method for easily quantifying a specific component in a sample without diluting or stirring the sample solution, and the following manufacturing method is disclosed. (Japanese Patent Laid-Open No. 3-20
2764). In this biosensor, an enzyme reaction layer consisting of a hydrophilic polymer, a redox enzyme and an electron acceptor is formed on an electrode system formed on an insulating substrate, and the redox enzyme, the electron acceptor and the sample solution are formed. The substrate concentration is measured by electrochemically detecting a change in the substance concentration during the reaction with the electrode system. As the method for producing the enzyme reaction layer, a hydrophilic polymer solution is applied onto the electrode system and then dried by heating, and then a mixed solution of a hydrophilic polymer, a redox enzyme and an electron acceptor is applied thereon, A process of forming by heating and drying at a constant temperature is disclosed.

[0003]

According to the conventional method for producing a biosensor as described above, the surface of the reaction layer loses smoothness during the production of the reaction layer, and therefore the sample solution is supplied to the biosensor. There was a problem that bubbles might be generated on the electrode at that time, and an accurate response could not be obtained.

[0004]

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 hydrophilic polymer layer on the electrode system. Provided is a method for producing a biosensor in which a reaction layer is formed by further providing a solution containing an oxidoreductase on the hydrophilic polymer layer and then drying the solution at two or more temperatures. Here, the reaction layer is dried under the condition that the temperature of the drying end point is higher than the temperature of the drying start point after dropping a solution containing an oxidoreductase or an electron acceptor on the hydrophilic polymer layer. It is preferably formed by The temperature at the drying start point is in the range of 20 ° C to 40 ° C, and the temperature at the drying end point is 45 ° C to 10 ° C.
It is preferably in the range of 0 ° C.

[0005]

According to the present invention, by further increasing the smoothness of the surface of the reaction layer, it is possible to extremely reduce the probability of generation of bubbles at the time of supplying the sample liquid, and as a result, it is possible to obtain a highly reliable biosensor. be able to. The above effect can be obtained even when the amount of the oxidoreductase added in the reaction layer is large, or when the amount of the electron acceptor contained is large, and when the reaction layer contains a pH buffer. It can be applied to biosensors having various reaction layer configurations. Furthermore, since the amount of water contained in the reaction layer during the production of the biosensor can be further reduced, a biosensor having high storage reliability can be obtained.

[0006]

EXAMPLES The present invention will be described below with reference to examples. Example 1 FIG. 1 is a plan view of a lactate sensor manufactured as an example of the biosensor of the present invention, excluding the reaction layer, and FIG.
FIG. 3 is a sectional view of the lactate sensor. The method for producing the lactate sensor will be described below. On an insulating substrate 1 made of polyethylene terephthalate, silver paste is printed by screen printing to form leads 2 and 3. Next, using a conductive carbon paste containing a resin binder, the measuring electrode 4 of the electrode system and the insulating layer 6 made of an insulating paste are sequentially formed by printing. The insulating layer 6 is the measurement electrode 4
The area of the exposed portion is constant and the leads 2 and 3 are partially covered. Finally, the counter electrode 5 of the electrode system is printed by using a conductive carbon paste containing a resin binder.

Next, a 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer is spread on the electrode system consisting of the measuring electrode 4 and the counter electrode 5 and dried to obtain CMC. Form the layers. Continuing, C
An aqueous solution of lactate oxidase (hereinafter abbreviated as LOD) as an oxidoreductase was developed on the MC layer and dried at 30 ° C. for 5 minutes in a warm air dryer, and then continuously for 5 minutes to 5 minutes.
The temperature is raised to 0 ° C. to form the reaction layer 7. In the above reaction layer forming step, when the LOD aqueous solution is dropped onto the CMC layer, the CMC layer is once dissolved, and the reaction layer 7 is formed in a state of being mixed with the oxidoreductase in the subsequent drying process. However, since it is not accompanied by stirring or the like, it is not in a completely mixed state, and the electrode system surface is in a state of being covered only with CMC.
That is, since oxidoreductase and the like do not come into contact with the surface of the electrode system, it is possible to prevent adsorption of proteins and the like on the surface of the electrode system. Adsorption of proteins on the electrode surface results in a reduction of the active electrode surface area, resulting in reduced sensor response.

In the lactate sensor produced as described above,
When 10 μl of a lactic acid aqueous solution is supplied as a sample solution by dropping onto the reaction layer 7, the reaction layer 7 is promptly dissolved in the sample solution. One minute after supplying the sample solution, a pulse voltage of +1.0 V was applied to the measurement electrode 4 in the anode direction based on the counter electrode 5 of the electrode system, and the current value after 5 seconds was measured. A response current value proportional to the lactic acid concentration of is obtained. The measurement principle of this lactate sensor will be described below. When the reaction layer 7 is dissolved in the sample solution, lactic acid in the sample solution is oxidized by LOD, and at the same time, oxygen dissolved in the sample solution is reduced to generate hydrogen peroxide. Then, by applying the above-mentioned pulse voltage, an oxidation current of the generated hydrogen peroxide is obtained, and this current value is proportional to the concentration of lactic acid as a substrate.

The reaction layer was dried under the conditions of 30 ° C. without raising the temperature.
After 10 minutes, the reaction layer could not be completely dried,
Further, it was necessary to extend the drying time. Meanwhile, 5
When the reaction layer was dried at 0 ° C. for 10 minutes, it was possible to sufficiently dry the reaction layer, but the smoothness of the reaction layer surface was deteriorated, and therefore, when the reaction layer was dissolved in the sample solution, bubbles were not formed. Occurrence was sometimes seen. The formation of bubbles leads to a reduction of the active electrode area, and consequently a factor that prevents an accurate response from being obtained. Also, the drying temperature condition is 30 ° C.
Even when the temperature was raised from 30 ° C. to 50 ° C. for 5 minutes instead of 50 ° C. and dried for 4 minutes continuously, the same effect as above was obtained.
Further, the above effect could be obtained even when the temperature was raised to 50 ° C. and then returned to 25 ° C. That is, the temperature raising process with time is not necessarily required, and the drying conditions may be set at at least two kinds of temperatures. By the above reaction layer drying conditions, the smoothness of the reaction layer surface can be enhanced, and as a result, a lactic acid sensor having highly accurate response characteristics can be obtained.

[Embodiment 2] In the same manner as in Embodiment 1, an electrode system consisting of leads 2, 3, a measuring electrode 4 and a counter electrode 5 shown in FIG. To form. Next, a 0.5 wt% aqueous solution of CMC was developed on the electrode system consisting of the measurement electrode 4 and the counter electrode 5,
Dry to form the CMC layer. Next, the CMC
A mixed aqueous solution of LOD and potassium ferricyanide as an electron acceptor is developed on the layer, and the mixture is heated in a warm air drier for 30 minutes.
After drying at 8 ° C. for 8 minutes, the temperature was raised to 50 ° C. over 5 minutes to form reaction layer 7.

In the lactate sensor produced as described above,
When 10 μl of a lactic acid aqueous solution is supplied as a sample solution by dropping onto the reaction layer 7, the reaction layer 7 is promptly dissolved in the sample solution. One minute after supplying the sample solution, a pulse voltage of +0.5 V was applied to the measuring electrode 4 in the anode direction based on the counter electrode 5 of the electrode system, and the current value after 5 seconds was measured. A response current value proportional to the lactic acid concentration of is obtained. The measurement principle of this lactate sensor will be described below. When the reaction layer 7 is dissolved in the sample solution, lactic acid in the sample solution is oxidized by LOD and at the same time potassium ferricyanide is reduced to produce potassium ferrocyanide. Next, by applying the above-mentioned pulse voltage, an oxidation current of the produced potassium ferrocyanide is obtained, and this current value is proportional to the concentration of lactic acid as a substrate. As in Example 1, a lactic acid sensor having highly accurate response characteristics can be obtained under the above reaction layer drying conditions.

[Embodiment 3] FIG. 3 is an exploded perspective view of a lactate sensor manufactured as an embodiment of the biosensor of the present invention, excluding the reaction layer, and FIG. 4 is a sectional view of the lactate sensor. The method for producing the lactate sensor will be described below. In the same manner as in Example 1, the electrode system including the leads 2, 3, the measuring electrode 4, and the counter electrode 5 and the insulating layer 6 are formed on the insulating substrate 1 by screen printing, as in FIG. Next, 0.5 wt% of CMC was placed on the electrode system consisting of the measuring electrode 4 and the counter electrode 5.
% Aqueous solution is spread and dried to form a CMC layer. Then, a mixed aqueous solution of LOD and potassium ferricyanide was developed on the CMC layer, and the mixture was placed in a warm air drier for 30 minutes.
After drying at 8 ° C. for 8 minutes, the temperature is raised to 50 ° C. over 5 minutes to form the reaction layer 7.

After forming the reaction layer 7 as described above,
The cover 9 and the spacer 8 are adhered to each other in a positional relationship shown by a chain line in FIG. When the cover is attached, the sample solution is easily introduced into the reaction layer 7 portion by a simple operation of bringing the sample solution into contact with the sample supply hole 10 at the tip of the sensor due to the capillary phenomenon in the space created by the cover and the spacer. The supply amount of the sample solution depends on the space volume generated by the cover and the spacer, and therefore does not need to be quantified in advance. Further, the evaporation of the sample liquid during the measurement can be minimized, and the measurement accuracy can be further improved. When 3 μl of a lactic acid aqueous solution as a sample solution is supplied from the sample supply hole 10 to the lactic acid sensor manufactured as described above, the sample solution quickly reaches the air holes 11 and the reaction layer 7 on the electrode system is dissolved.

One minute after supplying the sample solution, the counter electrode 5 of the electrode system is used as a reference and the measuring electrode 4 is +0.5 toward the anode.
When a pulse voltage of V was applied and the current value was measured 5 seconds later, a response current value proportional to the concentration of lactic acid in the sample solution was obtained. By the above reaction layer drying conditions, the smoothness of the reaction layer surface can be enhanced, and as a result, a lactic acid sensor having highly accurate response characteristics can be obtained.

[Embodiment 4] A glucose sensor manufactured as an embodiment of the present invention will be described. The method for manufacturing the glucose sensor will be described below. In the same manner as in Example 1, the electrode system including the leads 2 and 3, the measurement electrode 4 and the counter electrode 5 shown in FIG. 1 and the insulating layer 6 are formed on the insulating substrate 1 by screen printing. Next, a 0.5 wt% aqueous solution of CMC is spread on the electrode system composed of the measurement electrode 4 and the counter electrode 5 and dried to form a CMC layer. Subsequently, a solution of glucose oxidase (hereinafter abbreviated as GOD) as an oxidoreductase and potassium ferricyanide in a phosphate buffer solution (pH = 5.6) was developed on the CMC layer and developed in a warm air dryer. After drying at 30 ° C. for 5 minutes, the temperature is raised to 80 ° C. over 5 minutes and the reaction layer 7
To form.

When 10 μl of an aqueous glucose solution as a sample solution is supplied by dropping onto the reaction layer 7 to the glucose sensor manufactured as described above, the reaction layer 7 is promptly dissolved in the sample solution. 1 minute after supplying the sample solution,
A pulse voltage of +0.5 V is applied to the measurement electrode 4 in the anode direction based on the counter electrode 5 of the electrode system, and the current value after 5 seconds is measured to obtain a response current value proportional to the glucose concentration in the sample solution. To be By the above reaction layer drying conditions, the smoothness of the reaction layer surface can be enhanced, and as a result, a glucose sensor having highly accurate response characteristics can be obtained.

[Embodiment 5] A fructose sensor manufactured as an embodiment of the present invention will be described. The method for producing the fructose sensor will be described below. Example 1
In the same manner as above, the insulating layer 6 and the electrode system including the leads 2 and 3, the measurement electrode 4 and the counter electrode 5 shown in FIG. 1 are formed on the insulating substrate 1 by screen printing. Next, a 1 wt% aqueous solution of polyvinylpyrrolidone (hereinafter abbreviated as PVP) is spread on the electrode system composed of the measurement electrode 4 and the counter electrode 5 and dried to form a PVP layer. Subsequently, a fructose dehydrogenase (hereinafter abbreviated as FDH) as a redox enzyme and potassium ferricyanide were dissolved in a McIlvaine buffer solution (pH = 6) on the PVP layer, and a solution was developed to obtain a constant temperature and a constant humidity. After drying for 10 minutes in an atmosphere (20 ° C., relative humidity 0 to 25%),
The reaction layer 7 is formed by rapidly transferring it into a hot air dryer at 70 ° C. and further drying for 5 minutes.

When 10 μl of a fructose aqueous solution is supplied as a sample solution by dropping onto the reaction layer 7 to the fructose sensor produced as described above, the reaction layer 7 is immediately dissolved in the sample solution. Three minutes after the sample solution was supplied, a pulse voltage of +0.5 V was applied to the measurement electrode 4 in the anode direction based on the counter electrode 5 of the electrode system, and the current value after 5 seconds was measured. A response current value proportional to the fructose concentration of is obtained. By the above reaction layer drying conditions, the smoothness of the reaction layer surface can be enhanced, and as a result, a fructose sensor having highly accurate response characteristics can be obtained.

In the above examples, the drying conditions of the reaction layers of various biosensors were set respectively, but the conditions are not necessarily limited to these conditions, and as a result of various studies, the starting point of drying is from 20 ° C to 40 ° C. The suitable end point of drying is between 45 ° C. and 100 ° C. where the enzyme activity is not significantly damaged. Further, the temperature increasing process with time is not necessarily required, and it suffices to set the drying conditions by at least two kinds of temperatures. Furthermore, within the range where the effect of the present invention can be obtained, the temperature is raised once from the drying start point. It is also possible to finish the drying by lowering the temperature after heating. In the above examples, lactate oxidase, glucose oxidase, and fructose dehydrogenase were used as oxidoreductases, but not limited to these.
The same effect can be obtained by using cholesterol oxidase, urease or the like.

In the examples, carboxymethylcellulose and polyvinylpyrrolidone were used as the hydrophilic polymer, but the hydrophilic polymer is not limited to these, and other cellulose derivatives, specifically, hydroxyethyl cellulose, hydroxypropyl cellulose, Methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose may be used, and further, polyvinyl alcohol, gelatin and its derivatives, acrylic acid and its salts, methacrylic acid and its salts, starch and its derivatives, maleic anhydride and Similar effects were obtained using the salt. On the other hand, as the electron acceptor, various ferrocene derivatives, p-benzoquinone, phenazine methosulfate, methylene blue and the like can be used in addition to potassium ferricyanide shown in the above examples. Furthermore, although the two-electrode system including the measurement electrode and the counter electrode has been described in the above embodiment, the three-electrode system including the reference electrode enables more accurate measurement.

[0021]

As described above, according to the present invention, a biosensor having excellent responsiveness can be obtained by a simple operation.

[Brief description of drawings]

FIG. 1 is a plan view of a lactate sensor according to an embodiment of the present invention, excluding a reaction layer.

FIG. 2 is a cross-sectional view of the lactate sensor.

FIG. 3 is an exploded perspective view of a lactic acid sensor according to another embodiment of the present invention, excluding a reaction layer.

FIG. 4 is a sectional view of the lactate sensor.

[Explanation of symbols]

1 Insulating substrate A few leads 4 measuring poles 5 opposite poles 6 insulating layers 7 Reaction layer 8 spacers 9 cover 10 Sample supply hole 11 air holes

─────────────────────────────────────────────────── --- Continuation of the front page (56) References JP-A-5-196596 (JP, A) JP-A-4-295755 (JP, A) JP-A-6-88805 (JP, A) (58) Field (Int.Cl. ⁷ , DB name) G01N 27/327

Claims (5)

(57) [Claims] 1. A biosensor for quantifying a substrate by reducing an electron acceptor with an electron generated during a reaction between a substrate and a redox enzyme and electrically measuring the reduction amount of the electron acceptor. A method of manufacturing an electrode substrate comprising a measuring electrode and a counter electrode on an insulating substrate, providing a hydrophilic polymer layer on the electrode system, and oxidizing the hydrophilic polymer layer. After dropping the solution containing reductase,
A method for producing a biosensor, comprising a step of producing a reaction layer by drying at least two temperatures. 2. A biosensor for quantifying the substrate by reducing the electron acceptor with an electron generated during the reaction between the substrate and the oxidoreductase and electrically measuring the reduction amount of the electron acceptor. A method of manufacturing an electrode substrate comprising a measuring electrode and a counter electrode on an insulating substrate, providing a hydrophilic polymer layer on the electrode system, and oxidizing the hydrophilic polymer layer. After dropping the solution containing reductase,
A method for producing a biosensor, comprising a step of producing a reaction layer by drying under a condition that a temperature at a drying end point is higher than a temperature at a drying start point. 3. A biosensor for quantifying the substrate by reducing the electron acceptor with an electron produced during the reaction between the substrate and the oxidoreductase and electrically measuring the reduction amount of the electron acceptor. A method of manufacturing an electrode substrate comprising a measuring electrode and a counter electrode on an insulating substrate, providing a hydrophilic polymer layer on the electrode system, and oxidizing the hydrophilic polymer layer. A method for producing a biosensor, comprising a step of producing a reaction layer by dropping a solution containing a reductase and an electron acceptor and then drying the solution under a condition in which a temperature at a drying end point is higher than a temperature at a drying start point. 4. The method for producing a biosensor according to claim 2, wherein the temperature at the drying start point is in the range of 20 ° C. to 40 ° C., and the temperature at the drying end point is in the range of 45 ° C. to 100 ° C. 5. The bio according to claim 1, 2 or 3, wherein the oxidoreductase is one selected from the group consisting of lactate oxidase, glucose oxidase, fructose dehydrogenase, cholesterol oxidase and urate oxidase. Sensor manufacturing method.