JP2000046782A - Biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the response and reproducibility of an electrode type biosensor by improving adhesive properties of an electrode of a reaction detector in a reagent part reacting with a specimen component to be applied to the sensor. SOLUTION: An additive containing a protein or its derivative of a biopolymer is blended with a reagent part 10 of the biosensor, a thickness of the part 10 is suppressed, and adhesive properties of the part 10 with surfaces of electrodes 4, 5 for carrying the part 10 can be enhanced. Thus, the response reproducibility of the sensor is upgraded, further a linear correlativity of the concentration of the component and sensor response is improved, and the biosensor having higher reliability can be realized.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a so-called electrode-type biosensor, and more particularly, to a method in which a sample is administered in order to improve responsiveness and reproducibility in specifically qualifying or quantifying components in the sample. The present invention relates to a biosensor having an improved reagent section.

[0002]

2. Description of the Related Art In recent years, the development of a biosensor has enabled a simple measurement system for directly administering a sample to a sensor without dilution. Hereinafter, a conventional biosensor technology will be described by taking glucose measurement as an example.

[0003] Initially, as a method for measuring glucose, a biosensor combining glucose oxidase (hereinafter abbreviated as "GOD") as an enzyme for oxidizing glucose and an oxygen electrode or a hydrogen peroxide electrode was developed. . In this biosensor, when β-D-glucose reacts with GOD of an oxidase to convert it into δ-gluconolactone, oxygen becomes an electron acceptor and hydrogen peroxide is generated. The glucose is measured by measuring the amount of oxygen consumed by the oxygen electrode or measuring the amount of hydrogen peroxide produced by the hydrogen peroxide electrode.

[0004] However, when the measurement is performed by the electrode method using the oxygen electrode or the hydrogen peroxide electrode, the detection of glucose cannot be performed stably because the measurement is strongly dependent on the concentration of oxygen dissolved in the specimen sample.

For the purpose of solving this problem, the use of an enzyme reaction detection reagent (hereinafter, referred to as “mediator”) for detecting an enzyme reaction instead of oxygen as an electron acceptor allows a sample sample to be sampled without depending on dissolved oxygen. It has become possible to realize a biosensor capable of stably measuring glucose detection.

As shown in FIG. 1, a biosensor manufactured using a mediator has an electrode system having a measurement electrode 4 connected to a lead 2 and a counter electrode 5 connected to a lead 3 on an insulating substrate 1. A detection section 9 is formed, and a reagent section 10 containing an oxidoreductase and a mediator is formed on the electrode system detection section 9 as shown in FIG. 1 (b). The qualitative or quantitative detection of the administered sample is performed by the electrode system detecting section 9. Here, when glucose is used as the specimen sample, for example, GOD is used as the oxidoreductase, and potassium ferricyanide is used as the mediator, for example. In FIG. 1A, reference numeral 6 designates an exposed area of the electrodes 4 and 5 and an insulating layer which covers unnecessary portions of the leads 2 and 3 between the electrodes 4 and 5, reference numeral 7 denotes a spacer, and reference numeral 8 denotes a spacer. It is a cover.

In this biosensor, potassium ferricyanide as a mediator is consumed as an electron acceptor during the enzymatic reaction, and this potassium ferricyanide consumption is detected from the leads 2 and 3 through the electrodes 4 and 5 of the detecting section 9. The glucose value is measured by measuring the current value obtained.

[0008]

By the way, in the biosensor having the above structure, the detecting section 9 for detecting the enzyme-mediator reaction is used.
The adhesion between the electrodes 4 and 5 and the reagent section 10 and the uniform solubility of the reagent section 10 are important factors that greatly affect the performance of the biosensor. Hereinafter, the relationship between the mediator concentration and the sensor response characteristics in the biosensor will be discussed.

FIG. 4 is a graph showing the sensor response depending on the mediator concentration in the biosensor for measuring glucose produced by the above configuration. FIG. 5 shows the reagent part thickness and the sensor response reproducibility depending on the mediator concentration. It is a graph shown.

In the above biosensor, GO is added to the enzyme.
D. Reagent part of potassium ferricyanide in mediator 1
5 microliters (hereinafter abbreviated as “μl”) of these mixed solutions are applied onto the surfaces of the electrodes 4 and 5,
After drying, the reagent part 10 was formed.

When the concentration of the enzyme is fixed at 200 units / milliliter (hereinafter abbreviated as U / ml) and the concentration of the mediator is increased, as shown in FIG. 4, the concentration of the mediator becomes 25 mmol / l (hereinafter abbreviated as mM). )), The correlation coefficient (r) was 0.9831, but by increasing the mediator concentration to 150 mM,
It shows that it improves to 931. That is, it is understood that as the mediator concentration increases, the response sensitivity of the sensor increases accordingly, and the linear correlation between the glucose concentration and the current value also improves.

However, as shown in FIG. 5, as the mediator concentration is increased, the thickness of the reagent section 10 is increased to cause bulges and irregularities. As a result, the adhesion between the reagent section 10 and the electrodes 4 and 5 is increased. Decrease. In particular, this phenomenon is remarkable when the mediator concentration exceeds 50 mM (see the line graph connecting ● in the figure). Then, a coefficient of variation of the sensor response (hereinafter, referred to as an index of reproducibility of the sensor response)
Abbreviated as "CV". ) Is 5 to 10% when the mediator concentration is 125 mM or less, as shown by the bar graph in FIG.
As shown in FIG. 4, the mediator concentration was set to 150 with a good linear correlation of the sensor response.
When increased to mM, the CV deteriorates to nearly 25% and the sensor becomes very unreliable.

From the above, when the concentration of the mediator in the reagent section 10 is high, the sensor response sensitivity is improved, but the adhesion between the reagent section 10 and the electrodes 4 and 5 is reduced due to the bump or unevenness of the reagent section 10. In addition, there is a problem that the sensor performance is remarkably deteriorated due to partial melting failure.

Furthermore, due to the poor adhesion of the reagent part 10 itself, enzymes and mediators in the reagent part 10 are peeled off from the surfaces of the electrodes 4 and 5 during manufacture, handling, or storage of the sensor. A large error occurs in the amount of the loaded reagent that has been optimized, and as a result, the sensor response characteristic is significantly reduced.

Therefore, the present invention suppresses the bulging and unevenness of the reagent portion in order to stabilize both the responsiveness and reproducibility of the biosensor, and reduces the adhesion between the reagent portion and the surface of the electrode supporting the reagent portion. It aims to provide what is good.

[0016]

According to a first aspect of the present invention, there is provided a biosensor in which an electrode-based detecting portion having a working electrode and a counter electrode is formed on an insulating substrate, and an oxidizing device is formed on the electrode-based detecting portion. In a biosensor in which a reagent portion containing a reductase and an enzyme reaction detection reagent is formed, and the qualitative or quantitative detection of a sample sample administered to the reagent portion is performed by the electrode-based detection portion, the biosensor is contained in the reagent portion. It is characterized by having an additive containing a high molecular protein or a derivative thereof.

According to a second aspect of the present invention, in the biosensor, the biopolymer protein or the derivative thereof contained in the additive does not have a redox catalytic activity. It is characterized by the following.

The biosensor according to claim 3 of the present invention is the above-described biosensor, wherein the additive is gelatin, collagen, elastin, casein, peptone or a derivative thereof, or any combination thereof. It is characterized in that a mixture is used.

[0019]

Embodiments of the present invention will be described below. As shown in FIG. 1, the biosensor according to the embodiment of the present invention has almost the same configuration as that described in the section of the related art, but includes a biopolymer protein or It has an additive containing a derivative.

As the biomacromolecule protein or its derivative contained in the above-mentioned additives, those having no redox catalytic activity are preferably used, for example, gelatin, collagen, elastin, casein, peptone or derivatives thereof, or A mixture in which these are arbitrarily combined is used.

The oxidoreductase contained in the reagent section includes, for example, glucose oxidase (GOD),
Cholesterol oxidase, lactate oxidase, uricase, galactose oxidase, alcohol oxidase, choline oxidase, ascorbate oxidase, glucose dehydrogenase, cholesterol dehydrogenase, alcohol dehydrogenase and the like are used.

As the mediator contained in the reagent part, an electron acceptor of an organic or inorganic compound is used, for example, a metal cyano complex such as potassium ferricyanide, a quinone derivative such as benzoquinone, ferrocene, or ferrocene. Derivatives and the like are used.

The biosensor having the above configuration is manufactured, for example, as follows. First, silver paste is printed on the insulating substrate 1 made of polyethylene terephthalate by screen printing to form leads 2 and 3. Next, by printing a conductive carbon paste, an electrode system detecting unit 9 including the measuring electrode 4 and the counter electrode 5 is formed on the leads 2 and 3.
To form Further, an insulating paste is printed to define an exposed area of the electrodes 4 and 5 and to cover unnecessary portions of the leads 2 and 3 between the electrodes 4 and 5 to form an insulating layer 6.
Then, after a solution containing an enzyme and a mediator is appropriately supported on both the electrodes 4 and 5, the reagent portion 10 is formed by drying. Finally, the cover 8 and the spacer 7 are bonded in a positional relationship shown by a dashed line in FIG. 1 to manufacture an electrode-type biosensor.

Next, the operation of the biosensor will be described. First, the reagent section 1 in which the sample is carried on the detection section 9
Administer directly to 0. Then, the components in the sample sample undergo an enzymatic reaction with the oxidoreductase contained in the reagent section 10, and during this enzymatic reaction, the mediator becomes an electron acceptor and is dissociated and consumed. At this time, cations and anions generated by the dissociated mediator move to the electrodes 4 and 5 to generate an electromotive force. This electromotive force is detected from leads 2 and 3,
By measuring the current value, the components in the sample can be specifically qualitatively or quantitatively determined.

In the biosensor according to the present embodiment, an additive containing a protein of a biopolymer or a derivative thereof is blended in the reagent part. Since such an additive is a polymer substance and has a large cohesive force, In addition, the oxidoreductase and the mediator in the reagent section 10 can be carried with good adhesion. Therefore, according to the biosensor according to the present embodiment, an additive containing a biopolymer protein or a derivative thereof is blended in the reagent portion, and the agglutination of the additive causes the reagent portion 10 to protrude and become uneven. , The adhesion between the surfaces of the electrodes 4 and 5 carrying the reagent section 10 is enhanced, and peeling and falling off of enzymes and mediators from the surfaces of the electrodes 4 and 5 can be prevented. This has the effect that the sensor reproduction performance is greatly improved.

Further, as the biosensor of the present embodiment, a biopolymer protein or its derivative contained in the above-mentioned additive which does not have a redox catalytic activity is used, so that enzymatic and chemical The oxidation-reduction reaction can prevent the oxidation-reduction reaction with an unspecified component of the sample, and can detect only the sensor response corresponding to the reaction with the specified component. This has the effect that the sample can be measured.

Next, an embodiment will be described. (1) Preparation of Reagent Part The reagent part 10 supported on the electrodes 4 and 5 shown in FIG. 1 was prepared by adding a GOD of 200 U / ml and a mediator of potassium ferricyanide to 150 mM.
Gelatin was blended at various ratios of ３3 W / V%, and 5 μl of this mixture was dried under the surface of the electrodes 4 and 5 to obtain 0%.
A plurality of biosensors including the reagent section 10 in which gelatin was mixed at various ratios of ３3 W / V% were produced.

(2) Reagent thickness and sensor response reproducibility FIG. 2 shows the thickness and sensor response reproducibility of the reagent portion 10 of the glucose measuring biosensor having the gelatin-containing reagent portion 10 prepared above. It is a graph shown.

As is clear from the line graph in FIG. 2, the thickness of the reagent portion 10 depends on the concentration of gelatin mixed in the reagent portion 10, and the thickness of the reagent portion 10 decreases as the gelatin concentration increases. It can be seen that the bumps are suppressed and the unevenness is reduced. That is, with respect to the reagent part thickness, the reagent part 10 formed by appropriately applying 150 mM potassium ferricyanide has a reagent part thickness of about 540 μm to about 260 μm due to the blending effect of gelatin of about 0.25 W / V%.
From FIG. 5, this is improved to a reagent thickness of 75 mM or less potassium ferricyanide without gelatin.

Also, the CV which is the reproducibility of the sensor response
Regarding (%), referring to the bar graph in FIG. 2, when gelatin was not blended, it was 20% or more, but when gelatin was blended at about 1.5 W / V%, CV became 5%.
To about 2.0 W /
It is understood that CV is stabilized to about 2% by blending V% or more.

(3) Sensor Response Performance FIG. 3 shows a reagent section 10 containing gelatin by the above-described preparation.
6 is a graph showing the response performance of a biosensor provided with a biosensor. In FIG. 5, the reagent part 10 contains no gelatin,
0.50 W / V% blended, 1.00 W / V% blended, 2.0
Relationship between glucose concentration and current value when 0 W / V% blended or 2.75 W / V% blended (sensor response performance)
Is shown.

From FIG. 3, it can be seen that, by blending gelatin at about 2.0 W / V% or more, an extremely high linear correlation was obtained between the glucose concentration and the current value, and the correlation coefficient (r) was Gelatin 2.75 from 0.9931 at the time
It can be seen that the ratio can be improved to 0.9996 when W / V% is blended, and that the slope of the straight line is significantly improved as compared with the case where gelatin is not blended.

It is a well-known fact that gelatin itself does not have a redox catalytic activity, and the increase in the current value shown in FIG. 3 also improves the adhesion between the electrodes 4, 5 and the reagent portion 10. It is clear that this is due to

From the above examples, a reagent formed by drying 5 μl of a reagent solution containing 1.5 to 3.0 W / V% gelatin on the surfaces of the electrodes 4 and 5 of the biosensor under appropriate conditions. By applying the unit 10, a biosensor having high sensor response performance and high sensor response reproducibility can be realized.

The amounts of the biopolymers, enzymes, reagents, etc., the amounts supported on the sensor, and the methods shown in the above examples are merely examples. It is not limited to the amount, ratio, method and the like, but can be changed according to the purpose.

[0036]

As described above, according to the biosensor of the present invention, an additive containing a biopolymer protein or a derivative thereof is blended in the reagent portion. Since the substance is a substance and has a large cohesive force, the redox enzyme and the enzyme reaction detection reagent (mediator) in the reagent section can be carried with good adhesion. Therefore, the cohesive action of this additive suppresses the protrusion and unevenness due to the mediator concentration in the reagent part, increases the adhesion to the detection part carrying the reagent part, and peels the enzyme or the mediator from the detection part surface. And falling off, and as a result,
This has an effect that the sensor response performance and the sensor reproduction performance are greatly improved.

Further, in the biosensor of the present invention, the use of a biopolymer protein or a derivative thereof having no redox catalytic activity as the additive in the above-mentioned additive enables enzymatic and chemical redox reactions. Can prevent an oxidation-reduction reaction with an unspecified component of the sample sample, so that only a sensor response corresponding to the reaction with the specified component can be detected. Can be measured.

[Brief description of the drawings]

FIG. 1 is an exploded perspective view showing an electrode type biosensor.

FIG. 2 is a graph showing the effect of reducing the thickness of the reagent portion and the effect of improving the sensor response reproducibility by incorporating gelatin into the reagent portion in the electrode-type biosensor according to the embodiment of the present invention.

FIG. 3 is a graph showing the effect of improving the sensor response linear correlation by mixing gelatin in a reagent part in the electrode type biosensor according to the embodiment of the present invention.

FIG. 4 is a graph showing sensor response performance depending on a mediator concentration in a conventional electrode-type biosensor.

FIG. 5 is a graph showing an increase in the thickness of a reagent portion and a deterioration in sensor response reproducibility depending on a mediator concentration in a conventional electrode-type biosensor.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Insulating substrate 2, 3 Lead 4 Measurement electrode (working electrode) 5 Counter electrode 6 Insulating layer 7 Spacer 8 Cover 9 Detecting part 10 Reagent part

Claims (3)

[Claims] An electrode-based detection section having a working electrode and a counter electrode is formed on an insulating substrate, and a reagent section containing an oxidoreductase and an enzyme reaction detection reagent is formed on the electrode-system detection section. A biosensor for qualitatively or quantitatively detecting a specimen sample administered to the reagent part by the electrode-based detection part, wherein the reagent part has an additive containing a biopolymer protein or a derivative thereof. A biosensor characterized in that: 2. The biosensor according to claim 1, wherein the biopolymer protein or the derivative thereof contained in the additive does not have a redox catalytic activity. 3. The biosensor according to claim 1, wherein the additive is gelatin, collagen, elastin, casein, peptone or a derivative thereof, or a mixture of any combination thereof. Biosensor.