JP3267907B2 - Biosensor and Substrate Quantification Method Using It - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for quantifying a substrate contained in a sample such as blood, urine or fruit juice with high accuracy, quickly and simply, and a method for quantifying a substrate using the same.

[0002]

2. Description of the Related Art As an example of a method for quantifying a substrate, a method for quantifying glucose will be described. As a method for electrochemically quantifying glucose, a method in which glucose oxidase is combined with an oxygen electrode or a hydrogen peroxide electrode is generally known (for example, Shuichi Suzuki, "Biosensor", Kodansha). Glucose oxidase selectively oxidizes β-D-glucose as a substrate to D-glucono-δ-lactone using oxygen in the reaction system as an electron carrier. With this reaction, oxygen is reduced to hydrogen peroxide. Glucose can be quantified by measuring the amount of oxygen consumed at this time with an oxygen electrode or measuring the amount of generated hydrogen peroxide with a hydrogen peroxide electrode using a platinum electrode or the like. However, in the above-described method, the measurement is greatly affected by the concentration of dissolved oxygen depending on the measurement target, and the measurement itself becomes impossible under the condition without oxygen.

[0003] Therefore, without using oxygen as an electron carrier,
Glucose sensors of the type using metal complexes or organic compounds such as potassium ferricyanide, ferrocene derivatives and quinone derivatives as electron carriers have been developed. In this type of biosensor, a desired amount of glucose oxidase and an electron carrier can be stably supported on the electrode, and the electrode system and the reaction layer can be integrated in a state close to a dry state. . JP-A-3-202768
In this publication, a biosensor of this type is disclosed. This biosensor forms an electrode system consisting of a working electrode and a counter electrode on an insulating substrate by a method such as screen printing, and forms a reaction layer containing an oxidoreductase and an electron carrier on the electrode system. The cover and the spacer are combined with the insulating substrate.

[0004] By using this biosensor, the substrate in a sample can be quantified as follows. First, a sample solution is supplied to the reaction layer to dissolve the reaction layer, and the substrate in the sample solution reacts with the enzyme in the reaction layer. With this enzymatic reaction, the electron carriers in the reaction layer are reduced. After the enzymatic reaction, the reduced electron carrier is electrochemically oxidized,
The substrate in the sample is quantified by measuring the oxidation current value obtained at this time. In such a biosensor, various specific components can be quantified by using the corresponding oxidoreductase. In addition, such biosensors have been receiving much attention in recent years because they are disposable and can easily measure the concentration of a substrate simply by introducing a sample into a sensor chip inserted into a measuring instrument.

[0005]

As described above, the substrate in a sample is quantified from an oxidation current value obtained by oxidizing a reduced form of an electron carrier resulting from a series of enzymatic reactions on a working electrode. Can be. However, the sample may contain an easily oxidizable substance, such as ascorbic acid or urea, which is oxidized at the same time as oxidizing the reduced form of the electron carrier on the working electrode to generate an oxidation current. When these easily oxidizable substances are contained in the sample, a positive error may be given to the measurement result.

When an oxidation current value is measured by a two-electrode system having only a working electrode and a counter electrode, it is necessary to have an oxidant reduced on the counter electrode in response to the oxidation reaction on the working electrode. However, when the substrate concentration is high, most of the electron carriers are reduced as a result of the enzymatic reaction, so that the oxidized product reduced on the counter electrode is insufficient. As a result, the reduction reaction at the counter electrode becomes a rate-determining process, and adversely affects the obtained oxidation current value. An object of the present invention is to provide a biosensor which can eliminate the above-mentioned inconveniences and can quantify the substrate concentration in a sample with high accuracy. Further, the present invention provides a method for quickly and easily quantifying a substrate with high accuracy using this biosensor.

[0007]

A biosensor according to the present invention comprises an insulating substrate, an electrode system comprising a working electrode and a counter electrode formed on the substrate, and at least an oxidoreductase formed on the electrode system. Comprising a reaction layer containing an electron carrier, and reducing the electron carrier at the working electrode
Biosensor that measures substrate concentration in a sample based on current
And wherein the counter electrode is made of at least one kind of electrolytically oxidizable metal. Further, the method for quantifying a substrate according to the present invention comprises the steps of: using the above biosensor, adding a sample to a reaction layer thereof, and reacting the substrate with an enzyme; A step of applying a current to the working electrode and a step of measuring a current value flowing between the working electrode and the counter electrode.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for quantifying a substrate according to the present invention, as described above, a sample is first added to a reaction layer to react a substrate with an enzyme. With this enzymatic reaction, the electron mediator in the reaction layer is reduced according to the substrate concentration. Next, a potential for reducing the electron carrier remaining in the reaction system without being reduced by the enzymatic reaction is applied to the working electrode, and the reduction current is measured to determine the substrate concentration. If the amount of the electron carrier in the reaction layer is kept constant, the above-mentioned reduction current corresponds to the substrate concentration in the sample. Therefore, if a calibration curve is prepared by measuring the reduction current value of a standard solution containing a known amount of the substrate in advance, the substrate concentration in the sample can be determined based on the calibration curve.

As described above, according to the present invention, since the substrate is quantified from the reduction current, even if an oxidizable substance is mixed in the sample, it is not affected. In addition, since the counter electrode is made of a metal that can be electrolytically oxidized, even if the substrate concentration in the sample is low and the number of reductants of the electron carrier is small, when measuring the reduction current at the working electrode, the counter electrode is a rate-limiting process. Will not be. The counter electrode can be formed by printing the metal as it is on a substrate, for example, in the form of a paste, and thus the sensor manufacturing process can be simplified. The counter electrode may be formed from a mixture of the metal and carbon. Silver or copper is suitable as the metal that can be oxidized electrolytically. Further, iron, zinc, or the like can also be used.

In addition, the electrode system surface is coated with a hydrophilic polymer layer so that enzymes and electron carriers do not come into contact with the electrode system surface formed on the substrate, or so that proteins do not adsorb to the electrode system surface. Is preferred. Examples of the hydrophilic polymer forming such a layer include carboxymethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, gelatin and its derivatives, acrylic acid and its salts, methacrylic acid and its salts, starch and its derivatives, maleic anhydride and Its salts and cellulose derivatives such as hydroxypropylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose and carboxymethylethylcellulose can be used. As the electron carrier to be included in the reaction layer, potassium ferricyanide, p-benzoquinone, phenazine methosulfate, methylene blue, a ferrocene derivative, or the like can be used.

Further, as the oxidoreductase, glucose oxidase, glucose dehydrogenase, lactate oxidase, lactate dehydrogenase, fructose dehydrogenase, uricase, cholesterol oxidase, cholesterol oxidase, cholesterol esterase and the like can be used. Further, a combination of a plurality of the above enzymes, for example, glucose oxidase and invertase, glucose oxidase and invertase and mutarotase, or fructose dehydrogenase and invertase can be used. Further, a lecithin layer may be formed on the reaction layer in order to smoothly supply the sample solution.

[0012]

The present invention will be described below in more detail with reference to specific examples. FIG. 1 is an exploded perspective view of a two-electrode type glucose sensor excluding a reaction layer. Leads 2 and 3 are formed by printing a silver paste on the insulating substrate 1 made of polyethylene terephthalate by screen printing. Next, the working electrode 4 is formed by printing a conductive carbon paste containing a resin binder on the substrate 1. This working electrode 4 is in contact with the lead 2. Further, an insulating paste is printed on the substrate 1 to form an insulating layer 6. The insulating layer 6 covers the outer periphery of the working electrode 4, thereby keeping the area of the exposed portion of the working electrode 4 constant. Then, a silver paste is printed so as to be in contact with the leads 3 to form a ring-shaped counter electrode 5.

The insulating substrate 1 and the cover 9 provided with the air holes 11 and the spacer 10 are bonded together in a positional relationship as shown by a dashed line in FIG. 1 to produce a biosensor. The spacer 10 is provided with a slit 13 for forming a sample liquid supply path between the substrate and the cover. 1
Reference numeral 2 corresponds to the opening of the sample liquid supply path. FIG.
FIG. 3 is a longitudinal sectional view of a main part of the biosensor according to the present invention, excluding a spacer and a cover. A reaction layer 7 containing enzymes and an electron carrier is formed on an electrically insulating substrate 1 on which an electrode system is formed as shown in FIG. 1, and a lecithin layer 8 is formed on the reaction layer 7.

Example 1 A mixed aqueous solution of glucose oxidase (EC 1.1.3.4, hereinafter abbreviated as GOD) and potassium ferricyanide was dropped on an electrode system on a substrate 1 shown in FIG. 1 and dried. Reaction layer 7 was formed. Next, a toluene solution of lecithin was dropped on the reaction layer 7 and dried to form a lecithin layer 8. Then, the substrate 1, the cover 9 and the spacer 10 were adhered in a positional relationship as shown by a dashed line in FIG. 1 to produce a glucose sensor.
3 μl of the standard glucose aqueous solution is applied to the opening 1 of the sample liquid supply path.
Supplied from 2. The sample liquid reaches the air hole 11 part,
The reaction layer 7 on the electrode system dissolved. When the reaction layer 7 is dissolved, glucose in the sample solution is oxidized to gluconolactone by GOD. By this enzymatic reaction, ferricyanide ions are reduced to ferrocyanide ions.

After a certain period of time from the supply of the sample solution, a voltage of -0.8 V was applied to the working electrode 4 with respect to the counter electrode 5. By the application of this voltage, a reduction reaction of potassium ferricyanide not reduced by the enzymatic reaction occurs at the working electrode. Then, the silver at the counter electrode is oxidized to silver ions. Then, the current value after 5 seconds from the application of the voltage was measured. The obtained current value decreased with increasing glucose concentration. In other words, since the counter electrode was made of silver, the reductant was sufficiently present in the reaction system, indicating that the obtained current value was dependent on the reduction reaction of ferricyanide ions on the working electrode. The measurement accuracy of the obtained current response value was high.

<< Embodiment 2 >> On the electrode system of the substrate 1 shown in FIG.
Carboxymethyl cellulose (hereinafter abbreviated as CMC)
The aqueous solution was dropped and dried to form a CMC layer. This CM
A reaction layer and a lecithin layer were formed on the C layer in the same manner as in Example 1. Then, a glucose sensor was prepared in the same manner as in Example 1, and the response to the glucose standard solution was measured. As a result, the same response characteristics as in Example 1 were obtained.

Example 3 A glucose sensor was produced in the same manner as in Example 1 except that the counter electrode 5 in FIG. 1 was formed using a copper paste, and the response to a glucose standard solution was measured. The same response characteristics as described above were obtained.

Example 4 A glucose sensor was prepared in the same manner as in Example 2 except that the counter electrode 5 in FIG. 1 was formed using a mixed paste of silver and carbon, and the response to a glucose standard solution was measured. Thus, the same response characteristics as in Example 2 were obtained.

Example 5 A current response value was measured in the same manner as in Example 2 except that an aqueous glucose solution containing a known amount of ascorbic acid was used as a sample solution. As a result, the same response characteristics as in Example 2 using the standard glucose aqueous solution containing no ascorbic acid were obtained.

Comparative Example 1 Using the same sensor as in Example 2 and a glucose standard aqueous solution containing ascorbic acid, a voltage of 0.5 V was applied to the working electrode with reference to the counter electrode, and the current value after 5 seconds was measured. Then, the oxidation current value was measured. As a result, as the amount of ascorbic acid in the standard aqueous solution increases,
The current response value also increased.

[0021]

As described above, according to the present invention, a substrate in a sample can be quantified with high accuracy quickly and easily.

[Brief description of the drawings]

FIG. 1 is an exploded perspective view of a glucose sensor used in one embodiment of the present invention, excluding a reaction layer.

FIG. 2 is a longitudinal sectional view of a main part of the glucose sensor, excluding a spacer and a cover.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 Insulating substrate 2, 3 lead 4 Working electrode 5 Counter electrode 6 Insulating layer 7 Reaction layer 8 Lecithin layer 9 Cover 10 Spacer 11 Air hole 12 Sample liquid supply channel opening 13 Slit

Continuation of the front page (56) References JP-A-8-278276 (JP, A) JP-A-6-130024 (JP, A) (58) Fields studied (Int. Cl. ⁷ , DB name) G01N 27 / 327 G01N 27/416

Claims (5)

(57) [Claims] 1. A insulating substrate, comprising a reaction layer containing the effects are formed on the substrate electrode and the electrode system consisting of a counter electrode, and at least oxidoreductase and an electron mediator is formed on the electrode system The electron transfer at the working electrode
A biosensor for measuring a substrate concentration in a sample based on a reduction current of a body , wherein the counter electrode is made of at least one kind of electrolytically oxidizable metal. 2. An insulating substrate, an electrode system comprising a working electrode and a counter electrode formed on the substrate, and a reaction layer containing at least an oxidoreductase and an electron carrier formed on the electrode system. A biosensor, wherein the counter electrode comprises a mixture of at least one kind of electrolytically oxidizable metal and carbon. 3. The method according to claim 1, wherein the metal is silver or copper.
Or the biosensor according to 2. 4. The biosensor according to claim 1, wherein the reaction layer further contains a hydrophilic polymer. 5. A step of using the biosensor according to claim 1 or 2 to react a substrate with an enzyme by adding a sample to the reaction layer, and applying a potential to reduce an electron carrier not reduced in the step. A method for quantifying a substrate, comprising a step of applying a voltage to a pole and a step of measuring a current value between the counter electrode and the working electrode.