JPH0688805A - Biosensor - Google Patents

Abstract

PURPOSE:To obtain a highly reliable biosensor which can quickly and easily determine the sucrose content of a sample with high accuracy. CONSTITUTION:In the biosensor which is provided with an electrode system incorporating a measuring electrode 4 and counter electrode 5 on an insulating substrate 1 and a reactive layer 7 containing a hydrophilic polymer, electron acceptor, and enzyme adjacent to the electrode system, the enzyme is constituted of a combination of invertase, mutarotase, and glucose oxidase or invertase and fructose dehydrogenase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a biosensor for easily quantifying sucrose in a sample.

[0002]

2. Description of the Related Art Sensing technology using enzymes has been widely applied to medical analysis, food analysis, etc. because of its high selectivity, simplicity and speed. An enzyme sensor has been developed instead of a refractometer or liquid chromatographic method for the analysis of sugar concentration, and there is an example of an enzyme sensor using an immobilized enzyme membrane and a hydrogen peroxide electrode (K. Inoue, New Food Industry Vol. 28, No. 11, 45
-48 (1986)). This enzyme sensor can measure various substrates by selecting the type of enzyme to be immobilized. Here, sucrose measurement will be described. The enzyme sensor part mainly comprises a hydrogen peroxide electrode and an immobilized enzyme film provided on the electrode with a polymer film sandwiched therebetween. The enzymes used are saccharase, mutarotase and glucose oxidase. The measurement system includes a sample chamber having the enzyme sensor in a temperature control block, and is used for sample dilution, stirring, temperature control, oxygen supplementation,
The system such as sugar detection is automatically controlled by a program. When a sample solution containing sucrose is injected into the sample chamber, the sample solution reacts with each of the immobilized enzymes to generate hydrogen peroxide and gluconolactone. The sucrose concentration can be determined from the current value when hydrogen is electrochemically oxidized.

[0003]

In such a conventional enzyme sensor, since the sample chamber is provided with the enzyme sensor, hydrogen peroxide produced on the enzyme sensor diffuses in all directions. Therefore, in order to prevent a measurement error, it is necessary to decompose the backward flowing hydrogen peroxide by including a catalase enzyme in the buffer solution. Also,
Since the reaction is carried out in a solution, it is necessary to dilute, stir and control the temperature, and the measurement system is complicated and lacks in simplicity.
The present invention solves the above problems, and an object of the present invention is to provide a biosensor capable of quantifying sucrose in a sample with high accuracy, quickly and easily. The present invention is
Moreover, it aims at providing the highly reliable biosensor which can quantify the sucrose in a sample with high precision.

[0004]

A biosensor of the present invention comprises an electrode system including a measuring electrode and a counter electrode formed on an insulating substrate, and a reaction layer in contact with the electrode system,
The reaction layer contains at least an electron acceptor, an enzyme and a hydrophilic polymer, and a combination of invertase, mutarotase and glucose oxidase as the enzyme,
Alternatively, a combination of invertase and fructose-dehydrogenase is used. In the biosensor of the present invention, the reaction layer is composed of at least two layers including an enzyme-containing layer and an electron acceptor-containing layer.

Further, the present invention provides a structure in which the electron acceptor and the enzyme in the reaction layer are included in a hydrophilic polymer, or at least one of the electron acceptor and the enzyme and the electrode system are separated by the hydrophilic polymer. To do. Further, the present invention is
In the biosensor having at least two reaction layers, a hydrophilic polymer film soluble in a solvent that does not dissolve an enzyme is provided between the two layers forming the reaction layer.

Here, the inventory in the reaction layer is
Lutase, mutarotase and glucose oxidase
The carrying amount of each is 1 U / cm²Above 1U / cm²Since
Above and 5U / cm²The above is preferable. Well
In addition, invertase and fructo in the reaction layer
The loading amount of sucrose dehydrogenase is 1 U / cm.
2 or more and 5 U / cm²The above is preferable. Book
The biosensor of the invention is contained in the reaction layer,
Is separate from the reaction layer, but
Hold the reagent that will be the component of the buffer solution at the contact position.
It

[0007]

In order to quantify the sucrose in the sample solution using the biosensor of the present invention, the sample solution is supplied to the reaction layer in contact with the electrode system of the sensor. This sample solution dissolves the hydrophilic polymer in the reaction layer, and the enzyme and electron acceptor contained in the reaction layer come into contact with the sample. Then, in the sensor in which the reaction layer carries invertase, mutarotase and glucose oxidase as enzymes, the sucrose in the sample solution is hydrolyzed by invertase, and fructose and α-glucose are produced. α-
Glucose is rapidly converted into the optical isomer β-glucose by the catalytic action of mutarotase.
-Glucose undergoes oxidation by glucose oxidase. An electron acceptor, such as potassium ferricyanide, is reduced to potassium ferrocyanide by the electrons transferred by the oxidation reaction by glucose oxidase.

On the other hand, in the biosensor in which the reaction layer carries invertase and fructose dehydrogenase as enzymes, sucrose in the sample solution is hydrolyzed into fructose and α-glucose by invertase, and fructose is oxidized by fructose dehydrogenase. 5-keto-fructose is produced. Then, potassium ferricyanide is reduced to potassium ferrocyanide by the electrons transferred by the oxidation reaction by fructose dehydrogenase.

As described above, the reduced form of the electron acceptor produced by the oxidation reaction by glucose oxidase or fructose dehydrogenase is then oxidized by the application of a voltage with the counter electrode as the reference electrode. The sucrose can be quantified by the oxidation current. Further, the reaction layer contains a hydrophilic polymer, and this polymer is dissolved in the sample solution to provide a field for the reaction between the enzyme, the electron acceptor and the like contained in the reaction layer and the sample. Therefore, this polymer substantially blocks contact of enzymes and electron acceptors with the surface of the electrode system, so that the adsorption of proteins on the surface of the electrode system and the presence of substances with oxidizing ability such as electron acceptors. It becomes difficult for the characteristics of the electrode system to change due to chemical action. As a result, a highly accurate sensor response can be obtained. Therefore, it is preferable that the enzyme and the electron acceptor in the reaction layer are contained in the hydrophilic polymer, or that at least one of the electron acceptor and the enzyme is separated from the electrode system by the hydrophilic polymer.

Furthermore, if the reaction layer is divided into at least two layers, that is, a layer containing an enzyme and a layer containing an electron acceptor, the activity of the enzyme is not impaired by the electron acceptor, so that the performance of the enzyme can be improved by storage. A highly reliable biosensor that does not deteriorate can be obtained. If a hydrophilic polymer film soluble in a solvent that does not dissolve the enzyme is provided between the two layers, the effect of separating the enzyme and the electron acceptor is further increased.

When the reaction layer has a single layer structure,
Compared with the multi-layer structure, the manufacturing process can be simplified, and since it can be easily dissolved in the sample liquid, the sensor response speed can be shortened and the variation in response value can be reduced. Moreover, the influence of the pH of the sample solution is mitigated,
In order to obtain a stable sensor response, it is preferable that the reaction layer contains a buffer solution or a reagent which is a component thereof in advance.

Instead of including the reagent which is a component of the buffer solution in the reaction layer, the reagent can be held at a position separate from the reaction layer and in contact with the sample solution supplied to the reaction layer. With this configuration, since the reaction layer does not contain a reagent serving as a component of the buffer solution, the reaction layer surface is kept smooth,
It has the effect of suppressing the deterioration of the response due to the generation of bubbles and the variation. In addition, since the reagent that is a component of the buffer solution is isolated from the enzyme in the reaction layer, it is possible to increase the concentration of the reagent without considering the influence that the enzyme activity is inhibited by the reagent and the storage reliability is reduced. it can. By increasing the reagent concentration, the buffering action becomes stronger, the influence of the pH of the sample solution is mitigated, and a stable sensor response can be obtained. As the reagent that is a component of the buffer solution, phosphate, citric acid, acetic acid-acetate, or the like can be used in addition to phosphate. Next, with respect to the amount of enzyme supported, when the amount is small, the reaction rate is reduced and the measurement takes a long time. The preferable loading amount of each enzyme in the reaction layer is
Invertase and mutarose are each 1 U / cm ² or more, and glucose oxidase and fructose dehydrogenase are each 5 U / cm ² or more.

The hydrophilic polymer used in the present invention is not limited to the carboxymethyl cellulose of the examples, and other cellulose type, vinyl alcohol type, vinylpyrrolidone type, gelatin type, acrylate type, starch type. , Maleic anhydride type, acrylamide type and the like may be used. As the electron acceptor, p-benzoquinone, ferrocene and the like can be used in addition to potassium ferricyanide in the examples.

As described above, the biosensor of the present invention is
Sucrose can be quantified by a simple operation of supplying a sample solution to the reaction layer in contact with the electrode system and measuring the enzymatic reaction of the reaction layer with the electrode system. Further, since an enzyme reaction system is used, it has high substrate selectivity and high accuracy can be obtained by combining a reaction layer containing an enzyme and an electrode system. Furthermore, depending on the structure of the reaction layer, the response speed can be increased or the storage stability can be improved.

[0015]

EXAMPLES The present invention will be described below with reference to examples. Example 1 FIGS. 1 and 2 show a sucrose sensor manufactured as an example of the biosensor of the present invention. Hereinafter, a method for manufacturing this sensor will be described. First, on the insulating substrate 1 made of polyethylene terephthalate,
Print silver paste by screen printing and read leads 2 and 3
To form. Next, a measurement electrode 4 of the electrode system is formed by using a conductive carbon paste containing a resin binder, and subsequently an insulating layer 6 made of an insulating paste is formed by printing. The insulating layer 6 has an area of the exposed portion of the measurement electrode 4 (about 1
mm ² ) is constant, and the leads 2 and 3 are partially covered. Finally, the counter electrode 5 is formed by printing using the same carbon paste as the measuring electrode.

Next, a 0.25 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer is dropped on the electrode system and dried to form a CMC layer. On the other hand, invertase (EC
3.2.1.26: hereinafter referred to as INV) 250 U, mutarotase (EC 5.1.3.3: hereinafter referred to as MUT) 2
50 U and glucose oxidase (EC1.1.
3.4: hereinafter abbreviated as GOD) 750 U and 33 mg of potassium ferricyanide as an electron acceptor, CMC0.2
Phosphate buffer containing 5 wt% (0.2 M: K ₂ HPO ₄ −
0.2M: KH ₂ PO ₄ ; pH = 7.4) Dissolve in 1 ml. 4 μl of this mixed solution was dropped on the CMC layer,
The reaction layer 7 is formed by drying in a warm air dryer at 50 ° C. for 10 minutes. The reaction layer has a substantially circular shape with a diameter of about 3.6 mm, and substantially coincides with the outer peripheral portion of the counter electrode 5.

In the above reaction layer forming step, when a mixed solution of a phosphate, an enzyme and an electron acceptor is dropped on the CMC layer, the CMC layer made of a hydrophilic polymer is once dissolved and the enzyme is dried in the subsequent drying process. The reaction layer 7 is formed in a mixed state with the above. 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 the enzyme and the electron acceptor do not come into contact with the surface of the electrode system, adsorption of protein on the surface of the electrode system, potassium ferricyanide,
It is difficult for the characteristics of the electrode system to change due to the chemical action of a substance having an oxidizing ability such as H ₂ PO ₄ ⁻ . as a result,
A biosensor having a highly accurate sensor response can be obtained. After forming the reaction layer 7 as described above, the cover 9 having the air holes 11 and the spacers 8 having the grooves forming the sample supply holes 10 are bonded to the substrate 1 to assemble the sensor. When a transparent material is used for the cover, the state of the reaction layer and the introduction state of the sample solution can be confirmed very easily from the outside.

Further, when the cover is attached, the sample liquid is easily introduced into the reaction layer portion by a simple operation of bringing the sample liquid into contact with the sample supply hole 10 at the tip of the sensor due to the capillary phenomenon in the space formed by the cover and the spacer. In order to smoothly supply the sample solution, it is preferable to further spread a toluene solution of lecithin on the surface from the sample supply section to the reaction layer and dry it, if necessary. 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. Furthermore, evaporation of the sample liquid during measurement can be minimized, and highly accurate measurement can be performed. When 3 μl of the sucrose aqueous solution as a sample solution was supplied to the sucrose sensor manufactured as described above from the sample supply hole 10, the sample solution quickly reached the air hole 11 portion by the capillary phenomenon and the reaction layer on the electrode system was dissolved.

After a certain period of time from the supply of the sample solution, the counter electrode 5 of the electrode system is used as a reference for the measurement electrode 4 in the anode direction.
When a pulse voltage of 0.5 V was applied and the current value was measured after 5 seconds, a response current value proportional to the sucrose concentration in the sample solution was obtained. When the reaction layer 7 is dissolved in the sample solution, sucrose in the sample solution is hydrolyzed by INV to produce fructose and α-glucose. When α-glucose is left as an aqueous solution, it reaches equilibrium with β-glucose which is an optical isomer, but this equilibrium transfer is catalyzed by MUT, and α-glucose produced by INV rapidly becomes β-glucose, which is oxidized by GOD. Receive. The electrons transferred by the oxidation reaction by GOD reduce potassium ferricyanide to potassium ferrocyanide. Next, by applying the above-mentioned pulse voltage, an oxidation current of the produced potassium ferrocyanide is obtained, and this current value corresponds to the concentration of sucrose as a substrate.

Example 2 A 0.25 wt% aqueous solution of CMC is dropped on the electrode system prepared in the same manner as in Example 1 and dried to form a CMC layer. On the other hand, the enzyme INV25
0U, MUT250U and GOD750U, CMC
Phosphate buffer containing 0.25 wt% (0.2 M: K ₂ H
_{PO 4 -0.2M: KH 2 PO 4} ; pH = 7.4) 1ml
4 μl of this mixed solution is dropped on the CMC layer and dried in a warm air dryer at 50 ° C. for 10 minutes to form an enzyme layer. Subsequently, 4 μl of a 2 wt% ethanol solution of polyvinylpyrrolidone (hereinafter abbreviated as PVP) is dropped on the enzyme layer and dried at room temperature for about 20 minutes to form a PVP layer. Finally, 190 mg of potassium ferricyanide powder was dispersed in 0.5 wt% ethanol solution of purified soybean lecithin, and 3 μl of the solution was dropped on a PVP membrane and left to dry at room temperature for 2 to 3 hours to form a potassium ferricyanide layer. To do. After that, a sensor was assembled using a cover and a spacer in the same manner as in Example 1, and the sucrose aqueous solution was measured. As a result, a response current value proportional to the sucrose concentration in the sample solution was obtained.

[Embodiment 3] FIG. 2 is a sectional view of a biosensor of this embodiment. Insulating substrate 1 as in Example 1
After forming the leads 2 and 3, the electrode system and the insulating layer 6 on the top, a 0.25 wt% aqueous solution of CMC is dropped and dried to form a CMC layer. Next, INV250U, MUT
250 U and GOD 750 U with 0.25 wt of CMC
% Solution of 1% in water was dropped onto the CMC layer and dried in a warm air dryer at 50 ° C. for 10 minutes to form a reaction layer 7. The outer peripheral portion of the reaction layer has a diameter of about 3.6 mm, which is substantially the same as the diameter of the counter electrode. Next, a phosphate buffer solution (0.5
M: K ₂ HPO ₄ −0.5 M: KH ₂ PO ₄ ; pH = 7.
5 μl of 4) is dropped on the inside of the cover 9, that is, on the surface facing the electrode system, and dried for 13 minutes in a warm air dryer at 50 ° C. to form a layer 12 containing a reagent serving as a component of the buffer solution.

After forming the reaction layer 7 and the layer 12 as described above, the cover 9 and the spacer 8 are combined to form a sensor. In Example 3, since the reaction layer does not contain a reagent serving as a component of the buffer solution, the surface of the reaction layer is kept smooth, and there is an effect of suppressing a decrease or variation in response due to bubble generation. In addition, since the reagent that is a component of the buffer solution is supported on a place other than the reaction layer, it is possible to increase the concentration of the reagent without considering the effects that the reagent inhibits the enzyme activity and lowers the storage reliability. Is also possible. By increasing the reagent concentration, the buffer action becomes stronger, and the p
The effect of H is mitigated, and a stable sensor response can be obtained.

Further, in the above embodiment, phosphate is used as a reagent which is a component of the buffer solution, but the present invention is not limited to this, and phosphate-citric acid or acetic acid-acetate is used. Even if the same effect is obtained. Further, in Examples 1 and 2, the buffer salt concentration of 0.2M,
In Example 3, 0.5 M was used, but when the concentration of these buffers was examined, the buffering ability decreased when the concentration of the buffer was lower than 0.01 M, and the pH of the sample solution It became easily affected, and the responsiveness of the sensor decreased significantly. Further, when the concentration of the buffer solution is higher than 0.8 M, peeling or cracking may affect the layer formation, the dissolution rate of the reaction layer by the sample solution may decrease, and the response time of the sensor may increase. It had an impact. Therefore, the concentration of the buffer solution suitable for the sensor reaction is 0.01 M or more and 0.8.
It is M or less, and particularly preferably 0.1 M or more and 0.8 M or less.

[Embodiment 4] The same as Embodiment 1 except for the structure of the reaction layer 7. A 0.5 wt% aqueous solution of CMC is dropped on the electrode system manufactured in the same manner as in Example 1 and dried to form a CMC layer. Next, INV2 as an enzyme
50 U, fructose dehydrogenase (EC1.1.
99.11: hereinafter abbreviated as FDH) 1000 U and 33 mg of an electron acceptor potassium ferricyanide, CMC
Phosphate-citrate buffer containing 0.5 wt% (0.2
M: Na ₂ HPO ₄ −0.1 M: C ₃ H ₄ (OH) (COO
H) ₃ ; pH = 5.0) Dissolve in 1 ml, add 4 μl of this mixed solution onto the CMC layer, and dry in a warm air dryer at 50 ° C. for 10 minutes to form a reaction layer 7. The outer peripheral portion of the reaction layer has a diameter of about 3.6 mm, which is substantially the same as the diameter of the counter electrode. As in Example 1, phosphate, citric acid,
The mixed solution of INV, FDH and the electron acceptor and the CMC layer formed first are not in a completely mixed state, but the electrode system surface is in a state of being covered with only CMC. That is, to such an enzyme and an electron acceptor is not in contact with the surface of the electrode system, adsorption or the protein to the electrode system surface, potassium ferricyanide, H ₂ PO ₄ ^- electrode by a chemical action of a substance having an oxidizing ability, such as Changes in system characteristics are less likely to occur. As a result, a biosensor having a highly accurate sensor response can be obtained.

The response of the sucrose sensor produced as described above to an aqueous sucrose solution was measured in the same manner as in Example 1, and a response current value proportional to the sucrose concentration in the sample solution was obtained. In the above, when the reaction layer 7 is dissolved in the sample solution, sucrose in the sample solution is hydrolyzed into fructose and α-glucose by INV,
Fructose is oxidized by FDH to produce 5-keto-fructose. The electrons transferred by the oxidation reaction by FDH reduce potassium ferricyanide to potassium ferrocyanide. Next, by applying the above-mentioned pulse voltage, an oxidation current of the produced potassium ferrocyanide is obtained, and this current value corresponds to the concentration of sucrose as a substrate. In the above Examples 1 and 4, the enzyme, the electron acceptor and the hydrophilic polymer are mixed in the same solution and supported in the same layer, so that the manufacturing process can be simplified. In addition, since the reaction layer can dissolve the sample solution more easily than the multilayer structure, the sensor response speed can be shortened and the variation in the response value can be reduced.

[Embodiment 5] The same as Embodiment 1 except for the structure of the reaction layer 7. A 0.5 wt% aqueous solution of CMC is dropped on the electrode system manufactured in the same manner as in Example 1 and dried to form a CMC layer. Then INV250 as an enzyme
U, FDH 1000 U containing CMC 0.5 wt% phosphate-citrate buffer (0.2 M: Na ₂ HPO ₄ -0.1
M: C ₃ H ₄ (OH) (COOH) ₃ ; pH = 5.0) 1
4 ml of this mixed solution is added dropwise to the CMC layer and dried in a warm air dryer at 50 ° C. for 10 minutes to form an enzyme layer. Then polyvinylpyrrolidone (hereinafter PVP
2wt% ethanol solution of 4) on the enzyme layer
It is dropped and dried at room temperature for about 20 minutes to form a PVP layer.
Finally, 190 mg of potassium ferricyanide powder was dispersed in a 0.5 wt% ethanol solution of purified soybean lecithin, and 3 μl thereof was dropped on the PVP membrane and left to stand at room temperature for 2-3 hours to dry to form a potassium ferricyanide layer. To do. Then, a sensor was constructed by combining a cover and a spacer in the same manner as in Example 1, and the response to sucrose was measured. As a result, good linearity was obtained.

In Examples 2 and 5, the PVP layer was formed between the layer containing the enzyme and the layer containing the electron acceptor.
This is not absolutely necessary. By simply separating the reaction layer into a layer mainly containing an enzyme and a layer mainly containing an electron acceptor, storage reliability is significantly improved as compared with a mixed reaction layer. However, by providing the separation layer made of PVP, the electron acceptor and the enzyme can be sufficiently separated, and the storage reliability can be further improved. Here, PVP is used as the separation layer because PVP is a hydrophilic polymer and is also soluble in alcohol. Therefore, if an alcohol solution of PVP is dropped, the enzyme is not dissolved in alcohol and the enzyme is coated as the separation layer. This is because the substrate solution is an aqueous solution, so that when the substrate solution is supplied, PVP immediately has an affinity for water and the enzyme and the substrate can react. Therefore, the separation layer is not limited to PVP, and a hydrophilic polymer that is soluble in an organic solvent that does not dissolve an enzyme can obtain the same effect. In addition, although the concentration of the PVP film forming solution is 2 wt% in the above-mentioned example, when the concentration of PVP was examined, it was 4 w.
When the concentration is higher than t%, ethanol and PVP, which are the solvents of the potassium ferricyanide layer to be finally formed, are dissolved, and the potassium ferricyanide layer may not be completely dried or it may take a long time to interfere with sensor production. Came Therefore, the concentration of PVP is preferably 4 wt% or less.

[Embodiment 6] Similar to Embodiment 1, the insulating
Form the leads 2, 3 and electrode system and insulating layer 6 on the substrate 1.
After the completion, add 0.5 wt% CMC aqueous solution and dry.
Then, a CMC layer is formed. Then INV250U, F
DH1000U in 1 ml of 0.5 wt% CMC aqueous solution
Dissolve and add 4 μl of this mixed solution onto the CMC layer.
And dry in a warm air dryer at 50 ° C for 10 minutes to form a reaction layer.
Form 7. The outer circumference of the reaction layer is about 3.6 mm in diameter
Yes, it is approximately the same as the diameter of the counter electrode. Next, CMC0.
Phosphate-citrate buffer containing 25 wt% (0.5 M:
Na₂HPO_Four-0.5M: C 3H_Four(OH) (COOH)
₃PH = 5.0) inside the cover 9, that is, the electrode system
5 μl was dropped on the surface facing the plate, and then in a hot air dryer at 50 °
And dried for 13 minutes to form layer 12.

The reaction layers 7 and 12 are formed as described above.
After forming, cover 9 and spacer 8 are combined and
Configure the sensor. In the above embodiment, ferricyanation
The supported amount of potassium is 1.3 mg / cm Is 2. this
The amount of potassium ferricyanide carried was examined.
B, carrying amount is 2.7 mg / cm²More, smooth
Alternatively, it is difficult to form a reaction layer with sufficient strength.
The effects such as difficulty were seen. From this, ferry
The supported amount of potassium cyanide is 2.7 mg / cm²The following is
It is suitable. The mixture of potassium ferricyanide and enzyme
Such as increasing the concentration of hydrophilic polymer added to the combined solution,
When the viscosity of the mixed solution is increased, potassium ferricyanide
Loading amount of 6.5 mg / cm²Can be increased to
is there.

Regarding the combination of enzymes used,
Without being limited to the above examples, excellent sensor response was obtained even when an enzyme composed of a combination of INV and pyranose oxidase (EC 1.1.3.10) was used. Further, in the above-mentioned examples, the method of dissolving the enzyme and the electron acceptor in the sample solution was shown, but the method is not limited to this, and it can be applied to the case of being insolubilized in the sample solution by immobilization. it can. 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.

[0031]

As is apparent from the above description, according to the present invention, it is possible to obtain a highly reliable biosensor capable of quantifying sucrose with high accuracy, quickly and easily.

[Brief description of drawings]

FIG. 1 is a vertical sectional view of a biosensor according to an embodiment of the present invention.

FIG. 2 is an exploded perspective view of a biosensor according to an embodiment of the present invention with a reaction layer removed.

FIG. 3 is a vertical sectional view of a biosensor according to another embodiment of the present invention.

[Explanation of symbols]

 1 Insulating Substrate 2, 3 Lead 4 Measurement Electrode 5 Counter Electrode 6 Insulation Layer 7 Reaction Layer 8 Spacer 9 Cover 10 Sample Supply Hole 11 Air Hole 12 Layer Containing Reagents That Are Components of Buffer Solution

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Shiro Nankai 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd.

Claims (10)

[Claims] 1. An electrode system including a measuring electrode and a counter electrode formed on an insulating substrate, and a reaction layer in contact with the electrode system, wherein the reaction layer is at least an electron acceptor, an enzyme, and a hydrophilic property. A polymer, wherein the enzyme is invertase,
A biosensor characterized by being mutarotase and glucose oxidase. 2. An electrode system including a measuring electrode and a counter electrode formed on an insulating substrate, and a reaction layer in contact with the electrode system, wherein the reaction layer is at least an electron acceptor, an enzyme, and a hydrophilic property. A biosensor comprising a polymer, wherein the enzymes are invertase and fructose dehydrogenase. 3. The biosensor according to claim 1, wherein the reaction layer comprises at least two layers including an enzyme-containing layer and an electron acceptor-containing layer. 4. The biosensor according to claim 1, wherein the electron acceptor and the enzyme in the reaction layer are included in the hydrophilic polymer. 5. The biosensor according to claim 1, wherein the hydrophilic polymer separates at least one of an electron acceptor and an enzyme from the electrode system. 6. Between the two layers constituting the reaction layer,
The biosensor according to claim 3, further comprising a hydrophilic polymer film soluble in a solvent that does not dissolve the enzyme. 7. The amount of invertase, mutarotase and glucose oxidase carried in the reaction layer is 1 U / cm ² or more, 1 U / cm ² or more and 5 U / cm ^{2 respectively.}
The biosensor according to claim 1, which is the above. 8. The loading amount of invertase and fructose dehydrogenase in the reaction layer is 1 U / c, respectively.
The biosensor according to claim 2, which has m ² or more and 5 U / cm ² or more. 9. The biosensor according to claim 1, wherein a reagent serving as a component of the buffer solution is held at a position which is separated from the reaction layer and is in contact with the sample solution supplied to the reaction layer. 10. The biosensor according to claim 1, wherein the reaction layer contains a reagent serving as a component of a buffer solution.