JP3333183B2 - Biosensor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] 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, and the like because of its high selectivity, simplicity, and rapidity. For the analysis of sugar concentration, enzyme sensors have been developed instead of refractometers and liquid chromatography methods, 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 has a hydrogen peroxide electrode and an immobilized enzyme film provided on the electrode with a polymer film interposed therebetween. In addition, a saccharase, a rotatory photoenzyme, and glucose oxidase are used as enzymes. The measurement system includes a sample chamber having the enzyme sensor in a temperature control block, and dilutes the sample, agitates the sample, controls the temperature, supplements oxygen,
Sugar detection is a system that is automatically controlled by a program. When a sample solution containing sucrose is injected into the sample chamber, the sample solution and the immobilized enzymes react 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 generated on the enzyme sensor diffuses in all directions. Therefore, in order to prevent a measurement error, it is necessary to decompose the backflowing hydrogen peroxide by including a catalase enzyme in the buffer solution. Also,
Since the reaction is performed in a solution, dilution, stirring, and temperature control are required, and the measurement system is complicated and lacks simplicity.
An object of the present invention is to solve the above-mentioned problems, and to provide a biosensor capable of quantifying sucrose in a sample with high accuracy, quickly and easily. The present invention
It is another object of the present invention to provide a highly reliable biosensor capable of quantifying sucrose in a sample with high accuracy.

[0004]

A biosensor according to the present invention includes an electrode system including a measurement electrode and a counter electrode formed on an insulating substrate, and a reaction layer in contact with the electrode system.
Wherein the reaction layer contains at least an electron acceptor, an enzyme , a reagent serving as a component of a hydrophilic polymer and a buffer , and the enzyme uses a combination of invertase, mutarotase, and glucose oxidase, and the invertase in the reaction layer is used. , the supported amount of mutarotase and glucose oxidase respectively 1U / cm ² or more state, and are 1U / cm ² or more and 5U / cm ² or more, and
In addition, the loading amount of the reagent is 0.13 M or less with respect to the sample solution.
It is preferably not more than 1.06M . In the biosensor of the present invention, the reaction layer is composed of at least two layers: a layer containing an enzyme and a layer containing an electron acceptor.

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 is separated from the electrode system by the hydrophilic polymer. I do. Also, the present invention
In a biosensor comprising at least two reaction layers, a film of a hydrophilic polymer soluble in a solvent that does not dissolve the enzyme is provided between the two layers constituting the reaction layer.

[0006] The biosensor of the present invention holds a reagent as a component of a buffer solution at a position contained in the reaction layer or separated from the reaction layer but in contact with a sample solution supplied to the reaction layer. are doing.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In order to quantify sucrose in a sample solution using the biosensor of the present invention, a sample solution is supplied to a reaction layer in contact with an electrode system of the sensor. The hydrophilic polymer in the reaction layer is dissolved by this sample solution, and the enzyme and the electron acceptor contained in the reaction layer come into contact with the sample. Then, in a sensor in which the reaction layer carries invertase, mutarotase and glucose oxidase as enzymes, sucrose in the sample solution is hydrolyzed by invertase to produce fructose and α-glucose. α-Glucose is rapidly converted to optical isomer β-glucose by the action of mutarotase, and β-glucose is oxidized by glucose oxidase. An electron acceptor, for example, potassium ferricyanide, is reduced to potassium ferrocyanide by the electrons transferred in the oxidation reaction by glucose oxidase.

On the other hand, in a biosensor in which a reaction layer carries invertase and fructose dehydrogenase as enzymes, sucrose in a sample solution is hydrolyzed to 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 in the oxidation reaction by fructose dehydrogenase.

As described above, the reduced form of the electron acceptor generated by the oxidation reaction with glucose oxidase or fructose dehydrogenase is then oxidized by applying a voltage with the measurement electrode as the anode with respect to the counter electrode. Can quantify sucrose. Further, the reaction layer contains a hydrophilic polymer, and this polymer is dissolved in the sample solution to provide a site for a reaction between the sample and an enzyme or an electron acceptor contained in the reaction layer. Therefore, since this polymer substantially prevents enzymes and electron acceptors from coming into contact with the electrode system surface, it adsorbs proteins to the electrode system surface and removes oxidizing substances such as electron acceptors. It is difficult for the characteristics of the electrode system to change due to the chemical action. As a result, a highly accurate sensor response can be obtained. Therefore, it is preferable to adopt a configuration in which the enzyme or the electron acceptor in the reaction layer is included in the hydrophilic polymer, or a configuration in which at least one of the electron acceptor and the enzyme is separated from the electrode system by the hydrophilic polymer.

Further, when 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. A highly reliable biosensor that does not deteriorate can be obtained. If a hydrophilic polymer membrane 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 enhanced.

When the reaction layer has a single layer structure,
Compared with the multilayer structure, the manufacturing process can be simplified, and since it can be easily dissolved in a sample solution, there is an advantage that the response speed of the sensor can be reduced and the variation of the response value can be reduced. Further, in order to reduce the influence of the pH of the sample solution and obtain a stable sensor response, it is preferable that the reaction layer contains a buffer solution or a reagent as a component thereof in advance.

Instead of including the reagent as a component of the buffer solution in the reaction layer, the reagent may be separated from the reaction layer and held at a position where it comes into contact with the sample solution supplied to the reaction layer. With this configuration, the reaction layer does not contain a reagent that is a component of the buffer solution, so that the surface of the reaction layer is kept smooth,
This has the effect of reducing the response and variation due to the generation of bubbles. In addition, since the reagent, which 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 of the enzyme inhibiting the enzyme activity and reducing the storage reliability. it can. By increasing the concentration of the reagent, the buffering action becomes stronger, the influence of the pH of the sample solution is reduced, and a stable sensor response can be obtained. As a reagent serving as a component of the buffer solution, phosphate-citric acid or acetic acid-acetate can be used in addition to phosphate. Next, as for the amount of the enzyme to be carried, when the amount is small, the reaction speed is reduced and the measurement takes a long time. The preferred loading 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 carboxymethylcellulose of the examples, but may be other cellulose-based, vinyl alcohol-based, vinylpyrrolidone-based, gelatin-based, acrylate-based, starch-based. , Maleic anhydride, acrylamide, etc. may be used. As the electron acceptor, p-benzoquinone, ferrocene, and the like can be used in addition to the potassium ferricyanide in Examples.

As described above, the biosensor of the present invention comprises:
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 enzyme reaction of the reaction layer with the electrode system. In addition, since an enzyme reaction system is used, high precision can be obtained by a combination of a reaction layer containing an enzyme and an electrode system while having high substrate selectivity. Furthermore, depending on the configuration of the reaction layer, the response speed can be increased or the storage stability can be improved.

[0015]

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

Next, a 0.25 wt% aqueous solution of carboxymethylcellulose (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
3.2.2.126: hereinafter abbreviated as INV) 250 U, mutarotase (EC 5.1.3.3: hereinafter abbreviated as MUT) 2
50U and glucose oxidase (EC 1.1.
3.4: hereinafter abbreviated as GOD) 750 U and 33 mg of potassium ferricyanide as an electron acceptor were added to CMC 0.2
Phosphate buffer containing 5 wt% (0.2 M: K ₂ HPO ₄ −
0.2 M: 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 50 ° C. hot air drier for 10 minutes. The reaction layer is substantially circular with a diameter of about 3.6 mm, and substantially coincides with the outer periphery of the counter electrode 5.

In the above-mentioned reaction layer forming step, when a mixed solution of phosphate, enzyme and electron acceptor is dropped on the CMC layer, the CMC layer composed of the hydrophilic polymer is dissolved once and the enzyme is dried in the subsequent drying process. The reaction layer 7 is formed in a form mixed with the above. However, since no stirring or the like is involved, a completely mixed state is not obtained, and the surface of the electrode system is in a state covered with only CMC. That is, because the enzyme and the electron acceptor do not contact the electrode system surface, protein adsorption on the electrode system surface, potassium ferricyanide,
H ₂ PO ₄ ^- electrode system characteristics change due to chemical action of substances with oxidizing capability is less likely to occur, such as. as a result,
A biosensor having a highly accurate sensor response can be obtained. After forming the reaction layer 7 as described above, the sensor 9 is assembled by bonding the cover 9 having the air holes 11 and the spacer 8 having the grooves forming the sample supply holes 10 to the substrate 1. When a transparent material is used for the cover, the state of the reaction layer and the state of introduction of the sample solution can be checked very easily from outside.

Further, when the cover is mounted, the sample liquid is easily introduced into the reaction layer portion by a simple operation of only contacting the sample supply hole 10 at the tip of the sensor due to the capillary action in the space formed by the cover and the spacer. In order to smoothly supply the sample solution, it is preferable that a toluene solution of reticin is spread on the surface from the sample supply section to the reaction layer and dried as needed. Since the supply amount of the sample liquid depends on the space volume generated by the cover and the spacer,
There is no need to determine in advance. Furthermore, evaporation of the sample liquid during measurement can be minimized, and highly accurate measurement can be performed. When 3 μl of a sucrose aqueous solution was supplied as a sample solution from the sample supply hole 10 to the sucrose sensor prepared as described above, the sample solution quickly reached the air hole 11 portion by capillary action, and the reaction layer on the electrode system was dissolved.

After a certain time from the supply of the sample liquid, the measuring electrode 4 is moved in the direction of the anode to the measuring electrode 4 with reference to the counter electrode 5 of the electrode system.
When a pulse voltage of 0.5 V was applied and the current value after 5 seconds was measured, 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, the sucrose in the sample solution is hydrolyzed by INV to produce fructose and α-glucose. α-Glucose reaches equilibrium with the optical isomer β-glucose when left as an aqueous solution, but this equilibrium transfer is catalyzed by MUT, and α-glucose produced by INV quickly becomes β-glucose, and is oxidized by GOD. Receive. Potassium ferricyanide is reduced to potassium ferrocyanide by the electrons transferred in the oxidation reaction by GOD. Next, by the application of the pulse voltage, an oxidation current of the generated 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 an electrode system manufactured 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 by CMC
Phosphate buffer containing 0.25 wt% (0.2 M: K ₂ H
PO ₄ -0.2 M: KH ₂ PO ₄ ; pH = 7.4) 1 ml
And 4 μl of the mixture is dropped on the CMC layer, and dried in a 50 ° C. hot air drier 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 a 0.5 wt% ethanol solution of purified soybean lecithin, 3 μl of which was dropped on a PVP membrane, and left to dry at room temperature for 2 to 3 hours to form a potassium ferricyanide layer. I do. Thereafter, the sensor was assembled using the cover and the 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 an exploded perspective view of a biosensor of this embodiment. After forming the leads 2 and 3, the electrode system, and the insulating layer 6 on the insulating substrate 1 in the same manner as in the first embodiment, a 0.25 wt% aqueous solution of CMC is dropped and dried to form a CMC layer. Then, INV250U, M
UT250U and GOD750U are converted to CMC 0.25
The mixed solution dissolved in 1 ml of an aqueous solution of
4 μl is dropped on the 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 equal to the diameter of the counter electrode.
Next, a phosphate buffer containing 0.25 wt% of CMC (0.
_{5M: K 2 HPO 4 -0.5M:} KH 2 PO 4; pH = 7.
4) is dropped on the inside of the cover 9, that is, on the surface facing the electrode system, and dried in a 50 ° C. hot air drier for 13 minutes to form a layer 12 containing a reagent to be a component of a buffer solution.

After forming the reaction layer 7 and the layer 12 as described above, the sensor is constituted by combining the cover 9 and the spacer 8. 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 has 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 in a place other than the reaction layer, the concentration of the reagent should be increased without considering the effect such that the enzyme activity is inhibited by the reagent and storage reliability is reduced. Is also possible. By increasing the concentration of the reagent, the buffering action becomes stronger, and the p
The effect of H is reduced, and a stable sensor response can be obtained.

In the above embodiment, phosphate is used as a reagent as a component of the buffer solution. However, the present invention is not limited to this, and phosphate-citric acid or acetic acid-acetate may be used. The same effect can be obtained even if the same is performed. Further, in Examples 1 and 2, the concentration of the buffer salt was 0.2 M,
In Example 3, the buffer having a concentration of 0.5 M was used. However, the concentration of these buffers was examined. When the buffer concentration was lower than 0.01 M, the buffer capacity was reduced, and the pH of the sample solution was lowered. It became susceptible and the responsiveness of the sensor was significantly reduced. When the concentration of the buffer solution is higher than 0.8M, the layer formation is affected such as peeling or cracking, and the dissolution rate of the reaction layer by the sample liquid is reduced, and the response time of the sensor is increased. Affected. Therefore, the concentration of the buffer solution suitable for the sensor reaction is 0.01M or more and 0.8M or more.
M or less, especially 0.1 M or more and 0.8 M or less (i.e.,
That is, the amount of the reagent serving as a component of the buffer solution is
0.13M or more and 1.06M or less) .

Example 4 The structure of the present embodiment is the same as that of Example 1 except for the structure of the reaction layer 7. A 0.5 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. Next, INV2 as an enzyme
50 U, fructose dehydrogenase (EC 1.1.
99.11: hereinafter abbreviated as FDH) 1000 U and 33 mg of potassium ferricyanide as an electron acceptor were added to 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) dissolved in 1 ml, 4 μl of this mixed solution was dropped on the CMC layer, and dried in a 50 ° C. hot air drier 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 equal to 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 do not form a completely mixed state, and the surface of the electrode system is in a state of being covered only by the 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 unlikely to occur. As a result, a biosensor having a highly accurate sensor response can be obtained.

When the response of the sucrose sensor prepared as described above to an aqueous sucrose solution was measured in the same manner as in Example 1, 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, the sucrose in the sample solution is hydrolyzed by INV to fructose and α-glucose,
Fructose is oxidized by FDH to produce 5-keto-fructose. Potassium ferricyanide is reduced to potassium ferrocyanide by the electrons transferred in the oxidation reaction by FDH. Next, by the application of the pulse voltage, an oxidation current of the generated potassium ferrocyanide is obtained, and this current value corresponds to the concentration of sucrose as a substrate. In 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 dissolves the sample liquid more easily than the multilayer structure, the response speed of the sensor is reduced, and the variation of the response value can be reduced.

Example 5 The structure of the reaction layer 7 was the same as that of Example 1 except for the structure of the reaction layer 7. A 0.5 wt% aqueous solution of CMC is dropped on an electrode system manufactured in the same manner as in Example 1, and dried to form a CMC layer. Subsequently, INV250 was used as an enzyme.
U, 1000 U of FDH in a phosphate-citrate buffer containing 0.5 wt% of CMC (0.2 M: Na ₂ HPO ₄ -0.1
_{M: C 3 H 4 (OH} ) (COOH) 3; pH = 5.0) 1
Then, 4 μl of this mixed solution is dropped on the CMC layer, and dried in a 50 ° C. hot air drier for 10 minutes to form an enzyme layer. Subsequently, polyvinylpyrrolidone (hereinafter PVP)
4 μl of a 2 wt% ethanol solution of
The mixture is dropped and dried at room temperature for about 20 minutes to form a PVP layer.
Finally, 190 mg of potassium ferricyanide powder is dispersed in a 0.5 wt% ethanol solution of purified soybean lecithin, 3 μl of which is dropped on a PVP membrane and left to dry at room temperature for 2 to 3 hours to form a potassium ferricyanide layer. I do. Thereafter, a sensor was configured by combining the cover and the 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 is formed between the layer containing the enzyme and the layer containing the electron acceptor.
This is not necessary. Even if the reaction layer is simply separated into a layer mainly composed of an enzyme and a layer mainly composed of an electron acceptor, the storage reliability is greatly 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, so that the storage reliability can be further improved.
Here, the reason why PVP was used as the separation layer is that PVP is a hydrophilic polymer and also dissolved in alcohol, so if a PVP alcohol solution is dropped, the enzyme is coated as a separation layer without being dissolved in alcohol. Further, since the substrate solution is an aqueous solution, when the substrate solution is supplied, the PVP quickly has an affinity for water, and the enzyme and the substrate can react with each other. Therefore, the separation layer is not limited to PVP, and a hydrophilic polymer that is soluble in an organic solvent that does not dissolve the enzyme can achieve the same effect. In the above embodiment, the concentration of the PVP film forming solution is 2 wt%.
If the concentration is higher than wt%, ethanol and PVP, which are the solvents of the potassium ferricyanide layer formed last, will be dissolved, and the potassium ferricyanide layer will not be completely dried or will have a long time. I came. Therefore, the concentration of PVP is preferably 4 wt% or less.

[Embodiment 6] As in Embodiment 1, the insulating property
The leads 2 and 3, the electrode system, and the insulating layer 6 are formed on the substrate 1.
After completion, a 0.5 wt% aqueous solution of CMC is dropped and dried.
Then, a CMC layer is formed. Then, INV250U, F
DH1000U in 1ml of 0.5wt% aqueous solution of CMC
Dissolve and add 4 μl of this mixed solution onto the CMC layer
And dried in a warm air dryer at 50 ° C for 10 minutes.
7 is formed. The outer circumference of the reaction layer has a diameter of about 3.6 mm.
And almost coincides with the diameter of the counter electrode. Next, CMC0.
Phosphate-citrate buffer containing 25 wt% (0.5 M:
Na_TwoHPO_Four-0.5M: C _ThreeH_Four(OH) (COOH)
_ThreePH = 5.0) inside the cover 9, ie, the electrode system
5 μl is dropped on the surface opposite to
For 13 minutes to form layer 12.

After forming the reaction layer 7 and the layer 12 as described above, the sensor is constructed by combining the cover 9 and the spacer 8. In the above embodiment, the loading amount of potassium ferricyanide is 1.3 mg / cm ² . Examination of the supported amount of potassium ferricyanide revealed that if the supported amount was more than 2.7 mg / cm ^2, it was difficult to form a reaction layer having a smooth or sufficient strength. For this reason, the loading amount of potassium ferricyanide is preferably 2.7 mg / cm ² or less. In addition, such as increasing the concentration of the hydrophilic polymer to be added to the mixed solution of potassium ferricyanide and the enzyme,
If the viscosity of the mixed solution is increased, the amount of potassium ferricyanide carried can be increased to 6.5 mg / cm ² .

The combination of enzymes used is as follows:
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 1.1.1.3.10) was used. Further, in the above example, the method of dissolving the enzyme and the electron acceptor in the sample solution was described. it can. Further, in the above-described embodiment, a two-electrode system including a measurement electrode and a counter electrode has been described. However, if a three-electrode system including a reference electrode is used, more accurate measurement can be performed.

[0031]

As is clear from the above description, according to the present invention, a highly reliable biosensor capable of quantifying sucrose with high accuracy, quickly and easily can be obtained.

[Brief description of the drawings]

FIG. 1 is a longitudinal sectional view of a biosensor according to one embodiment of the present invention.

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

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

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 insulating substrate 2, 3 lead 4 measuring electrode 5 counter electrode 6 insulating layer 7 reaction layer 8 spacer 9 cover 10 sample supply hole 11 air hole 12 layer containing reagent to be a component of buffer solution

Continuation of the front page Examiner Koichi Kuroda (56) References JP-A-3-54447 (JP, A) JP-A-4-113262 (JP, A) JP-A-64-69944 (JP, A) JP-A-55 -101042 (JP, A) JP-A-2-83444 (JP, A) JP-A-1-134244 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27/327 JICST File (JOIS)

Claims (5)

(57) [Claims] An electrode system including a measurement electrode and a counter electrode formed on an insulating substrate, and a reaction layer in contact with the electrode system, wherein the reaction layer includes at least an electron acceptor, an enzyme ,
A hydrophilic polymer and a reagent serving as components of a buffer solution , wherein the enzymes are invertase, mutarotase, and glucose oxidase; and the amount of invertase, mutarotase, and glucose oxidase in the reaction layer is 1 U / cm ² or more and 1 U / cm ² or more, respectively. cm ² or more and 5U / cm ² or more der is, furthermore, 0.13 M supported amount of the reagent to the sample solution
A biosensor characterized by being at least 1.06M inclusive . 2. The biosensor according to claim 1, wherein said reaction layer comprises at least two layers: a layer containing an enzyme and a layer containing an electron acceptor. 3. The biosensor according to claim 1, wherein the electron acceptor and the enzyme in the reaction layer are included in the hydrophilic polymer. 4. 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. 5. The method according to claim 1, wherein the two layers constituting the reaction layer are:
The biosensor according to claim 2, wherein a film of a hydrophilic polymer soluble in a solvent that does not dissolve the enzyme is provided .