JPH0688804A - Fructose sensor - Google Patents

Abstract

PURPOSE:To quickly measure the fructose concentration of a sample liquid without using any liquid except the sample liquid by uniting the reactive sections of electrodes with reactive sections of enzyme, matrix, and electron acceptors on a substrate. CONSTITUTION:An electrode system (measuring electrode 4 and counter electrode 5) and insulating layer 6 are successively formed on an insulating substrate 1. Then a carboxymethylcellulose(CMC) layer is formed by dropping CMC onto the electrode system as a hydrophilic polymer and drying the polymer. When a mixed solution of an enzyme and electron acceptor is dropped onto the CMC layer, the CMC layer is once melted, is mixed with the enzyme, etc., and forms a reactive layer 7 during the succeeding drying process. However, the surface of the electrode system is coated with the CMC only. When an aqueous solution of fructose is supplied 10 as a sample solution to the layer 7, the aqueous solution is quickly brought to an air hole 11 section by a capillary action and the layer 7 dissolves in the aqueous solution. When a pulse voltage is applied for a fixed period of time across the electrode 4 toward the anode by using the electrode 5 as a reference after supplying the sample solution, a response current value is obtained proportionally to the fructose concentration of the sample solution.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fructose sensor capable of quickly and accurately quantifying fructose in a sample.

[0002]

BACKGROUND OF THE INVENTION Fructose (fructose) is known as a sugar widely contained in foods such as fruits, and various methods are used to measure its concentration. Among them, the method using a fructose sensor applying the enzyme electrode reaction has been variously attempted as a measurement method with excellent selectivity, rapid and simple, and there is an example of a fructose sensor using a dialysis membrane (Biosensors & Bioelectronics , 6 (19
91) 55-72.). This fructose sensor is a combination of a fructose dehydrogenase (hereinafter abbreviated as FDH) immobilized on a membrane and a dialysis membrane, and the measurement system as a whole is configured such that an electron acceptor is previously dissolved in a buffer solution. . A fructose-containing sample solution is dropped into this measurement system, and the fructose concentration is determined from the current value when the reduced electron acceptor produced by the enzymatic reaction is electrochemically oxidized.

[0003]

Since the fructose sensor having the above-mentioned structure uses various films, the delay of the response due to the diffusion becomes large. Further, since the measurement system is configured to contain a liquid, it is difficult to improve the operability of measurement, and maintenance of the measurement system is complicated.

[0004]

In order to solve the above problems, the present invention provides an electrode system consisting of a measuring electrode and a counter electrode on an insulating substrate, and a hydrophilic polymer and an enzyme on the electrode system. It is characterized in that a reaction layer mainly composed of an electron acceptor is provided.

[0005]

[Function] With the above structure, the electrode reaction part and the reaction part of the enzyme, the substrate and the electron acceptor can be integrated on the substrate, whereby the fructose concentration can be rapidly increased without using any liquid other than the sample liquid. Can be measured. That is, it is possible to obtain a fructose sensor which has a simple operation and high reliability.

[0006]

EXAMPLES The present invention will be described below with reference to examples.

(Example 1) FIG. 1 is a sectional view of a sensor manufactured as an example of the fructose sensor of the present invention. FIG. 2 is a diagram showing an exploded perspective view as seen from the obliquely upper direction of FIG. 1 (reaction layer is not shown).

A method of manufacturing the fructose sensor will be described below. Silver paste was printed on the insulating substrate 1 made of polyethylene terephthalate by screen printing to form leads 2 and 3. Further, an insulating layer 6 was sequentially formed by a printing method using an electrode system (measurement electrode 4, counter electrode 5) using a conductive carbon paste containing a resin binder and an insulating paste.

The insulating layer 6 has a constant area (about 1 mm ² ) of the exposed portion of the measuring electrode 4 and partially covers the leads 2 and 3.

Next, a 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer was dropped on the electrode system and dried to form a CMC layer. Subsequently, FDH as an enzyme was formed on the CMC layer.
(Toyobo fructose dehydrogenase) 1000U
And potassium ferricyanide 33m as electron acceptor
g of a phosphate-citrate buffer solution (0.2 M: Na ₂ HPO ₄ -0.1 M: C ₃ H ₄ (O
H) (COOH) ₃ ; pH = 5.0) 4 μl of a mixed solution dissolved in 1 ml was added dropwise to 1 in a warm air dryer at 50 ° C.
The reaction layer 7 was formed by drying for 0 minutes. 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.

When a mixed solution of phosphate, citric acid, FDH and an electron acceptor is dropped, the CMC layer formed first dissolves once and the reaction layer 7 is mixed with an enzyme in the subsequent drying process to form the reaction layer 7. Form. However, it is not completely mixed because it is not agitated and the electrode system surface is CM
The state is covered by only C. That is, since the enzyme and electron acceptor do not come into contact with the surface of the electrode system, adsorption of proteins on the surface of the electrode system and changes in the characteristics of the electrode system due to the chemical action of substances having an oxidizing ability such as potassium ferricyanide occur. It can be difficult,
As a result, a fructose sensor having a highly accurate sensor response can be obtained.

After forming the reaction layer 7 as described above,
The cover 9 and the spacer 8 were adhered so as to have a positional relationship shown by a dashed line in FIG. If 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. Since the supply amount of the sample solution depends on the space volume generated by the cover and the spacer, it is not necessary to quantify in advance. Furthermore, evaporation of the sample liquid during measurement can be minimized, and highly accurate measurement can be performed.

To the fructose sensor manufactured as described above, 3 μl of a fructose aqueous solution was supplied as a sample solution from the sample supply hole 10. The sample solution quickly reached the air holes 11 by the capillary phenomenon, and the reaction layer 7 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 and the measurement electrode 4 is moved to the anode direction +0.
When a pulse voltage of 5 V was applied and the current value was measured after 5 seconds, a response current value proportional to the fructose concentration in the sample solution was obtained.

When the reaction layer 7 is dissolved in the sample solution, fructose in the sample solution 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 potassium ferrocyanide produced is obtained, and this current value corresponds to the concentration of fructose which is the substrate.

(Embodiment 2) The composition of the reaction layer 7 is the same as that of the embodiment 1 except for the composition of the reaction layer 7. Therefore, only the reaction layer 7 will be described.

A 0.5 wt% CMC aqueous solution was dropped on the electrode system prepared in the same manner as in Example 1 and dried to form a CMC layer. Then, FDH as an enzyme was added to the CMC layer.
4 μl of a mixed solution prepared by dissolving 400 U in 1 ml of a phosphate-citrate buffer containing 0.5 wt% of CMC was added dropwise, and 5
The FDH layer was formed by drying for 20 minutes in a warm air dryer at 0 ° C. Then, a 2 wt% ethanol solution of polyvinylpyrrolidone (hereinafter abbreviated as PVP) was added to the FDH layer in an amount of 4 μl.
It was dropped and dried at room temperature for about 20 minutes to form a PVP layer.
Finally, 190 mg of potassium ferricyanide was dispersed in an ethanol solution containing 0.5 wt% of purified soybean lecithin, and P
3 μl was dropped on the VP layer and left at room temperature for 2 to 3 hours to be dried to form a potassium ferricyanide layer.

After that, a sensor was assembled using the cover and the spacer as in Example 1, and the response to fructose was measured. As a result, good linearity was obtained. Also,
When the storage reliability at 40 ° C. was evaluated, the initial responsiveness was maintained even after 1 month as compared with Example 1, and the like.
It has been found to have excellent storage properties. On the other hand, the reaction layer of the sensor of Example 2 has a multi-layered structure including at least two layers including a layer mainly containing an enzyme and a layer mainly containing an electron acceptor, whereas the sensor of Example 1 contains an enzyme. Since the electron acceptor is mixed to form a monolayer structure, the substrate solution and the reaction layer are rapidly dissolved, which is more advantageous than Example 2 in terms of the dissolution rate and the diffusion rate, and the substrate concentration can be reduced to about 10 seconds. It was possible to obtain a sensor response.

Although the PVP layer is formed in the above embodiment, this is not always necessary. By simply separating the reaction layer into a layer mainly containing an enzyme and a layer mainly containing an electron acceptor, the storage reliability was significantly improved as compared with the 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 it is PVP.
Is a hydrophilic polymer that dissolves in alcohol, so PVP
If the alcohol solution is added dropwise, the enzyme can be coated as a separation layer without dissolving the enzyme in alcohol,
Further, since the substrate solution is an aqueous solution, PVP promptly has an affinity for water when the substrate solution is supplied, and the enzyme and the substrate can react with each other. Therefore, the separation layer is not limited to PVP, and a hydrophilic polymer soluble in an organic solvent can also obtain the same effect. In the above embodiment, PV
Although the concentration of P is 2 wt%, when the concentration of PVP is examined, when the concentration becomes higher than 4 wt%, ethanol and PVP, which are the solvent of the potassium ferricyanide layer to be finally formed, are dissolved, The potassium cyanide layer did not dry completely, or it took a long time to interfere with the sensor fabrication. Therefore, the concentration of PVP was preferably 4 wt% or less.

In Examples 1 and 2 above, the FDH
Of 39.3 U / cm ² and potassium ferricyanide of 1.
3 mg / cm ² is loaded respectively. When the amounts of these enzymes and electron acceptors carried were examined, FD
With respect to H, when the supported amount is less than 5 U / cm ² , the reaction rate is lowered and the measurement takes a long time. Therefore, the supported amount of FDH was preferably 5 U / cm ² or more.

Further, when the amount of potassium ferricyanide supported was more than 2.7 mg / cm ^2, it was observed that the reaction layer could not be formed smoothly or with sufficient strength. From these, the amount of potassium ferricyanide supported is 2.7 mg / cm ^2.
The following were suitable: Furthermore, in Example 1, if the viscosity of the mixed solution is increased by increasing the concentration of the hydrophilic polymer added to the mixed solution of FDH and potassium ferricyanide,
The supported amount of potassium ferricyanide is 6.5 mg / cm ^2.
It was possible to increase up to. Further, in Example 2, the amount of potassium ferricyanide carried was 6.5 mg / cm 2, as in Example 1, by reducing the grain size of the potassium ferricyanide crystals and increasing the concentration of the dispersant.
It was possible to increase to ² .

In Examples 1 and 2 above, pH =
A phosphate-citrate buffer solution of 5.0 was used, but when the pH dependence of the catalytic reaction of FDH supported on the electrode was examined, pH = less than 2.5 and pH =
When it was larger than 8.5, the FDH was inactivated and the response of the sensor was significantly lowered. From these facts, the pH of the buffer solution used is preferably pH = 2.5 or more and pH = 8.5 or less, and particularly pH = 4.0 or more and pH = 6.0 or less,
A good linear relationship was obtained between the fructose concentration and the sensor response value.

Although fructose dehydrogenase (FDH) was used as the enzyme in Examples 1 and 2 above, the enzyme is not limited to this. An excellent sensor response was obtained even when an enzyme composed of a combination of hexokinase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase, or an enzyme composed of a combination of glucose isomerase and glucose oxidase was used in place of the FDH.

Although CMC was used as the hydrophilic polymer in Examples 1 and 2 above, it is not limited to this, and 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, specifically hydroxypropyl cellulose,
Similar effects were obtained by using methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose and carboxymethyl ethyl cellulose.

On the other hand, as the electron acceptor, as in Example 1 above.
Although potassium ferricyanide shown in and 2 is excellent in terms of stability and reaction rate, p-benzoquinone, ferrocene and the like can be used in addition to these.

Further, in the above-mentioned Examples 1 and 2, the method in which the enzyme and the electron acceptor are dissolved in the sample solution has been described, but the invention is not limited to this, and it can be obtained by immobilizing the enzyme and the electron acceptor in the sample solution by immobilization. The present invention can also be applied.

Further, in the above-mentioned Examples 1 and 2, the two-electrode system consisting of the measurement electrode and the counter electrode was described, but the three-electrode system with the addition of the reference electrode enables more accurate measurement.

[0029]

As described above, according to the present invention, a fructose sensor having high operability and reliability can be obtained, whereby the fructose concentration in the sample solution can be measured very easily.

[Brief description of drawings]

FIG. 1 is a sectional view of a fructose sensor according to an embodiment of the present invention.

FIG. 2 is an exploded perspective view of the same fructose sensor excluding a reaction layer.

[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

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

Claims (9)

[Claims] 1. An electrode system comprising a measuring electrode and a counter electrode formed on an insulating substrate, and a reaction layer formed on the electrode system, the reaction layer comprising at least a hydrophilic polymer, an enzyme and an electron acceptor. A fructose sensor characterized in that 2. An electrode system comprising a measuring electrode and a counter electrode formed on an insulating substrate, and a reaction layer formed on the electrode system, wherein the reaction layer is mainly composed of an enzyme and a hydrophilic polymer. A fructose sensor, which comprises at least two layers having an electron acceptor as a main component. 3. The fructose sensor according to claim 1, wherein the enzyme is fructose dehydrogenase, or a combination of hexokinase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase, or a combination of glucose isomerase and glucose oxidase. 4. The fructose sensor according to claim 3, wherein the loaded amount of fructose dehydrogenase is 5 U / cm ² or more. 5. The fructose sensor according to claim 1, wherein the electron acceptor is potassium ferricyanide. 6. The amount of potassium ferricyanide supported is 6.5.
The fructose sensor according to claim 4, which has a mg / cm ² or less. 7. The fructose sensor according to claim 1, wherein the reaction layer contains a reagent serving as a component of a buffer solution. 8. The fructose sensor according to claim 6, wherein the reagent which is a component of the buffer solution is a phosphate or a phosphate and citric acid. 9. The fructose sensor according to claim 7, wherein the pH of the buffer solution is pH = 2.5 or more and 8.5 or less.