JP3214562B2 - Reference electrode, biosensor and measuring device using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reference electrode used for electrochemically measuring a specific chemical substance in a solution by utilizing an enzymatic reaction or the like, and a biosensor using the same.

[0002]

2. Description of the Related Art The structure of a conventional reference electrode will be described with reference to FIG. FIG. 1 is a cross-sectional view and a top view of a reference electrode disclosed in Japanese Patent Application Laid-Open No. 9-182738. FIG.
Referring to (a), a polyimide layer PI1, an anode film M, and a silver layer S are formed on a cathode film K in this order. The silver layer S has a role of a reference electrode, and a polyimide layer PI2 is provided thereon as a protective layer. It is stated that a water-repellent photoresist may be provided as a protective layer instead of the polyimide layer PI2. It is described that providing such a protective layer on a reference electrode extends the life of the electrode.

[0003]

However, it has been difficult to obtain a sufficiently long service life with the above reference electrode. This is because a part of the surface of the reference electrode is in direct contact with the electrolyte, and silver chloride formed on the surface is easily dissociated in the electrolyte. According to the study of the present inventor, the period in which the reference electrode having the above structure operates while maintaining the initial performance is usually about two days.

In the above reference electrode, when a water-repellent photoresist is used as a material for the protective layer, the manufacturing process becomes complicated and the production cost increases, and there is room for improvement in this respect. This requires a process of repeating exposure and development using photolithography technology for the formation of a photoresist, and furthermore, forming an electrolyte injection port for injecting an electrolyte and a through hole for removing bubbles generated when the electrolyte is injected. It is necessary to keep it.

Further, when the above-mentioned reference electrode is applied to a biosensor, there is a problem that contaminants contained in a measurement sample adhere to the surface and the sensitivity is reduced. This will be described below.

A biosensor is a sensor having a function of converting a chemical substance in a solution into hydrogen peroxide or the like by a catalytic function of an enzyme, and measuring this by a redox reaction. In order to stabilize the characteristics of the biosensor and extend the service life, it is important to eliminate the influence of interfering substances and contaminants. The interfering substance is a chemical substance that affects the above-described redox reaction system and causes a positive error in the measurement result, and includes ascorbic acid, acetaminophen, and the like. On the other hand, a contaminant is a chemical substance that is adsorbed on the electrode surface and causes a negative error in the measurement result, and includes, for example, albumin, urea, urea compounds, creatinine, and the like.

However, the reference electrode according to the above prior art is
Since polyimide or photoresist was used as the material of the protective layer, contaminants easily adhered to the surface of the protective layer,
It was difficult to avoid a decrease in sensitivity after long-term use.

[0008]

According to the present invention, there is provided an electrode provided on an insulating substrate, and a protective layer formed so as to cover the electrode. And a reference electrode mainly comprising a polymer in which a pendant group containing at least a fluoroalkylene block is bonded to a fluorine-free vinyl polymer.

That is, the reference electrode of the present invention comprises an electrode (electrode layer) provided on an insulating substrate and a plurality of layers having various functions provided on the electrode.

[0010] In the present invention, the reference electrode is an electrode serving as a reference when measuring the electrode potential of the working electrode. The reference electrode of the present invention is suitably used when electrochemically measuring a specific chemical substance in a solution using an enzyme reaction, and is suitably used as a part of a biosensor or the like.

In the present invention, the polymer constituting the protective layer has a pendant group containing a fluoroalkylene block (fluoroalkylene unit). Therefore, adhesion of contaminants such as proteins and urea compounds is suppressed, and a reference electrode exhibiting stable characteristics even when used for a long time can be obtained. In addition, since the fluoroalkylene does not dissolve in most non-fluorinated solvents and detergents such as surfactants, a reference electrode having good chemical resistance can be obtained.

In addition, since this polymer has a vinyl polymer containing no fluorine as a main chain, it has good adhesion to an electrode or another organic polymer layer formed on the electrode. Therefore, no gap is formed between the protective layer and the electrode or the organic polymer layer formed on the surface thereof. Therefore, the surface of the electrode portion does not come into contact with the electrolytic solution, and the metal constituting the electrode portion can be prevented from being eluted into the electrolytic solution. Further, there is an advantage that the time required for cleaning the reference electrode can be reduced.

Furthermore, because of good adhesion to electrodes and the like,
The durability of the layer structure is improved, and a reference electrode that is unlikely to be damaged even when used for a long time can be obtained.

Here, in addition to the above-mentioned pendant group containing a fluoroalkylene block, other appropriate side chains and functional groups may be bonded. For example, -OH group, -COO
By having a functional group having an appropriate polarity such as an H group, the adhesion to an adjacent electrode or an organic polymer layer can be further enhanced.

The protective layer according to the present invention can produce a uniform thin film by a simple process such as a dipping method, a spin coating method and a spraying method, and is excellent in mass productivity. Further, according to the present invention, there is provided an electrode provided on an insulating substrate, and a protective layer formed so as to cover the electrode, wherein the protective layer is formed from a fluoroalcohol ester of polycarboxylic acid (A). A reference electrode is provided that is primarily characterized.

Further, according to the present invention, there is provided an electrode provided on an insulating substrate, and a protective layer formed so as to cover the electrode, wherein the protective layer is a fluoroalcohol of polycarboxylic acid (A). There is provided a reference electrode comprising an ester and an alkyl alcohol ester of a polycarboxylic acid (B).

According to the present invention, there is further provided an electrode provided on an insulating substrate, and a protective layer formed so as to cover the electrode, wherein the protective layer comprises an alkyl alcohol ester group and a fluoro alcohol ester group. And a reference electrode mainly comprising a polycarboxylic acid ester compound having the following formula:

These reference electrodes are composed of an electrode (electrode layer) provided on an insulating substrate and a plurality of layers having various functions provided on this electrode. Characterized by being formed of a polymer.

The reference electrode of the present invention uses a fluoroalcohol ester of polycarboxylic acid as a constituent material of the protective layer. Here, the fluoroalcohol ester of polycarboxylic acid is obtained by esterifying a part or all of the carboxyl groups of polycarboxylic acid with fluoroalcohol. Fluoroalcohol is one in which all or at least one of the hydrogen atoms in the alcohol is replaced with fluorine.

Since the constituent material of the protective layer has a fluoroalcohol ester group, adhesion of contaminants such as proteins and urea compounds is suppressed, and a reference electrode exhibiting stable characteristics even when used for a long time can be obtained. In addition, since the fluoroalcohol ester group does not dissolve in most non-fluorinated solvents and detergents such as surfactants, a reference electrode having good chemical resistance can be obtained.

The reference electrode of the present invention uses a polymer having a polycarboxylic acid main chain in the protective layer. Further, a fluoroalcohol is bonded to the main chain via an ester group. For this reason, the adhesiveness between the electrode and the organic polymer layer formed on the electrode is good, and no gap is formed between the electrode and the protective layer. Therefore, the surface of the electrode portion does not come into contact with the electrolytic solution, and the metal constituting the electrode portion can be prevented from being eluted into the electrolytic solution. Further, there is an advantage that the time required for cleaning the reference electrode can be reduced.

Furthermore, because of good adhesion to electrodes and the like,
The durability of the layer structure is improved, and a reference electrode that is unlikely to be damaged even when used for a long time can be obtained.

With respect to the main chain comprising the above polycarboxylic acid,
An appropriate functional group other than the fluoroalcohol ester group may be bonded. By having a functional group having an appropriate polarity, the adhesion to an adjacent electrode or an organic polymer layer formed on the electrode can be further increased.

Further, the protective layer is made of polycarboxylic acid (A)
In the case of a configuration comprising a fluoroalcohol ester of the above and an alkyl alcohol ester of the polycarboxylic acid (B), or a configuration mainly comprising a polycarboxylic acid ester compound having an alkyl alcohol ester group and a fluoro alcohol ester group, In addition to the effect, there is obtained an advantage that the stability at a high temperature is improved. The reference electrode and the biosensor including the same are sometimes stored and used at a relatively high temperature (for example, about 40 ° C.). When a normal reference electrode is left to stand at a high temperature and then used for measurement, the measurement sensitivity often fluctuates significantly when compared to when measured before standing. On the other hand, the reference electrode and the biosensor provided with the protective layer having the above-described configuration have almost no change in measurement sensitivity even when left at a high temperature, and are excellent in stability.

In the above-described reference electrode of the present invention, the electrode and the protective layer may be formed so as to be in direct contact with each other, or another layer may be interposed between them. For example, a configuration having a bonding layer mainly composed of a silane coupling agent between the electrode and the protective layer may be employed.

The protective layer in the present invention can produce a uniform thin film by a simple process such as a dipping method, a spin coating method and a spraying method, and is excellent in mass productivity. Further, according to the present invention, there is provided a biosensor using the above-mentioned reference electrode. Since the biosensor has a protective layer made of a polymer having the above-mentioned specific structure on the surface of the reference electrode, it has excellent long-term stability and can be used under a wide range of measurement conditions.

Further, according to the present invention, there are provided various measuring devices using the biosensor. That is, according to the present invention, there is provided a measuring instrument comprising: the biosensor; and a data notifying unit for notifying an electric signal obtained from the biosensor.

Also, the biosensor, an electrochemical measurement circuit for obtaining an electric signal from the biosensor, a data processing unit for calculating a measured value based on the electric signal, and a data notification for notifying the measured value And a measuring device comprising:

Since these measuring instruments have a biosensor having a working electrode of a specific structure, they have excellent long-term stability and can be used under a wide range of measuring conditions.
In addition, the operation method is simple, and even a person unfamiliar with the device can easily handle it.

Further, according to the present invention, the step of forming an electrode on an insulating substrate and the step of forming at least a fluoroalkylene block on a fluorine-free vinyl polymer directly or via another layer on the electrode. Applying a liquid containing a polymer having a pendant group bonded thereto, followed by drying and forming a protective layer.

In this method of manufacturing a reference electrode, a protective layer is formed by applying and drying a liquid containing a polymer having a specific structure as described above. For this reason, a protective layer excellent in stability at the time of repeated measurement, adhesion to an adjacent layer, durability and the like can be formed with good film thickness controllability. Further, since the liquid containing the polymer has a low viscosity, the protective layer can be easily formed with a small thickness. Specifically, after drying, a protective layer having a thickness of 0.01 to 3 μm can be favorably formed.

[0032]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, a "reference electrode" refers to an electrode which serves as a reference when measuring the potential of a working electrode as described above. The “biosensor” in the present invention includes the above-mentioned reference electrode, and usually has a configuration further including a working electrode and a counter electrode. Further, the “measuring device” in the present invention refers to a system including the above-described biosensor, and includes various means for notifying and processing an electric signal obtained from the biosensor. Hereinafter, the reference electrode, the biosensor, and the measuring device according to the present invention will be described in detail.

The first reference electrode according to the present invention has an electrode provided on an insulating substrate, and a protective layer formed so as to cover the electrode, and the protective layer is made of vinyl containing no fluorine. It is characterized by being mainly composed of a polymer in which a pendant group containing at least a fluoroalkylene block is bonded to a system polymer. Where "become the main"
"It means that the polymer is a main component constituting the protective layer, for example, that the content of the polymer in the protective layer is 50% by weight or more.

The “fluorine-free vinyl polymer” is
This is a part having a role of improving the adhesion to the electrode and the organic polymer layer formed on the electrode, and the structure is not particularly limited, but must not contain fluorine. When fluorine is contained in the polymer portion excluding the pendant group, the adhesion to the electrode or the organic polymer layer formed on the electrode is reduced, the adjustment of the solution becomes difficult, and the protective layer can be formed as a thin film. It becomes difficult.

The vinyl polymer containing no fluorine is a polymer having a main skeleton composed of carbon-carbon bonds. Preferred examples thereof include unsaturated hydrocarbons, unsaturated carboxylic acids,
And a homopolymer or a copolymer of one or more monomers selected from the group consisting of unsaturated alcohols. Of these, polycarboxylic acids are particularly preferred. By selecting such a polymer, the adhesion to an electrode or an organic polymer layer formed on the electrode becomes particularly good, and a protective layer having excellent durability can be obtained. Further, it is preferable that a fluoroalkylene block is bonded to the vinyl polymer via an ester group. Since the ester group has an appropriate polarity, the adhesion to the electrode and the organic polymer layer formed on the electrode is further improved.

The pendant group containing a fluoroalkylene block refers to a pendant group containing a fluoroalkylene as a constituent unit. Fluoroalkylene refers to an alkylene group in which some or all of the hydrogen atoms have been substituted with fluorine. The fluorine content of the pendant group, that is, x where the number of fluorine atoms contained in the pendant group is x and the number of hydrogen atoms contained in the pendant group is y
The value of / (x + y) is preferably from 0.3 to 1, more preferably from 0.8 to 1. By doing so, the adhesion of contaminants to the protective layer is effectively suppressed.

The number of carbon atoms of the pendant group is preferably 3 to
15, more preferably 5 to 10, most preferably 8 to 10. By doing so, the length of the pendant group becomes appropriate, good film-forming properties can be obtained, and adhesion of contaminants and the like is effectively prevented. In addition, the adhesiveness between the adjacent electrode and the polymer layer is well maintained.

The binding rate of the pendant group to the vinyl polymer, that is, the content of the pendant group is not particularly limited, and may be an appropriate value according to other polymer layer materials and applications. For example, 0.1 to 30%. By setting the content of the water-repellent pendant group in an appropriate range, the protection performance of the electrode surface is improved, and good adhesion to the adjacent polymer layer is obtained. Here, the binding ratio of the pendant group means a ratio of the pendant group to all the groups bonded to the carbon-carbon bond serving as the main skeleton of the vinyl polymer. For example, the vinyl polymer serving as the main chain is polyacrylic acid, and -COO
When 10% of the H groups are esterified to form pendant groups, the pendant group binding rate is 2.5% obtained by multiplying the -COOH group binding rate of 25% by the esterification rate of 10%.

The second reference electrode according to the present invention has an electrode provided on an insulating substrate, and a protective layer formed so as to cover the electrode. It is mainly composed of the alcohol ester (A). Note that “mainly” means that the polymer is a main component of the protective layer, for example, that the content of the polymer in the protective layer is 50% by weight or more.

The third reference electrode according to the present invention has an electrode provided on an insulating substrate and a protective layer formed so as to cover the electrode, and the protective layer is formed of a polycarboxylic acid. It is characterized by comprising a fluoro alcohol ester of (A) and an alkyl alcohol ester of polycarboxylic acid (B).

A fourth reference electrode according to the present invention has an electrode provided on an insulating substrate and a protective layer formed so as to cover the electrode, and the protective layer is formed of an alkyl alcohol ester. And a polycarboxylic acid ester compound having a group and a fluoroalcohol ester group. Note that “mainly” means that the polymer is a main component of the protective layer, for example, that the content of the polymer in the protective layer is 50% by weight or more.

The polycarboxylic acids (A) and (B) and the polycarboxylic acid constituting the polycarboxylic acid ester compound are polymers having a structural unit of a carboxylic acid such as acrylic acid, methacrylic acid, fumaric acid and itaconic acid. Say. For example, polymethacrylic acid, polyacrylic acid, a copolymer thereof, or the like can be given. The polycarboxylic acid (A) and the polycarboxylic acid (B) may be the same or different.

The polycarboxylic acid is a polymer having a structural unit of a carboxylic acid such as acrylic acid, methacrylic acid, fumaric acid, itaconic acid, and includes, for example, polymethacrylic acid or polyacrylic acid.

When the number of fluorine atoms contained in the fluoroalcohol ester group is x and the number of hydrogen atoms is y, x
The fluorine content of the fluoroalcohol ester group represented by / (x + y) is preferably 0.3 to 1, more preferably 0.8 to 1. By doing so, the adhesion of contaminants to the protective layer is effectively suppressed.

The carbon number of the fluoroalcohol constituting the fluoroalcohol ester is preferably 3 to 15, more preferably 5 to 10, and most preferably 8 to 10. By doing so, the length of the pendant group becomes appropriate, good film-forming properties can be obtained, and adhesion of contaminants and the like is effectively prevented. In addition, the adhesiveness between the adjacent electrode and the polymer layer is well maintained.

The esterification rate of the fluoroalcohol ester of the polycarboxylic acid is not particularly limited, and may be an appropriate value according to other polymer layer materials and applications. For example, 0.1 to 30%. The esterification ratio refers to a ratio at which the carboxyl group of the main chain polyacrylic acid is esterified. By setting the esterification rate within the above range, the content of the fluoroalcohol ester group having water repellency becomes appropriate, and the adhesion of contaminants to the protective layer is effectively suppressed, while the electrode portion and the organic polymer layer are formed. Good adhesion to the like is maintained.

In the present invention, the fluoroalcohol constituting the fluoroalcohol ester is preferably a primary alcohol. This is because adhesion of contaminants to the protective layer is effectively suppressed, and high chemical resistance to acids, alkalis, and various organic solvents can be obtained. For example, 1H, 1H-perfluorooctyl polymethacrylate or 1H, 1H, 2H, 2H-perfluorodecyl polyacrylate can be preferably used.

The protective layer may be composed of a fluoro alcohol ester of the polycarboxylic acid (A) and an alkyl alcohol ester of the polycarboxylic acid (B), or a protective layer having an alkyl alcohol ester group and a fluoro alcohol ester group. When the composition is mainly composed of a carboxylic acid ester compound, a reference electrode having good high-temperature stability can be obtained.

Preferred forms of the “fluoroalcohol ester” are as described above.

The alkyl alcohol in the alkyl alcohol ester moiety is a chain or cyclic alcohol represented by C _n H _{n + 2} OH (n is a natural number). n is an integer of 1 or more, preferably 2 to 10, more preferably 4
-8, most preferably 6. For example, a hexyl group, a cyclohexyl group and the like are preferably used. In this case, the stability when the reference electrode is left at a high temperature is further improved.

When the protective layer has a structure comprising a fluoroalcohol ester of the polycarboxylic acid (A) and an alkyl alcohol ester of the polycarboxylic acid (B), the fluorolayer of the polycarboxylic acid (A) with respect to the protective layer is formed. The content of the alcohol ester is preferably 50 to 99% by weight, more preferably 75 to 99% by weight, and most preferably 80 to 95% by weight. If the content is too low, the durability of the protective layer may decrease. On the other hand, if the content is too high, sufficient stability when left at high temperatures may not be obtained. On the other hand, the content of the alkyl alcohol ester of the polycarboxylic acid (B) in the protective layer is preferably 1
-50% by weight, more preferably 1-25% by weight, most preferably 5-20% by weight. If the content is too low, sufficient stability when left at high temperatures may not be obtained. If the content is too high, the durability of the protective layer may decrease. The alkyl alcohol ester of the polycarboxylic acid (B) is obtained by esterifying at least a part of the polycarboxylic acid (B) with the above-mentioned alkyl alcohol, and preferred examples include polyhexyl methacrylate. .

When the protective layer is composed of a polycarboxylic acid ester compound having an alkyl alcohol ester group and a fluoro alcohol ester group, the preferred forms of the respective ester groups are as described above, and various combinations of the ester groups are mentioned above. Can be adopted.
Although the ratio of the alkyl alcohol ester group to the fluoro alcohol ester group is not particularly limited, the value of a / b when the number of the fluoro alcohol ester groups in the ester compound is a and the number of the alkyl alcohol ester groups is b is , Preferably 50/50 to 99/1, more preferably 75/25 to 99/1, most preferably 80/20 to
95/5.

Examples of such polycarboxylic acid ester compounds include, for example, compounds having a repeating unit represented by the following formula (1). If the COO-R group is polyhexyl methacrylate, high-temperature stability is particularly improved. Also, the electrode protection performance is improved.

[0054]

Embedded image (N is an integer of 2 or more, X is an integer of 0 or more, Y is an integer of 1 or more.) As such a compound, a copolymer of 1H, 1H-perfluorooctyl methacrylate and cyclohexyl methacrylate, or , Acrylic acid 1H, 1H, 2H, 2H
-A copolymer of perfluorodecyl and cyclohexyl methacrylate. For example, a compound having a repeating unit of 1H, 1H, 2H, 2H-perfluorodecyl acrylate and cyclohexyl methacrylate, such as the following formula (2), is preferably used.

[0055]

Embedded image (N represents an integer of 2 or more.) The use of the above-described copolymer particularly improves high-temperature stability and also improves other characteristics such as electrode protection performance.

As described above, the protective layer of the reference electrode of the present invention is composed of a polymer having a specific structure, but may be composed of a mixture of two or more polymers having different structures and different molecular weights.

In the reference electrode described above, the thickness of the protective layer is preferably 0.01 to 3 μm, more preferably 0.01 to 1 μm, and most preferably 0.01 to 0.1 μm.
m. With such a thickness, sensitivity and responsiveness can be improved and the cleaning time can be shortened.

In the present invention, the molecular weight of the polymer constituting the protective layer is preferably from 1,000 to 50,000, more preferably from 3,000 to 30,000. The molecular weight here refers to a number average molecular weight, and is referred to as GPC (Gel Permiati).
on Chromatography). If the molecular weight is too large, it is difficult to adjust the solution, and it may be difficult to make the protective layer thinner. If the molecular weight is too small, adhesion of contaminants and the like may not be sufficiently prevented. When the protective layer is configured to include the fluoroalcohol ester of the polycarboxylic acid (A) and the alkyl alcohol ester of the polycarboxylic acid (B), it is desirable that each polymer has a molecular weight within the above range.

As described above, the reference electrode of the present invention is provided with the protective layer containing the fluorine-containing polymer.
This fluorine-containing polymer is characterized in that it has a specific structure as described above, thereby preventing the attachment of contaminants to the reference electrode and increasing the stability of measurement sensitivity during long-term use. ing. As a technique for utilizing a fluorine-containing polymer in a biosensor, a technique of disposing a Nafion membrane, which is an ion-exchange resin, above an immobilized enzyme layer of a working electrode is known (JP-A-8-50112).
The effects of the present invention cannot be obtained with a polymer having such a structure. Nafion is a cation exchange resin and has a structure in which a perfluoropolyalkylene ether side chain having a terminal sulfonic acid group is bonded to a perfluoromethylene main chain (formula (3)).

[0060]

Embedded image However, the Nafion membrane is intended to prevent the permeation of ionic interfering substances that interfere with the electrode reaction, and has almost no function of protecting the reference electrode.

The working electrode included in the biosensor of the present invention is preferably an enzyme electrode. An enzyme electrode refers to an electrode having an immobilized enzyme layer formed on the electrode surface. The immobilized enzyme layer is obtained by immobilizing an enzyme having a catalytic function using an organic polymer as a base material. The immobilized enzyme layer is formed by spin coating, for example, by dripping a solution containing various enzymes, a protein crosslinking agent such as glutaraldehyde, and albumin. Albumin protects various enzymes from the reaction of a cross-linking agent and serves as a substrate for proteins.
Enzymes include lactate oxidase, glucose oxidase, urate oxidase, galactose oxidase, lactose oxidase, sucrose oxidase, ethanol oxidase, methanol oxidase, starch oxidase, amino acid oxidase,
Enzymes that produce hydrogen peroxide or consume oxygen as products of the catalytic reaction, such as monoamine oxidase, cholesterol oxidase, choline oxidase, and pyruvate oxidase.

Here, hydrogen peroxide may be produced by using two or more enzymes simultaneously. For example, creatininase, creatinase, and sarcosine oxidase correspond to this. By using these enzymes, creatinine can be detected. Further, an enzyme and a coenzyme may be used simultaneously. For example, 3-hydroxybutyrate dehydrogenase and nicotinamide adenine dinucleotide (NA
D) corresponds to this. The use of these enzymes allows the detection of 3-hydroxybutyric acid. Further, an enzyme and an electron mediator may be used simultaneously. In this case, the electron mediator reduced by the enzyme is oxidized on the electrode surface, and an oxidation current value obtained at this time is measured. For example, glucose oxidase and potassium ferricyanide correspond to this. The use of these makes it possible to detect glucose.

As described above, the immobilized enzyme layer is not particularly limited as long as it contains at least an enzyme and has a function of converting a substance to be measured to hydrogen peroxide or the like which is an electrode sensitive substance.

FIG. 5 shows an example of a biosensor using the reference electrode of the present invention. In this example, a working electrode 7 is provided on a quartz substrate,
A counter electrode 8 and a reference electrode 9 are provided. The working electrode 7 and the counter electrode 8 are platinum electrodes, and the reference electrode 9 is a silver / silver chloride electrode. The coupling layer 4 and the protective layer 3 are formed on the counter electrode 8 in this order. On the working electrode 7 and the reference electrode 9,
The binding layer 4, the immobilized enzyme layer 10, and the protective layer 3 are formed in this order. The bonding layer 4 contains γ-aminopropyltriethoxysilane as a main component. Immobilized enzyme layer 10
May contain at least an enzyme and have a function of converting a substance to be measured into hydrogen peroxide or the like, which is an electrode sensitive substance. The protective layer 3 has a fluoroalcohol ester layer of methacrylic acid resin as a main component. The working electrode 7, the counter electrode 8, and the reference electrode 9 are each electrically connected to a measurement system.
By adopting such a structure, the service life of the biosensor can be extended and the characteristics can be stabilized. Although the figure shows an example of an amperometric type sensor, it goes without saying that the reference electrode of the present invention can also be applied to an ion sensitive field effect transistor type sensor.

The measuring device of the present invention is preferably provided with a biosensor detachably. This is because the electrode portion of the biosensor is consumed by use, and thus it is desirable to have a structure that can be easily replaced. Here, in addition to the form in which only the biosensor part is detachable, a form in which the wiring for connecting the biosensor to another part and the part including the biosensor are detachable may be used. . For example, in the measuring device having the configuration of FIG. 6, the wiring 54 between the biosensor 50 and the electrochemical measurement circuit unit 51 may be detachable.
The portion composed of the wiring 54 and the electrochemical measurement circuit section 51 may be detachable.

The data processing unit in the measuring device of the present invention
It has a function of calculating a measured value based on an electric signal obtained from a biosensor, and operates, for example, in a form of converting the electric signal into an analog signal and / or a digital signal and calculating a measured value. I do. The data processing unit may be configured to include various means, for example,
(A) clocking means, (b) time setting means for setting a measuring time, and time notifying means for notifying that the time set by the time setting means has come, and (c) operation explanation for explaining a method of operating the measuring instrument. Means, (d) a measured value storing means for storing the calculated measured value, (e) a password registering means for registering a password of a user of the measuring instrument, (f) a memo registering means for registering a memo, (g) Operation notification means for detecting a malfunction of the measuring instrument,
(H) calibration time reporting means for detecting and reporting the calibration time of the biosensor; (i) electrode replacement time reporting means for detecting and reporting the replacement time of the electrode portion of the biosensor;
It may be configured to include a part or all of (j) an abnormal current value notifying means for detecting and notifying an abnormal current value, and (k) a calibrating means for calibrating the biosensor. With such a configuration, the operability is further improved. Here, the processing results obtained by one or more of the above (a) to (k) are reported by, for example, a reporting unit.

The method of manufacturing a reference electrode according to the present invention comprises the steps of forming an electrode on an insulating substrate, and directly or through another layer, at least a fluoroalkylene is added to the fluorine-free vinyl polymer. Applying a liquid containing a polymer to which a pendant group containing a block is bonded, followed by drying to form a protective layer. Regarding preferred embodiments and the like of "a polymer in which a pendant group containing at least a fluoroalkylene block is bonded to a vinyl polymer containing no fluorine", the description of the first to fourth reference electrodes according to the present invention will be given. As described in the section, for example, fluoroalcohol esters of polycarboxylic acids are exemplified, and polymethacrylic acid 1H, 1H-perfluorooctyl, polyacrylic acid 1H, 1H, 2H, 2H-perfluorodecyl, or , Methacrylic acid 1H, 1H-
Copolymers of perfluorooctyl and cyclohexyl methacrylate, copolymers of 1H, 1H, 2H, 2H-perfluorodecyl acrylate and cyclohexyl methacrylate, and the like are included.

Hereinafter, embodiments of the present invention will be further described with reference to the drawings.

(First Embodiment) This embodiment will be described with reference to FIG. The reference electrode of the present embodiment has a structure in which an electrode 2 functioning as a reference electrode is provided on an insulating substrate 1, and a protective layer 3 made of a polyfluoro alcohol ester of methacrylic acid resin is formed thereon. .

As the material of the insulating substrate 1, a material mainly composed of a material having a high insulating property such as ceramics, glass, quartz and plastic can be used. water resistant,
It is preferable that the material is excellent in heat resistance, chemical resistance, and adhesion to electrodes.

The electrode 2 is easily formed on the insulating substrate 1,
Further, it is desirable to use a silver / silver chloride electrode which is easy to handle. As the structure of the silver / silver chloride electrode, because of its good sensitivity and strength, a laminate in which titanium, silver, and silver chloride are provided in this order, or titanium, platinum, silver, and silver chloride are provided in this order. Laminated bodies are preferred. The electrode 2 is formed by a sputtering method, an ion plating method,
It can be formed by a vacuum evaporation method, a chemical vapor deposition method, an electrolytic method, or the like. For example, a silver / silver chloride electrode can be prepared by forming a silver film by a sputtering method, and then immersing the silver film in an aqueous solution containing a chlorine compound having a higher ionization tendency than silver, for example, an iron chloride aqueous solution, and by an electrolytic method.

The fluoroalcohol ester of the methacrylic acid resin constituting the protective layer 3 means that a part or all of the methacrylic acid resin is esterified with a fluoroalcohol, and the fluoroalcohol means all or at least hydrogen in the alcohol. One is replaced by fluorine. For example, polymethacrylic acid 1H, 1H-
Perfluorooctyl or polyacrylic acid 1H, 1H, 2
H, 2H-perfluorodecyl can be used.
In the present invention, for example, polymethacrylic acid 1
H, 1H-perfluorooctyl refers to a polymer in which part or all of methacrylic acid is esterified with 1H, 1H-perfluorooctyl alcohol.

For the protective layer 3, for example, a fluoroalcohol ester solution of a methacrylic acid resin diluted with a perfluorocarbon solvent such as perfluorohexane is dropped on an immobilized enzyme layer 4 on which an enzyme having a catalytic function is immobilized. Can be formed by spin coating. The concentration of the methacrylic acid resin fluoroalcohol ester in the solution is preferably 0.1 to 5% by weight, and more preferably about 0.3% by weight, although it depends on the substance to be measured. When the content is in this range, good film-forming properties can be obtained, and adhesion of contaminants and the like can be effectively prevented.

The method of forming the protective layer 3 is not limited as long as a layer having a uniform thickness can be obtained, and a spray coating method or a dipping method other than the spin coating method can be used.

(Second Embodiment) This embodiment will be described with reference to FIG. In the reference electrode of the present embodiment, an electrode 2 functioning as a reference electrode is provided on an insulating substrate 1, a binding layer 4 made of a γ-aminopropyltriethoxysilane layer, and a polyfluoroalcohol ester layer of methacrylic acid resin are provided thereon. Has a structure in which the protective layer 3 is formed in this order. It is characterized in that a bonding layer 4 mainly composed of a silane coupling agent is provided between the electrode 2 and the protective layer 3.

The protection layer 3 is formed by the same method as in the first embodiment.

The bonding layer 4 has a role of improving the bonding strength between the electrode 2 and the protective layer 3. Further, it also has a function of modifying the surfaces of the insulating substrate 1 and the electrodes 2 to be hydrophilic, and increasing the uniformity of the thickness of the protective layer 3.

The bonding layer 3 mainly comprises a silane coupling agent. Examples of the type of the silane coupling agent include amino silane, vinyl silane, and epoxy silane. Among them, γ-aminopropyl triethoxy silane, which is a kind of amino silane, is preferable from the viewpoint of adhesion. The bonding layer 4 can be formed by, for example, spin-coating a silane coupling agent solution. At this time, the concentration of the silane coupling agent is preferably about 1 v / v% (vol / vol%). With such a concentration, good adhesion can be obtained.

(Third Embodiment) This embodiment will be described with reference to FIG. In the reference electrode of the present embodiment, an electrode 2 functioning as a reference electrode is provided on an insulating substrate 1, and a protective layer 3 mainly composed of polyfluoro alcohol ester of acrylic resin and polyhexyl methacrylate is formed thereon. It has a structure. A bonding layer mainly composed of a silane coupling agent may be provided between the protective layer 3 and the electrode 2 as needed. As the polyfluoro alcohol ester of an acrylic acid resin, for example, polyacrylic acid 1H, 1H, 2H, 2H-perfluorodecyl can be used.

The protective layer 3 is made of, for example, polyacrylic acid 1H, 1H, 2H, xylene hexafluoride as a solvent.
A mixed solution of 2H-perfluorodecyl and polyhexyl methacrylate can be dropped on the electrode 2 and formed by a spin coating method. At this time, the concentration of polyacrylic acid 1H, 1H, 2H, 2H-perfluorodecyl in the mixed solution depends on the substance to be measured.
The content is preferably 5% by weight, more preferably about 1% by weight. Also, the concentration of polycyclohexyl methacrylate is preferably 10% by weight or less, and more preferably about 1% by weight, although it depends on the substance to be measured. By adding polyhexyl methacrylate in this concentration range to 1H, 1H, 2H, 2H-perfluorodecyl polyacrylate, the physicochemical properties of the protective layer are improved. In particular, the strength against impact, scratches, twisting, etc. is improved.

(Fourth Embodiment) This embodiment shows an example of a method for manufacturing a reference electrode according to the present invention.

First, an electrode made of silver / silver chloride is formed on a substrate made of quartz.

Next, the electrode surface and the substrate surface are cleaned.
As a washing method, a method of washing with an organic solvent, an acid, or the like, a method of washing with an ultrasonic cleaner, and the like can be used in combination. As the organic solvent and the acid, those that do not damage the electrode material are used. As the organic solvent, a polar solvent is preferably used, and a ketone-based solvent such as acetone and an alcohol-based solvent such as isopropyl alcohol can be used. Also, as the acid,
Dilute sulfuric acid or the like is used. In addition, electrolytic cathode water can be used. Electrolytic cathode water refers to a liquid generated on the cathode side when pure water or the like is electrolyzed. Electrolytic cathode water has a high reducing property while being neutral to weakly alkaline, so that the potential of both the substrate surface and the surface of the adhered particles can be set to a negative potential while suppressing damage to the substrate and electrodes, and the desorbed particles Can be prevented from re-adhering.
Of the above, for example, a method of sequentially washing with acetone and dilute sulfuric acid is preferably used.

Next, a bonding layer is formed on the surface of the electrode.
As described above, a silane coupling agent such as γ-aminopropyltriethoxysilane is preferably used as a material constituting the bonding layer.

As a method of applying the coupling agent solution or the like,
A spin coating method, a spray method, a dipping method, a heated air flow method, or the like is used. The spin coating method is a method in which a liquid in which a constituent material of a bonding layer such as a coupling agent is dissolved or dispersed is applied by a spin coater. According to this method, a thin coupling layer can be formed with good film thickness controllability. The spray method is a method of spraying a coupling agent liquid or the like toward a substrate, and the dipping method is a method of dipping the substrate in the coupling agent liquid or the like. According to these methods, the bonding layer can be formed by a simple process without requiring a special device. The heated air flow method is a method in which a substrate is placed in a heated atmosphere and a vapor such as a coupling agent liquid flows through the substrate. According to this method as well, a thin coupling layer can be formed with good film thickness controllability.

After the application of the coupling agent solution or the like, drying is performed. The drying temperature is not particularly limited, but is usually room temperature (25
C) to 170C. The drying time is usually 0.5 to 24 hours, depending on the temperature. Drying may be performed in air, but may be performed in an inert gas such as nitrogen. For example, a nitrogen blowing method in which nitrogen is dried while spraying the substrate may be used.

After forming the binding layer, a protective layer is formed by applying a solution of a polycarboxylic acid in a fluoroalcohol ester or the like. As a method of applying the solution, a spin coating method, a dipping method, a spray method, a brush coating method, or the like is used, and among them, a spin coating method having excellent controllability of the film thickness is preferably used. When using the spin coating method, 0.01 to 3 μm
A protective layer made of a thin film having a thickness of about m can be formed with good film thickness controllability. Alternatively, a method may be used in which coating is performed by a dipping method in which the substrate is immersed in the above volume, and then drying is performed while blowing nitrogen gas. According to this method, the protective layer can be formed by a simple method.

After the application of the above solution, drying is performed. The drying temperature is desirably in a range that does not impair the activity of the enzyme in the immobilized enzyme layer, and is preferably in the range of room temperature (25 ° C) to 40 ° C. The drying time is usually 0.5 to 24 hours, depending on the temperature. Drying may be performed in air, but may be performed in an inert gas such as nitrogen. For example, a nitrogen blowing method in which nitrogen is dried while spraying the substrate may be used.

As described above, a reference electrode in which various layers having specific functions are formed on the electrode is manufactured.

(Fifth Embodiment) FIG. 12 shows an example of a biosensor according to the present invention. In this biosensor, a working electrode 7, a counter electrode 8, and a reference electrode 9 are formed on the same insulating substrate 1. On the working electrode 7 and the counter electrode 8, a binding layer 4, an immobilized enzyme layer 10, and a restricted permeation layer 11 are laminated in this order. On the reference electrode 9, a coupling layer 4 and a protective layer 3 are laminated in this order.

FIG. 1 shows an example of another biosensor according to the present invention.
3 is shown. In this biosensor, a working electrode 7 and a counter electrode 8 are formed on the same insulating substrate 1, and a reference electrode 9 is formed on another insulating substrate 1. On the working electrode 7 and the counter electrode 8, a binding layer 4, an immobilized enzyme layer 10, and a restricted permeation layer 11 are laminated in this order. On the reference electrode 9, a coupling layer 4 and a protective layer 3 are laminated in this order.

FIG. 1 shows an example of another biosensor according to the present invention.
It is shown in FIG. In this biosensor, a working electrode 7, a counter electrode 8, and a reference electrode 9 are formed on different insulating substrates 1, respectively. On the working electrode 7 and the counter electrode 8, a binding layer 4, an immobilized enzyme layer 10, and a restricted permeation layer 11 are laminated in this order. On the reference electrode 9, a coupling layer 4 and a protective layer 3 are laminated in this order.

As described above, the working electrode 7, the counter electrode 8, and the reference electrode 9 may be provided on the same substrate or different substrates.

(Sixth Embodiment) This embodiment shows an example of the measuring instrument of the present invention provided with a biosensor, an electrochemical measurement circuit section, a data processing section, and a data notification section. Hereinafter, description will be made with reference to FIGS. 6 and 7.

As shown in FIG. 6, the measuring device includes a biosensor 50, an electrochemical measurement circuit section 51, and a data processing section 5.
2 and the data notification unit 53 are connected by a wiring 54.

As the biosensor 50, for example, one having the reference electrode described in the first to fourth embodiments can be used. Since the biosensor 50 is a consumable item, it is preferable that the biosensor 50 be a detachable type that can be easily replaced.

The electrochemical measurement circuit section 51 uses a potentiostat in this embodiment, but is not particularly limited as long as it can apply a constant potential to the biosensor 50 and measure a current value.

The data processing section 52 has the configuration shown in FIG. 7, and includes a clock section 60, a time setting section 61, a time notifying section 62, an operation explanation section 63, a measured value storage section 64, a password registration section 65, Memo registration means 66, operation notifying means 67, calibration time notifying means 68, electrode replacement time notifying means 6
9, an abnormal current value notifying means 70 and a calibrating means 71 are included. Since the configuration includes the above-described units, the user of the measuring instrument can smoothly perform calibration, measurement, storage of measurement data, and the like of the electrodes. In the present embodiment, a personal computer (hereinafter, referred to as a personal computer) is used as the data processing unit 52. However, any data processing unit such as a microprocessor that can process signals from the electrochemical measurement circuit unit 51 is used. There is no particular limitation. The signal processed by the data processing unit 52 is converted into a measured value, and displayed by the data notifying unit 53 as the measured value.

In the present embodiment, the data notification unit 53 uses a display for a personal computer.
2 is not particularly limited as long as it has a function of notifying the data processed by step 2. The data processed by the data processing unit 52 includes the measurement value calculated by the data processing unit 52, the operation check and malfunction check of the biosensor 50, the abnormal current value detection result, the replacement time of the biosensor 50, the biosensor 50 calibration time and calibration procedure, date, time, clock, signal from the electrochemical measurement circuit unit 51 processed by the calculation unit of the data processing unit 52, operation means at the time of initial setting, and operation advice at the time of performing operation advice It is an operation explanation and the like. The means for informing is digital numerical value, analog numerical value, and voice. Other notification means include sound,
Light, vibration, color, light, graphics, and heat may be used, but digital numerical values and analog numerical values are preferably used.

The wiring 54 may be any electric wire that can connect them.

Next, each means (FIG. 7) included in the data processing section 52 will be described.

In this embodiment, the clock means 60 uses a clock built in a personal computer, but is not particularly limited as long as it has a function of indicating time to the arithmetic unit.

The time setting means 61 is a function for setting the time to be measured using the time measuring means 60. In the present embodiment, a part of the function of a clock built in the personal computer is used similarly to the time measuring means 60, but the arithmetic unit has a function of indicating time and a function of setting a time to be measured further. If so, there is no particular limitation. It is preferable that a plurality of times can be set. This is convenient when it is desired to perform a plurality of measurements a day.
It is more convenient if the use of the time setting means 61 can be selected.

The time notifying means 62 has a function of notifying at the time set by the time setting means 61. For example, if the setting is made by the time setting means 61 to notify every 12 hours, the measurer can know the measurement time from the time notifying means 62 every 12 hours.

The operation explanation means 63 has a function for explaining a method of operating the measuring instrument of the present invention and notes to be taken when performing the operation. Whether or not the operation explanation means 63 is used can be appropriately selected by setting.

The measurement value storage means 64 is a means for storing the measurement values obtained by the measuring instrument and other information, and a RAM (random access memory) is preferably used as a semiconductor storage element. Preferably, the measurement value storage means 64 can store a plurality of measurements. The measured value storage means 64 can store not only measured values but also various information processed in the data processing unit 52. Information to be stored is appropriately limited by setting.

The password registration means 65 has a function of restricting the use of the measuring device other than the specific person and the information of the measured values. By providing this, it is possible to protect the privacy of the user. Becomes The setting of the personal identification number is preferably a numerical value of four or more digits or an alphanumeric character in that high security can be secured. It is desirable that the password registration means 65 can register a plurality of passwords. In this way, the privacy of a plurality of users can be protected. In this embodiment, all information cannot be input / output unless a 4-digit password is entered. The use or non-use of the personal identification number registration means 65 can be appropriately changed by setting.

The memo registration means 66 includes a memo registration means capable of registering a memo, a memo item means for calling a registered memo group, a memo selection means for selecting a memo item to be registered from the called memo group, and a memo selection means. It is preferable to provide a memo calling means for calling the selected memo. In the present embodiment, such a configuration is employed, and for example, the weight, blood pressure, and body temperature at the time of measurement are registered as information of the measurer using the memo registration unit. Note that the memo registration means 6
6 can be changed as needed by setting.

The operation notifying means 67 is provided when the wiring 54 between the biosensor 50, the electrochemical measurement circuit section 51 and the data processing section 52 is disconnected or at least one of them is not connected. Has a function of notifying this. The use or non-use of the operation notifying means 67 can be appropriately changed by setting.

The calibration time notifying means 68 is provided for the biosensor 5.
This is a means for notifying the zero calibration time. When the biosensor is used to a certain extent, calibration is required. The calibration time notifying means 68 has a function of notifying the calibration time. The determination of the calibration time can be made based on the measurement time or the number of measurements. In the present embodiment, both or one of these can be set as a reference by setting.

The electrode replacement time notifying means 69 has a function of notifying the replacement time of the electrodes included in the biosensor 50. The determination of the replacement time can be made based on the measurement time, the number of measurements, the voltage drop of the battery, and the like. In this embodiment,
Depending on the setting, all or any one of them can be used as a reference.

The abnormal current value notifying means 70 causes the abnormal current to flow through the biosensor 50, the electrochemical measurement circuit section 51, the data processing section 52, and the wiring 54 connecting these elements, causing an unmeasurable state. This is a means for notifying that a part has been damaged.

The "notification" in the operation notifying means 67, the calibration time notifying means 68, the electrode replacement time notifying means 69, and the abnormal current value notifying means 70 is performed via, for example, the above-described data notifying section. The predetermined information is transmitted to the user of the measuring instrument.

The calibrating means 71 is used at the time of initial use or calibration, and has a function of explaining the calibration procedure of the biosensor 50 and calibrating the biosensor 50. The explanation of the calibration procedure and the like is performed via the calibration time notification means 68.

When the measuring device of the present embodiment is used, the service life of the biosensor, the calibration time, the operating procedure of the measuring device, and the like are displayed, so that even a person unfamiliar with the handling of the device can easily perform high-accuracy measurement. It can be carried out.

In this embodiment, as shown in FIG. 6, the biosensor 50, the electrochemical measurement circuit section 51, the data processing section 52, and the data notification section 53 are configured to be connected by the wiring 54. A configuration in which the electrochemical measurement circuit section 51 and the data notification section 53 are directly connected without providing the measurement circuit section 51 can also be employed. If you do this,
The analog signal obtained from the biosensor 50 is sent to the data notifying unit 53 as it is, and the measured value is displayed by a display method using a scale and a needle. In this case, it is convenient to attach a table or the like for converting the displayed value into a urine sugar level or a blood sugar level.

(Seventh Embodiment) This embodiment relates to FIG.
This shows an example of a measuring device further provided with a temperature sensor 56 and a pH sensor 57 in the measuring device of FIG. Hereinafter, description will be made with reference to FIG.

As shown in FIG. 8, the measuring device includes a biosensor 50, an electrochemical measurement circuit section 51, a data processing section 52, a data notification section 53, a temperature sensor 56,
The H sensor 57 is connected with the wiring 54.

In the data processing section 52, the temperature sensor 56 and p
The electric signal from the H sensor 57 is processed, and the temperature and pH are processed.
Is calculated. Then, the measured values of the specific components in the measurement sample calculated in the data processing unit 52 are the temperature and the pH.
And is displayed on the data notification unit 53.

The temperature sensor 56 is not particularly limited as long as it can obtain data in a format that can be processed by the data processing section 52, but is preferably a thermocouple thermometer or a resistance thermometer. The temperature sensor 56 measures the temperature of the measurement sample or the temperature of the measurement environment. When measuring the temperature of the measurement sample, the temperature sensor 56 is formed on a substrate provided with a biosensor including a reference electrode, for example. In this way, the temperature of the measurement sample can be measured with high accuracy, and accurate correction can be performed when measuring a specific component in the measurement sample. Alternatively, a configuration may be adopted in which a temperature sensor independent of the biosensor is provided, and the biosensor and the temperature sensor 56 are simultaneously immersed in the measurement sample. With this configuration, it is not necessary to replace the temperature sensor at the same time as the replacement of the biosensor, and the cost can be reduced. When measuring the temperature of the measurement environment, a configuration is provided in which a temperature sensor independent of the biosensor is provided, and the temperature sensor 56 is installed in the measurement environment. The temperature sensor 56 is installed inside the data notification unit 53 and the electrochemical measurement circuit unit 51, for example. This makes it possible to easily check whether the measurement environment is within the measurable temperature condition.

FIG. 15 shows an example of a measuring instrument in which a temperature sensor 56 is formed on a substrate provided with a biosensor. This measuring instrument has a working electrode 7, a counter electrode 8, and a reference electrode 9 formed on an insulating substrate 1.
Is provided. On the working electrode 7, the counter electrode 8, and the temperature sensor 56, the binding layer 4, the immobilized enzyme layer 10, and the restricted permeation layer 11 are formed in this order, and on the reference electrode 9, the binding layer 4, The protection layer 3 is formed. With such a configuration, it is possible to accurately correct the measured value based on the temperature.

As the pH sensor 57, a glass electrode or an ion-sensitive field-effect transistor can be preferably used, but it is not limited to these. For calibration of the pH sensor 57, a solution in which a pH indicator is previously dissolved in a calibration liquid used when configuring the biosensor 50 can be used. By doing so, the biosensor 50
And the calibration of the pH sensor 57 can be performed simultaneously. The pH indicator is preferably an oxalate solution or a phthalate solution used in a usual glass pH meter.

By using the measuring instrument of this embodiment, it becomes possible to accurately measure a specific component in a measurement sample having a wide temperature range and a wide pH range. The temperature of each measured sample,
This is because the measurement value of the biosensor can be corrected using the pH.

Although the temperature sensor 56 and the pH sensor 57 are connected to the data processing section 52 in this embodiment, they may be connected to the electrochemical measurement circuit section 51.

(Eighth Embodiment) This embodiment is different from the embodiment shown in FIG.
With respect to the measuring device, the communication processing unit connected to the data processing unit was further provided, and the data obtained by the data processing unit was transferred to the outside of the measuring device by the communication processing unit. Things. Hereinafter, description will be made with reference to FIG.

As shown in FIG. 9, the measuring instrument of this embodiment has a configuration in which a data processing section 52 and a communication processing section 58 are connected by a wiring 54. The communication processing unit 58 is a means for transmitting information on measured values to the outside. Usually, a modem is used, but it is not limited as long as it has a communication processing function. Communication lines used for transmission include, but are not limited to, telephone lines, infrared, wireless, and the like. Examples of the information transmitted include information processed by the data processing unit 52 and information displayed by the data notification unit 53. For example, the current value of the biosensor 50, password, measurement time, pH, temperature, memo content,
The measured value calculated by the data processing unit 52, the biosensor 5
0 replacement time, biosensor 50 calibration time, biosensor 50 operation check and malfunction check, abnormal current value,
The signal from the electrochemical measurement circuit unit 51 processed by the calculation unit of the data processing unit 52 is transmitted to the outside of the measuring instrument by the communication processing unit 58. The transmission destination is a server or a computer connected through a communication line or the like. The information to be transmitted can be appropriately selected by setting.

By using the measuring instrument of this embodiment, it becomes possible for a diabetic patient to measure his or her own urine sugar at home and transmit the measurement result to a medical institution such as a hospital through a telephone line. As a result, appropriate advice such as diet management and exercise management can be received from a medical institution, and the condition management of a diabetic patient at home becomes possible. Further, since data such as a malfunction of the biosensor can also be transmitted, it is possible to appropriately receive services such as repair and maintenance of the device from a manufacturer, for example.

(Ninth Embodiment) This embodiment relates to FIG.
FIG. 14 shows an example of a measuring device further provided with a printing unit 59 in the measuring device of FIG. Hereinafter, description will be made with reference to FIG.

As shown in FIG. 10, the measuring device of the present embodiment has a configuration in which a data processing section 52 and a printing section 59 are connected by a wiring 54. The printing unit 59 may be a thermal type, a thermal transfer type, a dot impact type, an ink jet type, or a laser beam dry type, but is not particularly limited. Desirably, a low-cost, simple thermal structure is preferred. Printing unit 5
The wiring 54 connected to the printing unit 5 is not limited to an electric wire,
Infrared rays may be used in consideration of a usage pattern when 9 is not used. The printing unit 59 only needs to be able to print the data displayed on the data notification unit 53, but it is also possible to limit the data to be printed by setting.

The use of the measuring instrument of the present embodiment not only makes it possible to print and save data such as measured values on paper, but also allows a diabetic patient to print a measurement result using a printing function. The doctor can bring the completed paper to the hospital and show the result to the doctor, and receive appropriate advice from the doctor who has watched the measurement result.

(Embodiment 10) This embodiment shows an example of a measuring device provided with an external storage unit 55 in addition to the measuring device of FIG. Hereinafter, description will be made with reference to FIG.

As shown in FIG. 11, the measuring instrument of this embodiment has a configuration in which a data processing section 52 and an external storage section 55 are connected by a wiring 54. As the external storage unit 55, a normal storage medium can be used, and a magnetic storage medium such as a floppy disk, a semiconductor storage medium such as a memory card, and an optical storage medium such as an optical disk are preferably used. This is because it can be easily attached and detached and can be obtained at a low price.

Using the measuring instrument of this embodiment, the measured data can be stored in a storage medium, so that the user can bring this storage medium to a hospital as needed. Then, the doctor at the hospital can analyze the stored measurement data and take appropriate medical measures for the diabetic patient or the like. Further, a large amount of measurement data can be stored for a long period of time. Further, since the management is performed by the password, the privacy of the patient can be protected, and one apparatus can be used by a plurality of persons. The contents to be stored can be appropriately selected by setting.

[0134]

The present invention will be described in more detail with reference to the following examples.

Example 1 First, four devices having silver / silver chloride electrodes having an area of 1 mm ² formed on a quartz substrate of 10 mm × 6 mm were manufactured.

Next, different solutions were spin-coated on these silver / silver chloride electrodes to form the following (1) to
The reference electrode of (4) was produced. (1) 22.5 w / v containing 1 v / v% glutaraldehyde
Electrode having an albumin layer formed by spin-coating a 2% albumin solution (2) Reference electrode having an acetylcellulose layer formed by spin-coating an acetylcellulose solution adjusted to 2 w / v% using acetone (3) Perfluoro A reference electrode in which a polyfluoroalcohol ester solution of methacrylic acid resin adjusted to 0.3% by weight using hexane to form a polyfluoroalcohol ester layer of methacrylic acid resin was used. (4) Using xylene hexafluoride 2.0% by weight
Reference electrode prepared by forming a polyfluoroalcohol ester layer of an acrylic resin by spin-coating a polyfluoroalcohol ester solution of an acrylic acid resin adjusted to the conditions of 3000 rpm and 30 rpm, respectively.
Seconds. After application by spin coating, drying is performed,
A protective layer was formed.

Glutaraldehyde, albumin, xylene hexafluoride and acetylcellulose were manufactured by Wako Pure Chemical Industries, Ltd., and acetone was manufactured by Kanto Chemical Co., Ltd. The polyfluoro alcohol ester of acrylic acid resin is polyacrylic acid 1H, 1H, 2H, 2H-
Perfluorodecyl was used. As the fluoroalcohol ester of the methacrylic acid resin, Florad 722 manufactured by Sumitomo 3M Limited was used. Florard 722 is a polymethacrylic acid 1H, 1H-perfluorooctyl,
Average molecular weight (Mn) is about 7,000 (GPC measurement value)
It is. As a diluent, perfluorohexane, Florad 726 manufactured by Sumitomo 3M Limited was used.

These four kinds of reference electrodes were measured by measuring the spontaneous potential in a TES (N-tris (hydroxymethyl) -methyl-2-aminoethanesulfonic acid) buffer solution containing 150 mM sodium chloride and having a pH of 7. The service life was evaluated. The time until the obtained spontaneous potential became unstable was defined as the continuous service life.

As a result, the reference electrode on which the albumin layer was formed had a continuous service life of about 24 hours, and the reference electrode on which the acetylcellulose layer was formed had a continuous service life of about 26 hours. The reference electrode on which the fluoroalcohol ester layer was formed exhibited a service life of about 2000 hours or more, and the reference electrode on which the acrylic resin polyfluoroalcohol ester layer was formed exhibited a service life of about 2400 hours or more.

Example 2 First, four devices having silver / silver chloride electrodes having an area of 1 mm ² formed on a quartz substrate of 10 mm × 6 mm were manufactured.

Next, different solutions were spin-coated on these silver / silver chloride electrodes to form four types of layers, and the following reference electrodes (1) to (4) were produced. (1) 22.5 w / v containing 1 v / v% glutaraldehyde
Electrode having an albumin layer formed by spin-coating a 2% albumin solution (2) Reference electrode having an acetylcellulose layer formed by spin-coating an acetylcellulose solution adjusted to 2 w / v% using acetone (3) Perfluoro A reference electrode in which a polyfluoroalcohol ester solution of methacrylic acid resin adjusted to 0.3% by weight using hexane to form a polyfluoroalcohol ester layer of methacrylic acid resin was used. (4) Using xylene hexafluoride 2.0% by weight
Reference electrode prepared by forming a polyfluoroalcohol ester layer of an acrylic resin by spin-coating a polyfluoroalcohol ester solution of an acrylic acid resin adjusted to the conditions of 3000 rpm and 30 rpm, respectively.
Seconds.

Glutaraldehyde is manufactured by Wako Pure Chemical Industries, Ltd.
The other reagents are the same as in Example 1.

These four kinds of reference electrodes were measured by measuring the spontaneous potential in a TES (N-tris (hydroxymethyl) -methyl-2-aminoethanesulfonic acid) buffer solution containing 150 mM sodium chloride and having a pH of 7. The service life was evaluated. The time until the obtained spontaneous potential became unstable was defined as the continuous service life.

As a result, the reference electrode on which the albumin layer was formed had a continuous service life of about 38 hours, and the reference electrode on which the acetylcellulose layer had been formed had a continuous use life of about 48 hours. The reference electrode on which the fluoroalcohol ester layer was formed exhibited a service life of about 3400 hours or more, and the reference electrode on which the acrylic resin polyfluoroalcohol ester layer was formed exhibited a service life of about 3400 hours or more.

Example 3 After forming a silver / silver chloride electrode having an area of 1 mm ² on a 10 mm × 6 mm quartz substrate, a 1 v / v% γ-aminopropyltriethoxysilane solution was further formed thereon. A polyfluoroalcohol ester solution of methacrylic acid resin adjusted to 0.3% by weight with perfluorohexane was spin-coated thereon to produce a reference electrode on which a polyfluoroalcohol ester layer of methacrylic acid resin was formed. The spin coating conditions were all 3000 rpm for 30 seconds. The reagents used are the same as in Example 1.

The prepared reference electrode was used to measure the current-potential characteristics of hydrogen peroxide in a pH 7 TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid) buffer containing 150 mM sodium chloride. Then, the obtained peak potential was evaluated. The working electrode and the counter electrode were platinum electrodes having an electrode area of 4 mm ² . Also,
Existing glass reference electrode (Toa Denpa Kogyo Co., Ltd.)
(GST-5422S). FIG. 3 shows the obtained current-potential characteristics. 0 to 4 mM in the figure
Indicates the concentration of hydrogen peroxide.

As a result, the peak for hydrogen peroxide of the reference electrode according to the present invention was observed at about 600 mV, and the existing reference electrode showed a peak at about 700 mV. The difference between the two, 100 mV, is calculated from the Nernst equation (−59.16 mV / decade, E _e = E ⁰ + (R
_{T / nF) loga o / a} r). That is, the potential almost coincided with the concentration difference between 150 mM and 3.3 M potassium chloride. In addition, the current-potential characteristics with respect to hydrogen peroxide were in good agreement except for the aforementioned peak shift.

Example 4 Two 10 mm × 6 mm quartz substrates were prepared, and a silver / silver chloride electrode having an area of 1 mm ² was formed on each of them. Then, a 1 v / v% γ-aminopropyltriethoxysilane solution was spin-coated thereon.
The spin coating conditions were 3000 rpm and 30 rpm, respectively.
Seconds.

Next, different solutions were spin-coated on these to produce the following reference electrodes (1) and (2). (1) A reference electrode in which a polyfluoroalcohol ester solution of methacrylic acid resin adjusted to 0.3% by weight using perfluorohexane was spin-coated to form a polyfluoroalcohol ester layer of methacrylic acid resin. (2) Xylene hexane 2.0% by weight using fluoride
Reference electrode prepared by forming a polyfluoroalcohol ester layer of an acrylic resin by spin-coating a polyfluoroalcohol ester solution of an acrylic acid resin adjusted to the conditions of 3000 rpm and 30 rpm, respectively.
Seconds. The reagents used are the same as in Example 1.

For the above two types of reference electrodes, 150 m
The decrease rate of the spontaneous potential with respect to the chloride ion concentration in a TES (N-tris (hydroxymethyl) -methyl-2-aminoethanesulfonic acid) buffer solution containing sodium chloride at pH 7 is expressed by the Nernst equation.
The evaluation was made in comparison with 9.16 mV / decade.

As a result, the spontaneous potential of the reference electrode according to the present invention was -59.4 mV / decade for the polyfluoro alcohol ester of methacrylic acid resin and 50% to 500 mM for chloride ion concentration of 50 to 500 mM. Fluoro alcohol ester reference electrode is -5
It decreased at 9.0 mV / decade, which was in good agreement with the Nernst equation. It was shown that it functioned normally as a reference electrode.

Example 5 Six devices having silver / silver chloride electrodes having an area of 1 mm ² formed on a quartz substrate of 10 mm × 6 mm were manufactured.

Next, on these silver / silver chloride electrodes, 1 v / v
% Γ-aminopropyltriethoxysilane solution was spin-coated to form a γ-aminopropyltriethoxysilane layer, and then the following reference electrodes (1) to (3) were prepared. (1) On the γ-aminopropyltriethoxysilane layer,
A reference electrode in which a protective layer made of polymethacrylic acid 1H, 1H-perfluorooctyl was formed by spin coating a polymethacrylic acid 1H, 1H-perfluorooctyl solution adjusted to 0.3% by weight using perfluorohexane. (2) On the γ-aminopropyltriethoxysilane layer,
Polyacrylic acid 1H, 1H, 2H, 2H-perfluorodecyl solution adjusted to 2% by weight using xylene hexafluoride was spin-coated to obtain polyacrylic acid 1H, 1
A reference electrode on which a protective layer made of H, 2H, 2H-perfluorodecyl is formed. (3) On the γ-aminopropyltriethoxysilane layer,
A mixed solution of polyacrylic acid 1H, 1H, 2H, 2H-perfluorodecyl adjusted to 2% by weight using xylene hexafluoride and polyhexyl methacrylate adjusted to 2% by weight was spin-coated to obtain polymethacryl. Cyclohexyl acid and polyacrylic acid 1H, 1H,
A reference electrode on which a protective layer containing 2H, 2H-perfluorodecyl is formed.

The conditions for spin coating were all 3000 rp.
m for 30 seconds. These three types of reference electrodes were separately prepared at 25 ° C. and 50 ° C. at pH 7 containing 150 mM sodium chloride (N-tris (hydroxymethyl).
Methyl 2-aminoethanesulfonic acid) buffer was used to measure the spontaneous potential to evaluate the continuous service life. The time until the obtained spontaneous potential became unstable was defined as the continuous service life.

As a result, polymethacrylic acid 1H, 1H-
The reference electrode on which the perfluorooctyl layer is formed,
The service life was 2,000 hours at 25 ° C. and 10 hours at 50 ° C. The reference electrode on which the polyacrylic acid 1H, 1H, 2H, 2H-perfluorodecyl layer is formed is 25 ° C.
And a service life of 1,200 hours at 50 ° C. A reference electrode on which a polyacrylic acid 1H, 1H, 2H, 2H-perfluorodecyl layer containing polyhexyl methacrylate is formed at 300C at 25 ° C.
It showed a service life of 0 hours and 2800 hours at 50 ° C.

(Embodiment 6) This embodiment shows an example of a measuring instrument having the configuration shown in FIG.

First, the procedure for manufacturing the biosensor portion of the measuring instrument according to this embodiment will be described. First 10mm x 6
A silver / silver chloride electrode (area 1 mm ² ) was formed on a 1 mm quartz substrate. Subsequently, a 1 v / v% γ-aminopropyltriethoxysilane solution was spin-coated on the entire surface to form a binding layer. On top of that, polyacrylic acid 1H, 1 adjusted to 2% by weight using xylene hexafluoride.
A mixed solution of H, 2H, 2H-perfluorodecyl and polyhexyl methacrylate adjusted to 2% by weight is spin-coated to obtain 1H, 1H, 2H, 2H-perfluorodecyl polyacrylate containing cyclohexyl polymethacrylate. A reference electrode having a layer was formed. The spin coating conditions were all 3000 rpm for 30 seconds.

Subsequently, a quartz substrate of 10 mm × 6 mm was prepared separately from the substrate on which the reference electrode was formed, and a working electrode (area of 7 mm ² ) made of platinum and a counter electrode (area of 4 mm ² ) were formed thereon.
Was formed, a 1 v / v% γ-aminopropyltriethoxysilane solution was spin-coated on the entire surface to form a binding layer. Thereafter, 22.5 w / w containing 56.5 U / μl glucose oxidase and 1 v / v% glutaraldehyde.
An immobilized enzyme layer was formed by spin-coating a v% albumin solution. Then, a 1.7% by weight polyfluoro alcohol ester solution of an acrylic acid resin was spin-coated thereon to form a restricted permeation layer. The polyfluoro alcohol ester of acrylic acid resin is polyacrylic acid 1H, 1
H, 2H, 2H-perfluorodecyl was used. The diluent used was xylene hexafluoride. The spin coating conditions were 3000 rpm and 30 seconds.

Using the biosensor having the electrodes formed as described above, a measuring instrument having the structure shown in FIG. 6 was manufactured. The electrode portion and the flexible substrate were connected by wire bonding, and the flexible substrate and the electrochemical measurement circuit portion were connected using a pin jack type electric wire.

The electrochemical measurement circuit is a potentiostat HOKUTODENKOPOTENTIOSTAT / GALVANOSTATH manufactured by Hokuto Denko.
A150G was used. As a data processing device, a personal computer PC-9821RaII23 manufactured by NEC Corporation was used. As the data notification unit 53, a display PC-KP531 manufactured by NEC Corporation was used. The electrochemical measurement circuit, the data processing device, and the data notification unit 53 were connected by a pin jack type electric wire.

Next, the operation of the measuring instrument of this embodiment will be described. The user dipped the above biosensor in 1 mM TES (N-tris (hydroxyl) -methyl-2-aminoethanesulfonic acid) buffer solution containing 150 mM sodium chloride at pH 7, and then turned on the device. Was. Then, [Time is set in the data notification section.
Please enter the current time. ] Was displayed. When the user operates the keys based on this display and inputs the current time, the data notification section displays the message "The current time has been input. ] Was displayed. If the time is not entered correctly, select [Set time again. Please enter the current time. ] Is displayed. Thus, the input current time is stored in the data processing unit.

Next, the data notification section displays [Preparing. Please wait for a little while. ] Was displayed. When the current value sent from the enzyme electrode stabilizes, the data notification section displays [Calibrate. Immerse the electrode in the calibration solution. ] Was displayed. Based on this display, the user can
Calibration was performed by immersion in a calibration solution of 200 mg / dl glucose.
Then, the data notification section displays [Calibration completed successfully.
After washing, return to buffer. ] Was displayed. The data processing unit determines whether or not calibration has been performed normally, and the result is displayed on the data notification unit. If the calibration was not performed successfully, the message [Calibration cannot be performed. Wash the electrode and immerse it in the calibration solution again. ] Or [The electrode is broken. Please replace. Is displayed. After the calibration was completed, the user dipped the enzyme electrode in the buffer to prepare for the measurement. If the electrode is not returned to the buffer for more than 10 seconds after the calibration is completed, a warning sound is generated.

Subsequently, the user selected the [start measurement] item displayed on the data notification section. Then, the data notification section displays [Start measurement. Immerse the electrode in urine. ] Was displayed. Based on this display, the user dipped the electrode in urine and started measurement. After 10 seconds from the start of measurement, the data notification section displays [Measurement completed successfully. The urine sugar level is XXmg / dl. ] Was displayed. The data processing unit determines whether or not the measurement has been performed normally, and the result is displayed on the data notification unit. If the calibration was not performed properly, [Measurement is not possible. Wash the electrode and immerse it again in urine. ] Or [The electrode is broken. Please replace. ] Is displayed on the data notification unit.

[0164] After the measurement is completed, the data notification section displays [Wash electrode and return to buffer. ] Was displayed. If the electrode is not returned to the buffer for more than 10 seconds after the end of the measurement, a warning sound is generated. After that, the user selected the item of “measurement end” in the data notification unit to end the measurement.

In the measuring instrument of this embodiment, the time to be measured can be set in advance. At the set time,
When the notification sound is generated, the data notification section displays [Start measurement. Immerse the electrode in urine. Is displayed. The set time can be set arbitrarily, and a plurality of times can be set.

At the time of input to the data processing unit in the measuring instrument of this embodiment, a notification sound is emitted when the input is accepted and when the input is not accepted. The notification light may be emitted instead of the notification sound. If an abnormal current occurs between the biosensor, the electrochemical measurement circuit, the data processing device, and the wires, the abnormal current notification means [An abnormal current was detected. ] Is displayed on the data notification unit. This display can prevent the device from being damaged.

Further, since the biosensor, the electrochemical measurement circuit, and the data processing device are all connected by pin-jack type electric wires, they can be easily attached and detached between them, and can be replaced as necessary. is there.

As described above, by using the measuring instrument of the present embodiment, measurement can be performed at a regular time, and furthermore, it is possible for anyone to easily handle the apparatus without causing an operation error.

(Embodiment 7) This embodiment shows an example of a measuring instrument having the configuration shown in FIG. This measuring device is obtained by adding a pH sensor and a temperature sensor to the measuring device of FIG.

As a temperature sensor and a pH sensor, a thermocouple type temperature sensor and an ion-sensitive field effect transistor type pH sensor were used, respectively. pH sensor, temperature sensor, electrochemical measurement circuit, data processing device,
And the data notification unit were connected by electric wires.

The operation of the measuring instrument according to the present embodiment will be described below. The user first immersed the enzyme electrode in 1 mM TES (N-tris (hydroxyl) -methyl-2-aminoethanesulfonic acid) buffer solution containing 150 mM sodium chloride at pH 7, and then turned on the apparatus. About 1
After a minute, the base current value of the enzyme electrode stabilized. In this state, the enzyme electrode was immersed in a 200 mg / dl glucose standard solution to calibrate the enzyme electrode. Since the glucose standard solution also contains a pH indicator, calibration of the pH sensor can be performed at the same time. The power of the apparatus is not turned off while the enzyme electrode is connected except when the electrode is replaced.

Subsequently, the user selected the item "start measurement" displayed on the data notification section. Then, [Start measurement. Immerse the electrode in urine. ] Was displayed on the data notification unit.

On the basis of this display, the user measured urine glucose of two diabetic patients as subjects continuously one by one.
In addition, the blood pressure and the body temperature of the subject were simultaneously input using the memo registration means at the time of measurement. As a result, 10 seconds after the first patient immersed the enzyme electrode in the urine, the data notification section displayed [The measurement was completed successfully. The urine sugar level is 80mg / dl. Is displayed, and at the same time, the message [Measurement is completed successfully. The urine sugar level is 80mg / dl. ] Was displayed. Then, about 20 seconds later, the second patient immersed the enzyme electrode in urine, and 10 seconds later, the data notification section displayed [The measurement was completed successfully. The urine sugar level is 180mg / dl. Is displayed, and at the same time, the message [Measurement is completed successfully. The urine sugar level is 180mg / dl. ] Was displayed. At this time, the existing clinical test equipment (Hitachi automatic analyzer 7050, glucose dehydrogenase method)
As a result of comparing the measured values with, the measured values almost match,
It showed high correlation.

As described above, by using the measuring instrument of this embodiment, the urine sugar of two persons could be continuously measured. In addition, it was possible for even a person with poor visual acuity to know his / her urine sugar level reliably. Furthermore, the body temperature and blood pressure that have been input using the memo registration means can be called and compared with the urinary sugar level, thereby enabling detailed disease state management. Also,
Since temperature and pH corrections were performed on the obtained measured values, it was possible to perform a measurement with high measurement accuracy comparable to that of an existing clinical test device.

(Embodiment 8) This embodiment shows an example of a measuring instrument having the configuration shown in FIG. This measuring device is obtained by adding a communication processing unit 58 to the measuring device of FIG. The communication processing unit 58 is provided by NEC
The modem terminal adapter PC-IT65S1P manufactured by Co., Ltd. was used. pH sensor, temperature sensor, electrochemical measurement circuit,
Each part of the data processing device, data notification unit, communication processing unit,
All were connected by electric wires.

Using this apparatus, urine sugar of one diabetic patient was twice daily for 30 days (after breakfast and after dinner, respectively).
After the time), the measurement was continuously performed, and the data after the measurement was transmitted to the hospital one by one using a telephone line.

As a result, the patient adhered to the measurement time of urine sugar, and the hospital was able to graph and analyze the urine sugar value sent thereto and manage the patient's condition appropriately.

(Embodiment 9) This embodiment shows an example of a measuring instrument having the configuration shown in FIG. This measuring device is obtained by adding a printing section 59 to the measuring device of FIG. As the printing unit 59, NEC
A laser printer Multiwriter2000X manufactured by Co., Ltd. was used. Data processing unit and printing unit are printer cable PC-CA2
Connected with 02. Hereinafter, a result of measurement using the measuring device of the present embodiment will be described.

Using this apparatus, urine glucose of 100 diabetic patients was measured continuously. Calibration was performed only once when the instrument was started. The same sample was also measured using an existing clinical test device, and the measurement results of the measuring device of this example and the existing clinical test device (Hitachi Automatic Analyzer 7050, glucose dehydrogenase method) were compared. As a result, the correlation coefficient was 0.96, and the regression equation was Y = 1.09X + 8.
It was 8. It has been clarified that the measuring device of the present embodiment can obtain the same measurement accuracy as that of the clinical test device. In addition, the measurement time when the measuring instrument of the present example was used was about 90 seconds per sample, and quick measurement was possible. Further, since the measuring instrument of the present example was provided with the printing section 59, the measurement result was printed quickly and could be confirmed. Patients could also bring a printed version of the measurements to the hospital for advice from a physician.

(Embodiment 10) This embodiment shows an example of a measuring instrument having the configuration shown in FIG. This measuring device is different from the measuring device of FIG.
Has been added. 3.5-inch optical disk unit PC-OD3 manufactured by NEC Corporation as an external storage unit
02R was used. The external storage device and the data processing unit were connected by an electric wire. Hereinafter, the operation of the measuring instrument of the present embodiment will be described.

The user first immerses the enzyme electrode in 1 mM TES (N-tris (hydroxyl) -methyl-2-aminoethanesulfonic acid) buffer solution containing 150 mM sodium chloride at pH 7, and then turns on the power of the apparatus. I put it.
After about one minute, the base current value of the enzyme electrode was stabilized. In this state, immerse the enzyme electrode in 200 mg / dl glucose standard solution,
Calibration of the enzyme electrode was performed.

Next, the user selects the item of "input of the number of persons to be measured" displayed on the data notification unit, and inputs the number of persons to be measured. ] Was displayed. When the user inputs the number of people to be measured based on this display, the data notification section will perform [Measurement of ○ people. (Yes, Y / No, N)]. If [Yes, Y] is selected, [Measurement of ○○ is possible. ] Was displayed. If [No, N], click [Enter the number of people to be measured again]. ] Is displayed,
The above procedure is repeated until [Yes, Y] is selected.

Next, the user selected the item of [PIN] displayed on the data notification unit, and then selected [Registration of PIN]. The data processing unit recognizes that the password input button has been pressed, and then registers the password in the data notification unit. Please enter a 4-digit number. ] Is displayed. When the user operates the keys based on this display and inputs a four-digit number, the data notification section displays "Please enter the same password. ] Was displayed. When the user enters the same number again, [Password accepted. ] Was displayed. The registration of the personal identification number is registered for the measurement person. Thus, the registered personal identification number is stored in the memory in the data processing unit.

Next, the data notification section displays [Start measurement. Enter your PIN and immerse the electrode in urine. ] Was displayed. Based on this display, the user entered the password and then dipped the electrode in urine to start the measurement. Then, the data notification section displays [Measurement completed successfully. The urine sugar level is XXmg / dl. ] Was displayed. If the measurement is not performed normally, [Cannot measure.
Wash the electrode and immerse it again in urine. ] Or [The electrode is broken. Please replace. ] Is displayed. If the correct password is not entered, [Start measurement again. Enter your PIN and immerse the electrode in urine. ] Is displayed. If the passwords do not match three times in a row, all the measurement data up to the previous time are erased and the setting returns to the initial setting.

[0185] After the measurement is completed normally, the data notification section displays [Wash electrode and return to buffer. ] Was displayed.

Next, the user clicks [Memo Registration] in the data notification section.
And select [Register memo? (Yes, Y / No, N)]. When [Y] was selected, the message [Enter your PIN] was displayed. After entering the PIN and entering the memo content, [Register memo? (Yes, Y / No, N)]. Enter [Y], enter a memo, and then select [Register memo again. (Yes, Y / No, N)]. The user entered [Y] and registered the memo. To stop these inputs, enter [N].
Also, when recalling, modifying, or deleting the registered memo, select [Enter the PIN and then specify the memo number. ] Is displayed. When the user inputs the personal identification number based on this display, the user can recall, modify, or delete the memo. If the security code is not entered correctly, [The security code does not match. Please enter your PIN again. ] Is displayed. If the passwords do not match three consecutive times, all the memo contents up to the previous time are erased and the settings are returned to the initial settings.

Next, the result of measurement using the measuring instrument of this embodiment will be described. Urine glucose of two diabetic patients was repeatedly measured twice a day for one week using the measuring instrument of this example. Calibration was performed only once when the instrument was started. The same sample was also measured using an existing clinical test device (Hitachi Automatic Analyzer 7050, glucose dehydrogenase method), and the measurement results of the measurement device of this example and the existing clinical test device were compared. As a result, the correlation coefficient was 0.955 (n = 28). It has been clarified that the measuring device of the present embodiment can obtain the same measurement accuracy as that of the clinical test device. In addition, since the patient name was input using the memo function, the data of the measured value was not confused, and furthermore, since the data was managed by the password, the privacy at the time of the measurement could be protected. In addition, the measured data could be displayed in a bar graph. Further, since the optical disk storing the data is portable, it was possible to manage the data with another personal computer and to analyze the data.

[0188]

As described above, the reference electrode and the biosensor using the same according to the present invention are provided with a protective layer mainly composed of a polymer having a specific structure on the electrode. It works.

The first effect is that the service life and the stability during use are significantly increased. This is because the electrode is covered with the protective layer, and the metal material constituting the electrode is prevented from being eluted into the electrolytic solution. For example, when the electrode is a silver / silver chloride electrode, dissociation of silver chloride and elution of silver ions are suppressed.

The second effect is that contaminants hardly adhere to the surface of the reference electrode, and stable sensitivity can be obtained even when used for a long time. The reason for this is that a protective layer mainly containing a polymer having a specific structure to which contaminants are hardly attached is provided.

A third effect is that the size of the reference electrode and the biosensor can be reduced as compared with the conventional case, and the manufacturing restrictions on these shapes can be reduced as compared with the conventional case. The reason for this is that a potassium chloride solution, a space for storing the solution, an inlet for the solution, a liquid junction, and the like are not required inside the electrode, and the electrode operates only when a certain concentration of chloride ions is present.

A fourth effect is that production can be performed in large quantities at low cost. The reason is that the main manufacturing process consists of producing an electrode by a sputtering method or the like and forming a protective layer or the like by a spin coating method, so that most of the existing semiconductor manufacturing processes can be used. is there.

A fifth effect is that handling is facilitated because the strength of the reference electrode is improved. This is because the protective layer formed on the surface of the reference electrode of the present invention is mainly composed of a polymer having a high strength and a specific structure excellent in adhesion to an electrode or the like.

Further, if the biosensor of the present invention is used as a urine glucose sensor for measuring glucose (urine glucose) in urine, it is impossible for a person having a urine glucose value within a normal range, which was impossible with a conventional sensor. In addition, it is possible to measure urinary glucose of a person who falls under the reserve army of diabetes, and to collect data useful for preventing diabetes.

In the method for producing an enzyme electrode of the present invention, the protective layer is formed by applying and drying a solution containing a polymer component having a specific structure, so that the stability at the time of repeated measurement and the adhesion to an adjacent layer are improved. A protective layer having excellent properties and durability can be formed with good film thickness controllability.

[Brief description of the drawings]

FIG. 1 is a sectional view showing an example of a reference electrode of the present invention.

FIG. 2 is a sectional view showing an example of a reference electrode according to the present invention.

FIG. 3 is a diagram showing characteristics of a reference electrode of the present invention.

FIG. 4 is a front view and a cross-sectional view of a conventional biosensor (urine sugar sensor).

FIG. 5 is a diagram showing a schematic structure of a biosensor (urine sugar sensor) of the present invention.

FIG. 6 is a diagram showing an example of the configuration of a measuring instrument of the present invention.

FIG. 7 is a diagram showing an example of a configuration of a data processing unit included in the measuring device of the present invention.

FIG. 8 is a diagram showing an example of a configuration of a measuring instrument of the present invention.

FIG. 9 is a diagram showing an example of the configuration of a measuring instrument of the present invention.

FIG. 10 is a diagram showing an example of a configuration of a measuring instrument of the present invention.

FIG. 11 is a diagram showing an example of the configuration of a measuring instrument of the present invention.

FIG. 12 is a diagram showing an example of the configuration of the biosensor of the present invention.

FIG. 13 is a diagram showing an example of the configuration of the biosensor of the present invention.

FIG. 14 is a diagram showing an example of the configuration of the biosensor of the present invention.

FIG. 15 is a diagram showing an example of the configuration of the biosensor of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Insulating substrate 2 Electrode 3 Protective layer 4 Bonding layer 8 Counter electrode 9 Reference electrode 10 Immobilized enzyme layer 11 Restriction permeation layer 40 Anode electrode 41 Opening 42B Lead wire 43 Cathode electrode 48A pad 48B pad 49 Glass substrate 50 Biosensor 51 Electrochemical measurement Circuit unit 52 Data processing unit 53 Data notification unit 54 Wiring 55 External storage unit 56 Temperature sensor 57 pH sensor 58 Communication processing unit 60 Clocking unit 61 Time setting unit 62 Time notification unit 63 Operation explanation unit 64 Measured value storage unit 65 Password registration Means 66 Memo registration means 67 Operation notification means 68 Calibration time notification means 69 Electrode replacement time notification means 70 Abnormal current value notification means 71 Calibration means P11 Polyimide layer P12 Polyimide layer K Cathode film M Anode film S Silver layer

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI G01N 27/30 311 G01N 27/30 353J 27/416 353U 27/46 B 336C

Claims (55)

(57) [Claims] An electrode provided on an insulating substrate, and a protective layer formed so as to cover the electrode, wherein the protective layer
A reference electrode mainly comprising a polymer in which a pendant group containing at least a fluoroalkylene block is bonded to a fluorine-free vinyl polymer. 2. The method according to claim 1, wherein the vinyl polymer is a homopolymer or a copolymer of one or more monomers selected from the group consisting of unsaturated hydrocarbons, unsaturated carboxylic acids, and unsaturated alcohols. The reference electrode according to claim 1, wherein 3. The reference electrode according to claim 1, wherein the vinyl polymer is a polycarboxylic acid. 4. The reference electrode according to claim 1, wherein the fluoroalkylene block is bonded to the vinyl polymer via an ester group. 5. The fluorine-containing pendant group represented by x / (x + y), where x is the number of fluorine atoms contained in the pendant group and y is the number of hydrogen atoms contained in the pendant group. The reference electrode according to any one of claims 1 to 4, wherein the ratio is 0.3 to 1. 6. The reference electrode according to claim 1, wherein the pendant group has 3 to 15 carbon atoms. 7. The polymer having a molecular weight of 1,000 to 5,
7. The reference electrode according to claim 1, wherein the reference electrode is 0000. 8. An electrode provided on an insulating substrate, and a protective layer formed to cover the electrode, wherein the protective layer comprises:
A reference electrode mainly comprising a fluoroalcohol ester of polycarboxylic acid (A). 9. An electrode provided on an insulating substrate, and a protective layer formed to cover the electrode, wherein the protective layer comprises:
A reference electrode comprising a fluoroalcohol ester of polycarboxylic acid (A) and an alkyl alcohol ester of polycarboxylic acid (B). 10. The reference electrode according to claim 9, wherein the polycarboxylic acid (B) is polymethacrylic acid, polyacrylic acid, or a copolymer of acrylic acid and methacrylic acid. 11. The alkyl alcohol ester of the polycarboxylic acid (B) is an ester compound in which at least a part of a carboxyl group of the polycarboxylic acid (B) is esterified with an alkyl alcohol having 2 to 10 carbon atoms. The reference electrode according to claim 9, wherein the reference electrode is provided. 12. The reference electrode according to claim 11, wherein the alkyl alcohol ester of the polycarboxylic acid (B) is polycyclohexyl methacrylate. 13. The reference according to claim 8, wherein the polycarboxylic acid (A) is polymethacrylic acid, polyacrylic acid, or a copolymer of acrylic acid and methacrylic acid. electrode. 14. When the number of fluorine atoms in the fluoroalcohol ester group contained in the fluoroalcohol ester of the polycarboxylic acid (A) is x and the number of hydrogen atoms is y, it is represented by x / (x + y). The reference electrode according to any one of claims 8 to 13, wherein the fluorine content of the fluoroalcohol ester group is 0.3 to 1. 15. The fluoroalcohol ester contained in the fluoroalcohol ester of the polycarboxylic acid (A) has a fluoroalcohol ester group having 3 to 1 carbon atoms.
15. The reference electrode according to claim 8, wherein the reference electrode is 5. 16. The reference according to claim 8, wherein the fluoroalcohol constituting the fluoroalcohol ester group contained in the fluoroalcohol ester of the polycarboxylic acid (A) is a primary alcohol. electrode. 17. The fluoroalcohol ester of the polycarboxylic acid (A) is polymethacrylic acid 1H, 1H-
2. The composition according to claim 1, which is perfluorooctyl.
7. The reference electrode according to 6. 18. The fluoroalcohol ester of the polycarboxylic acid (A) is polyacrylic acid 1H, 1H, 2
The reference electrode according to claim 16, wherein the reference electrode is H, 2H-perfluorodecyl. 19. The fluoroalcohol ester of the polycarboxylic acid (A) has a molecular weight of 1,000 to 50,000.
The reference electrode according to any one of claims 8 to 18, wherein 20. An electrode provided on an insulating substrate, and a protective layer formed so as to cover the electrode, wherein the protective layer is a polycarboxylic acid having an alkyl alcohol ester group and a fluoro alcohol ester group. A reference electrode mainly comprising an ester compound. 21. When the number of fluorine atoms in the fluoroalcohol ester group is x and the number of hydrogen atoms is y, x
21. The reference electrode according to claim 20, wherein a fluorine content of the fluoroalcohol ester group represented by / (x + y) is 0.3 to 1. 22. The reference electrode according to claim 20, wherein the fluoroalcohol constituting the fluoroalcohol ester group has 3 to 15 carbon atoms. 23. The reference electrode according to claim 20, wherein the fluoroalcohol constituting the fluoroalcohol ester group is a primary alcohol. 24. The reference electrode according to claim 23, wherein the fluoroalcohol ester group is polymethacrylic acid 1H, 1H-perfluorooctyl. 25. The reference electrode according to claim 23, wherein the fluoroalcohol ester group is a polyacrylic acid 1H, 1H, 2H, 2H-perfluorodecyl group. 26. The reference electrode according to claim 20, wherein the alkyl alcohol ester group has 2 to 10 carbon atoms. 27. The reference electrode according to claim 26, wherein said alkyl alcohol ester group is polyhexyl methacrylate. 28. The reference electrode according to claim 1, wherein the electrode is a silver / silver chloride electrode. 29. The silver / silver chloride electrode comprising titanium, silver,
29. The reference electrode according to claim 28, wherein the reference electrode comprises a laminate in which silver and silver chloride are provided in this order, or a laminate in which titanium, platinum, silver, and silver chloride are provided in this order. 30. The protective layer has a thickness of 0.01 to 3 μm.
30. The reference electrode according to claim 1, wherein m is m. 31. The reference electrode according to claim 1, further comprising a bonding layer mainly composed of a silane coupling agent between the electrode and the protective layer. A biosensor comprising the reference electrode according to any one of claims 1 to 31. 33. The biosensor according to claim 32, which is used for measuring glucose in urine. 34. A measuring device, comprising: the biosensor according to claim 32; and a data reporting unit that reports an electric signal obtained from the biosensor. 35. The biosensor according to claim 32 or 33, an electrochemical measurement circuit unit that obtains an electric signal from the biosensor, a data processing unit that calculates a measurement value based on the electric signal, A measuring device comprising: a data notifying unit for notifying a measured value. 36. The data processing section comprises: (a) clocking means; (b) time setting means for setting a measurement time; and time notifying means for notifying that the time set by the time setting means has come. ) Operation explanation means for explaining the operation method of the measuring instrument; (d) measured value storing means for storing the calculated measured value; (e) password registration means for registering the password of the user of the measuring instrument; (f) Memo registration means for registering memos,
(G) operation notifying means for detecting malfunction of the measuring instrument, (h)
A calibration time notifying means for detecting and notifying a calibration time of the biosensor; (i) an electrode replacement time notifying means for detecting and notifying an exchange time of an electrode portion of the biosensor; and (j) detecting and notifying an abnormal current value. An abnormal current value informing means, and (k) a calibrating means for calibrating the biosensor,
The measuring instrument according to claim 35, wherein the measuring instrument includes part or all. 37. The data notifying unit may further include (a) to (d) in addition to the measurement value calculated by the data processing unit.
37. The measuring device according to claim 36, wherein a processing result obtained by one or more of (k) means is notified. 38. The measuring instrument according to claim 36, wherein a plurality of times can be set by the time setting means. 39. The measuring instrument according to claim 36, wherein said password registration means can register a plurality of passwords. 40. The measuring device according to claim 36, wherein said measured value storage means can store a plurality of measured values. 41. A memo registration means for calling a memo group registered in advance, a memo selection means for selecting a memo item to be registered from the called memo group, and a memo for calling the memo selected by the memo selection means. 41. The measuring device according to claim 36, further comprising a calling unit, wherein a plurality of memos can be registered. 42. The apparatus according to claim 3, further comprising a temperature sensor, wherein the measured value is corrected using the temperature of the measurement sample or the measurement environment measured by the temperature sensor.
42. The measuring device according to any one of 6 to 41. 43. The measurement according to claim 36, further comprising a pH sensor, wherein the measurement value is corrected using a pH value of a measurement sample measured by the pH sensor. vessel. 44. A data processing apparatus further comprising a communication processing unit connected to the data processing unit, wherein the data obtained by the data processing unit is transferred to the outside of the measuring device by the communication processing unit. A measuring device according to any one of claims 36 to 43. 45. The printing apparatus according to claim 36, further comprising a printing unit connected to the data processing unit, wherein the printing unit prints data obtained by the data processing unit.
45. The measuring instrument according to any one of to 44. 46. An external storage unit connected to the data processing unit, wherein the data obtained by the data processing unit is stored by the external storage unit. The measuring device according to any one of the above. 47. The measuring device according to claim 46, wherein the external storage unit uses a removable storage medium. 48. The measuring instrument according to claim 47, wherein the storage medium is a semiconductor storage medium, a magnetic storage medium, or an optical storage medium. 49. A method of notifying the data notifying unit includes a digital numerical value, an analog numerical value, a graphic, a voice, a sound, a vibration, a heat,
49. The measuring device according to claim 34, wherein the measuring device is light. 50. The measuring instrument according to claim 34, wherein said biosensor is detachably provided. 51. forming an electrode on an insulating substrate;
A liquid containing a polymer in which a pendant group containing at least a fluoroalkylene block is bonded to a vinyl polymer containing no fluorine is applied to the electrode directly or via another layer, and then dried to form a protective layer. Forming a reference electrode. 52. The method according to claim 51, wherein after forming the electrode, a bonding layer mainly composed of a silane coupling agent is formed, and then the protective layer is formed. 53. The liquid containing the polymer is applied by a spin coating method.
3. The method for manufacturing a reference electrode according to item 2. 54. The method for manufacturing a reference electrode according to claim 51, wherein after applying the liquid containing the polymer by a dipping method, drying is performed while blowing nitrogen gas. 55. The method for manufacturing a reference electrode according to claim 51, wherein the thickness of the protective layer is 0.01 to 3 μm after drying.