JPH0721479B2 - Enzyme electrode, sensor using the same, quantitative analysis method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention comprises an enzyme immobilized on a conductive substrate and electrically responds to the catalytic activity of the enzyme in the presence of a specific substrate. It relates to an enzyme electrode.

[Conventional technology]

The advantages of current-sensing biosensors that incorporate an enzyme as a biocatalyst have been the
and Turner) “Biotechnology and Genetics English Review (Biotec.Genet.En
g.Rev.), G. Russell edition (ed.G.Russell) ”(1984,
Volume 1, Pages 89-120, Intercept, Newcastle-upon-Tyn
e)) or G. Davis "Biosensors" (1985, Volume 1, 161-178). These techniques are different in signal conversion method, and are roughly classified into (a) a type in which an electrical response caused by oxidation of a product of an enzymatic reaction occurring on the electrode surface is detected, and (b) an electron by a redox reagent. Are transferred from the enzyme to the electrode, which is called a "mediator participation type", and (c) a type which is called a "direct electron transport type" which does not require a mediator as described above. Hereinafter, the reactions of these types (a) to (c) will be outlined in order.

Regarding (a) above, this type of reaction is represented as follows, using a certain oxidase (for example, glucose oxidase, alcohol oxidase, etc.) that produces hydrogen peroxide as the reaction progresses.

Substrate + O ₂ (oxidase) Oxidation product + H ₂ O _{2 In} this method, hydrogen peroxide is oxidized on the surface of the potentiostatic electrode.

H ₂ O ₂ O ₂ ＋ 2H ⁺ ＋ 2e An electric signal is generated along with the transfer of electrons from hydrogen peroxide to the electrode. If the conditions are appropriately selected, the strength of the current generated by the enzymatic reaction is It becomes proportional to the concentration of.

Various devices have been proposed so far for quantifying glucose, but most of them are limited in reproducibility, response speed, and applicable range of glucose concentration. One of the commercial successes to some extent is the use of hydrogen peroxide in systems where glucose is the substrate and glucono-1,5-lactone is the product. Further, a method based on a physicochemical measurement such as a secondary reaction of hydrogen peroxide (for example, colorimetric analysis) or conductance is also known. However, these methods generally have a slow response speed and are very sensitive to the oxygen pressure in the sample. This oxygen pressure sometimes fluctuates greatly, and it is attempted to simplify the analysis. If the oxygen pressure is lowered, the linearity of the current response may be deteriorated, and a value lower than the expected value may occur near the upper limit. The same applies to indirect analysis methods that use substrates other than glucose.

Regarding (b) "Mediator-involved biosensor" In this type of reaction, the enzyme is always kept in the reduced type (high electron density type) by reacting the substrate whose concentration is to be measured with the enzyme. In order to put this type of reaction-based sensor into practical use, it is necessary to achieve electrical connection between an electron source (such as an electron-dense active site in the enzyme molecule) and the electrode body. However, the active site of the enzyme is generally buried in the crevices or folds in the macromolecular structure, and it is impossible or extremely difficult for the electrode body to approach such a site. Therefore, in order to perform reliable and sensitive signal conversion, electrical connection between the two is necessary, but it is difficult to actually achieve this. However, the use of electron carriers, or "mediators," here facilitates electron transfer between the enzyme and the electrode. When the "mediator" is in the oxidized form, it takes in electrons from the enzyme to become the reduced form, and then donates the electrons to the electrode to return itself to the oxidized form.

Recently, some biosensors using a carbon electrode on which glucose oxidase is immobilized have been reported, and the above-mentioned mediator can be applied here. For example, "Biosensors" reported by Jonsson and Gorton, in which the enzyme was covalently immobilized by the cyanuric chloride method and stably used for several months, "Biosensors", (1985, 1st Volume, 355-369
page). However, the N-methylphenazinium ion (phenazine methosulfate) used as a mediator in this sensor is unstable and easily lost by washing, and it is a serious drawback that it must be replaced every time it is used. In addition, although it has been demonstrated that the electrochemical conversion carried out via the mediator in this sensor antagonizes the reduction reaction of the enzyme well, this electrode is susceptible to the oxygen concentration. In addition to this, a biosensor using ferrocene or its derivative as a mediator and having an immobilized glucose oxidase electrode is used.
al.) et al. [Analyt Chem. 56, 667-673, 1984 and EP 0,078,636]. In this case, electron transfer to the electrode via the mediator proceeds as follows.

The details of this electrode reaction mechanism are not clear. In particular, it is not known how the insoluble reduced ferrocene carries the charge to the electrode and maintains the activity of the cyclic compound mediator. However, this conundrum may not be related to ionic ferrocene derivatives.
Furthermore, the response rate in this reaction is much slower than the potential response rate expected from the known reaction rate in the same enzyme reaction, and the life of the electrode is also limited. These are probably due to the limited stability of the enzyme.

The use of mediators for signal conversion has several attendant problems. The first is that the mediator easily leaks from the site containing the biocatalyst, the second is that the oxidized and / or reduced mediators are difficult to diffuse, and the third is that the mediator itself is inherently defective. It is stable.

Regarding "(c)" Direct Electron Transport Biosensor "" An attempt to create a biosensor without a mediator was suggested by Tarasevich in a recent review of biological electrocatalysis. Chemistry (Bioelectrochemistry) "
(Vol. 10, pp. 231-295, 1985).

Such a biosensor can be called a “reagentless type” or a “mediatorless type”. Some examples of mediatorless enzyme electrodes are introduced in the above-mentioned review article by Tarasevic. These electrodes structural units and is similar to methyl viologen NMP ⁺ TCNQ ^- organic conductive polymer is used comprising either or both of (N- methyl phenazinium tetracyano-4-quinodimethane) organic conducting salts such as , Which improve the performance of the electrodes and act as mediators. Many of the methods for converting electrons from redox proteins through such modified electrodes belong to this category.

The problem here is the inherent instability of the organic conductive polymer and the organic conductive salt. For example, the NMP / TCNQ modified electrode used as a biosensor for alcohol detection has a life half-life of about 15 days. This type of electrode is also susceptible to oxygen.

According to the reports so far, most of the examples use carbon substrate electrodes, but there are very few enzyme electrodes that can be said to be mediator-less in the true sense, and the fact is that there are many failures. Johnson and Gorton, supra, suggested in a recent paper on the use of glucose oxidase that the main cause of the problem is the immobilization of the enzyme itself. That is, when an enzyme is immobilized, electron transfer ability is apt to be hindered by various obstacles such as steric hindrance, and a mediator is inevitably necessary.

On the other hand, a rare example in which a highly active oxidase is immobilized on a carbon electrode or a platinum electrode has also been reported. For example, Ianiello et al. "Analyt. Chem." (Vol. 54, 1098-11)
01, 1982) is glucose oxidase and L-
Introducing a mediatorless sensor in which amino acids are covalently bonded on a carbon electrode by the cyanuric chloride method.

However, according to Ianiero and Yacynych, "Analyt. Chem."
(Vol. 53, pp. 2090-2095, 1981), the life half-life of this sensor is as short as 20 to 30 days. Moreover, the effect of oxygen has not been clarified.

A large number of biosensors, particularly glucose sensors, which function based on the above-described principle have been disclosed in the related art, and a typical selection method thereof has been widely accepted. Here, Matsushita Electric Industrial Co., Ltd.
The technology disclosed in the publication will be particularly examined. This publication discloses an indirect glucose electrode. here,
The hydrogen peroxide produced by the oxidation of glucose in the presence of glucose oxidase is oxidized on a platinum electrode,
An oxidation current proportional to the concentration of the sample substrate (glucose in this case) is obtained.

This electrode is composed of a conductive carbon substrate that supports an immobilized enzyme layer such as immobilized glucose oxidase.
The conductive substrate itself is formed of graphite particles containing a fluorocarbon resin as a binder in a proportion of 10% by weight or less, and a platinum thin film having a thickness of 1 μm or less is deposited on the substrate by electrolytic action or vapor deposition. . In the invention disclosed in Japanese Patent Laid-Open No. 56-163447, the inconvenience caused by immobilizing the enzyme directly on the surface of platinum is avoided, the response is as fast as 5 seconds, and the sensitivity is high. An enzyme electrode characterized by being persistent has been introduced. However, in recent experiments, the use of such an enzyme electrode has not led to the above-mentioned various effects.

[Problems to be Solved by the Invention]

Therefore, it is mainly used for glucose biosensors,
There remains a need for reliable, reproducible, fast response and sensitivity, and long-term stability enzyme electrodes.

The present invention has been proposed in view of such circumstances, using a novel carbon substrate as an enzyme electrode, and by immobilizing an enzyme such as glucose oxidase on the electrode in a more effective manner, the responsiveness is improved. The purpose of the present invention is to provide a current detection type sensor with excellent stability.

[Means for Solving the Problems]

The improved enzyme electrodes provided by the present invention do not require the use of mediators unless otherwise desired and also function in the presence of very low concentrations of dissolved oxygen. Further, the current density per cm ^{2 of the} enzyme electrode is several hundred microamperes in a 10 mM glucose solution, which is excellent in current response characteristics.
Since such current response characteristics are superior to conventional current detection biosensors, 0 to 100 when the electrode area is 1 mm ² or less.
It is also suitable for manufacturing microprobe biosensors that can generate a current of nA.

The electrodes can also be constructed with very small amounts of immobilized enzyme. That is, 1-2 without a protective film
Second, even with a protective film, it has a glucose response speed of 10 to 30 seconds, which is faster than any conventional glucose sensor. In addition, this electrode exhibits excellent stability even at room temperature if it is stored in a dipped state.

The response characteristics of this electrode were excellent even after several months of use. The applicable concentration range is wide, and it works at a much lower potential than usual (this electrode is 325 mV as compared to the usual 650 mV), and the background current value at the operating potential is very low.

The enzyme electrode or biosensor according to the present invention is prepared by uniformly mixing with a high-purity platinum group metal in advance in an enzyme electrode capable of electrically responding to the catalytic activity of an enzyme in the presence of a specific substrate, Alternatively, it is formed by molding carbon particles or graphite particles in which the platinum group metal is deposited or adsorbed on the surface of individual particles using a resin binder, and is a substantially non-uniform resin-fixed carbon layer or resin-bonded graphite layer. At least a part of the conductive support is composed of a porous conductive substrate layer having a platinum group metal uniformly dispersed therein, and an enzyme is immobilized or adsorbed on the surface of the porous conductive substrate layer. It consists of Therefore, the enzyme electrode according to the present invention is different from the laminated type uniform platinum-coated carbon electrode disclosed in Japanese Patent Laid-Open No. 56-163447 in the above-mentioned carbon particles or graphite particles bonded by a resin binder. And a platinum group element dispersed very uniformly in the layer. The layer of carbon particles bound by the resin binder is more preferably formed by using carbon particles in which colloidal platinum or palladium is previously deposited or adsorbed before being formed into a substrate. Further, polytetrafluoroethylene is particularly preferable as the resin binder used when the carbon particles having a platinum coating are molded into an electrode substrate in the present invention.

To discuss the structure of the enzyme electrode according to the present invention in a little more detail, first, the electrode is a conductive substrate having at least a layer in which carbon powder having platinum group elements such as platinum and palladium adsorbed on the surface in advance is bound with a resin binder. It is desirable to consist of

As the carbon powder, any one may be used as long as it does not hinder the subsequent immobilization of the enzyme. Those that have a lot on the surface are used. Such a carbon powder is in direct contrast to glassy carbon, which does not adsorb enzymes well. The particle size is 3 to 50 nm, more preferably 5 to 30 nm.

As a method for depositing platinum (or palladium) on the carbon particles, vapor deposition, electrochemical deposition, simple adsorption from a colloidal suspension or the like is preferable, but carbon particles and platinum group metal are simply mixed. May be. In any case, the platinum group metal proportion ranges from 1 to 20% by weight, more preferably from 5 to 15% by weight, based on the weight of carbon.
Is. However, the above range is not absolute, but is determined from a practical point of view. If the proportion of platinum group metal is less than 1% by weight, the signal strength will be reduced and measurement will be difficult unless a device having extremely high sensitivity is used. Also, if the proportion of platinum group metal is more than 20% by weight, the cost is high, but the response speed and sensitivity improvement effect is small. In fact, if the proportion of platinum group metal is extremely large, the sensitivity is rather reduced. . Therefore, to form a platinum group metal coating or a palladium coating on carbon particles,
For example, by oxidative decomposition of compounds such as chloroplatinic acid, or for example, British Patent No. 1,357,494, US Pat.
As disclosed in Nos. 44,193 and 4,166,143, colloidal platinum or palladium can be directly deposited on the surface of carbon particles by using a complex of platinum or palladium having a ligand that is easily oxidized. preferable.

The carbon particles thus coated with platinum or palladium are then molded using a suitable water repellent resin binder. As the water-repellent resin binder at this time, a fluororesin such as polytetrafluoroethylene is particularly preferable. By this, a completely self-supporting porous body mainly composed of the above-mentioned platinum-coated or palladium-coated carbon particles using a resin as a binder is formed, or more generally, a porous material composed of particles using a resin as a binder is formed. The layer is formed on the surface of a conductive substrate such as metal, carbon or graphite. Particularly preferable materials for the substrate for forming the platinum-coated carbon particle layer using a resin as a binder include, for example, U.S. Pat. No. 4,229,49.
Carbon paper disclosed in US Pat. No. 4,293,3
The open-pore type carbon cloth disclosed in No. 96 is mentioned.
In order to maximize the porosity, the amount of the resin used as the binder is set to the minimum necessary for ensuring the structure and stability of the electrode layer. The thickness of the electrode layer at this time is usually 0.1 to 0.5 mm or less, though there are exceptions. From the viewpoint of optimizing structural properties, mechanical strength, and porosity, the amount of resin binder is at least 5 to 10% by weight, and at most 80% by weight, based on the weight of the carbon powder with platinum coating or palladium coating.
It is usually selected in the range of 30-70%. If the amount of the resin binder is less than 5% by weight, the carbon particles and the graphite particles are insufficiently bound to each other. Conversely 80
If it exceeds 5% by weight, the content of carbon particles or graphite particles becomes relatively small and the output signal becomes very weak, so that a recording device having very high sensitivity is required.

As the resin, all kinds of resins including conductive and semi-conductive resins can be used, but synthetic fluororesin, particularly polytetrafluoroethylene is preferable. Here, considering the process of oxidation, this binder needs to be oxygen permeable. For this reason, the above binder must have the minimum oxygen solubility under atmospheric pressure, and the value is at least 2 × 10 ⁻³ cm ^{3 of} oxygen per 1 cm ³ of binder under normal temperature and normal pressure.
And

The binders that can be used and their oxygen solubilities are described in "The Polymer Handbook" [J.
Brand wrap and EH Immagut edition (Ed.J.Brandru
p and EH Immergut) (1st edition, 1967, published by Interscience) (Interscience)]

Solubility × 10 ² (cm ³ ) Polytetrafluoroethylene (PTFE) 0.276 Fluororesin other than PTFE 0.2 or more (variable) Polyethyl methacrylate 8.6 Polystyrene 18.2 (calculated value) Polyvinyl acetate 6.3 Polyvinyl chloride 2.92 Polycarbonate 0.51 Poly ( 4-Methylpentene-1) 24.3 Polyisoprene 10.3 Polychloroprene 7.5 Poly 1,3-butadiene 9.7 Silicone rubber 31.1 The preferred electrode substrate used in the present invention is Prototech (Massachusetts, USA) Prototech (US). There is a series of materials marketed under the trade name of Prototech) and used as electrocatalytic gas diffusion electrodes for fuel cells. The method for preparing these materials is described in the above-mentioned U.S. Pat.Nos. 4,044,193, 4,166,143, and 4,293,396.
, And U.S. Pat.No. 4,478,696,
It would be perfect if these references were available. The outline is that colloidal platinum with a particle size of 15 to 25Å (1.5 to 2.5 nm) is adsorbed on the surface of carbon particles with a particle size of 50 to 300 Å (5 to 30 nm). It acts as a nucleating agent and immediately forms platinum sol on the spot.

Next, the platinum coated carbon particles are molded on a conductive support such as carbon paper using a synthetic resin binder. At this time, it is more preferable that the synthetic resin binder is a fluororesin, particularly polytetrafluoroethylene.

Another technique is to impregnate preformed porous carbon cloth with platinum-coated carbon particles,
In particular, those bonded using polytetrafluoroethylene are disclosed in the aforementioned US Pat. No. 4,293,396.
On the other hand, the material used in the present invention is not limited to Prototech, and includes a similar material in which platinum-coated or palladium-coated carbon particles are molded using a resin binder. In particular, U.S. Pat.
A material such as a fuel cell electrode disclosed in No. 0, that is, carbon paper impregnated with a water repellent resin such as polytetrafluoroethylene is used as a support, and platinum black and carbon particles or carbon particles are formed thereon by screen printing or the like. It is considered that the one in which a catalyst layer formed by mixing a water-repellent resin such as polytetrafluoraethylene as a binder in a homogeneous mixture with graphite particles is also usable.

As a method for immobilizing an enzyme on the surface of a platinum-coated or palladium-coated carbon substrate having a resin binder as described above, a covalent bond method via carbodiimide or carbonyldiimidazole, 1,6-dinitro-3,4 Any conventionally known method such as a covalent bond method using difluorobenzene (DFDNB) and a cross-linking method using glutaraldehyde can be adopted.

As an example, the procedure for immobilizing glucose oxidase is shown below.

A. Carbodiimide treatment 1. Cut an electrode of appropriate size from the Prototech electrode sheet.

2. Immerse the cut electrode in ethanol for about 5 minutes to fully impregnate the binder and backing layer coated with polytetrafluoroethylene.

3. Pull up the electrode from ethanol and wash it completely with distilled water so that ethanol does not remain.

5 ml of a solution of p-methyltoluenesulfonate of 4-cyclohexyl-3- (2-morpholino) carbodiimide dissolved in 0.1M acetate buffer (pH 4.5) at a concentration of 0.15M
(Or less) and soak the electrode in this solution for 90 minutes at room temperature. At this time, you may shake gently using a shaker. When the electrode floats on the surface of the solution and is not soaked well, the above process is repeated from the above operation 2.

5. Pull up the electrode and wash it thoroughly with distilled water. Next, glucose oxidase is newly dissolved in an acetate buffer of pH 5.6 at a rate of 5.0 mg / ml, and the above electrode is immersed in this solution at room temperature for 90 minutes and gently shaken.

6. Pull the electrode out of the enzyme solution and wash it thoroughly with 0.1 M acetate buffer. The electrode is now complete.

7. Store the electrode in 0.1M acetate buffer (pH 5.6) at 4 ℃.

B. Carbonyldiimidazole treatment 1. Perform operation 1 above. The above operations 2 and 3 are omitted.

2. Dissolve N, N'-carbonyldiimidazole in anhydrous dimethylformamide at a rate of 40 mg / ml.

3. Immerse the electrode in this solution at room temperature for 90 minutes. Shake gently if necessary 4. Pull up the electrode and dry off excess carbonyldiimidazole solution. Next, this electrode is immersed in a newly prepared glucose oxidase solution for 90 minutes.

5. Perform steps 6 and 7 above.

C. DFDNB treatment 1. Perform steps 1 to 3 of A above.

2. Thoroughly wash the electrode with 0.1 M sodium borate solution (pH 8.5).

3.1,6-Dinitro-3,4-difluorobenzene is dissolved in methanol at a rate of 0.1021 g / 5 ml. The electrode is immersed in this solution for 10 minutes at room temperature.

4. Pull up the electrode and wash it thoroughly with sodium borate solution. Next, this electrode is immersed in a glucose oxidase solution at room temperature for 90 minutes.

5. Perform steps 6 and 7 above.

In the immobilization step, other bifunctional group-type reagents having various chain lengths can be used. For example, dimidates such as dimethylmalonimidate and dimethylsuberimidate are used.

Further, depending on the type of enzyme, it has been found that simply adsorbing the enzyme on the support of the platinum-coated or palladium-coated carbon particles fixed by a resin binder is effective even without using a crosslinking method or the like. And confirmed using glucose oxidase.

The surface layer of the immobilized enzyme can be physically protected by providing an appropriate porous protective film or protective film such as polycarbonate. Needless to say, however, the above-mentioned protective film / protective film must be capable of permeating an enzyme substrate such as glucose that is quantified. Since the response speed of the sensor becomes slightly slower when these protective films are provided, it cannot be said that there is a disadvantage in that, but the response speed of the enzyme electrode or the biosensor according to the present invention is the same even if such a protective film is provided. The response speed of the enzyme electrode is equal to or higher than that.

As described above, the present invention has the glucose oxidase electrode with glucose oxidase as the immobilized enzyme in mind, but it is of course possible to use other oxidoreductases. However, the use of other oxidoreductases does not always give the same effect as this case. This is not necessarily because the enzymes are ineffective by nature, but because of another factor. For example, when oxalic acid is quantified using oxalate oxidase, the substrate oxalic acid itself is electrochemically oxidized on the electrode substrate and masks most of the enzymatic action.

However, peroxide-forming enzymes such as lactate oxidase, galactose oxidase, cholesterol oxidase are preferred.

A complex enzyme system is also possible, for example a non-oxidase that acts on a first substrate to produce a second substrate for an oxidase and a concentration on the first substrate that acts on the second substrate. A system in combination with an oxidase that generates a proportional current can be considered. Examples of such a combination include a two-enzyme system consisting of β-galactosidase and glucose oxidase (for lactose quantification), a three-enzyme system consisting of β-glucan degrading enzyme, β-glucosidase and glucose oxidase (for quantification of β-glucan). , A three-enzyme system consisting of invertase, mutase and glucose oxidase (for sucrose quantification) and the like.

Further, as another application form of the sensor, first, a reagent that acts on the first substrate by a precursor reaction and uses the product as a substrate of the enzyme electrode according to the present invention (whether an enzyme or not) ) And processes are also possible.

Examples of such precursor reactions are common in the field of immune reactions. It will be easy for those skilled in the art to use the above-mentioned reaction-based enzyme electrode in constructing various sensors including an immunosensor.

However, the enzyme electrode according to the present invention is first applied as a biosensor for detecting or quantifying reducing substrates such as glucose present in clinical samples such as blood, serum, plasma, urine, sweat, tears, and saliva. With that in mind.

The following are possible applications in the non-clinical field.

(A) Fermentation process monitoring (b) Industrial process control (c) Environmental monitoring, ie control of pollution by exhaust gas and effluent (d) Food inspection (e) Veterinary applications, especially similar to the clinical applications mentioned above In various sensors such as biosensors that employ the enzyme electrode material according to the present invention, conventionally known materials can be used for other structural members, electric wires, non-conductive (insulating) supports, probes and the like. , Of course. However, dare to say, the electrode material is generally a thin sheet or wafer made of paper. In the case of a biosensor, an electrode material is usually mounted on an insulating support or a probe so that it can be immersed in a sample. The size of the electrode material in such cases is actually very small,
It rarely exceeds a few square millimeters. The electrical contact with the electrode material can be made by various methods. For example, it is conceivable to bring this electrode material into contact with an electrical contact or terminal made of a suitable conductor such as platinum or silver.

In this case, the electrode material needs to have sufficient thickness and strength to be self-supporting without an insulating support or carrier for holding it, and the electric wire is directly connected to the surface of the electrode material.

As the support, other than carbon paper, a semiconductor surface such as a field effect transistor or a non-conductive surface can be used. When the latter is used, the electrical connection is made directly to the resin binder-containing carbon particle layer or graphite layer having the platinum group element coating.

〔Example〕

The following examples illustrate the preparation and properties of enzyme electrode materials according to the present invention.

Example 1 (prior art as a comparative example) First, an enzyme electrode was prepared according to the prior art. That is, carbon black particles (Vulcan
XC-72) is a commercially available graphitized carbon paper coated with 10% by weight of polytetrafluoroethylene as a binder. A porous carbon paper is used as a conductive substrate, and a platinum thin film (thickness less than 1 μm is formed on the conductive substrate by electrolytic deposition. ) Was formed. Various types of such platinum-coated carbon paper were prepared.

Next, Aspergillus nige
The glucose oxidase derived from r) was immobilized by the above-mentioned carbodiimide treatment and glutaraldehyde crosslinking. That is, the surface of the platinum-coated electrode was first treated with an aqueous solution of glucose oxidase and then dried, and the precipitated glucose oxidase was crosslinked with glutaraldehyde at room temperature (25 ° C).

This electrode material was cut out into a disk shape having a diameter of 2 mn to obtain a test sample.

Example 2 (Glucose Electrode) Glucose oxidase derived from Aspergillus niger was placed on a platinum-coated carbon paper marketed by Prototech (Massachusetts, USA) under the trade name of "Prototech". It was fixed. Here, the platinum-coated carbon particles (Vulcan XC-72) used in the platinum-coated carbon paper are treated with hydrogen peroxide as disclosed in Example 1 of the above-mentioned US Pat. No. 4,044,193. Colloidal platinum (particle size 1.5 to 2.5 nm) is deposited on the surface of carbon particles (nominal particle size 30 nm) by oxidative decomposition of platinum (II) sulfite complex. The carbon particles are further shaped and bonded to the surface of commercially available graphitized carbon paper using about 50% by weight of polytetrafluoroethylene. The final platinum content is 0.24 mg / cm ² (7.5-10% by weight with respect to the carbon particles).

Various kinds of such prototech electrodes were prepared, and glucose oxidase was immobilized thereon by the various methods described above, that is, carbodiimide treatment, carbonyldiimidazole treatment or DNDFB treatment.

In addition to the above-mentioned electrodes, those in which glucose oxidase was immobilized on a prototech electrode by glutaraldehyde crosslinking or simple adsorption were also prepared. In the latter case, glucose oxidase was newly dissolved in acetate buffer (pH 5.6) at a rate of 5.0 mg / ml, and the prototech electrode was immersed in this solution for 90 minutes at room temperature. Even without such a method, the electrode substrate is immersed in an enzyme solution to form an anode,
The enzyme can be easily adsorbed by performing electrophoresis for a minute.

Example 3 A layer of carbon particles coated with platinum in advance was formed on carbon paper by using polytetrafluoroethylene as a binder (US Pat. No. 4,044,193, cited above). The enzyme was immobilized.

Lactate oxidase Galactose oxidase Glucose oxidase / β-galactosidase In order to clarify the advantages of the present invention as compared to the prior art and the properties of the enzyme electrode material, each enzyme electrode prepared in each of the above examples was modified with an improved rank oxygen electrode system [ The current response characteristics were examined by setting the cells in a cell equipped with Rank Brothers (Bottisham, Cambridge). This system consists of the attached drawings and Analytica Kimika Actor (Analytica Ch
imica Acta) (183, pp. 59-66, 1986). In this system, an enzyme electrode (diameter 5 mm) having a carbon paper according to the present invention as a support is adopted in place of the membrane, and the enzyme electrode is further supported by a button type platinum electrode. The counter electrode (platinum foil) was inserted through the cell cover and a silver-silver chloride electrode was used as the reference electrode. Separately from this, a two-electrode system experiment was also conducted in which the counter electrode and the reference electrode were integrally configured as a silver chloride ring surrounding the enzyme electrode. in this case,
A protective film is newly provided, but a stirrer is not provided. Usually, the pH 7.0 buffer solution used in the test is stirred by a magnetic stirrer, and the potential of the working electrode is set to 600 mV with respect to the reference electrode using a potentiostat. However, in the case of a two-electrode system, this potential was 325 mV. Wait long enough for the current background value to decrease, then
The substrate solution was injected via syringe.

The current response was recorded on the chart.

Hereinafter, the experimental results will be described in detail with reference to the drawings. here,
The contents of the drawings are as follows.

FIG. 1 is a characteristic diagram showing the current response characteristics of a glucose oxidase electrode according to the present invention and a glucose oxidase electrode using a conventional carbon electrode material in comparison.

FIG. 2 is a characteristic diagram showing the stability of the glucose oxidase electrode according to the present invention.

FIG. 3 is a characteristic diagram showing the relationship between the current response characteristic and the glucose concentration of the glucose oxidase electrode according to the present invention.

FIG. 4 is a characteristic diagram showing changes in current response characteristics of the glucose oxidase electrode according to the present invention when oxygen pressure is changed.

FIG. 5 is a characteristic diagram showing the stability of the glucose oxidase electrode according to the present invention when stored at room temperature.

FIG. 6 is a characteristic diagram showing the stability of a glucose oxidase electrode according to the prior art as a comparative example when stored at room temperature.

FIG. 7 is a characteristic diagram showing the current response characteristics of the glutaraldehyde cross-linked glucose oxidase electrodes according to the present invention and the prior art in comparison.

FIG. 8 is a characteristic diagram showing the current response characteristics of the carbodiimide-treated glucose oxidase electrodes according to the present invention and the prior art in comparison.

FIG. 9 is a characteristic diagram showing the current response characteristics of the carbodiimide-treated lactate oxidase electrode according to the present invention and the prior art in comparison.

FIG. 10 is a characteristic diagram showing current response characteristics of the galactose oxidase electrode according to the present invention.

FIG. 11 is a characteristic diagram showing current response characteristics of the lactate oxidase electrode according to the present invention.

FIG. 12 shows glucose oxidase / β- according to the present invention.
It is a characteristic view which shows the current response characteristic of a galactosidase composite electrode.

FIG. 13 is a characteristic diagram showing current response characteristics of the glucose oxidase electrode according to the present invention in which polyvinyl acetate is used in place of polytetrafluoroethylene as a binder for platinum-coated carbon particles.

FIG. 14 is a characteristic diagram showing current response characteristics of a glucose oxidase electrode according to the present invention in which glucose oxidase is immobilized on a carbon particle layer with a palladium coating formed on carbon paper using polytetrafluoroethylene as a binder. is there.

FIG. 15 is a schematic sectional view of an improved rank electrochemical cell for measuring the operating characteristics of the enzyme electrode according to the present invention.

FIG. 16 is a schematic cross-sectional view showing a two-electrode system experimental apparatus used in some measurements.

Most of the above data was obtained using the electrochemical cell shown in FIG. This cell is composed of two main parts, a base (1) and an annular jacket (2) that contains a water storage chamber (h) and controls the temperature of the cell by circulating water. It is connected to each other by a dedicated joint ring (3) that is cut. At the center of the base (1), there is a platinum contact (d), on which the enzyme-immobilized carbon paper electrode test disk (a) is placed. This test disc (a) is fixed in place by rubber O-rings (e) and (f) when the two main parts are combined.

Adjustable joint ring (g) from the top of the cell filled with enzyme solution
The stopper (4) with a mark is inserted, and the stopper (4) is inserted.
A counter electrode made of platinum (b) and a silver-silver chloride reference electrode (c) are attached therethrough. As described above, the potential of the working electrode was adjusted to 600 mV at the time of measurement, and the current output was measured with the apparent surface area of the electrode in contact with the substrate solution being 0.14 cm ² . The measurement results are shown as the current density in each of the above characteristic diagrams, that is, the current output per unit area of the electrode (a) in contact with the substrate.

In the configuration shown in FIG. 16, the platinum contact (B) is surrounded by the reference / counter electrode composite electrode (C) through the insulating sleeve (G). The porous polycarbonate membrane mounted on the O-ring is for properly holding the test disc (E) (carbon paper electrode on which enzyme is immobilized) on the platinum contact. The sample chamber (F) is open so that the sample can be dropped on the polycarbonate film. The working electrode potential is set to 325 mV and the current is detected by the potentiostat (A). Working electrode potential 325 mV
The two-electrode system, which is defined as, is superior to the three-electrode system in which the same potential is 600 mV in terms of ease of use and lower background current value. However, no matter which electrode system is selected, there is no significant difference in various characteristics such as storage stability, operation stability, linearity of response, and oxygen concentration dependency of the enzyme electrode according to the present invention.

The results of this experiment will be described in more detail below.

Linearity and time dependence of current response characteristics Fig. 1 shows an example of current response characteristics when glucose is continuously added in a three-electrode system equipped with a stirrer and the final concentration is changed up to 35 mM. It is shown. Electrodes A, B, and C all have glucose oxidase immobilized by the method A described above.
Is a platinum coated activated carbon support according to the present invention, that is, an electrode material (trade name: Prototech) in which platinum coated carbon particles are coated on carbon paper using polytetrafluoroethylene as a binder, and electrode B is a graphite rod. The electrode C is formed by cutting off a commercially available carbon paper having no platinum coating, and the electrode C is formed by cutting the electrode. As seen from this figure, the current response of the electrodes B and C is low level and has little fluctuation as a whole, which is close to the result of the mediator type electrode often seen in the conventional literature. On the other hand, the current response of the electrode A is more reliable and stable, and the response time is about 1 second. (Here, the sharp protrusion at the head of the signal in the early stage of the response has no particular significance because it is caused by the infusion operation of glucose. The plateau has a meaning as a signal depending on the glucose concentration. The current response of these three types of electrodes showed high linearity with respect to glucose concentration as shown in FIG. 2 (however, in FIG. 2, the results of electrode A and electrode C were shown. Only shown). Therefore, for example, when the blood glucose concentration is directly measured, the applicable concentration range can be widened (0 to 30 mM).

The glucose oxidase at this electrode A is
Similar results are obtained when immobilized by the method of 1), suggesting that this method achieves good linearity over a wider concentration range.

As is clear from FIG. 2, the response characteristics of the electrode A remained virtually unchanged after 23 days, but the other electrodes (electrode C
Only shown. The response characteristics of) deteriorated with time. Similar behavior is also observed when the other enzyme immobilization methods described above are adopted, and by any method, when the same activated carbon material as the electrode A is used, an electrode with excellent responsiveness and high stability is obtained. However, a good electrode was not obtained when an inert carbon material was used. The response time of electrode A is
It remained unchanged for 23 days, but the other electrodes had an initial response time of 23
It was ~ 30 seconds, while it increased to 2-3 minutes after 8 days. An active electrode such as the electrode A generally causes a slight decrease in responsiveness on the first day, but is stable thereafter and hardly changes with time. Furthermore, the response characteristic of electrode A is pH
After storage in the buffer of 5.6 at 4 ° C. for 6 months or more, there was almost no change during the first few days. After 6 months, the response characteristics gradually decreased, but after 12 months, the initial value was 70.
% Responsiveness was maintained.

As described above, FIGS. 1 and 2 show the current output of the enzyme electrode in the unit of μA / cm ² when the potential of the working electrode in the three-electrode system as shown in FIG. 15 is 600 mV. .

FIG. 5 shows the results of examining the stability of the above electrodes stored for a longer period of time. That is, the glucose oxidase electrode on which the enzyme was immobilized by carbodiimide treatment was stored at room temperature in an acetate buffer of pH 5.6 for 180 days,
The response characteristics were investigated using 5 mM glucose as a substrate. For comparison, the results of the electrode according to the prior art (Example 1) are shown in FIG. The results of FIG. 5 are obtained from the measurement of the working electrode potential of 600 mV in the three-electrode system, and the results of FIG. 6 are obtained of the measurement of the working electrode potential of 325 mV in the two-electrode system. It is a thing.

Further, FIGS. 7 to 9 show the results of comparison between the prior art (Example 1) and the present invention using various electrodes having different enzymes and different immobilization methods. All of these measurements were performed with a two-electrode system and a working electrode potential of 325 mV.

FIGS. 10 to 12 show the response characteristics when galactose, lactate, and lactose were used as substrates, respectively.
All of these measurements were performed with a three-electrode system and a working electrode potential of 600 mV.

Reuse of Electrode: Possibility of Repeated Measurement The enzyme electrode according to the present invention has a long service life which cannot be achieved by conventional electrodes while the substrate is continuously supplied. This was demonstrated by the following stringent string of tests.

First, the glucose oxidase electrode (Example 2) was set in a closed cell equipped with a stirrer, which was filled with a glucose solution, and the current was detected with an initial concentration of 5 mM and an initial current of 100 μA. When the electrode is continuously used for 18 hours in this state, the signal gradually decreases to 10 μA or less. The integrated value of the detected currents is about 75% of the value theoretically calculated that two electrons are produced from one molecule of glucose.

When the glucose solution was exchanged for the same electrode and the above experiment was immediately repeated, the value of the initial current was restored, and the continuously supplied substrate was similarly reduced.

Moreover, in a continuous experiment, the glucose concentration was maintained at 5 mM by continuously circulating the solution from a large storage tank, and the current generation was continued for another 5.7 days. During 100 hours, the current output gradually decreased and settled down to 45 μA, and this state continued for 40 hours. This is because the enzyme that was not tightly bound dropped from the electrode substrate during this period (however, there was no correlation between the current output and the stirring speed), or other factors worked. Is considered to be.

After performing the above-mentioned long-term test, the current response characteristics of the enzyme electrode in a certain glucose concentration range were measured according to the method described above. Although the signal amplification was lower than that when using a new substrate solution, it showed a very sharp step change in the glucose concentration range of 0 to 30 mM, and this enzyme electrode was long under a constant substrate concentration. It was confirmed that the current response characteristics did not deteriorate even when used for a period of time. This result was confirmed by three experiments in which the enzyme electrode was stored at 4 ° C. for 1 week, after 8 weeks, or for an additional 4.7 days under a constant substrate concentration. The response characteristics did not change with glucose concentration.

From these test results, the enzyme electrode
It can be seen that it can be used for 250 hours (15,000 minutes or more) or has an extremely long life. The working life of the enzyme electrode in the prior art is much shorter than that of the enzyme electrode according to the present invention, often it is only a few hours [Turner "Proceedings Biotech"]. (Vol. 85, 19
1985, (Europe), Online Publications, Pinner, (Online Publications, Pinne
r) UK, pp. 181-192]. For example, the half-life of glucose electrodes using glucose oxidase coupled with ferrocene is generally on the order of 24 hours (Turner, supra), Cass et al (Analytical Chemistry, (Analyt. Chem. ) Volume 56, 667-673
Page, 1984] (supra), presents a stable electrode that can be used for 50 hours. This electrode had a standard deviation of 1% or less even after conducting 50 consecutive measurements on a glucose solution having a concentration of 50 mM.

Applicability to continuous measurement The final response signal level (45 μA for 5 mM glucose solution) confirmed in the above repeated use test did not change even after aeration of the glucose solution, and also after storage for several weeks. There wasn't.

The current output of this electrode was measured using a glucose solution that had been sterilized under conditions that did not cause a decrease in glucose concentration due to bacterial contamination, and as a result, it was confirmed that it could be stably taken out for 12 hours. The signal level at this time was constant over the entire measurement period. This electrode has been "conditioned" in a solution that has been stirred or circulated for several days, which may have been successful. As described above, continuous measurement of glucose is possible by appropriately conditioning or cleaning the electrodes.

Reproducibility within a batch The enzyme electrode according to the present invention can withstand repeated use and exhibits high responsiveness to glucose as long as it is clean and provided with appropriate preparation conditions. When several sets of electrodes of the same size were made by the same method, their performances were very close to each other, and even if the current response characteristics were measured under the same conditions, only a few percent error occurred. Further, all the electrodes thus produced had a very long life and high reliability as compared with the conventional electrodes as described above. The electrodes of the present invention do not change in performance during storage and can be used for weeks, whereas conventional electrodes often have varying performance within a single batch. For example, the Turner mentioned above states that there are only a few glucose oxidase electrodes that have an extremely long life span, with a half-life of 600 hours in a batch, but the majority of the electrodes have a short half-life of 24 hours. There is. Therefore, reliability cannot be guaranteed even if these electrodes are used for significantly longer than 24 hours.

Oxygen concentration dependence of current response performance In order to investigate the effect of dissolved oxygen, the test cell was improved and a new oxygen electrode was provided in addition to the glucose electrode. In a series of experiments, dissolved oxygen was removed from the system by blowing argon. Under such conditions, the above-mentioned electrode A sensitively responds to the addition of glucose, and this response mechanism is almost independent of the dissolved oxygen concentration, and rather the surface structure of the electrode combined with a suitable enzyme immobilization method. It was suggested that this is due to the peculiar property of. Such results have not been reported in the past.

Furthermore, the potential of the electrode A prepared by the above method A is set to 60
An experiment was conducted to set the output signal to 0 mV and measure the output signal while continuously blowing in argon. At this time, the oxygen concentration in the sample was also measured at the same time.

The results are shown in FIG. From this figure, it is clear that the current signal (upper graph) is almost constant and does not substantially depend on the oxygen concentration in the sample (lower graph). In another experiment, it was confirmed that this current signal hardly changed for 10 minutes even when the oxygen concentration rapidly decreased. On the other hand, in the electrode B prepared by the above-mentioned method B, the current signal drops within 5% within 3 minutes,
During this period, the oxygen concentration decreased by 90%. In this case, when oxygen was introduced into the system again, the current response was recovered, albeit relatively slowly. Since these electrodes work only in a limited glucose concentration range when argon is continuously blown in, an extremely small amount of oxygen is required to express a part of the enzyme function of extracting hydrogen from the substrate. Seem. In addition, it cannot be denied that a very small amount of oxygen, which cannot be detected by the enzyme electrode, is adsorbed on the electrode and plays a role.

Enzyme immobilization amount As a result of measuring the glucose reduction rate and the maximum current density, respectively, the amount of enzyme immobilized on the electrode A without deactivation was about 7 μg of active enzyme immobilized per 1 cm ² of the electrode surface area. It turned out to be equal to (There have been few references discussing the immobilized amount of glucose oxidase in biosensors in the past.) With each of the above-mentioned immobilization methods, an electrode with extremely high activity was produced even when the enzyme solution was diluted 10 times or more. I knew I could do it.

Temperature dependence of current response characteristics The glucose concentration range was set to 0 to 30 mM, and the current response characteristics of the electrode A were examined in the temperature range of 10 to 37 ° C. The temperature coefficient was 2-3% per 1 ° C. This corresponds to the activation energy of Arrhenius (about 24 kJ / mol). On the other hand, the temperature coefficient of the biosensor coupled with ferrocene (see the literature by Cass et al., Supra) is reported to be 4% per 1 ° C.

pH dependence A slight pH dependence of current responsiveness was observed, but pH 7.0-8.0
It can be considered that there is substantially no pH dependence in the region (2) except when the glucose concentration is very high (25 mM or more).

Current Response Characteristics of Electrodes with Protective Film In an experimental system with solution stirring, it was found that a polycarbonate film can be used with almost no effect on the signal waveform and size. The response time is approx.
It was 20 seconds.

Application to Whole Blood Sample Analysis An electrode with a polycarbonate overcoat was suitable for direct measurement of blood glucose concentration. The signal interfered by 0.2 mM ascorbic acid was 2.5% when measured on a glucose solution with a concentration of 5 mM.

Application to various analytical biosensors As described above, good results were obtained by applying the enzyme electrode according to the present invention to a rank type cell using an improved Clark electrode. It has been found that very good results can be obtained in the applied form.

As an example, the above electrode was connected to an electric wire and enclosed in a glass tube to prepare a probe having a diameter of 2 mm which is generally used conventionally. Put glucose test solution in a container such as a beaker, stir it, and insert the probe as described above together with the reference electrode and counter electrode into it to perform reliable concentration measurement without removing dissolved oxygen. You can When the measurement was performed using the above-mentioned probe or a probe having the same structure and smaller than this, it was found that the current responsiveness is approximately proportional to the apparent area or weight of the electrode when the glucose concentration is the same.

Further miniaturization (area about 0.25 ~ 0.50mm ² , weight 30 ~ 60μ
The electrode used in g) and the probe used were also prepared. Here, the connection part of the electric wire was covered with a plastic sleeve, and this probe was incorporated into a catheter needle having a diameter of 1.5 mm. This catheter needle can be used as a probe-type sensor that penetrates through a rubber seal and is inserted into a container such as a fermentor containing a test liquid or a waste liquid reservoir to measure the glucose concentration in the liquid. With this configuration, the electrode is protected by the catheter needle during insertion, and the catheter needle can be kept clean.

A current signal of 1 to 10 μA is usually taken out from the small electrode as described above, but it is possible to perform precise measurement in the range of 1 to 100 nA depending on the device devised. Since the enzyme electrode that takes out the signal current in such a range is extremely small (area: about 0.005 mm ² , weight: 1 μg), it should be incorporated into a thin needle-type microprobe used in biometrics. use.

The operating mechanism of the enzyme electrode according to the present invention is not necessarily clear, but some conclusions can be drawn from the experimental results.

So far, the active groups formed on the surface of carbon by performing surface oxidation at high temperature participate in the same crosslinking reaction as in the case of immobilization of the enzyme, and the number and type of such surface groups are platinum (or It is known that white metal elements such as palladium) increase when present as thin-layer surface catalysts [Kinoshita and Stonehart, “Modern Aspects of Electrochemistry”].
Electrochemistry ”(No. 12, Ed. Bockris and Conway, Ed. Bockris and Conway, Plenum Press, New York, pages 183-266, 1)
977]. It is clear that different immobilization methods result in different enzyme binding states. For example, it has been reported so far that various amino acid residues in the enzyme are used for immobilization, but when the material activated by cyanuric chloride and the enzyme are bound to each other, It is known that only the lysine residue of the enzyme is used for immobilization (see Ianiero and Yashinich, “Analytical Chemistry”, Vol. 53, 2090-2095, 1981, supra). Changes in the tertiary structure of the enzyme due to immobilization are also not considered to be the same for all immobilization methods, and such variability is responsible for the large differences in enzyme activity and stability in this type of study. It seems that there is.

The electrode substrate material used in the present invention has an extremely non-uniform property, unlike a layered uniform material as disclosed in Japanese Patent Laid-Open No. 56-163447 mentioned above. There is. This brings diversity to the cross-linking structure and increases the possibility that various orientations appear in the three-dimensional structure. Even if no cross-linking agent is used, surface adsorption is strong. Oxygen can enter into the pores of such a carbonaceous substrate, which serves to increase the surface area of the enzyme and to adopt a conformation that does not affect stability and activity. This is because the enzyme is bound to a relatively flat surface with a much smaller surface area, such as platinum, glassy carbon, or graphite, and as a result, the conventional conformation in which the conformation that the enzyme can take is limited. This is a big difference from the style. Moreover, the responsiveness of the enzyme electrode according to the present invention is extremely fast at 1 to 2 seconds, and not only the enzyme activity is high, but also the electrode itself has a large number of electron accepting sites, so that the electron transfer is performed very quickly. Tells that. This means that the platinum-coated carbon particles are present in high density over a very large area in the microstructure,
This is because the platinum on the surface and the active site of the enzyme can be successfully brought close to each other.

In the enzyme electrode according to the present invention, in order to examine what influence the amount of the resin binder has, whether other resins can be used as the binder, and whether other white metal elements can be applied, Glucose oxidase electrodes with varying amounts of resin binder, glucose oxidase electrodes using polyvinyl acetate as a binder, or glucose oxidase electrodes using palladium as a platinum group element were prepared.

Regarding the amount of resin binder, the present inventors actually
70% by weight (value based on carbon particles or graphite particles that are uniformly mixed with the platinum group metal, or the platinum group metal is deposited or adsorbed on the surface of each particle)
Experiments were conducted in the range of.

When polytetrafluoroethylene was 30% by weight, the current output was 106 μA for a glucose concentration of 4.8 mmol, and it increased to 589 μA at a glucose concentration of 33.3 mmol. On the other hand, when polytetrafluoroethylene was 70% by weight, the current output decreased to 65 μA for a glucose concentration of 4.8 mmol, and even when the glucose concentration was 33.3 mmol, it increased to 330 μA.

There are two possible causes for the decrease in current output when the amount of resin binder is increased. First, as the amount of the hydrophobic binder increases, the hydrophobicity of the electrode itself also increases and the wetting of the test solution decreases. Secondly, since polytetrafluoroethylene is a non-conductor, increasing the amount thereof decreases the conductivity of the electrode and weakens the output signal.

Further, for a glucose oxidase electrode using polyvinyl acetate as a binder, the above-mentioned method A is adopted as a method for immobilizing glucose oxidase, and the platinum-coated carbon paper electrode on which the glucose oxidase is immobilized is made of polytetrafluoroethylene as a binder. The procedure was as described in Example 2 above, except that 50% by weight of polyvinyl acetate was used instead of fluoroethylene.

When the potential of this electrode was set to 325 mV and the measurement was carried out by the improved rank electrode system as well, a substantially linear current response characteristic was achieved as shown in FIG.

Further, regarding a glucose oxidase electrode using palladium as a platinum group element, the above-mentioned method A is adopted as a method for immobilizing glucose oxidase, and the palladium-coated carbon paper electrode on which the glucose oxidase is immobilized is a colloidal palladium carbon. After depositing on the surface of particles (nominal particle size 30 nm, trade name Vulcan XC-72),
It was prepared by forming a thin layer having a thickness of 0.1 mm on conductive carbon paper using 50% by weight of polytetrafluoroethylene as a binder.

A disk with a diameter of 2 mm was cut out from this palladium-coated carbon paper electrode and set on a platinum contact of a two-electrode cell as shown in Fig. 16, and the potential for 325 mV was examined for the response to glucose. The results are shown in FIG. 14, and in this case as well, a nearly linear response characteristic was confirmed with respect to the glucose concentration.

By analogy with the fact that the platinum group elements used in the gas diffusion electrode exhibit behaviors very similar to each other as disclosed in the above-mentioned US Pat. It is believed that other platinum group elements, such as rhodium and rhodium, can substitute for platinum and palladium.

Finally, we will describe examples of application to cholesterol electrodes and sucrose electrodes, which are considered to have great practical significance.

Example 4 (Cholesterol Electrode) The platinum-coated carbon paper manufactured by Prototech Co., which was used in Example 2 above, was mixed with phosphate buffer (pH 7.4) and cholesterol oxidase (EC1.1.3.6. The solution dissolved in the concentration was impregnated at 22 ° C. for 3 hours.

Next, the platinum-coated carbon paper which adsorbed cholesterol oxidase was cut out into a disk with a diameter of 1.5 mm to form an electrode, which was set in a two-electrode system cell as shown in FIG. Was set to a potential of 340 mV, and the responsiveness to a cholesterol standard solution was examined.

The above cholesterol standard solution is a phosphate buffer solution (pH 7.4) containing 5% surfactant (Triton X-100, registered trademark).
Add cholesterol at a rate of 50,100,200 mg / dl to
It was prepared by heating to 70 ° C. after ultrasonic stirring.

The responsiveness of the above electrodes to cholesterol is shown in the table below.

Example 5 (Sucrose Electrode) This sucrose electrode is an enzyme to which a complex enzyme system composed of three kinds of enzymes is applied, and these enzymes convert α-glucose into invertase, which converts α-glucose into α-glucose. A mutase that converts to a mixture of β-glucose and glucose oxidase that acts on β-glucose to produce an output signal proportional to the initial concentration of sucrose.

The platinum coated carbon paper manufactured by Prototech Co. used in Example 2 was impregnated with an enzyme mixed solution prepared by dissolving the following enzymes in a phosphate buffer (pH 7.0) at room temperature for 90 minutes.

Glucose oxidase (EC1.1.3.4.) 6mg / ml Invertase (EC3.2.1.26.) 6mg / ml Mutarotase (EC5.1.3.3.) 2mg / ml Next, with the platinum coating adsorbing these enzymes. Cut out carbon paper in the shape of a disk with a diameter of 5 mm to make an electrode.Set this in a three-electrode cell as shown in Fig. 15 and set the silver-
The response to an aqueous solution containing sucrose was investigated by setting the potential of 400 mV to the silver chloride electrode. The contact area of the electrode with the test solution is 0.16 cm ² .

Further, the same measurement was carried out for an electrode prepared by simultaneously immobilizing these three kinds of enzymes on the platinum-coated carbon paper by carbodiimide treatment or glutaraldehyde crosslinking method.

The response curves of these three types of electrodes to the sucrose solution are shown in FIG.

From FIG. 17, it was found that almost linear response was obtained when any of adsorption, carbodiimide treatment and glutaraldehyde crosslinking was performed.

〔The invention's effect〕

The present invention provides a current detection type sensor having excellent responsiveness and stability by using a novel carbon substrate as an enzyme electrode and immobilizing an enzyme such as glucose oxidase on the electrode by a more effective method. be able to. The improved enzyme electrodes provided by the present invention do not require mediators unless otherwise desired and function in the presence of very low levels of dissolved oxygen. Not only that, the current density per square cm in a glucose solution having a concentration of 10 mM is several hundred microamperes, which is excellent in current response characteristics. Since such current response characteristics are superior to those of the conventional current detection type biosensor, it is also suitable for manufacturing a microprobe type biosensor that exhibits a current output of 0 to 100 nA when the electrode area is 1 mm or less.

The electrodes can also be constructed with very small amounts of immobilized enzyme. That is, 1-2 without a protective film
Second, with a protective film, it has a faster glucose response characteristic than that of any conventional glucose sensor, which is 10 to 30 seconds. In addition, this electrode exhibits excellent stability even at room temperature if it is stored in a dipped state.

The response characteristics of this electrode were excellent even after several months of use. The applicable concentration range is wide, and it works even at a potential much lower than usual (this electrode is 325 mV as compared with the normal 650 mV), and the background current value at the operating potential is very low.

[Brief description of drawings]

FIG. 1 is a characteristic diagram showing the current response characteristics of a glucose oxidase electrode according to the present invention and a glucose oxidase electrode using a conventional carbon electrode material in comparison. FIG. 2 is a characteristic diagram showing the stability of the glucose oxidase electrode according to the present invention. FIG. 3 is a characteristic diagram showing the relationship between the current response characteristic and the glucose concentration of the glucose oxidase electrode according to the present invention. FIG. 4 is a characteristic diagram showing changes in current response characteristics of the glucose oxidase electrode according to the present invention when oxygen pressure is changed. FIG. 5 is a characteristic diagram showing the stability of the glucose oxidase electrode according to the present invention when stored at room temperature. FIG. 6 is a characteristic diagram showing the stability of a glucose oxidase electrode according to the prior art as a comparative example when stored at room temperature. FIG. 7 is a characteristic diagram showing the current response characteristics of the glutaraldehyde cross-linked glucose oxidase electrodes according to the present invention and the prior art in comparison. FIG. 8 is a characteristic diagram showing the current response characteristics of the carbodiimide-treated glucose oxidase electrodes according to the present invention and the prior art in comparison. FIG. 9 is a characteristic diagram showing the current response characteristics of the carbodiimide-treated lactate oxidase electrode according to the present invention and the prior art in comparison. FIG. 10 is a characteristic diagram showing current response characteristics of the galactose oxidase electrode according to the present invention. FIG. 11 is a characteristic diagram showing current response characteristics of the lactate oxidase electrode according to the present invention. FIG. 12 shows glucose oxidase / β- according to the present invention.
It is a characteristic view which shows the current response characteristic of a galactosidase composite electrode. FIG. 13 is a characteristic diagram showing current response characteristics of the glucose oxidase electrode according to the present invention in which polyvinyl acetate is used in place of polytetrafluoroethylene as a binder for platinum-coated carbon particles. FIG. 14 is a characteristic diagram showing current response characteristics of a glucose oxidase electrode according to the present invention in which glucose oxidase is immobilized on a carbon particle layer with a palladium coating formed on carbon paper using polytetrafluoroethylene as a binder. is there. FIG. 15 is a schematic sectional view of an improved rank electrochemical cell for measuring the operating characteristics of the enzyme electrode according to the present invention. FIG. 16 is a schematic cross-sectional view showing a two-electrode system experimental apparatus used in some measurements. FIG. 17 is a characteristic diagram showing current response of the sucrose electrode to which the present invention is applied.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Jeremy Richard Mason, London, London, W3-3, Flyers Place Lane 45, (72) Inventor, Christopher Frank Thurston, London, W5, Runley, London Road 26 (72) Inventor John Ling Stealing London W3, London 3, Twifo Avenyu 78 (72) Inventor David Robert Deceaser United Kingdom, Surrey GU 22, 8Aue, Old Walking, Pound Fee Ludo Court No. 4 (56) Reference JP-A-55-129745 (JP, A) JP-B-58-53745 (JP, B2) JP-B-59-12135 (JP, B2) White matter, nucleic acid, enzyme ", Vol. 30, No. 4 (1985), p. 247 ~ 276

Claims (14)

[Claims] 1. A conductive support comprising a layer of carbon particles or graphite particles bound to a resin binder and containing platinum or a platinum group metal, and an oxidoreductase immobilized or adsorbed on the conductive support, In an enzyme electrode capable of electrically responding to the catalytic activity of an enzyme in the presence of a specific substrate, the conductive support has a platinum group metal on the surface of 1 to 20% by weight based on carbon or graphite. Consisting of a porous layer in which carbon particles or graphite particles having a particle size of 3 to 50 nm pre-adsorbed with are bound by a resin binder,
The platinum group metal is uniformly dispersed in this porous layer, and the resin binder is 2 × 10 ^-3 cm ³ per cm ³ under normal temperature and pressure.
It has the above enzyme solubility and is present in the porous layer at a ratio of 5 to 80% by weight based on the carbon particles or graphite particles. The redox enzyme is a carbon particle or graphite particle having a platinum group metal adsorbed on the surface. An enzyme electrode characterized by being an oxidoreductase that is directly impregnated into a porous layer in contact with a substrate and directly transports electrons to a conductive layer without passing through a mediator or an electron transfer agent. 2. The enzyme electrode according to claim 1, wherein the platinum group metal is platinum or palladium. 3. The enzyme electrode according to claim 1, wherein the resin binder is a fluorocarbon resin or polyvinyl acetate. 4. The enzyme electrode according to claim 3, wherein the resin binder is polytetrafluoroethylene. 5. The enzyme electrode according to claim 1, wherein the redox enzyme is glucose oxidase. 6. A porous layer formed by binding carbon particles or graphite particles, which are preliminarily adsorbed on the surface of platinum group metal particles, with a resin binder is deposited on the surface of a conductive piece made of carbon paper. The enzyme electrode according to any one of claims 1 to 5, which is characterized. 7. The porous layer has a particle size of 5 to 30 nm in which colloidal platinum having a particle size of 1.5 to 2.5 nm is pre-adsorbed on the surface of carbon particles.
7. The enzyme electrode according to any one of claims 1 to 6, further comprising platinum carbon powder composed of the carbon particles described above. 8. The enzyme electrode according to any one of claims 1 to 7, wherein the oxidoreductase is simply adsorbed on the porous layer without having a covalent bond. 9. The enzyme electrode according to claim 1, wherein the surface of the conductive support is protected by a microporous membrane that is permeable to the substrate. 10. The enzyme electrode according to claim 8, wherein the microporous membrane is a polycarbonate membrane. 11. A sensor incorporating the enzyme electrode according to any one of claims 1 to 7. 12. A method for quantitative analysis of a substrate to be analyzed in a sample, wherein the substrate to be analyzed itself is used as it is, or the substrate to be analyzed shows reactivity with an oxidoreductase, which allows measurement in proportion to its concentration. A quantitative analysis method, which comprises contacting with the enzyme electrode according to any one of claims 1 to 9 after converting into a substrate species capable of generating a different electric current. 13. The quantitative analysis method according to claim 12, wherein the sample is a clinical sample. 14. The quantitative analysis method according to claim 13, wherein the sample is a blood sample, the analytical agent is glucose, and the redox enzyme is glucose oxidase.