JPS63307350A - Enzyme electrode - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention consists of an enzyme immobilized on a conductive substrate, which electrically responds to the catalytic activity of the enzyme in the presence of a specific substrate. This relates to enzyme electrodes.

[Conventional technology]

The advantages of current-detecting biosensors incorporating enzymes as biocatalysts have been demonstrated so far by Aston and Turner (Ast.
on a'and Turner) rBiotechnology and Genetics English Review (Biotec, Genet, Eng, Re
v, ), G, Russell & W (ed, 'G, Ru5s
ell) J (1984, Volume 1, pp. 89-120, Intercept, Newcastle-upon
on-Tyne>> or G, Davis
G,) r Biosensors J
(1985, Vol. 1, pp. 161-178). These technologies differ in their signal conversion methods, and can be roughly divided into (a) types in which electrical responses caused by the oxidation of enzymatic reaction products that occur on the electrode surface are detected; and (b) types in which electrical responses are detected by a redox reagent. (c) A type called "mediator-involved type," in which electrons are transported from the enzyme to the electrode, and (c) a type called "direct electron transport type," which does not require a mediator as described above. Below, the outlines of these types (a) to (c) of reactions will be described in order.

This type of reaction is expressed as follows, taking for example certain oxidases (such as glucose oxidase, alcohol oxidase, etc.) that produce hydrogen peroxide as the reaction progresses. It will be done.

Substrate +02-.(Oxidase)-.-) Oxidation product +H,O.

In this method, hydrogen peroxide is oxidized at the surface of a potentiostatic electrode.

Ht Ot −−−−−−−−−−−−−−>
Ot + 2 H" + 'l eAn electrical signal is generated as electrons move from hydrogen peroxide to the electrode, and if conditions are appropriately selected, the strength of the current generated by the enzymatic reaction will be similar to that of the analyte. becomes proportional to the concentration of

Various devices have been proposed so far as devices for quantifying glucose, but most of them have limitations in reproducibility, response speed, and applicable range of glucose concentration. One example that has achieved some success on a commercial basis is the use of hydrogen peroxide as described above in a system using glucose as a substrate and glucono-1,5-lactone as a product. Also known are methods based on secondary reactions of hydrogen peroxide (for example, colorimetric analysis) and physicochemical measurements such as conductance. However, these methods generally have a slow response speed and are highly sensitive to the oxygen pressure in the sample.

This oxygen pressure sometimes fluctuates greatly, and if the oxygen pressure is lowered in an attempt to simplify the analysis, there is a risk that the linearity of the current response will deteriorate and a value lower than the expected value will appear near the upper limit. . The same can be said for indirect analytical methods using substrates other than glucose.

Top *(b) 2 and 2 in the "Mezoie biosensor" In this type of reaction, the enzyme is always kept in a reduced form (high electron density type) by reacting with the substrate whose concentration is to be measured. drooping For a sensor based on this type of reaction to be practical, it is a requirement to achieve an electrical connection between the electron source (such as an electron-dense active site within an enzyme molecule) and the electrode body. However, since the active sites of enzymes are generally buried in crevices and folds within the macromolecular structure, it is impossible or extremely difficult for the electrode body to access these sites. Therefore, although electrical connection between the two is necessary for reliable and sensitive signal conversion, this is difficult to achieve in practice. However, if an electron carrier, ie, a "mediator" is used here, electron transfer between the enzyme and the electrode becomes easy. When this "mediator" is in its oxidized form, it takes electrons from the enzyme to become its reduced form, and then returns to its oxidized form by donating electrons to the electrode.

In recent years, several biosensors using carbon electrodes on which glucose oxidase is immobilized have been reported, and the above-mentioned mediator can be applied here. For example, Jonsson and Gorton reported an example in which enzymes were covalently immobilized using the cyanuric chloride method and used stably for several months in the ``Biosensors'' method.
) J, (1985 1st %, 355-369
However, the N-methylphenazinium ion (phenazine methosulfate) used as a mediator in this sensor is unstable and easily lost during cleaning, and must be replaced every time it is used. This is a serious drawback. Additionally, although it has been demonstrated that the electrochemical conversion performed via the mediator in this sensor is well-competent with the oxygen reduction reaction, this electrode is sensitive to oxygen concentration. A biosensor that uses its derivative as a mediator and has an immobilized glucose oxidase electrode has been developed using Cass
C analytical chemistry (Analyt, Chew, ) reported by et al.
Volume 56.

Pages 667-673, 1984 and European Patent No. 0
．． No. 078.636], in which case the electron transfer to the electrode via the mediator proceeds as follows.

Glucose + Enzyme [oxidized form] −・・−・・・・
...-) Glucono-II 5-lactone decazyme [reduced form] Enzyme [reduced form] + 7 erosene [oxidized form] (ferridinium ion) ......-) Enzyme [oxidized form] ÷ Ferrocene [reduced form] ] Ferrocene [reduced form] ・−・・ (Electrons are transferred to the electrode) , −−−・−・・−・Ciferidinium ion The details of this electrode reaction mechanism are not clear, especially how It is undeniable that the insoluble reduced form of ferrocene carries charge to the electrode and maintains the activity of the cyclic compound mediator, although this conundrum may not be relevant to ionic ferrocene derivatives. Furthermore, the response speed in this reaction is slower than the potential response speed expected from the known reaction speed 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.

Several problems are associated with the use of mediators for signal conversion. The first is that the mediator tends to leak out from the site containing the biocatalyst, the second is that the oxidized, reduced, or both forms of the mediator are difficult to diffuse, and the third is that the mediator itself is inherently inert. It's a stable thing.

Regarding biosensors, Tarasevich (Tarasevich) suggests in his recent review on biological electrocatalysis, "Bioelectrocatalysis". Chemistry (Bioelec
trochemistry) J (Volume 10, 23
1-295, 1985).

Such a biosensor can be called a "reagent-less type" or a "mediator-less type." Several examples of mediator-less enzyme electrodes are introduced in the review by Tarasevi Sochi mentioned above. These electrodes contain structural molecules similar to methyl viologen and NMP"TCNQ" (N
Organic electroconductive polymers containing one or both organic conductive salts, such as -methylzenadinium tetracyano-4-quinodimethane), have been used to improve electrode performance and act as mediators. There is.

Many of the methods for converting electrons from redox proteins via such modified electrodes belong to this category.

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

Looking at the reports to date, most of them use carbon substrate electrodes, but there are only a few enzyme electrodes that can be said to be truly mediatorless, and the reality is that there are many failures. As suggested by Johnson and Gorton, supra, in a recent paper on the use of glucose oxidase, the main cause of the problem lies in the immobilization of the enzyme itself. In other words, when an enzyme is immobilized, its electron transfer ability is likely to be inhibited by various obstacles including steric hindrance, and a mediator is inevitably required.

On the other hand, rare cases have been reported in which highly active oxidases were immobilized on carbon or platinum electrodes. For example, Ianiello et al.
) J (No. 54S, page 10984101, 19
(1982) introduced a mediator-less sensor in which glucose oxidase and L-amino acids are covalently bonded onto a carbon electrode by the cyanuric chloride method.

However, Ianniello and Yacynych
According to “Analytical Chemistry”
t.

Chem,) J (Vol. 53, pp. 2090-2095, 1981), the half-life of this sensor is 20
It is short at ~30 days. Furthermore, the influence of oxygen has not been clarified.

Many biosensors, particularly glucose sensors, which function based on the above-mentioned principle have been disclosed in the past, and representative selection methods thereof are also widely accepted. Here, Matsushita Electric Industrial Co., Ltd.
The technique disclosed in Japanese Patent No. 447 will be particularly considered. This publication discloses an indirect glucose electrode. Here, hydrogen peroxide produced by oxidizing glucose in the presence of glucose oxidase is oxidized on a platinum electrode to obtain an oxidation current proportional to the concentration of the substrate (glucose in this case) in the sample.

This electrode is composed of a conductive carbon substrate supporting a layer of an immobilized enzyme such as immobilized glucose oxidase.

This conductive substrate itself is made of graphite particles containing fluorocarbon resin as a binder at a proportion of 10% by weight or less, and a thin platinum film with a thickness of 1 μm or less is deposited on the substrate by electrolytic action or vapor deposition. . The invention disclosed in Japanese Patent Publication No. 56-163447 mentioned above avoids the inconveniences associated with directly immobilizing enzymes on the surface of platinum, and has a fast response of 5 seconds and a high sensitivity that lasts. An enzyme electrode that is characterized by its unique characteristics is introduced. However, even when such enzyme electrodes were used in recent experiments, the various effects described above could not be obtained.

(Problem to be solved by the invention) Therefore, it is mainly used for glucose biosensors,
There remains a need for enzyme electrodes that are reliable, reproducible, fast response times and sensitive, and have long-term stability.

The uninvented invention was proposed in view of the above circumstances, and uses a new carbon substrate as an enzyme electrode and immobilizes an enzyme such as glucose oxidase on the electrode in a more effective manner to improve responsiveness. .

The purpose of this invention is to provide a current detection type sensor with excellent stability.

[Means to solve the problem]

The improved enzyme electrode provided by the present invention eliminates the need for a mediator unless specifically desired and also functions in the presence of very low concentrations of dissolved oxygen. Further, the current density per enzyme electrode 1 is several hundred microamperes in a 10 mM glucose solution, and the current response characteristics are excellent. This current response characteristic is superior to conventional current detection biosensors, so even if the electrode area is 1 mm or less, O
It is also suitable for manufacturing a microprobe type biosensor that can generate a current of ~100 nA.

The electrodes can also be constructed using very small amounts of Sonodaka fluorine. That is, in the case of no protective film, 1 to 2
It has faster glucose response characteristics than any conventional glucose sensor, with a response time of 10 to 30 seconds even with a protective film. Furthermore, this electrode exhibits excellent stability even at room temperature if stored in a immersed state.

The response characteristics of this electrode were excellent even after several months of use. The applicable concentration range is wide, and even at a much lower potential than usual, ataL (32 mV compared to the normal 650 mV)
5 mV), 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 a highly pure 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. or a substantially non-uniform resin-fixed carbon layer or resin-fixed graphite formed by molding carbon particles or graphite particles in which the platinum group metal is precipitated or adsorbed onto the surface of individual particles using a resin binder. At least a part of the conductive support is constituted by a porous conductive substrate layer in which a platinum group metal is uniformly dispersed, and the enzyme is immobilized or adsorbed on the surface of the porous conductive substrate layer. It is made up of Therefore, the enzyme electrode according to the present invention differs from the Seireki-type homogeneous platinum-coated carbon electrode disclosed in the above-mentioned Japanese Patent Application Publication No. 56-163447, in which carbon particles or graphite are bonded together by a resin binder. It has at least a highly non-uniform layer of particles in which the platinum group element is extremely uniformly dispersed. It is more preferable that the layer of carbon particles bound by the resin binder is formed using carbon particles on which colloidal platinum or palladium has been precipitated or adsorbed before being molded into a substrate. In the present invention, polytetrafluoroethylene is particularly preferred as the resin binder used when forming the platinum-coated carbon particles into an electrode substrate.

Discussing the structure of the enzyme electrode according to the present invention in more detail, first, the electrode is a conductive substrate having at least a layer in which carbon powder, on which a platinum group element such as platinum or palladium has been adsorbed in advance, is bonded with a resin binder. It is desirable that the

Any carbon powder may be used as long as it does not interfere with the subsequent immobilization of the enzyme. Those with a large amount on the surface are used. Such carbon powder is in stark contrast to glass-covered carbon, which cannot adsorb enzymes well. Particle size is 3-50 nm, more preferably 5-30 nm
It is.

Preferred methods for depositing platinum (or palladium) on the carbon particles include vapor deposition, electrochemical deposition, or simple adsorption from a colloidal suspension. It's okay. In each case, the proportion of platinum group metal ranges from 1 to 20% by weight, more preferably from 5 to 15% by weight, based on the weight of carbon.

However, the above range is not absolute, but is determined from a practical standpoint. If the proportion of platinum group metal is less than 1% by weight, the signal intensity will decrease and measurement will be difficult unless a very sensitive device is used. Furthermore, if the proportion of platinum group metal is greater than 20% by weight, the effect of improving response speed and sensitivity will be small in spite of the high cost, and in fact, if the proportion of inner pot group metal is extremely high, the sensitivity will actually decrease. do. Therefore, formation of platinum or palladium coatings on carbon particles can be achieved by oxidative decomposition of compounds such as chloroplatinic acid, or by e.g.
As disclosed in Nos. 044,193 and 4.166, 143, colloidal platinum or palladium is directly applied to carbon particles using a platinum or palladium complex having an easily oxidized ligand. Preferably, it is deposited on the surface.

The carbon particles coated with platinum or palladium in this manner are then molded using a suitable water-repellent resin binder. As the IΩ aqueous resin binder at this time, a fluororesin such as polytetrafluoroethylene is particularly preferable. As a result, a completely self-supporting porous body mainly composed of the above-mentioned platinum-coated or palladium-coated carbon particles is formed using the resin as a binder, or
Alternatively, more generally, a porous layer made of particles with resin as a binder is formed on the surface of a conductive substrate made of metal, carbon, graphite, or the like. Particularly preferable materials for the substrate on which the platinum-coated carbon particle layer is formed using resin as a binder include, for example, U.S. Pat.
Examples include carbon paper as disclosed in US Pat. No. 29,490 and open-pore carbon cloth as disclosed in US Pat.

In order to increase the porosity as much as possible, the amount of resin used as a binder is minimized to ensure the structural properties and stability of the electrode layer. Although there are some exceptions, the thickness of the electrode layer at this time is generally about 0.1 to 0.5 mm or less. Structural Properties 1 From the viewpoint of optimizing mechanical strength and porosity, the amount of the resin binder is at least 5 to 10% by weight, and at most 80% by weight, based on the weight of the platinum-coated or palladium-coated carbon powder. It is usually in the range of 30 to 70%. If the amount of the resin binder is less than 5% by weight, it is insufficient to bind carbon particles and graphite particles to each other. On the other hand, if it exceeds 80% by weight, the content of carbon particles or Graph 141 particles becomes relatively too small and the output signal becomes very weak, requiring a recording device with very high sensitivity.

All kinds of resins can be used, including conductive and semiconductive ones, but synthetic fluororesins, particularly polytetrafluoroethylene, are preferred. Here, in consideration of the oxidation process, it is necessary that this binder is permeable to oxygen i3. For this reason, the above binder must have an oxygen solubility of 1 less under atmospheric pressure, and the value is less per 1 cm' of binder at room temperature and normal pressure (also known as oxygen 2
0-'cm'.

The binders that can be used and their oxygen solubility are listed in The Polymer Handbook.
ndbook) J (J, brand wrap and E,
H, Immergut 1i (Ed, J, Brand
rup and E, Il, fmergut) (1st edition, 1967, published by Interscience (Int
The following is an excerpt from erscience))).

""J"
18.2 (Calculated value) Polyvinyl acetate
6.3 Polyvinyl chloride
2. '92 polycarbonate
0.51 poly (4-methylpentene-1)
24.3 Polyisoprene 1O0
3 Polychloroprene 7.5 Poly 1
．． 3-Butadiene 9.7 Silicone rubber
31.1 Preferred electrode substrates for use in the present invention include those manufactured by Protochik (Massachusetts, USA);
There is a series of materials commercially available under the trade name ototech that are used as electrocatalytic gas diffusion electrodes in fuel cells. Methods for preparing these materials are described in U.S. Pat.
No. 44,193, No. 4.166, No. 143, No. 4.
No. 293.396, and U.S. Pat. No. 4,478.69.
6, and it would be perfect if these references could be obtained.

To give an overview, the particle size is 50 to 300 nm (5 to 30 nm).
) Particle size 15-25 (1,5-2.) on the carbon particle surface.
5 nm) colloidal platinum is adsorbed, and the mode of this adsorption is such that, for example, the carbon particles themselves serve as a nucleating agent and a platinum sol is immediately formed on the spot.

Next, the platinum-coated carbon particles are molded onto 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 a pre-formed porous carbon cloth with platinum-coated carbon particles,
In particular, bonding using polytetrafluoroethylene is disclosed in the aforementioned U.S. Pat. No. 4,293,396. In contrast, the materials used in the present invention are not limited to protodic materials, but also include similar materials in which platinum-coated or palladium-coated carbon particles are molded using a resin binder. In particular, materials such as the electrodes of fuel cells disclosed in U.S. Pat. It is also possible to use a catalyst layer formed by printing or the like in which a homogeneous mixture of platinum black and carbon particles or graphite particles is mixed with a water-based resin such as polytetrafluoroethylene as a binder.

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, covalent bonding via carbodiimide or carbonyldiimidazole, 2L1,6-sinitro3.
Any conventionally known method can be employed, including a covalent bonding method using 4-difluorobenzene (DFDNB), a crosslinking method using glutaraldehyde, and the like.

- As an example, the procedure for immobilization of glucose oxidase is shown below.

A. Force L Revoshii Middle 1. Cut out an electrode of an appropriate size from a prototic 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. Remove the electrode from the ethanol and thoroughly wash it with distilled water so that no ethanol remains.

4. 1-cyclohexyl-3-(2-morpholino)carbodiimide p-methyltoluenesulfonate at 0.
5mj of solution dissolved in 1M acetate buffer (pH 4,5) to a concentration of 0.15M! (or less),
The electrode is immersed in this solution for 90 minutes at room temperature. At this time, you can use a shaker to gently shake.
If the electrode floats to the surface of the solution and is not immersed well, the above process is repeated from step 2 above.

5. Pull up the electrode and clean it thoroughly with distilled water.

Next, glucose oxidase is freshly dissolved at a rate of 5.0 mg/ml in an acetate buffer of pH 5.6, and the above electrode is immersed in this solution at room temperature for 90 minutes, followed by gentle shaking.

6. Remove the electrode from the enzyme solution and wash thoroughly with 0.1M acetate buffer. The electrode is now complete.

7. Preserve the electrode in 0.1 M acetate buffer (pH 5, 6).
) at 4°C.

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

2.N,N'-carbonyldiimidazole is dissolved in anhydrous dimethylformamide at a rate of 40 mg/mn.

3. Immerse the electrode in this solution for 90 minutes at room temperature. Glance gently if necessary.

4. Pull up the electrode and dry and remove excess carbonyldiimidazole solution. The electrode is then immersed in a freshly prepared glucose oxidase solution for 90 minutes.

5. Perform operations 6 and 7 above.

once once ヱdanΣllight 1 (2) Perform operation A or operation 3 above.

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

3. Dissolve 1.6-sinitro 3.4-difluorobenzene in methanol at a ratio of 0.1021 g/5 ml. The electrodes are immersed in this solution for 10 minutes at room temperature.

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

5. Perform operations 6 and 7 above.

Other bifunctional reagents having various chain lengths can be used in this immobilization step, such as cimidates such as dimethylmalonimidate and dimethylsherimidate.

Additionally, depending on the type of enzyme, it has been found that it is effective to simply adsorb the enzyme onto a platinum-coated or palladium-coated carbon particle support that is fixed with a resin binder without using a crosslinking method. This has been confirmed using glucose oxidase.

The surface layer of the immobilized enzyme can usually be physically protected by providing a suitable porous protective film or protective membrane such as polycarbonate. However, it goes without saying that the above-mentioned protective film/protective membrane must be able to permeate the enzyme substrate such as glucose to be quantified.

Providing these protective films slightly slows down the response speed of the sensor, which can be considered a disadvantage, but the response speed of the enzyme electrode or biosensor according to the present invention is lower than that of the conventional technology even with such a protective film. The response speed is equivalent to or faster than that of the enzyme electrode.

As described above, the present invention is directed to a glucose oxidase electrode in which glucose oxidase is an immobilized enzyme, but it is of course possible to use other oxidoreductases (oxidoreductases). However, when using other oxidoreductases, it is not always possible to achieve the same effect as the present case. This is not necessarily because these enzymes are inherently ineffective, but is due to other factors. For example, when oxalate is quantified using oxalate oxidase, the substrate oxalate itself is electrochemically oxidized on the electrode substrate, masking most of the enzymatic action.

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

Complex enzyme systems are also possible, for example a non-oxidase that acts on a first substrate to generate a second substrate for the oxidase, and a non-oxidase that acts on said second substrate to raise the concentration of the first substrate. A system in combination with oxidase that generates a proportional current is considered. Examples of such combinations include a two-enzyme system consisting of β-galactosidase and glucose oxidase (for lactose determination), and a three-enzyme system consisting of β-glucan degrading enzyme, β-glucosidase, and glucose oxidase (for β-glucan determination). , a three-enzyme system (for sucrose determination) consisting of invertase, mutase, and glucose oxidase.

Furthermore, as another application form of the sensor, a reagent (which may or may not be an enzyme) that first acts on the first 51 through a precursor reaction and uses the product as a substrate for the enzyme electrode according to the present invention is proposed. ) and processes are also possible.

Many examples of such precursor reactions are found in the field of immune reactions. Those skilled in the art will easily be able to use enzyme electrodes based on the above-mentioned reactions in constructing various sensors including immunosensors.

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. This was done with this in mind.

Possible applications to non-clinical fields include the following.

(a) monitoring of fermentation processes; (b) industrial process control; (C) environmental monitoring, i.e. control of contamination by exhaust gases and effluents; (d) food testing (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 (insulated) supports, probes, etc. There is no need to go into detail, but the electrode material is generally a thin sheet or wafer of paper.

In the case of biosensors, electrode materials are typically mounted on an insulating support or probe that can be immersed into a sample. The size of the electrode material in such cases is actually very small, rarely exceeding a few square mm. Electrical contact with the electrode material can be made in various ways, for example by bringing the electrode material into opposing 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 stand on its own without an insulating support or carrier to hold it, and the electric wire is directly connected to the surface of the electrode material.

As the support, in addition to carbon paper, a semiconductor surface such as a field effect transistor, a non-conductive surface, etc. can be used. If the latter is used, the electrical connection is made directly to the layer of resin-bound carbon particles or graphite with a coating of platinum group elements.

〔Example〕

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

lJ (becomes ') First, an enzyme electrode was prepared according to the conventional technique. That is, a porous carbon paper in which carbon black particles (Palcan XC-72) with a nominal particle size of 30 nm were coated with 10% polytetrafluoroethylene as a binder on a commercially available graphitized carbon paper was used as a conductive substrate.
On top of this is electrolytic deposition, platinum mW! ji (thickness l
jIm) was formed.

Various types of such platinum-coated carbon papers were prepared.

Next, Aspergillus niger
Glucose oxidase derived from P. IIus niger was immobilized by the aforementioned carbodiimide treatment and glutaraldehyde crosslinking. That is, the electrode surface coated with platinum was first treated with an aqueous solution of glucose oxidase, 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 with a diameter of 2 mm, and used as a test sample.

2 Glucose) From Protochik (Massachusetts, USA)
Aspergillus n.
Glucose oxidase derived from P. iger) was immobilized. Here, the platinum-coated carbon particles (Palcan XC-72) used in the platinum-coated carbon paper are as disclosed in Example 1 of the aforementioned U.S. Pat. No. 4,044.193. Oxidative decomposition of the platinum(II) sulfite M complex with hydrogen peroxide produces colloidal platinum (particle size 1.
5 to 2.5 nm) to carbon particles (nominal particle size 3'On
m).

These carbon particles are further molded 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 B/cm'' (7.5-10 weight percent relative to carbon particles).

Various types of such prototic electrodes were prepared, and glucose oxidase was immobilized thereon by the various methods described above, ie, carbodiimide treatment, carbonyl dimidazole treatment, or DNDFB treatment.

In addition to the electrodes described above, we also created prototic electrodes in which glucose oxidase was immobilized by glutaraldehyde crosslinking or simple adsorption.

In the latter case, glucose oxidase should be dissolved in acetic acid solution (
(pH 5, 6) at a rate of 5.0 mg/m A, and a prototic electrode was placed in this solution at room temperature for 90 minutes.
It was immersed for 0 minutes. Also, even if you do not use this method,
The enzyme can also be simply adsorbed by immersing the electrode substrate in an enzyme solution to serve as an anode and performing electrophoresis for 60 minutes.

A layer of carbon particles coated with platinum in advance was formed on carbon paper using polytetrafluoroethylene as a binder (U.S. Pat. No. 4,044,193 mentioned above), and then the following was formed on the carbon paper coated with platinum using the carbodiimide treatment described above. The enzymes listed in were immobilized.

Lactate oxidase Galactose oxidase Glucose oxidase/β-galactosidase In order to demonstrate the advantages of the present invention over the prior art and the properties of the enzyme electrode materials, each of the separately prepared enzyme electrodes described above was used as an improved rank oxygen electrode system. Rank Brothers Bottisham, Cambridge
) ) was installed in a cell equipped with a 1000 MHz battery, and the current response characteristics were investigated. This system is based on the attached drawings and the power of analysis, your power, and the actors (Δanalytica Chimica
Acta) (Volume 183, pages 59-66, 19
As illustrated in 1986). In this system, instead of the membrane, an enzyme electrode (diameter 5 mm) using the carbon paper according to the present invention as a support is used, and this enzyme electrode is further supported by a button-shaped platinum electrode. A counter electrode (platinum foil) was inserted through the cover of the cell, and a silver-silver chloride electrode was used as a reference electrode. Separately, we also conducted experiments with a two-electrode system in which the counter electrode and reference electrode were integrally constructed as a silver chloride ring surrounding the enzyme electrode. In this case, a protective film is newly provided, but a stirring device is not provided. Normally, the pH 7.0 buffer solution used in the test is stirred with a magnetic stirrer, and the potential of the working electrode is adjusted to GOOmV with respect to the reference electrode using a potentiometer.
Set it so that However, in the case of a two-electrode system, this potential was set to 325 mV. Wait until the background value of the current is low before injecting from the ik thermothermal solution syringe.

The current response was recorded on a chart.

The results of this experiment will be explained in detail below using drawings.

Here, the contents of the drawing are as follows.

FIG. 1 is a characteristic diagram showing a comparison of 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.

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

FIG. 3 shows the current response characteristics of the glucose oxidase electrode according to the present invention and glucose? ; It is a characteristic diagram showing the relationship with degree.

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 stored at room temperature as a comparative example.

FIG. 7 is a characteristic diagram showing a comparison of the current response characteristics of the glutaraldehyde crosslinked glucose oxidase electrode according to the present invention and the prior art.

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

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

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

FIG. 11 is a characteristic diagram showing the 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 diagram showing the current response characteristics of a galactosidase composite electrode.

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

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

FIG. 15 is a schematic cross-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 experimental apparatus used in some of the measurements.

Most of the above data is No. 1! V! This was obtained using the electrochemical cell shown in I. This cell is the base (1)
, and an annular jacket (2) containing a water storage chamber (h) and controlling the temperature of the cell by circulating water.
It consists of two main parts, both of which are connected to each other by a special threaded joint (3). The above base (
In the center of 1) is a platinum contact (d), on which a test disk (a) of a carbon paper electrode on which enzymes are immobilized is placed. This test disc (a) is held in place by rubber O-rings (e) and (f) when the two main parts are assembled.

An adjustable ring (g
) is inserted, and the stopper (4) is inserted.
), through which a platinum-type counter electrode (b) and a silver-silver chloride reference electrode (c) are mounted. As mentioned above, during measurement, the potential of the working electrode was adjusted to 600 mV, and the current output was determined by adjusting the apparent surface area of the electrode in contact with the substrate solution to 0.14 cm.
The measurement results were expressed as the current density, that is, the current output per unit area of the electrode (a) in contact with the substrate in each of the above characteristic diagrams.

In the configuration shown in FIG. 16, the platinum contact (B) is surrounded by a reference/counter electrode composite electrode (C) via an insulating sleeve (G). A porous polycarbonate membrane mounted on the O-ring is used to properly hold the test disk (E) (carbon paper electrode with immobilized enzyme) on the platinum contact. The sample room (F) is; 5r! It is open-molded,
The potential of the zero working electrode, which allows the sample to be dropped onto the polycarbonate membrane, is set at 325 mV, and the current is detected by a botenshirostat (8). A two-electrode system with a working electrode potential of 325 mV has a working electrode potential of 600 mV.
It is superior to the three-electrode system with V in terms of ease of use and low background current value. However, no matter which electrode system is selected, the storage stability of the enzyme electrode according to the present invention
There are no major differences in the operating stability, response linearity, oxygen concentration dependence, and other characteristics.

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

Figure 1 shows the current response characteristics when glucose is added continuously in a three-electrode system equipped with a stirrer and the final concentration is varied up to 35mM. This is an example. Electrodes A, B,
Electrode A is a platinum-coated activated carbon support according to the present invention, in which platinum-coated carbon particles are immobilized with polytetrafluoroethylene. Electrode material (trade name: Brototec) coated on carbon paper as a binder; Electrode B is a conductive support made of sliced graphite rods; Electrode C is a conductive material made by cutting commercially available carbon paper that does not have a platinum coating. Each of them consists of a sexual support. Looking at this figure, the current responses of electrodes B and C are generally low level and less variable, and are close to the results of mediator type electrodes commonly seen in the prior literature. In contrast, the current response to the electrodes is more reliable and stable, with a response time of about 1 second. (Here, the sharp protrusion seen at the head of the signal at the beginning of the response is due to the glucose injection operation and has no particular significance.The flat part has the meaning of a signal that depends on the glucose concentration. It is a part.

) The current responses of these three types of electrodes all showed high linearity with respect to glucose concentration, as shown in Figure 2 (However, Figure 2 only shows the results for electrodes A and C).
. Therefore, the applicable concentration range can be widened, for example, when directly measuring the blood glucose concentration (
0-30mM).

Glucose oxidase to this electrode is
Similar results were obtained when immobilized by the method described above, suggesting that this method achieves good linearity over a wider concentration range.

As is clear from FIG. 2, the response characteristics of electrode A remained virtually unchanged even after 23 days, but the response characteristics of the other electrodes (only electrode C is shown in the figure) deteriorated over time. Similar behavior is observed when other enzyme immobilization methods mentioned above are used, and when using the same activated carbon material as electrode A, an electrode with excellent responsiveness and high stability can be obtained using any of the methods. However, good electrodes could not be obtained when inert carbon materials were used. Also, the response time for the electrode remained unchanged for 23 days, but the response time for the other electrodes increased from an initial response time of 23 to 30 seconds to 2 to 3 minutes after 8 days. Active electrodes such as electrode A generally exhibit a slight decrease in responsiveness on the first day, but thereafter become stable and show little change over time. Furthermore, the response characteristics of Electrode A hardly changed during the first few days even after being stored in a pH 5.6 buffer at 4° C. for more than 6 months. Although the response characteristics gradually decreased after 6 months, 70% of the initial value was maintained even after 12 months.

Above, Figures 1 and 2 show the current output of the enzyme electrode in units of μA/cm when the potential of the working electrode is 600 mV in the three-electrode system shown in Figure 15. .

FIG. 5 shows the results of storing the above electrode for a longer period of time and examining its stability. That is, a glucose oxidase electrode on which the enzyme had been immobilized by carbodiimide treatment was stored for 180 days in an acetate buffer at pH 5 or 6 at room temperature, and then its 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 in Figure 5 were obtained from measurements with a working electrode potential of 600 mV in a three-electrode system, and the results in Figure 6 were obtained from measurements with a working electrode potential of 325 mV in a two-electrode system. It is something.

Furthermore, FIGS. 7 to 9 show the results of a comparison between the prior art (Example 1) and the present invention using various electrodes with different enzymes and different immobilization methods. All these measurements were carried out at a voltage of 325 mV in one working electrode chamber of the two-electrode system.

FIGS. 10 to 12 show the response characteristics when galactose, lactate, and lactose are used as substrates, respectively. All of these measurements are performed using a three-electrode system.

The working electrode potential was 600 mV.

Snowball L: 1゛LJ, 1'''' The enzyme electrode according to the present invention has a long lifespan that has not been achieved with conventional electrodes while the substrate is continuously supplied. This was demonstrated through the following series of rigorous tests.

First, the glucose oxidase electrode (Example 2) was set in a closed cell with a stirring device filled with a glucose solution, and current was detected at an initial concentration of 5 m M + initial current of 100 μA. When the electrode is continuously used in this state for 18 hours, the signal gradually decreases to 10 μA or less.

The integrated value of the detected current is calculated from 1 molecule of glucose to 2
The theoretically calculated value of approximately 7
It is 5%.

Immediately repeating the above experiment by exchanging the glucose solution for the same electrode restored the initial current value and the continuously delivered substrate decreased as well.

Also, in one continuous experiment, the glucose concentration was maintained at 5mM by continuously circulating the solution from a large reservoir, and the current generation was sustained for an additional 5.7 days. 1
During 00 hours, the current output gradually decreased and settled at 45 μA, and this state remained for 40 hours. This may be due to enzymes that were not tightly bound falling off the electrode substrate during this period (although no correlation was observed between current output and stirring speed), or other factors. This is thought to be the result.

After performing the above long-term test, the current response characteristics of the enzyme electrode over a range of glucose concentrations were measured according to the method described above. Although the signal amplification was lower than when fresh substrate solution was used, it showed a very sharp step-like change in the glucose concentration range of 0 to 30 mM, and the enzyme electrode showed a very sharp step-like change in the glucose concentration range of 0 to 30 mM. It was confirmed that the current response characteristics did not deteriorate even when used for a period of time. This result was obtained after the enzyme electrode was stored at 4°C for one week.

The i1
1, and no change in response characteristics depending on glucose concentration was observed in any of the experiments.

These test results show that this enzyme electrode can be used for a total of at least 250 hours (more than 15,000 minutes), or has an extremely long life.

The operating life of enzyme electrodes in the prior art is much shorter than that of enzyme electrodes according to the invention, often only a few hours [Turnet Proc.
ceedings Biotech) J (Volume 85,
1985.

(Europe) +Online Publications, Bina+ (Online Pub
lications, Pinner) UK, 181
-192 pages]. For example, the half-life of a glucose electrode using glucose oxidase coupled with ferrocene is generally about 24 hours (Turner).
1 supra), Casset al. (Analytical Chemistry.

(Analyt, Chem,) Volume 56, 667
~Page 673, 1984] (cited above) announced that the same electrode could be used stably for 50 hours. This electrode is 50mM? 50 for glucose solution at s degree
The standard deviation was less than 1% even after repeated measurements.

The final response signal level (45 μA for a 5 mM glucose solution) observed in the repeated use test described above did not change when the glucose solution was aerated, and it did not change even after several weeks of storage. There wasn't.

The current output of this electrode was measured using a sterilized glucose solution under conditions that would not cause a decrease in glucose concentration due to bacterial contamination, and it was confirmed that the current output could be stably obtained for 12 hours. The signal level at this time was constant throughout the measurement period. The electrodes were "conditioned" in a solution that was stirred or circulated over several days, which may have led to good results. In this way, continuous measurement of glucose 1 is also possible if the electrodes are properly conditioned or cleaned.

As long as the enzyme electrode is clean and suitable for birch preparation, the enzyme electrode of the present invention can withstand repeated use.1. When we created several pairs of electrodes of the same size using the same method that showed high responsiveness to glucose, their performances were very similar, and even when their current response characteristics were measured under the same conditions, The error was only a few percent. Furthermore, all the electrodes made in this way had a much longer lifespan and higher reliability than conventional electrodes, as mentioned above. The electrodes of the present invention do not change in performance during storage and can be used for many weeks, whereas conventional electrodes often exhibit variable performance even within a single batch. For example, Turner mentioned above that there are a very small number of glucose oxidase electrodes in one batch that have an extremely long half-life of 600 hours, but the majority of electrodes have a short half-life of 24 hours. ing. Therefore, authenticity cannot be guaranteed even if these electrodes are used for significantly more than 24 hours.

Ieme,・'no,? mr white' -
In order to investigate the effects of dissolved oxygen, the test cell was modified and added an oxygen electrode in addition to the glucose electrode. In a series of experiments, dissolved oxygen was removed from the system by blowing argon. Under these conditions, the electrode A described above responds sharply to the addition of glucose, and this response mechanism is almost independent of the dissolved oxygen concentration, but rather depends on the surface of the electrode combined with an appropriate enzyme immobilization method. It was suggested that this is due to properties specific to the structure. Such results have not been reported in the past.

Furthermore, the potential of electrode A created by method A described above was increased to 6
An experiment was conducted in which the output signal was measured while continuously blowing argon at a voltage of 00 mV.

At this time, the oxygen concentration in the sample was also measured at the same time.

The results are shown in FIG. Looking at this diagram, we see that the current signal (
It is clear that the graph (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 changes for 10 minutes even if the oxygen concentration rapidly decreases.

On the other hand, in electrode B prepared by method B described above, the current signal decreased within 5% within 3 minutes, and the oxygen concentration decreased by 90% during this period. In this case, when oxygen was reintroduced into the system, the current response recovered, albeit relatively slowly. Continuing to blow argon will cause these electrodes to work only within a limited range of glucose concentrations;
It appears that oxygen at the pole 11 is required to express part of the enzyme's function of extracting hydrogen from the substrate. Furthermore, the possibility cannot be denied that a trace amount of oxygen that cannot be detected by the enzyme electrode is adsorbed by the electrode and plays some role.

As a result of measuring the rate of decrease in glucose and the maximum current density, the amount of enzyme immobilized on electrode A without being deactivated was approximately 7 μg of active enzyme per 1 cm of electrode surface area. It was found that the amount of glucose oxidase immobilized in a biosensor has been discussed in almost no literature.In each of the above-mentioned immobilization methods, the enzyme solution is diluted 10 times or more. It was found that electrodes with very high activity could still be created.

Part 2 of 1 The raw glucose concentration range was set to 0 to 30 mM, and the current response characteristics of electrode A were investigated in the temperature range of 10 to 37°C.

The temperature coefficient was 2-3% per degree Celsius. This corresponds to the Arrhenius activation energy (approximately 24 kJ 1 mol). On the other hand, in a biosensor coupled with ferrocene (see the above-mentioned literature by Cass et al.), the temperature coefficient is reported to be 4% per 1°C.

A slight pH dependence of current response was observed, but at pH 7
, 0 to 8.0, it can be considered that there is virtually no pH dependence, except when the glucose concentration is very high (25 mM or higher).

・9 Clouds with i, ・, Machi It was found that in an experimental system involving stirring of an iM solution, a polycarbonate membrane could be used with almost no effect on the signal waveform and magnitude. In the system without solution stirring, the response time was about 20 seconds.

The electrode with a cardiac polycarbonate protective membrane to whole blood volume was suitable for direct measurement of blood concentration of glucose. 5m M 974
When measured on a glucose solution of 0.2mM
The signal interfered with by ascorbic acid was 2.5%.

As mentioned above, good results were obtained when the enzyme electrode of the present invention was applied to a rank cell using an improved Clark electrode. It was found that very good results were obtained in terms of morphology as well.

As an example, the above electrode was connected to an electric wire and encapsulated in a glass bamboo to create a probe with a diameter of 2 mm, which is commonly used in the past. If a glucose test solution is placed in a container such as a beaker, stirred, and a probe like the one described above is inserted into the container along with a reference electrode and a counter electrode, highly reliable concentration measurements can be made without removing dissolved oxygen. Can be done.

When measurements were performed using the above probe or an even smaller probe with the same structure, it was found that current responsiveness is roughly proportional to the apparent area or weight of the electrode if the glucose concentration is the same.

We also created a probe using a smaller electrode (approximately 0.25 to 0.50 mm in area and 30 to 60 μg in weight).Here, the wire connection was covered with a plastic sleeve, and the probe was made with a diameter of 1.5 mm. It was assembled into a 5 mm catheter needle, which penetrated a rubber seal into the fermentation tank containing the test liquid.

It can be inserted into a container such as a waste liquid reservoir and used as a probe type sensor 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 also be kept clean.

Normally, a current signal of 1 to 10 μA is extracted from the small electrodes mentioned above, but depending on the device's ingenuity, a current signal of 1 to 100 μA can be extracted.
It is also possible to perform precise measurements within the range of A. Enzyme electrodes that extract signal currents in this range are extremely small (approximately 0.005 mm in area and 1 μg in weight), so they cannot be incorporated into thin nail-shaped microprobes such as those used in biological measurements. Use with.

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

It has been reported that active groups formed on the carbon surface by surface oxidation at high temperatures participate in a cross-linking reaction similar to that used for enzyme immobilization, and that the number and type of such surface groups are different from that of platinum (or It is known that it increases when a platinum metal element such as palladium is present as a thin layer surface catalyst [Kinoshita and Stonehart].
Modern Aspects of Electrochemistry (
Modern Aspects of Electr
ochen+1stry) J (No, 12. Bockris and Conway (Ed, Bockris an
d Conway) + Plenum.

Plenum Press, New York, pp. 183-266, 1977]. It is clear that the binding state of the enzyme differs depending on the immobilization method. For example, in many cases reported so far, various amino acid residues in enzymes are used for immobilization, but when a material activated with cyanuric chloride and fil element are combined, It is known that only the enzyme residue is used for immobilization (Ianniello and Yasinich, “Analytical Chemistry”, 5).
Volume 3, pages 2090-2095.

(1981), changes in the tertiary structure of the enzyme due to immobilization are also unlikely to be the same for all immobilization methods, and such variability may result in large differences in enzyme activity and stability in this type of study. This seems to be the cause of this.

The electrode substrate material used in the present invention has extremely non-uniform properties, unlike the laminated uniform material disclosed in the aforementioned Japanese Patent Application Laid-open No. 56-163447. There is. This brings diversity to the crosslinked structure and increases the possibility that various orientations will appear in the three-dimensional structure.

Even when no crosslinking agent is used, surface adsorption is strong.

The pores in these carbonaceous plates allow oxygen to enter, which increases the surface area of the enzyme and helps it adopt a conformation that does not interfere with stability and activity. This point is
There are significant differences with traditional bonding modes in which enzymes are bound to relatively flat surfaces with much smaller surface areas, such as platinum, glassy carbon, or graphite, thereby limiting the possible conformations of the enzyme. That's the difference. Moreover, the responsiveness of the enzyme electrode according to the present invention is extremely fast at 1 to 2 seconds, and not only is the enzyme activity high, but the electrode itself has many electron accepting sites, so electron transfer is extremely rapid. That's telling. This is because the platinum-coated carbon particles are present at a high density over a very wide area in the microstructure, allowing the surface platinum and the active site of the enzyme to successfully approach each other.

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

Regarding the amount of resin binder, the present inventors actually calculated 3
0-70 double star 9A (uniformly mixed with platinum group metals or
Alternatively, the value is based on carbon particles or graphite particles in which the platinum group metal is precipitated or adsorbed on the surface of each particle. ) experiments were conducted on the range of

When polytetrafluoroethylene is 30% by weight, 4.
The current output for a glucose concentration of 8 mmol is 106
μA, and at a glucose concentration of 33.3 mmol, 58
It rose to 9μ8. 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 increased only to 330 μA even when the glucose concentration was 33.3 mmol.

There are two possible reasons why the current output decreases as the amount of resin binder increases. Firstly, as the amount of the hydrophobic binder increases, the hydrophobicity of the electrode itself also increases, reducing its wettability to the test solution. Second, since polytetrafluoroethylene is a non-conductor, increasing the amount thereof will reduce the conductivity of the electrode, resulting in a weak output signal.

In addition, for the glucose oxidase electrode using polyvinyl acetate as a binder, the above-mentioned method was adopted as the method for immobilizing glucose oxidase, and the platinum-coated carbon paper electrode on which the glucose oxidase is immobilized was made using polyvinyl acetate as the binder. It was prepared substantially according to the method of Example 2 above, except that 50% by weight polyvinyl acetate was used in place of fluoroethylene.

When the potential of this electrode was set to 325 mV and measurement was similarly performed using an improved rank electrode system, a substantially linear current response characteristic was achieved as shown in FIG. 13.

Furthermore, for the glucose oxidase electrode that uses palladium as the platinum group element, the above-mentioned method is adopted as the method for immobilizing glucose oxidase, and the palladium-coated carbon paper electrode on which the glucose oxidase is immobilized uses colloidal palladium on carbon. Particles (nominal particle size 3゜r+m, 1 name Palcan XC-72)
After depositing on the surface, the palladium-coated carbon particles were coated with 50% by weight of polytetrafluoroethylene as a binder on conductive carbon paper to a thickness of 0.
It was created by molding into a thin layer of 1 mm.

A disk with a diameter of 2 mm was cut out from this palladium-coated carbon paper electrode and set on the platinum contact of a two-electrode cell as shown in FIG. 16, and the responsiveness to glucose was examined at a potential of 325 mV.

The results are as shown in Section 14 (2I), and in this case as well, a nearly linear response characteristic to the glucose concentration was confirmed.

No. 4,293, cited above, shows that the platinum group elements used in gas diffusion electrodes behave very similarly to each other.
By analogy with what is disclosed in No. 396 and the like, it is considered that other platinum group elements such as ruthenium and rhodium can be substituted for platinum and palladium in the enzyme electrode according to the present invention.

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

4 (Cholesterol band) The platinum-coated carbon paper manufactured by Protochik used in Example 2 above was mixed with cholesterol oxidase (EC 1, 1, 3° 6,) in phosphate buffer (pH 7, 4).
It was immersed in a solution dissolved at a concentration of 1/ml at 22°C for 3 hours.

Next, the platinum-coated carbon paper that had adsorbed cholesterol oxidase was cut out into a disc shape with a diameter of 1.5 squares to be used as an electrode, and this was set in a two-electrode cell as shown in Figure 16, and silver-chloride was removed. 340+w for silver electrode
The potential was set to V, and the responsiveness to a cholesterol standard solution was examined.

The above cholesterol standard solution has a surface activity of 5%; T'l
Phosphate buffer (pH 7) containing Lydon X-100)
,4) Add cholesterol to 50.100.200■7d
The mixture was added at a ratio of 1 liter, ultrasonic stirring, and then heated to 70°C.

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

Table 5 (Sucrose) This sucrose electrode uses a complex enzyme system consisting of three types of enzymes: invertase, which converts sucrose into α-glucose; mutase, which converts the β-glucose into a mixture, and glucose oxidase, which acts on the β-glucose to generate an output signal proportional to the initial concentration of sucrose.

The platinum-coated carbon paper manufactured by Protochik used in Example 2 above was impregnated with an enzyme mixed solution in which the following enzymes were dissolved in phosphate buffer (pH 7.0) at room temperature for 90 minutes.

Glucose oxidase (EC1, 1, 3, 4,)
6mg/ml inhertase (IiC3,2,
1,26,) 6 IIf/m It mutarotase
(EC5,1,3,3,) 2 try/m1
Next, the platinum-coated carbon paper on which these enzymes have been adsorbed is cut out into a disc shape with a diameter of 5 mm to serve as an electrode, and this is set in a three-electrode cell as shown in Fig. 15 to inject silver chloride. Set to a potential of 400IIl■ with respect to the electrode,
The response to an aqueous solution containing sucrose was investigated. Note that the contact area of the electrode with the test solution is 0.16 cd.

Similar measurements were also performed on electrodes prepared by simultaneously immobilizing these three types of enzymes on the platinum-coated carbon paper using carbodiimide treatment or glutaraldehyde crosslinking.

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

From this Figure 17, adsorption and carbodiimide treatment.

It was found that almost linear response was obtained in any case of glutaraldehyde crosslinking.

〔Effect of the invention〕

The present invention provides a current detection sensor with 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 in a more effective manner. be able to. The improved enzyme ItFi provided by the present invention does not require the use of a mediator unless specifically desired and also functions in the presence of very low levels of dissolved oxygen. Not only 10mM
The current density per square centimeter in a glucose solution of 100° C. is several hundred microamperes, and the current response characteristics are excellent.

Such current response characteristics are superior to conventional current detection type biosensors, so the electrode area is 1 mm or less and O~1
It is also suitable for manufacturing a microprobe type biosensor that exhibits a current output of 00 nA.

The bridge can also be constructed using very small amounts of immobilized enzyme. That is, in the case of no protective film, 1 to 2
seconds, 10 seconds if there is a protective film. ~30 seconds, which is faster than any conventional glucose sensor. Furthermore, this electrode exhibits excellent stability even at room temperature if stored in a immersed state.

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

[Brief explanation of drawings]

FIG. 1 is a characteristic diagram showing a comparison of 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. 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 characteristics and 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 stored at room temperature as a comparative example. FIG. 7 is a characteristic diagram showing a comparison of the current response characteristics of the glutaraldehyde crosslinked glucose oxidase electrode according to the present invention and the prior art. FIG. 8 is a characteristic diagram showing a comparison of the current response characteristics of the carbodiimide-treated glucose oxidase electrode according to the present invention and the prior art. FIG. 9 is a characteristic diagram showing a comparison of the current response characteristics of the carbodiimide-treated lactate oxidase electrode according to the present invention and the conventional technique. FIG. 10 is a characteristic diagram showing the current response characteristics of the galactose oxidase electrode according to the present invention. FIG. 11 is a characteristic diagram showing the 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 diagram showing the current response characteristics of a galactosidase composite electrode. FIG. 13 is a characteristic diagram showing the current response characteristics of a glucose oxidase electrode according to the present invention in which polyvinyl acetate is used instead of polytetrafluoroethylene as a binder for carbon particles with a platinum VL film. FIG. 14 is a characteristic diagram showing the current response characteristics of the glucose oxidase electrode according to the present invention, in which glucose oxidase is immobilized on a palladium-coated carbon particle layer formed on carbon paper using polytetrafluoroethylene as a binder. be. FIG. 15 is a schematic cross-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 experimental apparatus used in some of the measurements. FIG. 17 is a characteristic diagram showing the current responsiveness of the sucrose electrode to which the present invention is applied. Patent applicant: Cambridge Life'Sciences BLC I, Patent attorney: Akira Koike ((lh2 people) Class L2-S4 Friends /rnmolL-'7'tb
Cor Fig. 10 Roughchie) 517t/mrnolL Fig. 11 Lacto-also 4fl/rnmolL-'5
Io 15 20 30
40'A=+-z: @fi-/mmolL Nse a'''IT r7i ri-+t- course 5*I/mmolL-'14th
Figure 15 Figure j6

Claims (21)

[Claims] (1) In an enzyme electrode capable of electrically responding to the catalytic activity of an enzyme in the presence of a specific substrate, a high purity platinum group metal is homogeneously mixed in advance, or the platinum group metal is mixed with individual platinum group metals. It is formed by molding carbon particles or graphite particles precipitated or adsorbed on the surface of particles using a resin binder, and the carbon particles or graphite particles are formed in a substantially nonuniform resin-fixed carbon layer or resin-fixed graphite layer. At least a part of the conductive support is constituted by a porous conductive substrate layer in which a platinum group metal is uniformly dispersed in a proportion of 1 to 20% by weight, and the surface of the porous conductive substrate layer An enzyme electrode is an enzyme that has an enzyme immobilized or adsorbed on it. (2) The proportion of the resin binder is 5 to 80% by weight based on the carbon particles or graphite particles in which the platinum group metal is uniformly mixed or the platinum group metal is precipitated or adsorbed on the surface of each particle. The enzyme electrode according to claim 1, characterized in that: (3) The enzyme electrode according to claim 1 or 2, wherein the platinum group metal is platinum or palladium. (4) The resin binder is 2 per cm^2 at room temperature and normal pressure.
4. The enzyme electrode according to claim 1, wherein the enzyme electrode is a resin binder having an oxygen solubility of x10^-^3cm^2 or more. (5) The resin binder is fluororesin, polyvinyl acetate,
Polyethyl methacrylate, polystyrene, polyvinyl chloride, polycarbonate, poly(4-methylpentene-1)
), polyisoprene, polychloroprene, poly-1,3
The enzyme electrode according to claim 4, wherein the enzyme electrode is at least one of -butadiene and silicone rubber. (6) The enzyme electrode according to any one of claims 1 to 5, wherein the enzyme immobilized or adsorbed on the surface of the porous conductive substrate layer is an oxidoreductase. (7) The enzyme electrode according to claim 6, wherein the oxidoreductase is any one of glucose oxidase, galactose oxidase, lactate oxidase, and cholesterol oxidase. (8) The enzyme electrode according to any one of claims 1 to 5, wherein the enzyme immobilized or adsorbed on the surface of the porous conductive substrate layer is a complex enzyme system. (9) The enzyme electrode according to claim 8, wherein the composite enzyme system is a combination of glucose oxidase and β-galactosidase, or a combination of β-glucosidase and glucose oxidase. (10) The enzyme electrode according to any one of claims 1 to 9, wherein the porous conductive substrate layer is bonded to a conductive base to serve as a conductive support. (11) The enzyme electrode according to claim 10, wherein the conductive base is conductive carbon paper. (12) A claim characterized in that the porous conductive substrate layer is a resin-fixed carbon layer or a resin-fixed graphite layer formed by molding carbon particles or graphite particles on which a platinum group metal has been precipitated or adsorbed using a resin binder. The enzyme electrode according to claims 1 to 11. (13) The particle size of the carbon particles or graphite particles is 5 to 30 nm, and the particle size of the platinum group metal adsorbed to the surface thereof is 1.5 to 2.5 nm. Enzyme electrode. (14) The enzyme electrode according to any one of claims 1 to 13, wherein the surface of the conductive support is protected by a microporous membrane that is permeable to the substrate. (15) The enzyme electrode according to claim 14, wherein the microporous membrane is a polycarbonate membrane. (16) In an enzyme electrode capable of electrically responding to the catalytic activity of an enzyme in the presence of a specific substrate, the platinum group metal is homogeneously mixed in advance with a highly purified platinum group metal, or the platinum group metal is mixed with individual platinum group metals. It is formed by molding carbon particles or graphite particles precipitated or adsorbed on the surface of the particles at room temperature and normal pressure using a resin binder having an oxygen solubility of 2 x 10^-^3 cm^2 or more per cm^2, At least a portion of the conductive support is constituted by a porous conductive substrate layer in which a platinum group metal is uniformly dispersed in a substantially nonuniform resin-fixed carbon layer or resin-fixed graphite layer, and the porous An enzyme electrode in which an enzyme is immobilized or adsorbed on the surface of a highly conductive substrate layer. (17) In an enzyme electrode capable of electrically responding to the catalytic activity of an enzyme in the presence of a specific substrate, the palladium is homogeneously mixed with high-purity palladium in advance, or the palladium is applied to the surface of individual particles. Porous material formed by molding precipitated or adsorbed carbon particles or graphite particles using a resin binder, with palladium uniformly dispersed in a substantially non-uniform resin-fixed carbon layer or resin-fixed graphite layer. An enzyme electrode in which at least a part of a conductive support is constituted by a conductive substrate layer, and an enzyme is immobilized or adsorbed on the surface of the porous conductive substrate layer. (18) In an enzyme electrode capable of electrically responding to the catalytic activity of an enzyme in the presence of a specific substrate, the platinum group metal is homogeneously mixed with a highly purified platinum group metal in advance, or the platinum group metal is mixed with individual platinum group metals. It is formed by molding carbon particles or graphite particles precipitated or adsorbed on the particle surface using a resin binder, and the platinum group metal is uniformly distributed in the substantially nonuniform resin-fixed carbon layer or resin-fixed graphite layer. At least a part of the conductive support is constituted by a dispersed porous conductive substrate layer, and any one of galactose oxidase, lactate oxidase, and cholesterol oxidase is immobilized on the surface of the porous conductive substrate layer. Enzyme electrode made by adsorption or oxidation. (19) In an enzyme electrode capable of electrically responding to the catalytic activity of an enzyme in the presence of a specific substrate, the platinum group metal can be homogeneously mixed with a high purity platinum group metal in advance, or the platinum group metal can be mixed with individual platinum group metals. It is formed by molding carbon particles or graphite particles precipitated or adsorbed on the particle surface using a resin binder, and the platinum group metal is uniformly distributed in the substantially nonuniform resin-fixed carbon layer or resin-fixed graphite layer. An enzyme electrode, wherein at least a part of a conductive support is constituted by a dispersed porous conductive substrate layer, and a composite enzyme system is immobilized or adsorbed on the surface of the porous conductive substrate layer. (20) Claim 20 characterized in that the composite enzyme system consists of glucose oxidase and β-galactosidase.
The enzyme electrode described. (21) The enzyme electrode according to claim 20, wherein the composite enzyme system consists of invertase, mutase, and glucose oxidase.