JPS5816698B2 - Enzyme electrode and its manufacturing method - Google Patents

Enzyme electrode and its manufacturing method

Info

Publication number
JPS5816698B2
JPS5816698B2 JP53084482A JP8448278A JPS5816698B2 JP S5816698 B2 JPS5816698 B2 JP S5816698B2 JP 53084482 A JP53084482 A JP 53084482A JP 8448278 A JP8448278 A JP 8448278A JP S5816698 B2 JPS5816698 B2 JP S5816698B2
Authority
JP
Japan
Prior art keywords
enzyme
enzyme electrode
electron
conductive substance
electrode
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
JP53084482A
Other languages
Japanese (ja)
Other versions
JPS5510584A (en
Inventor
中村研一
南海史朗
飯島孝志
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Panasonic Holdings Corp
Original Assignee
Matsushita Electric Industrial Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Matsushita Electric Industrial Co Ltd filed Critical Matsushita Electric Industrial Co Ltd
Priority to JP53084482A priority Critical patent/JPS5816698B2/en
Publication of JPS5510584A publication Critical patent/JPS5510584A/en
Publication of JPS5816698B2 publication Critical patent/JPS5816698B2/en
Expired legal-status Critical Current

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Classifications

    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02EREDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30Hydrogen technology
    • Y02E60/50Fuel cells
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P70/00Climate change mitigation technologies in the production process for final industrial or consumer products
    • Y02P70/50Manufacturing or production processes characterised by the final manufactured product

Description

【発明の詳細な説明】 本発明は、酵素の特異的触媒作用を受ける基質に対して
電気化学的活性を有し、基質の濃度を迅速かつ簡便に測
定することができ、しかも連続使用、繰り返し使用ので
きる酵素電極を得ることを目的とする。
DETAILED DESCRIPTION OF THE INVENTION The present invention has electrochemical activity toward a substrate that is subject to specific catalytic action of an enzyme, can quickly and easily measure the concentration of the substrate, and can be used continuously and repeatedly. The aim is to obtain a usable enzyme electrode.

本発明は、また酸素電極などと組み合わせることにより
、基質のもつ化学エネルギーを電気エネルギーに変換す
る電池に用いられる酵素電極に関する。
The present invention also relates to an enzyme electrode used in a battery that converts the chemical energy of a substrate into electrical energy by combining it with an oxygen electrode or the like.

酵素の有する特異的触媒作用を工業的に利用する試みの
一例として、酵素反応系と電気化学反応系を結びつける
ことにより、酵素と特異的に反応する物質である基質の
濃度を検出することが試みられている。
As an example of an attempt to industrially utilize the specific catalytic action of enzymes, an attempt was made to detect the concentration of a substrate, which is a substance that specifically reacts with enzymes, by linking an enzymatic reaction system and an electrochemical reaction system. It is being

酵素反応を電気化学反応として扱うには、例えば、酵素
反応系にこれと共役する適当なレドックス化合物を介在
させ、このレドックス化合物の酸化還元反応を電気化学
的に検出する方法が用いられている。
In order to treat an enzyme reaction as an electrochemical reaction, for example, a method is used in which an appropriate redox compound conjugated with the enzyme reaction system is interposed, and the redox reaction of this redox compound is electrochemically detected.

具体的)こは酵素との共役反応で還元(又は酸化)され
たレドックス化合物を電気化学的に酸化(又は還元)し
、基質濃度をこのとき流れる電流として検出することが
できる。
Specifically, a redox compound that has been reduced (or oxidized) in a coupled reaction with an enzyme is electrochemically oxidized (or reduced), and the substrate concentration can be detected as the current flowing at this time.

しかし高価な酵素やレドックス化合物を溶解した状態で
使用するため、これらを測定毎に使い捨てることになり
、また測定操作も煩雑である。
However, since expensive enzymes and redox compounds are used in a dissolved state, they must be discarded after each measurement, and the measurement operation is also complicated.

これらの問題を解決し、酵素などの繰り返し使用を可能
とし、実用的な酵素電極とするためには、酵素、レドッ
クス化合物を集電体としての電子伝導性物質とともに一
体固定化する必要がある。
In order to solve these problems, enable repeated use of enzymes, and create a practical enzyme electrode, it is necessary to integrally immobilize enzymes and redox compounds together with an electron conductive substance as a current collector.

本発明者らは、これら酵素、レドックス化合物を一体固
定化した酵素電極を得る方法について種種検討した結果
、電子伝導性物質として例えばカーボン粉末と不溶性レ
ドックス化合物の混合物をプレヌ成型し、この成型体上
に酵素を固定化する方法、あるいは前記混合物中に予め
酵素を固定化したカーボン粉末を混合しておき、その後
成型体とする方法を見出した。
The present inventors investigated various methods for obtaining an enzyme electrode in which these enzymes and redox compounds are integrally immobilized. As a result, the present inventors formed a mixture of, for example, carbon powder and an insoluble redox compound as an electron conductive substance by plain molding, and deposited the molded body on this molded body. We have discovered a method in which enzymes are immobilized in the mixture, or a method in which carbon powder on which enzymes have been immobilized is mixed in advance into the mixture and then molded.

こうして得られた酵素電極は、基質濃度を迅速かつ簡便
に測定しうるものであった。
The thus obtained enzyme electrode was able to measure substrate concentration quickly and easily.

本発明は、この酵素電極を改良し、酵素およびレドック
ス化合物の使用量を大幅に減少させ、しかも高性能の酵
素電極を提供するものである。
The present invention improves this enzyme electrode, significantly reduces the amount of enzyme and redox compound used, and provides a high-performance enzyme electrode.

すなわち、本発明の酵素電極は、電子伝導性物質からな
る第1の層と、酸化還元酵素と共役する不溶性レドック
ス化合物と電子伝導性物質との混合物からなり、内部に
酸化還元酵素を固定化した第2の層とで構成したことを
特徴とする。
That is, the enzyme electrode of the present invention consists of a first layer made of an electron-conductive substance, a mixture of an insoluble redox compound conjugated with an oxidoreductase, and an electron-conductivity substance, and the oxidoreductase is immobilized therein. It is characterized by being composed of a second layer.

ここで、第2の層には、必要に応じて酵素とともその補
酵素を固定化する。
Here, an enzyme and its coenzyme are immobilized on the second layer as necessary.

この構成によれば、以下に説明するように、きわめて簡
便なる製造法で酵素電極を得ることができる。
According to this configuration, the enzyme electrode can be obtained by an extremely simple manufacturing method, as described below.

第1図は本発明による酵素電極の構成例を示す。FIG. 1 shows an example of the structure of an enzyme electrode according to the present invention.

図中1は電子伝導性物質からなる第1の層、2は固定化
された酵素と、この酵素と共役する不溶性レドックス化
合物と電子伝導性物質とからなる第2の層であり、両者
は一体成型により構成される。
In the figure, 1 is a first layer made of an electron conductive substance, and 2 is a second layer made of an immobilized enzyme, an insoluble redox compound conjugated with this enzyme, and an electron conductive substance, and both are integrated. Constructed by molding.

層2は基質と酵素およびレドックス化合物の間の反応を
行なわせる部分であり、層1は層2の集電体および基体
の役割を果たす。
Layer 2 is the part that allows the reaction between the substrate, enzyme, and redox compound to take place, and layer 1 serves as a current collector and substrate for layer 2.

これら2層の構成としては第2図に示すように、反応層
を両側に設けるなど必要に応じて組み合わせることがで
きる。
As shown in FIG. 2, these two layers can be combined as necessary, such as by providing reaction layers on both sides.

このようにして、必要最小限量の酵素、不溶性レドック
ス化合物で電極を構成することができる。
In this way, the electrode can be constructed using the minimum necessary amount of enzyme and insoluble redox compound.

次に酵素電極を用いた測定方法について述べる。Next, a measurement method using an enzyme electrode will be described.

第3図に本発明による酵素電極を用いて基質濃度を測定
する場合の測定系を示す。
FIG. 3 shows a measurement system for measuring substrate concentration using the enzyme electrode according to the present invention.

図中3は記録計、4はポテンショヌタット、5は参照極
、6は塩橋、7は対極、8は上記の酵素電極9を装着し
た電極ホルダー、10は基質を含むpH5,6のリン酸
緩衝液である。
In the figure, 3 is a recorder, 4 is a potentiostat, 5 is a reference electrode, 6 is a salt bridge, 7 is a counter electrode, 8 is an electrode holder equipped with the enzyme electrode 9 described above, and 10 is a phosphor containing a substrate at pH 5 and 6. It is an acid buffer.

なお酵素電極9は、層2が緩衝液と接触するようにホル
ダー8に装着され、層1には例えば白金のリードが付け
られる。
Note that the enzyme electrode 9 is attached to the holder 8 so that the layer 2 is in contact with the buffer solution, and a lead made of platinum, for example, is attached to the layer 1.

酵素電極9を緩衝液に浸漬後、電極電位を参照極に対し
一定電位に保持し、基質の濃度変化に伴うレドックス化
合物の酸化還元電流の変化量を検出する。
After the enzyme electrode 9 is immersed in the buffer solution, the electrode potential is maintained at a constant potential with respect to the reference electrode, and the amount of change in the redox current of the redox compound due to the change in the concentration of the substrate is detected.

このとき、溶液中に基質が存在しない場合にも、電子伝
導性物質や固定化に用いた試料酵素、あるいはレドック
ス化合物中の不純物などの酸素還元に伴う残余電流が流
れる。
At this time, even if no substrate is present in the solution, a residual current flows due to oxygen reduction of the electron conductive substance, the sample enzyme used for immobilization, or impurities in the redox compound.

この残余電流の大小は基質、酵素、レドックス化合物の
反応に基づく応答電流のS/N比を決定する。
The magnitude of this residual current determines the S/N ratio of the response current based on the reaction of the substrate, enzyme, and redox compound.

これに関して、電極性能を向上するには、レドックス化
合物、酵素の量を必要最小限度とし、これらを電子伝導
性物質とともに最適な構成とする必要がある。
In this regard, in order to improve electrode performance, it is necessary to reduce the amounts of redox compounds and enzymes to the minimum necessary, and to optimize the composition of these together with electron-conducting substances.

またこうすることにより高価な酵素、レドックス化合物
の有効利用を図ることができる。
Moreover, by doing so, it is possible to effectively utilize expensive enzymes and redox compounds.

電子伝導性物質としては、酸化還元に対して安定な金属
あるいは酸化スズなどの導電性金属酸化物を用いること
ができる。
As the electron conductive substance, a metal that is stable against redox or a conductive metal oxide such as tin oxide can be used.

特にカーボンは化学的に安定な良導電性物質であり、加
えて酵素反応を阻害することもないなど電子伝導性物質
として好ましい。
In particular, carbon is a chemically stable and highly conductive substance, and in addition, it does not inhibit enzymatic reactions, so it is preferable as an electron conductive substance.

次にこの酵素電極の製造法について説明する。Next, a method for manufacturing this enzyme electrode will be explained.

まず、粉末状とした電子伝導性物質と不溶性レドックス
化合物を十分混合する。
First, a powdered electronic conductive substance and an insoluble redox compound are thoroughly mixed.

この混合物上に酵素、必要ならば補酵素をも含めてグル
クルアルデヒドなどの架橋試薬を用いて固定化する。
The enzyme, including coenzymes if necessary, is immobilized onto this mixture using a cross-linking reagent such as glucuraldehyde.

次に、得られた電子伝導性物質、不溶性レドックス化合
物、固定化酵素からなる混合物の少量と電子伝導性物質
とを例えばプレス成型などにより一体成型する。
Next, a small amount of the obtained mixture consisting of the electron conductive substance, insoluble redox compound, and immobilized enzyme is integrally molded with the electron conductive substance by, for example, press molding.

この場合成型体の強度を上げるためには、結着剤を用い
ても良い。
In this case, a binder may be used to increase the strength of the molded product.

また、酵素の固定化については、上記方法以外に、電子
伝導性物質と酵素を直接化学結合するなど、各種の方法
を用いることができる。
In addition to the above methods, various methods can be used to immobilize the enzyme, such as direct chemical bonding of the electron conductive substance and the enzyme.

以下本発明についてその実施例により説明する。The present invention will be explained below with reference to Examples.

電子伝導性物質としてのアセチレンブラック、黒鉛など
のカーボン粉末と、不溶性レドックス化合物としてのク
ロルアニルを十分に混合する。
Carbon powder such as acetylene black or graphite as an electron conductive substance and chloranil as an insoluble redox compound are thoroughly mixed.

次にこの混合物上にグルタルアルデヒドを用いて、酸化
還元酵素であるグルコーヌオキシダーゼを固定化する。
Next, glucone oxidase, which is an oxidoreductase, is immobilized on this mixture using glutaraldehyde.

こうして得られたカーボン、クロルアニル、固定化グル
コーヌオキシダーゼの混合物の少量とカーボン粉末をプ
レス成型により一体成型する。
A small amount of the thus obtained mixture of carbon, chloranil, and immobilized glucone oxidase and carbon powder are integrally molded by press molding.

−り記の酵素電極を用いて、グルコーヌ濃度を2X10
”−4モル/lとしたときの電流値の変化を第4図に示
す。
- Using the enzyme electrode described above, increase the concentration of glucone to 2x10
FIG. 4 shows the change in current value when the current value is -4 mol/l.

またグルコーヌ濃度と電流増加量の関係を第5図に示す
Furthermore, the relationship between the glucone concentration and the amount of increase in current is shown in FIG.

図より明らかなごとく、この酵素電極は、基質の添加に
対して迅速に応答し、かつ残余電流も少なく、基質濃度
変化に対する応答直線性も良好であるなど、優れた特注
を有する。
As is clear from the figure, this enzyme electrode has excellent custom features, such as rapid response to substrate addition, low residual current, and good response linearity to changes in substrate concentration.

酵素が、アルコール脱水素酵素などのように、補酵素を
必要とする場合には、酵素とともに補酵素をも、電子伝
導性物質と不溶性レドツクヌ化合物の混合物上に固定化
しておくと、前記同様に良好な応答特性が得られた。
When an enzyme requires a coenzyme, such as alcohol dehydrogenase, if the coenzyme is immobilized together with the enzyme on a mixture of an electron conductive substance and an insoluble redotsukunu compound, it can be processed in the same way as above. Good response characteristics were obtained.

レドツクヌ化合物としては、クロルアニルの他にブロム
アニルあるいは各種レドツクヌポリマーなどの不溶性レ
ドツクヌ化合物を用いても良い。
As the redotsukunu compound, in addition to chloranil, insoluble redotsukunu compounds such as bromoanil or various redotsukunu polymers may be used.

以上述べた如く、本発明によれば、酵素、レドツクヌ化
合物の有効利用を図り、きわめて容易に優れた性能を有
する酵素電極を得ることができる6
As described above, according to the present invention, it is possible to effectively utilize enzymes and Redotsukunu compounds, and to obtain enzyme electrodes with excellent performance very easily.

【図面の簡単な説明】[Brief explanation of the drawing]

第1図は本発明の酵素電極の構成例を示す図、第2図は
他の構成例を示す図、第3図は基質濃度の測定系を示す
図、第4図は酵素電極のグルコーヌに対する応答特注を
示す図、第5図はグルコーヌ濃度と電流増加量との関係
を示す。 1・・・・・・第1の層、2・・・・・・第2の層。
Fig. 1 shows an example of the structure of the enzyme electrode of the present invention, Fig. 2 shows another example of the structure, Fig. 3 shows a substrate concentration measurement system, and Fig. 4 shows how the enzyme electrode reacts to glucone. FIG. 5, a diagram showing the response customization, shows the relationship between the glucone concentration and the amount of increase in current. 1...First layer, 2...Second layer.

Claims (1)

【特許請求の範囲】 1 電子伝導性物質からなる第1の層と、酸化還元酵素
と共役する不溶性レドックス化合物と電子伝導性物質と
の混合物からなり、内部に酸化還元酵素を固定化した第
2の層とを有することを特徴とする酵素電極。 2 電子伝導性物質からなる第1の層と、酸化還元酵素
と共役する不溶性レドックス化合物と電子伝導性物質と
の混合物からなり、内部に酸化還元酵素とその補酵素と
を固定化した第2の層とを有することを特徴とする酵素
電極。 3 酸化還元酵素と共役する不溶性レドックス化合物と
電子伝導性物質との混合物に架橋試薬により酸化還元酵
素を固定化する工程と、前記酸化還元酵素を固定化した
混合物の層さ電子伝導性物質の層とを一体に成型する工
程とを有する酵素電極の製造法。
[Scope of Claims] 1. A first layer made of an electron-conductive substance, and a second layer made of a mixture of an insoluble redox compound conjugated with an oxidoreductase and an electron-conductivity substance, in which the oxidoreductase is immobilized. An enzyme electrode characterized in that it has a layer of. 2. A first layer made of an electron-conductive substance, and a second layer made of a mixture of an insoluble redox compound conjugated with an oxidoreductase and an electron-conductivity substance, in which the oxidoreductase and its coenzyme are immobilized. An enzyme electrode characterized by having a layer. 3 Immobilizing the oxidoreductase on a mixture of an insoluble redox compound conjugated with the oxidoreductase and an electron conductive substance using a crosslinking reagent, and a layer of the electron conductive substance containing the mixture in which the oxidoreductase is immobilized. and a step of integrally molding the enzyme electrode.
JP53084482A 1978-07-10 1978-07-10 Enzyme electrode and its manufacturing method Expired JPS5816698B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP53084482A JPS5816698B2 (en) 1978-07-10 1978-07-10 Enzyme electrode and its manufacturing method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP53084482A JPS5816698B2 (en) 1978-07-10 1978-07-10 Enzyme electrode and its manufacturing method

Publications (2)

Publication Number Publication Date
JPS5510584A JPS5510584A (en) 1980-01-25
JPS5816698B2 true JPS5816698B2 (en) 1983-04-01

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Country Status (1)

Country Link
JP (1) JPS5816698B2 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0117914Y2 (en) * 1981-09-18 1989-05-24

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Publication number Publication date
JPS5510584A (en) 1980-01-25

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