【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）こ検出する方法が用いられている。To treat an enzyme reaction as an electrochemical reaction, for example, a method is used in which an appropriate redox compound that is conjugated with the enzyme reaction system is interposed, and the redox reaction of this redox compound is detected electrochemically. .
具体的に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),
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.
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々検討した結果、電子伝導性物
質として例えばカーボン粉末を用い、これと不溶性レド
ックス化合物との混合物をプレス成型し、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。As a result of various studies on how to obtain an enzyme electrode in which these enzymes and redox compounds are integrally immobilized, we found that carbon powder, for example, is used as the electron conductive material, a mixture of this and an insoluble redox compound is press-molded, and a mixture of this and an insoluble redox compound is press-molded. We have found a method of immobilizing the enzyme, or a method of mixing carbon powder on which the enzyme has been immobilized in advance in the mixture, and then forming a molded body.
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て、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、電子伝導性物質と不磨性レドックス化合
物とからなる第２の層と、第２の層上に固定化された酵
素からなる第３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 includes a first layer made of an electron-conductive substance, a second layer made of an electron-conductive substance and a non-polishing redox compound, and an enzyme electrode immobilized on the second layer. It is characterized by comprising a third layer consisting of an enzyme.
ここで、前記不活性レドックス化合物：ま前記酵素と共
役するものであり、必要に応じて第３の層には、第２の
層上に固定化した補酵素を加える。Here, the inactive redox compound is one that is conjugated with the enzyme, and if necessary, a coenzyme immobilized on the second layer is added to the third layer.
第１図は本発明による酵素電極の構成の一例を示す。FIG. 1 shows an example of the structure of an enzyme electrode according to the present invention.
図において、１は電子伝導性物質からなる層、２は不溶
性レドックス化合物と電子伝導性物質からなる層、３は
固定化された酵素を含む層であり、不磨性レドックス化
合物は前記酵素と共役する。In the figure, 1 is a layer made of an electron conductive substance, 2 is a layer made of an insoluble redox compound and an electron conductive substance, and 3 is a layer containing an immobilized enzyme, and the non-polishing redox compound is conjugated with the enzyme. do.
これらの３層は一体成型により構成されている。These three layers are constructed by integral molding.
層２と３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と層３の集電
体および基体の役割を果たす。Layers 2 and 3 are the parts that allow reactions between the substrate, enzyme, and redox compound to take place, and layer 1 serves as a current collector and substrate for layers 2 and 3.
これら３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 three 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４は記録計、５はポテンショスタット、６は参照極
、７は塩橋、８は対極、９は上記の酵素電極１０を装着
した電極ホルダー、１１は基質を含むｐＨ５，６のリン
酸緩衝液である。In the figure, 4 is a recorder, 5 is a potentiostat, 6 is a reference electrode, 7 is a salt bridge, 8 is a counter electrode, 9 is an electrode holder equipped with the enzyme electrode 10 described above, and 11 is a phosphoric acid containing a substrate at pH 5 and 6. It is a buffer solution.
なお酵素電極１０は、固定化された酵素を含む層３が緩
衝液１１と接触するようにホルダー９に装着され、電子
伝導性物質の層１には例えば白金のリードが付けられる
。The enzyme electrode 10 is attached to the holder 9 so that the layer 3 containing the immobilized enzyme is in contact with the buffer solution 11, and the layer 1 of the electron conductive material is attached with leads made of, for example, platinum.
酵素電極を浸漬後、電極電位を参照極に対し一定電位に
保持した後、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 is immersed, the electrode potential is maintained at a constant potential with respect to the reference electrode, and then the amount of change in the redox current of the redox compound due to the change in the concentration of the substrate is detected.
基質の単位濃度当たりの電流変化量が大きいほど、また
、基質濃度と電流変化量の間の直線関係が基質濃度のよ
り広範囲にわたって成立するほど酵素電極の性能が良い
と言える。It can be said that the performance of the enzyme electrode is better as the amount of change in current per unit concentration of substrate is larger, and the linear relationship between the substrate concentration and the amount of change in current is established over a wider range of substrate concentrations.
本発明の酵素電極においては、固定化酵素層が試料溶液
に接しており、基質は酵素層内を拡散して電極内部へ達
し、反応にあずかる。In the enzyme electrode of the present invention, the immobilized enzyme layer is in contact with the sample solution, and the substrate diffuses within the enzyme layer to reach the inside of the electrode and participate in the reaction.
すなわち、酵素層により基質の移動が制御されるので、
比較的高濃度の基質に対しても先に述べた直線関係が維
持される。In other words, the movement of the substrate is controlled by the enzyme layer, so
The previously mentioned linear relationship is maintained even for relatively high concentrations of substrate.
また一方、この酵素電極では、少量の酵素、レドックス
化合物を電子伝導性物質とともに最適な構成としている
ので、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。On the other hand, in this enzyme electrode, a small amount of an enzyme and a redox compound are optimally configured together with an electron conductive substance, so that expensive enzymes and redox compounds can be used effectively.
電子伝導性物質としては酸化還元に対して安定な金属や
、カーボン、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, metals that are stable against redox, carbon, or conductive metal oxides such as tin oxide can be used.
特にカーボンは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 stable and highly conductive substance and 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.
次にこの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 this mixture and an electronically conductive substance are integrally molded, for example, by press molding.
この場合、成型体の強度を上げるために、適当な結着剤
を用いてもよい。In this case, an appropriate binder may be used to increase the strength of the molded product.
こうして得られた、電子伝導性物質から成る層と、電子
伝導性物質と手芸性レドックス化合物とからなる層の２
層から構成される成型体において、不溶性レドックス化
合物を含有する層の上に、酵素、必要ならば補酵素をも
含めて固定化する。The two layers thus obtained, one consisting of an electronically conductive substance and the other layer consisting of an electronically conductive substance and a handicraft redox compound.
In a molded body composed of layers, enzymes, including coenzymes if necessary, are immobilized on the layer containing the insoluble redox compound.
酵素の固定化には、グルタルアルデヒドなどの架橋試薬
による固定化法など、各種の方法を用いることができる
。Various methods can be used to immobilize the enzyme, such as an immobilization method using a crosslinking reagent such as glutaraldehyde.
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、不容性レドックス化合物としてのブ
ロムアニルを十分混合する。Carbon powder such as acetylene black or graphite as an electron conductive substance and bromoanil as an insoluble redox compound are thoroughly mixed.
次にこ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。Next, a small amount of this mixture and carbon powder are integrally molded by press molding.
得られた成型体のブロムアニルを含有する層の上へ、酸
化還元酵素であるグルコースオキシダーゼをグルタルア
ルデヒドにより固定化する。Glucose oxidase, which is an oxidoreductase, is immobilized onto the bromoanil-containing layer of the obtained molded body using glutaraldehyde.
この本発明による酵素電極をＡとする。比較のための酵
素電極として、カーボン粉末と、予めグルコースオキシ
ダーゼを固定化したカーボン粉末と、ブロムアニルとを
十分混合した後、プレス成型したものを作製した。This enzyme electrode according to the present invention is designated as A. As an enzyme electrode for comparison, carbon powder, carbon powder on which glucose oxidase had been immobilized in advance, and bromoanil were thoroughly mixed and then press-molded.
この酵素電極をＢとする。This enzyme electrode is designated as B.
上記の酵素電極を用いて、グルコース濃度を２×１０
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, the glucose concentration was adjusted to 2 × 10
FIG. 4 shows the change in current value when expressed as mol/l.
また、グルコース濃度と電流増加量の関係を第５図に示
す。Further, FIG. 5 shows the relationship between the glucose concentration and the amount of increase in current.
図より明らかなごとく、酵素電極Ａは固ポ化酵素層を有
するもののＢと同様、基質の添加に対し迅速に応答し、
かつ基質濃度変化に対する応答直線性が向上するなど、
優れた特性を有する。As is clear from the figure, enzyme electrode A, which has a solidified enzyme layer, responds quickly to the addition of a substrate, similar to electrode B.
and improved linearity of response to changes in substrate concentration.
Has excellent properties.
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 the enzyme requires a coenzyme, such as alcohol dehydrogenase, good response characteristics were obtained by immobilizing the coenzyme together with the enzyme.
レドックス化合物としては、ブロムアニルの他にクロル
アニル、あるいは各種レドックスポリマーなどの不溶性
レドックス化合物を用いてもよい。As the redox compound, in addition to bromoanil, insoluble redox compounds such as chloranil or various redox polymers may be used.
以上述べたごとく、本発明によれば、酵素、レドックス
化合物の有効利用をはかり、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。As described above, according to the present invention, it is possible to effectively utilize enzymes and redox compounds, and to obtain an enzyme electrode with excellent performance very easily.
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スに対する応答特性を
示す図、第５図はグルコース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、３
・・・・・・第３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 glucose. FIG. 5, a diagram showing response characteristics, shows the relationship between glucose concentration and current increase amount. 1...First layer, 2...Second layer, 3
...Third layer.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP53084481AJPS5816697B2 (en) | 1978-07-10 | 1978-07-10 | Enzyme electrode and its manufacturing method |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP53084481AJPS5816697B2 (en) | 1978-07-10 | 1978-07-10 | Enzyme electrode and its manufacturing method |
| Publication Number | Publication Date |
|---|---|
| JPS5510583A JPS5510583A (en) | 1980-01-25 |
| JPS5816697B2true JPS5816697B2 (en) | 1983-04-01 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP53084481AExpiredJPS5816697B2 (en) | 1978-07-10 | 1978-07-10 | Enzyme electrode and its manufacturing method |
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