JPH06105237B2 - Enzyme and mediator immobilization method - Google Patents
Enzyme and mediator immobilization methodInfo
- Publication number
- JPH06105237B2 JPH06105237B2 JP63252422A JP25242288A JPH06105237B2 JP H06105237 B2 JPH06105237 B2 JP H06105237B2 JP 63252422 A JP63252422 A JP 63252422A JP 25242288 A JP25242288 A JP 25242288A JP H06105237 B2 JPH06105237 B2 JP H06105237B2
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- mediator
- immobilizing
- response
- acid
- 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 - Lifetime
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Description
【発明の詳細な説明】 産業上の利用分野 本発明は、電解重合法を用いた酵素の固定化方法に関
し、特に導電性高分子膜中への酵素及びメディエーター
の固定化方法に関する。TECHNICAL FIELD The present invention relates to a method for immobilizing an enzyme using an electropolymerization method, and more particularly to a method for immobilizing an enzyme and a mediator in a conductive polymer film.
従来の技術 従来、バイオセンサなどを構成するために必要な酵素や
メディエーターの固定化方法としては、種々の方法が試
みられている。例えば、酵素については、グルタルアル
デヒドなどの固定化試薬を用いる共有結合法や、光反応
性樹脂を用いて高分子マトリックス中に固定化する包括
固定法などがある。また、メディエーターについては、
化学修飾した電極表面にメディエーター分子を結合させ
る方法、あるいは、メディエーターをポリマー化するな
どの固定化方法がある。2. Description of the Related Art Conventionally, various methods have been attempted as methods for immobilizing enzymes and mediators necessary for constructing biosensors and the like. For example, with respect to the enzyme, there are a covalent bond method using an immobilizing reagent such as glutaraldehyde and an entrapping immobilizing method in which a photoreactive resin is used to immobilize the enzyme in a polymer matrix. For mediators,
There is a method of binding a mediator molecule to the chemically modified electrode surface, or an immobilization method such as polymerizing the mediator.
発明が解決しようとする課題 この様な従来の構成では、メディエーターの電気化学的
酸化還元は電極近傍に固定化されたものに限定されるの
は当然であり、充分な電子伝達の役割を担うことができ
ない。一方、固定化反応にともない酵素活性が低下し、
さらに固定化された酵素とメディエーター間の電子伝達
反応も双方が近接している部分に限られる。この様に、
従来の固定化方法では、酵素や補酵素の活性保持や、メ
ディエーターを介した酵素〜電極反応系を構成する上で
課題がある。Problems to be Solved by the Invention In such a conventional configuration, it is natural that the electrochemical redox of the mediator is limited to that immobilized in the vicinity of the electrode, and it plays a role of sufficient electron transfer. I can't. On the other hand, the enzyme activity decreases with the immobilization reaction,
Furthermore, the electron transfer reaction between the immobilized enzyme and mediator is also limited to the area where both are in close proximity. Like this
The conventional immobilization method has problems in maintaining the activity of enzymes and coenzymes and in constructing an enzyme-electrode reaction system via a mediator.
課題を解決するための手段 本発明は上記課題を解決するため、少なくとも酵素、メ
ディエーター及び電解により高分子膜を形成するモノマ
ーを含有する電解液を用い、電解重合により電極基体上
に生成する導電性高分子膜中へ前記酵素とともにメディ
エーターを固定化するものである。Means for Solving the Problems To solve the above problems, the present invention uses an electrolytic solution containing at least an enzyme, a mediator, and a monomer to form a polymer film by electrolysis, and conducts electroconductivity generated on an electrode substrate by electrolytic polymerization. This is to immobilize the mediator together with the enzyme in the polymer membrane.
作用 本発明によれば、電解重合反応時にメディエーターのア
ニオン基がドーピングされて導電性高分子膜が形成さ
れ、同時に酵素あるいはさらに加えて補酵素が上記膜中
にトラップされる。このため、きわめて容易にかつ温和
な条件下で酵素とメディエーターを導電性高分子膜中に
固定化することができ、酵素と電極の間の電子移動を円
滑に行なわせることが可能となる。Action According to the present invention, the anionic group of the mediator is doped during the electropolymerization reaction to form a conductive polymer film, and at the same time, an enzyme or additionally a coenzyme is trapped in the film. Therefore, it is possible to immobilize the enzyme and the mediator in the conductive polymer film extremely easily and under mild conditions, and it is possible to smoothly carry out electron transfer between the enzyme and the electrode.
実施例 以下、本発明を実施例により説明する。Examples Hereinafter, the present invention will be described with reference to Examples.
（実施例１） 酵素としてグルコースオキシダーゼ（GOD）、メディエ
ーターとしてフェロセンカルボン酸、電解重合用のモノ
マーとしてピロールをそれぞれ用いた場合について説明
する。Example 1 A case where glucose oxidase (GOD) is used as an enzyme, ferrocenecarboxylic acid is used as a mediator, and pyrrole is used as a monomer for electrolytic polymerization will be described.
GOD1mg/ml、ピロール0.2mol/l、フェロセンカルボン酸1
x10-4mol/lをそれぞれ含有する25℃のリン酸緩衝液中に
電極基体として白金板を浸漬し、飽和カロメル電極に対
し0.8Vの電位に設定して電解した。析出電気量（電解電
気量）200ミリクーロン（mC）／cm2の場合に得られた電
解重合膜をａとする。また、上記の全く同様の方法によ
り、析出電気量が100mC/cm2の場合に得られた電解重合
膜をｂ、400mC/cm2の場合に得られた電解重合膜をｃと
する。GOD 1mg / ml, pyrrole 0.2mol / l, ferrocenecarboxylic acid 1
A platinum plate was immersed as an electrode substrate in a phosphate buffer solution containing x10 -4 mol / l at 25 ° C, and electrolysis was performed by setting the potential to 0.8 V with respect to the saturated calomel electrode. The electrolytic polymerization film obtained when the amount of deposited electricity (electrolytic amount of electricity) was 200 millicoulombs (mC) / cm 2 was designated as a. Further, by exactly the same method described above, b the resulting electrolytic polymerization film when deposition quantity of electricity of 100 mC / cm 2, the electrolytic polymerization film was obtained when the 400mC / cm 2 and c.
上記、a,b,c3種類の白金板上に得られた電解重合膜につ
いてグルコースに対する応答を調べた。測定はリン酸緩
衝液中に上記の電解重合膜を形成した白金板を浸漬した
後、、参照極の飽和カロメル電極に対して＋0.4Vに設定
し、グルコースを加えた際の電流増加量（応答電流）を
測定した。The response to glucose was investigated for the electropolymerized membranes obtained on the above a, b, and c types of platinum plates. The measurement was carried out by immersing the platinum plate on which the above electropolymerized film was formed in a phosphate buffer, then setting it to +0.4 V with respect to the saturated calomel electrode of the reference electrode, and increasing the amount of current when glucose was added ( The response current) was measured.
第１図にａ、ｂ、ｃ各々の場合についてグルコース濃度
と応答電流の関係を、また、第２図にｃについて高濃度
のグルコースに対する応答を示す。図より明かなごと
く、本発明になる固定化膜をセンサに適用した場合、そ
の応答濃度域を電解重合時の析出電気量で容易に制御す
ることができる。FIG. 1 shows the relationship between glucose concentration and response current for each of a, b, and c, and FIG. 2 shows the response to high glucose for c. As is clear from the figure, when the immobilization film of the present invention is applied to a sensor, its response concentration range can be easily controlled by the amount of deposited electricity during electrolytic polymerization.
一方、第３図に析出電気量とグルコース濃度20mmol/lに
対する応答電流の関係を示すが、200mC/cm2付近で最大
の応答が得られた。析出電気量が200mC/cm2を越えると
膜中のイオン種や基質であるグルコースの拡散が遅くな
るために応答電流が低下するものと考えられる。しかし
ながら、第３図に示すように応答濃度域を飛躍的に増大
することができる。On the other hand, FIG. 3 shows the relationship between the amount of deposited electricity and the response current with respect to the glucose concentration of 20 mmol / l. The maximum response was obtained at around 200 mC / cm 2 . It is considered that when the amount of electricity for deposition exceeds 200 mC / cm 2 , the response current decreases because the diffusion of ionic species in the film and glucose as a substrate slows down. However, the response concentration range can be dramatically increased as shown in FIG.
比較のために、メディエーターのフェロセンカルボン酸
を膜中に有しないGOD固定化膜を前記同様に作製した。
この膜のグルコースに対する応答を、酸素を飽和したリ
ン酸緩衝液を用いた場合、およびフェロセンカルボン酸
を溶解したリン酸緩衝液を用いた場合についてそれぞれ
前記同様に検討したところ、いずれも応答電流が50％以
下、応答も大幅に遅い等の特性を示した。For comparison, a GOD-immobilized membrane having no mediator ferrocenecarboxylic acid in the membrane was prepared in the same manner as above.
The response of this membrane to glucose was examined in the same manner as above for the case of using a phosphate buffer solution saturated with oxygen and for the case of using a phosphate buffer solution in which ferrocenecarboxylic acid was dissolved. The characteristics were 50% or less and the response was significantly slow.
（実施例２） 酵素として、GOD、β−フルクトシダーゼ、ムタロター
ゼをそれぞれ1mg/ml、ピロール0.2mol/l、ナフトキノン
スルホン酸1x10-4mol/lをそれぞれ含有する25℃のリン
酸緩衝液中に電極基体として白金板を浸漬し、実施例１
と同様にして酵素、メディエーター固定化膜を作製し
た。得られた電解重合膜について、しょ糖に対する応答
を実施例１と同様に測定したところ、前記グルコースの
場合と同様に良好な応答が得られた。(Example 2) In a phosphate buffer at 25 ° C containing GOD, β-fructosidase, and mutarotase at 1 mg / ml, pyrrole at 0.2 mol / l, and naphthoquinonesulfonic acid at 1x10 -4 mol / l, respectively, as enzymes. Example 1 was immersed in a platinum plate as an electrode substrate.
An enzyme- and mediator-immobilized membrane was prepared in the same manner as in. The response of the obtained electropolymerized membrane to sucrose was measured in the same manner as in Example 1. As a result, a good response was obtained as in the case of glucose.
（実施例３） 酵素としてアルコールデヒドロゲナーゼ、補酵素として
ニコチンアミドアデニンジヌクレオチド（NAD）、メデ
ィエーターとしてベンゾキノンスルホン酸、電極基体と
してカーボン電極を用いた以外は実施例１と同様の方法
により電解重合膜を作製し、得られた膜のエタノールに
対する応答を実施例１と同様に測定したところ、前記グ
ルコースの場合と同様に良好な応答が得られた。Example 3 An electrolytic polymer film was prepared in the same manner as in Example 1 except that alcohol dehydrogenase was used as an enzyme, nicotinamide adenine dinucleotide (NAD) was used as a coenzyme, benzoquinone sulfonic acid was used as a mediator, and a carbon electrode was used as an electrode substrate. When the response of the produced membrane to ethanol was measured in the same manner as in Example 1, a good response was obtained as in the case of glucose.
以上に述べた実施例においては、メディエーターとして
フェロセンカルボン酸、ベンゾキノンスルホン酸、ナフ
トキノンスルホン酸を用いた場合について示したが、こ
れらに限定されることはない。メディエーターとして
は、用いる酵素の活性中心またはさらに添加した補酵素
の酸化還元電位よりも貴な酸化還元電位を有するもので
あればよい。メディエーターのアニトン基が電解重合で
形成される高分子膜中にドーパントとして取り込まれる
ことにより膜に導電性が付与さる。このため、この様な
状態で固定化されたメディエーターは、同時に膜中に包
括固定された酵素や補酵素との間の酸化還元反応に極め
て効率よく働くものと考えられる。Although the examples described above show the case where ferrocenecarboxylic acid, benzoquinonesulfonic acid, or naphthoquinonesulfonic acid is used as the mediator, the mediator is not limited to these. Any mediator may be used as long as it has a redox potential nobler than that of the active center of the enzyme used or the coenzyme added thereto. Conductivity is imparted to the film by incorporating the anitone group of the mediator as a dopant into the polymer film formed by electrolytic polymerization. Therefore, it is considered that the mediator immobilized in such a state works very efficiently in the redox reaction between the enzyme and the coenzyme entrapped and immobilized in the membrane at the same time.
また、電解により高分子膜を形成するモノマーとしては
実施例に示したピロール以外にピロール誘導体、あるい
はチオフェンやチオフェン誘導体も同様に用いることが
できる。Further, as the monomer for forming the polymer film by electrolysis, a pyrrole derivative, or thiophene or a thiophene derivative can be similarly used in addition to the pyrrole shown in the examples.
さらに、酵素としては上記実施例に示したグルコースオ
キシダーゼなどに限定されることはなく、アルコールオ
キシダーゼやコレステロールオキシダーゼなど種々の酵
素を用いることができる。また、単一の酵素に限られる
ことはなく、複合酵素系にも適用できることは実施例に
示した通りである。さらに、アルコールデヒドロゲナー
ゼなどのように補酵素を必要とする酵素系については、
その補酵素も同時に固定化することにより、メディエー
ターを介しての効果的な電子伝達を行なわせることが可
能である。Furthermore, the enzyme is not limited to the glucose oxidase shown in the above examples, and various enzymes such as alcohol oxidase and cholesterol oxidase can be used. Further, as shown in the examples, the present invention is not limited to a single enzyme and can be applied to a complex enzyme system. Furthermore, for enzyme systems that require coenzymes such as alcohol dehydrogenase,
By immobilizing the coenzyme at the same time, it is possible to carry out effective electron transfer via the mediator.
発明の効果 以上のように本発明によれば、きわめて容易に酵素とメ
ディエーターを導電性高分子膜中に固定化することがで
き、酵素と電極の間の電子移動を極めて円滑に行なわせ
ることが可能となる。EFFECTS OF THE INVENTION As described above, according to the present invention, the enzyme and the mediator can be immobilized in the conductive polymer film very easily, and the electron transfer between the enzyme and the electrode can be carried out very smoothly. It will be possible.
第１図および第２図は本発明の一実施例になるグルコー
スセンサの応答特性図、第３図はその析出電気量と応答
特性を示す図である。1 and 2 are response characteristic diagrams of a glucose sensor according to an embodiment of the present invention, and FIG. 3 is a diagram showing the amount of deposited electricity and the response characteristic.
フロントページの続き (72)発明者 飯島 孝志 大阪府門真市大字門真1006番地 松下電器 産業株式会社内 (56)参考文献 特開 平１−32160（ＪＰ，Ａ)Front page continuation (72) Inventor Takashi Iijima 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (56) Reference JP-A-1-32160 (JP, A)
Claims (4)
により高分子膜を形成するモノマーを含有する電解液を
用い、電解重合により、電極基体上に生成する導電性高
分子膜中へ前記酵素とともにメディエーターを固定化す
ることを特徴とする酵素及びメディエーターの固定化方
法。1. An electrolysis solution containing at least an enzyme, a mediator, and a monomer that forms a polymer film by electrolysis, and the mediator is fixed together with the enzyme in a conductive polymer film formed on an electrode substrate by electrolytic polymerization. A method for immobilizing an enzyme and a mediator, which comprises:
りも貴な酸化還元電位を有する請求項１に記載の酵素及
びメディエーターの固定化方法。2. The method for immobilizing an enzyme and a mediator according to claim 1, wherein the mediator has a redox potential nobler than the redox potential of the enzyme.
酸、ベンゾキノンスルホン酸、ナフトキノンスルホン酸
から選ばれた少なくとも一種からなる請求項２に記載の
酵素及びメディエーターの固定化方法。3. The method for immobilizing an enzyme and a mediator according to claim 2, wherein the mediator comprises at least one selected from ferrocenecarboxylic acid, benzoquinonesulfonic acid and naphthoquinonesulfonic acid.
体、あるいはチオフェンまたはチオフェン誘導体から選
ばれたものである請求項１に記載の酵素及びメディエー
ターの固定化方法。4. The method for immobilizing an enzyme and a mediator according to claim 1, wherein the monomer is selected from pyrrole or a pyrrole derivative, or thiophene or a thiophene derivative.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63252422A JPH06105237B2 (en) | 1988-10-06 | 1988-10-06 | Enzyme and mediator immobilization method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63252422A JPH06105237B2 (en) | 1988-10-06 | 1988-10-06 | Enzyme and mediator immobilization method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0299850A JPH0299850A (en) | 1990-04-11 |
| JPH06105237B2 true JPH06105237B2 (en) | 1994-12-21 |
Family
ID=17237140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63252422A Expired - Lifetime JPH06105237B2 (en) | 1988-10-06 | 1988-10-06 | Enzyme and mediator immobilization method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH06105237B2 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5264104A (en) * | 1989-08-02 | 1993-11-23 | Gregg Brian A | Enzyme electrodes |
| US5320725A (en) * | 1989-08-02 | 1994-06-14 | E. Heller & Company | Electrode and method for the detection of hydrogen peroxide |
| US5264105A (en) * | 1989-08-02 | 1993-11-23 | Gregg Brian A | Enzyme electrodes |
| US5262035A (en) * | 1989-08-02 | 1993-11-16 | E. Heller And Company | Enzyme electrodes |
| GB9019126D0 (en) * | 1990-09-01 | 1990-10-17 | Cranfield Biotech Ltd | Electrochemical biosensor stability |
| DE10221435B4 (en) | 2002-05-14 | 2004-10-28 | Isabella Dr. Moser | Enzyme electrode arrangement, a method for the production thereof and a biosensor arrangement comprising this enzyme electrode arrangement |
| JP2007027019A (en) * | 2005-07-21 | 2007-02-01 | Ebara Corp | Anode for biological power generation and method and device for biological power generation utilizing same |
-
1988
- 1988-10-06 JP JP63252422A patent/JPH06105237B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0299850A (en) | 1990-04-11 |
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