JP2777569B2 - Enzyme function electrode - Google Patents
Enzyme function electrodeInfo
- Publication number
- JP2777569B2 JP2777569B2 JP63319412A JP31941288A JP2777569B2 JP 2777569 B2 JP2777569 B2 JP 2777569B2 JP 63319412 A JP63319412 A JP 63319412A JP 31941288 A JP31941288 A JP 31941288A JP 2777569 B2 JP2777569 B2 JP 2777569B2
- Authority
- JP
- Japan
- Prior art keywords
- electrode
- enzyme
- fiber
- present
- plating method
- Prior art date
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Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、フレキシビリ性を持ち、表面積が大きく、
軽く、カットが容易で、応用範囲の広い、バイオセンサ
ー、バイオリアクター用酵素機能電極に関する。DETAILED DESCRIPTION OF THE INVENTION (Industrial application field) The present invention has flexibility, a large surface area,
The present invention relates to an enzyme-functional electrode for biosensors and bioreactors that is light, easy to cut, and has a wide range of applications.
（従来の技術） バイオセンサー用の酵素機能電極は、被測定液中の生
化学物質濃度の測定手段として、酵素を利用して測定対
象の生化学物質を反応させ、その生成物の濃度を電気的
に測定し、間接的に酵素反応に寄与する基質を定量する
ために用いられている。(Conventional technology) An enzyme-functional electrode for a biosensor is a means for measuring the concentration of a biochemical substance in a liquid to be measured, by reacting a biochemical substance to be measured using an enzyme, and measuring the concentration of the product by using an enzyme. It is used for quantitatively measuring and quantitatively determining a substrate that indirectly contributes to an enzymatic reaction.
従来、上記バイオセンサーに使用される酵素電極とし
ては、作用電極及び対照電極よりなるものが知られてい
る。この作用電極は、白金よりなる下地電極に測定対象
となる生化学物質を基質とする酵素を固定化した高分子
膜と、この酵素による酵素反応の生成物の一つを選択し
て透過する膜とからなるものである。Conventionally, as an enzyme electrode used in the biosensor, an enzyme electrode comprising a working electrode and a control electrode is known. The working electrode consists of a polymer membrane in which an enzyme whose substrate is a biochemical substance to be measured is immobilized on a base electrode made of platinum, and a membrane that selectively transmits one of the products of an enzyme reaction by the enzyme. It consists of:
（発明が解決しようとする課題） 上述したように、通常、酵素電極には白金よりなる下
地電極が使用されている。例えば、特開昭62−235557号
は下地電極表面に高分子膜を形成しこの高分子膜中に、
被測定検体中に含まれ測定対象生化学物質を基質とする
酵素と、この酵素による反応に伴い酸化又は還元される
酸化還元物質とを固定してなる作用電極と、この作用電
極と共に被測定検体中に浸漬される対照電極とよりなる
酵素電極を開示するものであるが、下地電極に白金板そ
のものを使用している。その他特開昭62−215861号、特
開昭62−144061号等に種々の酵素機能電極が開示されて
いるが、これらに開示された酵素電極は、白金板そのも
のをカソードとし、酵素透過膜、酵素固定化膜及び半透
膜積層して形成された酵素電極である。(Problem to be Solved by the Invention) As described above, usually, a base electrode made of platinum is used for the enzyme electrode. For example, JP-A-62-235557 discloses a method of forming a polymer film on the surface of a base electrode,
A working electrode comprising an enzyme containing a biochemical substance to be measured as a substrate and a redox substance which is oxidized or reduced by the reaction of the enzyme, and a working electrode together with the working electrode; An enzyme electrode comprising a control electrode immersed therein is disclosed, but a platinum plate itself is used as a base electrode. Others JP-A-62-215861, JP-A-62-144061 and the like disclose various enzyme functional electrodes, but the enzyme electrodes disclosed therein, the platinum plate itself as a cathode, an enzyme permeable membrane, It is an enzyme electrode formed by laminating an enzyme immobilized membrane and a semipermeable membrane.
上述した電極、白金電極等は、非測定検体容器形体に
応じて電極の形を変えたい場合、平板でフレキシビリ性
に欠けるため自由に曲げることができず不都合である。
またカットが容易に出来ない点で加工性も悪い。また白
金電極等の表面はフラットで表面積が小さいので反応性
が小さく、感度が弱いと言う問題点も持っている。When it is desired to change the shape of the electrode, the platinum electrode and the like according to the shape of the non-measurement sample container, the electrode and the platinum electrode are inconvenient because they are flat and lack flexibility and cannot be freely bent.
In addition, workability is poor in that cutting cannot be easily performed. Further, the surface of the platinum electrode or the like is flat and has a small surface area, so that the reactivity is low and the sensitivity is low.
又最近酵素電極のバイオリアクターとしての利用とし
て、電極表面に酵素を直接固定化して、電極に加える電
圧を変化させて、反応を外部から制御することが行われ
るようになってきた。この際、バイオリアクターでは反
応性が重要である。この場合も白金電極は表面積が小さ
いため反応性が小さく、又感度が弱いと言う問題点があ
る。Recently, as an application of an enzyme electrode as a bioreactor, an enzyme has been directly immobilized on the electrode surface, the voltage applied to the electrode is changed, and the reaction is externally controlled. At this time, reactivity is important in the bioreactor. Also in this case, there is a problem that the platinum electrode has a small surface area and thus has low reactivity and low sensitivity.
バイオリアクター酵素電極を反応塔に望む形状で充填
できること、又充填量が多くなると軽いということが要
求されるが、この点でも白金電極等は問題が多い。It is required that the bioreactor enzyme electrode can be filled into the reaction tower in a desired shape, and that the more the filling amount is, the lighter it is, the platinum electrode and the like have many problems.
（課題を解決するための手段） 本発明の目的は上記従来技術の欠点を解決することに
ある。(Means for Solving the Problems) An object of the present invention is to solve the above-mentioned drawbacks of the prior art.
本発明の酵素機能電極は非金属性繊維である植物繊
維、動物繊維、人造繊維または無機繊維よりなる布はく
の電極部の表裏全体に金属をメッキして電極としこれに
酵素を直接固定化したものであり、非金属繊維布はくの
持つフレキシビリ性、表面凹凸性、軽量性、カット容易
性と金属の持つ電極性能とが極めて有効に結合されて、
従来技術の欠点が解消されるという効果を示す。The enzyme-functional electrode of the present invention is a non-metallic fiber made of vegetable fiber, animal fiber, artificial fiber or inorganic fiber. The flexibility of the non-metallic fiber fabric, surface irregularities, light weight, ease of cut and the electrode performance of the metal are very effectively combined,
This has the effect of eliminating the disadvantages of the prior art.
本発明の酵素機能電極のフレキシビリ性はJIS1096剛
軟性Ａ法（45゜カンチレバー法）によれば10mm〜130mm
の軟らかいものであるる。尚通常の白金電極は200mm以
上で実質上測定不可能である。The flexibility of the enzyme-functional electrode of the present invention is from 10 mm to 130 mm according to the JIS 1096 rigid-soft A method (45 ° cantilever method).
Is soft. In addition, a normal platinum electrode cannot be measured practically at 200 mm or more.
本発明に用いられる布はくは、綿、麻等の植物繊維、
羊毛、絹等の動物繊維、キュプラ、アセテート、ポリエ
ステル、ポリアミド、ポリアクリロニトリル、ポリウレ
タン、ポリプロピレン等の人造繊維、無機繊維の何れで
もよく、これら各々の単独あるいは二種以上の混紡、混
織の形でも差し支えない。布はくの形態は織物、編物、
レース、網、不織布、紙等である。好ましい繊度は１デ
ニール〜100デニールであるがこれに限定されるもので
はない。１デニール以下であってもさしつかえないが１
デニール以下の細い糸は製造が技術的に高度となりコス
ト高となる。又1000デニール以上では硬くなり本発明の
目的であるフレキシビリ性を持った電極を得ることが困
難となる。Cloth used in the present invention, cotton, vegetable fibers such as hemp,
Animal fibers such as wool, silk, etc., artificial fibers such as cupra, acetate, polyester, polyamide, polyacrylonitrile, polyurethane, polypropylene, etc., and inorganic fibers may be used. No problem. Cloths are woven, knitted,
Lace, net, non-woven fabric, paper, etc. The preferred fineness is from 1 denier to 100 denier, but is not limited thereto. Even if it is less than 1 denier, it may be 1
The production of fine yarns of denier or less is technically advanced and costly. On the other hand, if the denier is more than 1000 denier, it becomes difficult to obtain an electrode having flexibility, which is the object of the present invention.
本発明で電極に使用する金属としては金、銀、白金等
があるが、特に金と銀が好ましい。これらは直接非金属
繊維布はく上にメッキしてもよいが、ニッケル、銅等で
メッキした上にこれらをメッキすることも好ましい。メ
ッキ方法としては無電解メッキ法が好ましい。予め無電
解メッキ法で金属をメッキしたのち電解メッキを行って
も良い。The metal used for the electrode in the present invention includes gold, silver, platinum and the like, and gold and silver are particularly preferable. These may be plated directly on the non-metallic fiber cloth, but it is also preferable to plate them with nickel, copper or the like and then plate them. As a plating method, an electroless plating method is preferable. Electroplating may be performed after plating a metal by an electroless plating method in advance.
メッキ厚は0.05μが好ましいが、これらは限定されな
い。0.05μ以下では同電性不良、被膜強度不良となりや
すく、１μ以上では本発明の目的であるフレキシビリ性
を損なう恐れがあり、又コストが高くなり好ましくな
い。かくして安価に強固な金属化が達成される。The plating thickness is preferably 0.05 μm, but these are not limited. If it is 0.05 μm or less, poor electrical conductivity and poor film strength are apt to occur, and if it is 1 μm or more, the flexibility, which is the object of the present invention, may be impaired, and the cost increases, which is not preferable. Thus, strong metallization is achieved at low cost.
金属化布はく上への酵素の固定化は適宜の固定化法に
よって行ないうる。通常は光硬化性樹脂等の被膜形成性
樹脂の溶液又は分散液に酵素又は酵素液を含有させて塗
布する方法等によって行なわれる。好ましい方法の１つ
はポリウレタンのプレポリマー分散液を用いる方法であ
る。The enzyme can be immobilized on the metallized cloth by an appropriate immobilization method. Usually, it is carried out by a method in which an enzyme or an enzyme solution is contained in a solution or a dispersion of a film-forming resin such as a photo-curable resin and applied. One preferred method is to use a polyurethane prepolymer dispersion.
本発明に用いられる酵素は反応に電子の授受を行うも
のであれば何れの酵素であってもよく、グルコースオキ
シダーゼ、ガラクトースオキシダーゼ、アルコールオキ
シダーゼ、コレステロールオキシダーゼ等のオキシタゼ
酵素、グルコースデヒドロゲナーゼ、アルコールデヒド
ロゲナーゼ等のデヒドロゲナーゼ酵素、その他ジアホラ
ーゼ、カタラーゼ等が挙げられる。The enzyme used in the present invention may be any enzyme as long as it transfers electrons to the reaction, such as glucose oxidase, galactose oxidase, alcohol oxidase, oxidase enzymes such as cholesterol oxidase, glucose dehydrogenase, and alcohol dehydrogenase. Dehydrogenase enzymes, diaphorase, catalase and the like can be mentioned.
（実 施 例） 以下に、本発明の実施例について説明する。(Examples) Examples of the present invention will be described below.
（Ａ）電極の作製 次に述べるのはグルコースセンサー用電極、及びグル
コノラクトンリアクター電極を作製する場合の一実施例
である。(A) Production of Electrode The following is an example of producing an electrode for a glucose sensor and a gluconolactone reactor electrode.
○メッキ布はくの作製 下記処方にしたがい繊度50デニール（フイラメント数
24本）ポリエステルフイラメント糸よりなるタフタ（経
密度170本／吋、緯密度85本／吋）にニッケルメッキを
まず行いメッキ厚0.3μのニッケルメッキ布を得た。○ Preparation of plated cloth According to the following formula, fineness of 50 denier (number of filaments)
First, nickel plating was performed on a taffeta (a density of 170 threads / inch, a weft density of 85 threads / inch) made of polyester filament yarn to obtain a nickel-plated cloth having a plating thickness of 0.3 μm.
・触媒附与処理 処 方 塩化パラジウム 0.3g/L 塩化第一錫 15.0g/L 塩 酸 200cc/L 液 温 30℃ 処理時間 10分間 充分水洗後、80℃で熱風乾燥した。・ Catalyst treatment Treatment Palladium chloride 0.3 g / L Stannous chloride 15.0 g / L Hydrochloric acid 200 cc / L Temperature 30 ° C Treatment time 10 minutes After thoroughly washing with water, hot air drying was performed at 80 ° C.
・活性化処理 次いで塩酸200cc/の水溶液中で50℃で３分間処理 ・無電解ニッケルメッキ法 処方 硫酸ニッケル 25g/L 次亜リン酸ナトリウム 25g/L ピロリン酸ナトリウム 50g/L pH 10〜11 液温 50℃ 時間 10分間 次にアトメックス無電解メッキ液（日本エンゲルハル
ド（株））15倍希釈溶液に上記のニッケルメッキ布を浸
漬し90℃、10分間金メッキを行いメッキ厚0.5μの金メ
ッキ布を得た。・ Activation treatment Next, treatment at 50 ° C for 3 minutes in an aqueous solution of hydrochloric acid 200cc / ・ Electroless nickel plating method Prescription Nickel sulfate 25g / L Sodium hypophosphite 25g / L Sodium pyrophosphate 50g / L pH 10-11 Liquid temperature Next, immerse the above nickel-plated cloth in a 15-fold diluted solution of Atomex electroless plating solution (Nihon Engelhard Co., Ltd.) and gold-plate at 90 ° C for 10 minutes to obtain a gold-plated cloth with a plating thickness of 0.5μ. Was.
○固定化溶液の作製 特開昭62−263668号の手法で固定化を行った。アイゼ
ラックス1020（保土谷化学）500mgに、アイゼラックス
０（保土谷化学）12.5mgとアイゼラックスＰ（保土谷化
学）10mgをミツクスしたものを加えて充分に混合した。
次に、グルコースオキシダゼII（東洋紡）120U/mgを30m
g加えて充分に混合した。Preparation of immobilization solution Immobilization was carried out by the method described in JP-A-62-263668. A mixture of 12.5 mg of Izelux 0 (Hodogaya Chemical) and 10 mg of Izelux P (Hodogaya Chemical) was added to 500 mg of Izelux 1020 (Hodogaya Chemical) and mixed well.
Next, glucose oxidase II (Toyobo) 120 U / mg for 30 m
g was added and mixed well.
○酵素固定化電極の作製 金メッキ布はく（112mm2）に固定化溶液（30mg）を塗
布し、25℃で24時間乾燥し製膜固定化した。○ Preparation of enzyme-immobilized electrode An immobilization solution (30 mg) was applied to gold-plated cloth (112 mm 2 ) and dried at 25 ° C. for 24 hours to form a film.
（Ｂ）サイクリックボルタンメトリー 性能のチェックを、三極法似よるボルタンメトリーで
行った。測定条件は以下の通りである。(B) Cyclic voltammetry The performance was checked by voltammetry similar to the triode method. The measurement conditions are as follows.
装置 ポテンショスタット （日厚計測NPGFZ−2501A） 記録計 （GRAPHTEC WX1100） 参照電極（銀／塩化銀電極） 対照電極（白金板） 電解質 0.1M クエン酸バッフアー（pH5.4） 0.1M 塩化カリウム 0.5mM フエロセンカルボン酸（東京化成） 結果 Ｉセンシング性 本発明の酵素機能電極を、上記電解液に浸漬し、0.02
V/Sの電位掃引速度で−0.1V〜0.5Vの掃引電位間のサイ
クリックボルタモグラムを記録した 図１に示すように
グルコース無添加（Ａカーブ）、グルコース100mM添加
（Ｂカーブ）でカーブに差が見られ応答性を示した。Equipment Potentiometer (Nissan Measurement NPGFZ-2501A) Recorder (GRAPHTEC WX1100) Reference electrode (silver / silver chloride electrode) Reference electrode (platinum plate) Electrolyte 0.1M Citric acid buffer (pH5.4) 0.1M Potassium chloride 0.5mM Erosene carboxylic acid (Tokyo Kasei) Result I Sensing properties The enzyme-functional electrode of the present invention was immersed in
A cyclic voltammogram was recorded between the sweep potentials of -0.1 V to 0.5 V at a potential sweep speed of V / S. As shown in FIG. 1, the curves differed when glucose was not added (A curve) and glucose was added 100 mM (B curve). Was observed, indicating responsiveness.
図２はグルコース添加量を変化させ、0.5Vでの電流値
とそれぞれの無添加との差を求めプロットしたものであ
る。直線性が見られ、本発明の電極はグルコース濃度測
定用バイオセンサーとして有用なことが判明した。FIG. 2 is a graph in which the difference between the current value at 0.5 V and the respective values without addition was determined and the glucose addition amount was varied. Linearity was observed, indicating that the electrode of the present invention was useful as a biosensor for measuring glucose concentration.
II布状電極特性 本発明の酵素機能電極はその表面写真（図３）に示し
たように表面積が大きい。また布状電極であるのでフレ
キシビリ性が大きく、軽くかつカットの容易な電極が得
られた。フレキシビリ性はJIS1096剛軟性Ａ法（45゜カ
ンチレバー法）で測定し80mmのものが得られた。II Cloth-like electrode characteristics The enzyme-functional electrode of the present invention has a large surface area as shown in its surface photograph (FIG. 3). In addition, since the electrode is a cloth-like electrode, the electrode has high flexibility and is light and easily cut. Flexibility was measured by the JIS 1096 rigidity A method (45 ° cantilever method), and a value of 80 mm was obtained.
電極表面積を変化させてサイクリックボルタンメント
リーを測定したのが図４である。（１）は表面積56mm2
であり、（２）はその２倍の112mm2である。0.5Vでの電
流値を測定したところ、面積同様（２）は（１）の２倍
となった。FIG. 4 shows the measurement of the cyclic voltammentary by changing the electrode surface area. (1) is 56mm 2 surface area
(2) is 112 mm 2 which is twice as large. When the current value at 0.5 V was measured, (2) was twice as large as (1) as in the area.
上記結果で明らかなように、本発明の酵素機能電極が
バイオリアクター、バイオセンサーに使用した場合、そ
の表面積が大きいことにより高生産性、高感度なものが
得られる。As is clear from the above results, when the enzyme-functional electrode of the present invention is used for a bioreactor or a biosensor, a high productivity and high sensitivity can be obtained due to its large surface area.
III直接固定化特性 本発明の酵素機能電極は酵素を電極面積に固定化して
あるので、電極での電子の移動を、電極に加えられる電
圧を変化させる事によって、反応を外部から制御するこ
とが可能となった。III Direct Immobilization Characteristics Since the enzyme-functional electrode of the present invention has the enzyme immobilized on the electrode area, the transfer of electrons at the electrode can be externally controlled by changing the voltage applied to the electrode. It has become possible.
（発明の効果） 本発明の酵素機能電極は、表面積が大きいことにより
高生産性、高感度のバイオリアクター、バイオセンサー
を与える。又フレキシビリ性を持ち、且つカット等の加
工が容易で酵素機能電極の形を自由に変えられるので応
用の広い特長を与える。更に、酵素を直接固定化してあ
るので、反応を外部から制御可能である。(Effect of the Invention) The enzyme functional electrode of the present invention provides a bioreactor and a biosensor with high productivity and high sensitivity due to its large surface area. In addition, it has flexibility and is easy to process such as cutting, and can freely change the shape of the enzyme-functional electrode, so that it has a wide range of applications. Further, since the enzyme is directly immobilized, the reaction can be externally controlled.
図１は本発明の実施例の酵素機能電極を用いてバッファ
ー中でサイクリクボルタンメトリーを行った図である。 図２は本発明の実施例の酵素機能電極を用いてグルコー
ス応答性を測定して作成した検量線である。 図３は本発明の実施例の酵素機能電極表面の繊維の形状
を示す電子顕微鏡写真であり、Ａは固定化前、Ｂは固定
化後を示す。 図４は本発明の実施例の酵素機能電極の表面積を変化さ
せてサイクリックボルタンメトリーを行った図である。FIG. 1 is a diagram in which cyclic voltammetry was performed in a buffer using an enzyme-functional electrode according to an example of the present invention. FIG. 2 is a calibration curve prepared by measuring glucose responsiveness using the enzyme functional electrode of the example of the present invention. FIG. 3 is an electron micrograph showing the shape of the fiber on the surface of the enzyme-functional electrode according to the example of the present invention, wherein A shows before immobilization and B shows after immobilization. FIG. 4 is a diagram in which cyclic voltammetry was performed by changing the surface area of the enzyme functional electrode of the example of the present invention.
Claims (4)
維よりなる布はくの電極部の表裏全体に金属メッキした
電極に酵素を直接固定化してなるフレキシビリ性を持
ち、表面積が大きく、軽く且つカットが容易な酵素機能
電極。The present invention has the flexibility of immobilizing an enzyme directly on an electrode metal-plated on the entire front and back of an electrode portion of a fabric fiber made of plant fiber, animal fiber, artificial fiber or inorganic fiber, and has a large surface area and light weight. An enzyme function electrode that is easy to cut.
機能電極。2. The enzyme functional electrode according to claim 1, wherein the metal is gold or silver.
を上層としてなる請求項１記載の酵素機能電極。3. The enzyme functional electrode according to claim 1, wherein the metal comprises nickel or copper as a lower layer and gold or silver as an upper layer.
キ法とそれに引き続いての電解メッキ法との組合せから
なる請求項１記載の酵素機能電極。4. The enzyme functional electrode according to claim 1, wherein the plating method comprises an electroless plating method or a combination of an electroless plating method and a subsequent electrolytic plating method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63319412A JP2777569B2 (en) | 1988-12-20 | 1988-12-20 | Enzyme function electrode |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63319412A JP2777569B2 (en) | 1988-12-20 | 1988-12-20 | Enzyme function electrode |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02165046A JPH02165046A (en) | 1990-06-26 |
| JP2777569B2 true JP2777569B2 (en) | 1998-07-16 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63319412A Expired - Fee Related JP2777569B2 (en) | 1988-12-20 | 1988-12-20 | Enzyme function electrode |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2777569B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2386960C2 (en) * | 2004-05-14 | 2010-04-20 | БАЙЕР ХЕЛТКЭР ЭлЭлСи | Voltammetric system for analysing biological substances |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6375655A (en) * | 1986-09-19 | 1988-04-06 | Olympus Optical Co Ltd | Enzyme electrode apparatus |
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1988
- 1988-12-20 JP JP63319412A patent/JP2777569B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02165046A (en) | 1990-06-26 |
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