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JPH0661266B2 - Immobilized enzyme thin film - Google Patents

Immobilized enzyme thin film


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JPH0661266B2 JP7036286A JP7036286A JPH0661266B2 JP H0661266 B2 JPH0661266 B2 JP H0661266B2 JP 7036286 A JP7036286 A JP 7036286A JP 7036286 A JP7036286 A JP 7036286A JP H0661266 B2 JPH0661266 B2 JP H0661266B2
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JPS62228274A (en )
恒雄 千葉
益男 相澤
寛明 篠原
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【発明の詳細な説明】 (産業上の利用分野) 本発明は固定化酵素薄膜に係り、更に具体的には、グルコースオキシダーゼの酵素活性を保持したアニリンの重合体からなる固定化酵素薄膜に関する。 DETAILED DESCRIPTION OF THE INVENTION (INDUSTRIAL FIELD) The present invention relates to immobilized enzyme film, and more particularly, relates to an immobilized enzyme membrane comprising a polymer of aniline holding the enzymatic activity of the glucose oxidase.

(従来の技術) 従来、酵素の固定化方法としては、包括法、架橋化法、 (Prior Art) Conventionally, as a method for immobilizing enzymes, entrapment method, cross-linking method,
共有結合法等が知られている。 Covalent bonding method, and the like are known. 更に、最近、固定化酵素薄膜として、例えば電気化学重合法により、グルコースオキシダーゼ・ポリピロール導電性酵素薄膜が作られた。 Further, recently, as an immobilized enzyme film, for example by electrochemical polymerization method, glucose oxidase polypyrrole conductive enzyme film was made. ところがこのようなポリピロール酵素薄膜は、その導電性ゆえ、電極被覆膜として使用する場合、選択性に劣るという欠点があつた。 However such polypyrrole enzymes films, its conductivity because, when used as electrode covering film, there has been a disadvantage of poor selectivity. すなわち、酵素活性を発現させる設定電位において、ポリピロール薄膜表面で種々の分子が反応を起こすという問題点があつた。 That is, in the set potential to express enzyme activity, various molecules polypyrrole thin film surface has been made is a problem that it reacts.

(発明の目的) 本発明の目的は、選択性に優れ、かつ簡便で強固な固定化酵素薄膜を提供することにある。 Object of the INVENTION An object of the present invention is to provide a highly selective and simple robust immobilized enzyme membrane. また、酵素透過性の優れた固定化酵素薄膜を提供することにある。 Another object is to provide an enzyme permeability excellent immobilized enzyme membrane.

(発明の構成) 本発明の目的は、グルコースオキシダーゼを固定化してなるアニリンの重合体からなる固定化酵素薄膜によつて達成される。 An object of the present invention (structure of the invention), is by connexion achieved immobilized enzyme membrane comprising a polymer of aniline comprising immobilized glucose oxidase.

以下詳細に本発明を説明する。 The following details illustrate the present invention.

本発明で使用するグルコースオキシダーゼは、グルコースを酸化してD−グルコン酸を生ずる反応を触媒する公知の酵素である。 Glucose oxidase for use in the present invention is a known enzyme that catalyzes a reaction resulting oxidized to D- gluconic acid with glucose.

本発明の固定化酵素薄膜は、アニリンとグルコースオキシダーゼを、電気化学重合法などの常法を用いて固定化することにより製造される。 Immobilized enzyme film of the present invention, the aniline and glucose oxidase, is prepared by immobilizing using conventional methods, such as electrochemical polymerization.

すなわち、例えば、電解液中にアニリンおよびグルコースオキシダーゼを加え、Ag・AgCl電極を参照電極とした電解セル内において、定電位あるいは定電流電気化学重合法により、一定温度で行なわれる。 That is, for example, aniline and glucose oxidase was added in the electrolytic solution, in the electrolytic cell in which a reference electrode Ag · AgCl electrode, by a constant potential or constant current electrochemical polymerization is carried out at a constant temperature. ここで使用される電解液に制限はなく、例えば0.1mM以上の硫酸あるいは適当濃度のリン酸緩衝液等が選ばれるが、酸性度が上がるほど固定化酵素薄膜の酵素活性は良くなる傾向にある。 Here limited to the electrolytic solution to be used is not, for example, phosphate buffer solution of sulfuric acid or an appropriate concentration of more than 0.1mM is selected, the enzymatic activity the better trend of the immobilized enzyme film as acidity increases is there. また、アニリンおよびグルコースオキシダーゼの濃度はそれぞれ例えば、25mM〜1Mおよび0.1mg/ml Further, each concentration of aniline and glucose oxidase, for example, 25MM~1M and 0.1 mg / ml
〜50mg/mlの範囲が適当である。 Range to 50 mg / ml are suitable. 更に、電解セルおよび作用極・対極に制限はなく、それぞれ例えばガラスセルおよび白金電極等が使用できる。 Furthermore, not limited to the electrolytic cell and the working electrode, counter electrode, respectively, for example a glass cell and a platinum electrode or the like can be used. 定電位で重合する場合、良質な膜を得るため設定電位は0.5VvsAg・AgCl When the polymerization at a constant potential, setting the potential for obtaining a good quality film 0.5VvsAg · AgCl
以上であることが望ましい。 Greater than or equal it is desirable. また、重合温度は、例えば通常5℃〜50℃の間から選ばれる。 The polymerization temperature is selected from between, for example, usually 5 ° C. to 50 ° C..

このようにして得られる固定化酵素薄膜は、選択性の高い酵素活性を有し、更に酵素膜は電極表面に強固に付着して容易にはがれず、酸素透過性も良い。 Thus immobilized enzyme film obtained has a high enzymatic activity selective, further enzymes film is not easily peeled off firmly attached to the electrode surface may be oxygen permeability.

(実施例) 以下に実施例を示し、本発明の固定化酵素薄膜について更に詳細に説明するが、本発明はその要旨を越えない限り以下の実施例に限定されるものではない。 EXAMPLES the following examples, but will be described in more detail immobilized enzyme film of the present invention, the present invention is not limited to the following Examples unless exceeding the gist thereof.

実施例1 実験装置の概略を図1に示す。 A schematic of the Example 1 experimental apparatus shown in FIG. 白金電極を作用極および対極とし、Ag・AgCl電極を参照電極としたガラス製電解セルを用い、0.1Mアニリンとグルコースオキシダーゼ10mgを含む0.1MH 2 SO 4溶液2ml中で、一定温度において、120秒間、0.9V(対Ag・AgCl)の定電位をかけて電気化学重合を行なつた。 A platinum electrode as a working electrode and a counter electrode, using a glass electrolysis cell as a reference electrode Ag · AgCl electrode, in 0.1MH 2 SO 4 solution 2ml containing 0.1M aniline and glucose oxidase 10 mg, at a constant temperature, 120 seconds, 0.9V (vs. Ag · AgCl) row electrochemical polymerization over a constant potential of Natsuta.

この時、白金電極表面は薄縁色に変色した。 In this case, the platinum electrode surface was discolored Usuberi color. 終了後、白金電極表面に酵素固定化ポリアニリン膜が生成され、かつ酵素活性が保持されていることを確かめるために、ペルオキシダーゼ法を採用した。 After completion, the generated enzyme immobilized polyaniline film on Pt electrode surface, and to make sure that the enzyme activity is retained and adopted peroxidase method. すなわち、電極を洗浄後乾燥し、1Mグルコース0.3ml、0.1Mフエノール0.3ml、0.1M4−アミノアンチピリン0.3ml、 In other words, the electrode was dried after washing, 1M glucose 0.3 ml, 0.1 M phenol 0.3 ml, 0.1M4- aminoantipyrine 0.3 ml,
0.5mg/mlペルオキシダーゼ0.3mlおよび0.1M 0.5mg / ml peroxidase 0.3ml and 0.1M
リン酸緩衝液1.8mlの混合溶液中に浸漬した。 It was immersed in a mixed solution of phosphate buffer 1.8 ml. 約15 About 15
分間放置した後、503nmの可視光スペクトルを測定した。 After standing min was measured visible light spectrum of 503 nm. 可視光スペクトルの吸光度から、グルコースオキシダーゼの酵素活性量を求めると、276ng/cm 2であつた。 From the absorbance in the visible light spectrum, when determining the amount of enzyme activity of glucose oxidase, Atsuta at 276ng / cm 2.

尚、ポリアニリン膜の酸素透過性を調べるため、前述の方法においてアニリンのみを使用して白金電極表面にポリアニリン膜を生成させ、これを0.1Mリン酸緩衝液中、0〜−0.6V(対Ag/AgCl 2 )の電位域で常法に従い電位走査を行つたところ、第2図に示すように、酸素ガスの通気により還元電流は大きく増大し、膜中を酸素が透過し、白金電極上で電気化学還元されることが分つた。 Incidentally, in order to examine the oxygen permeability of the polyaniline film, to produce a polyaniline film on the platinum electrode surface using only aniline in the process described above, which in 0.1M phosphate buffer, 0 to-0.6V ( where having conducted the usual manner potential scan at a potential range of pair Ag / AgCl 2), as shown in FIG. 2, reduction current by aeration of oxygen gas greatly increases, through the membrane oxygen permeates, platinum electrode it was divide that is electrochemically reduced at the top.

実施例2 0.1Mリン酸緩衝液2mlに、0.1Mアニリンおよびグルコースオキシダーゼ9.9mgを溶かし、実施例1と同様な方法で電気化学重合を行なつた。 Example 2 0.1M phosphate buffer 2 ml, dissolved 0.1M aniline and glucose oxidase 9.9 mg, row electrochemical polymerization in the same manner as in Example 1 Natsuta. この時、白金電極表面は薄黄銅色となつた。 In this case, the platinum electrode surface has decreased and the thin brass color. 終了後、ペルオキシダーゼ法によりグルコースオキシダーゼの活性量を求めると、 After completion of the seek active amount of glucose oxidase peroxidase method,
115ng/cm 2であつた。 Atsuta at 115ng / cm 2.

(発明の効果) 本発明によれば、酵素活性を保持し、選択性が高く、更に酸素透過性が良い強固な固定化酵素薄膜が得られる。 According to the present invention (Effect of the Invention), it retains the enzymatic activity, high selectivity, further oxygen permeability good firm immobilized enzyme film obtained.
本発明の固定化酵素薄膜はバイオセンサーや、臨床検査用のグルコース検出装置、あるいはグルコン酸生成のバイオリアクター等への応用が可能である。 Immobilized enzyme film of the present invention can be applied and biosensors, glucose detection device for clinical examination, or to a bioreactor or the like of gluconic acid production.

更に、電気化学重合法などを用いれば、微小な電極の表面に容易に固定化酵素薄膜を作製できるため、ミクロなパターンを有したバイオ素子を形成する有力な手段となりうる。 Furthermore, the use of such electrochemical polymerization method, since the easily immobilized enzyme film on the surface of fine electrodes can be made, can be an effective means for forming a bio-element having a microscopic pattern.


図1は、実施例で使用した電解装置の概略図を示す。 Figure 1 shows a schematic view of an electrolytic apparatus used in Example. 図2は、実施例1で製造したポリアニリン膜の酸素透過性を示す。 Figure 2 shows the oxygen permeability of the polyaniline film prepared in Example 1. 作用極 対極 参照極 塩橋 ガラス製電解セル 電解液 KCl飽和溶液 Working electrode counter electrode reference electrode salt bridge glass electrolysis cell electrolyte KCl saturated solution

Claims (1)

    【特許請求の範囲】 [The claims]
  1. 【請求項1】グルコースオキシダーゼを固定化してなるアニリンの重合体からなることを特徴とする固定化酸素薄膜 1. A fixing oxygen thin film characterized by comprising a polymer of aniline comprising immobilized glucose oxidase
JP7036286A 1986-03-28 1986-03-28 Immobilized enzyme thin film Expired - Lifetime JPH0661266B2 (en)

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JP7036286A JPH0661266B2 (en) 1986-03-28 1986-03-28 Immobilized enzyme thin film

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JP7036286A JPH0661266B2 (en) 1986-03-28 1986-03-28 Immobilized enzyme thin film

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JPS62228274A true JPS62228274A (en) 1987-10-07
JPH0661266B2 true JPH0661266B2 (en) 1994-08-17



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Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6428556A (en) * 1987-07-23 1989-01-31 Bridgestone Corp Enzyme electrode
JP2570621B2 (en) * 1994-06-27 1997-01-08 日本電気株式会社 A manufacturing method thereof, and a cell array forming method for cell culture substrate and
US6413410B1 (en) 1996-06-19 2002-07-02 Lifescan, Inc. Electrochemical cell
CA2236850C (en) * 1995-11-16 2004-06-01 Alastair Mcindoe Hodges Electrochemical cell

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