JPH0661266B2 - Immobilized enzyme thin film - Google Patents
Immobilized enzyme thin filmInfo
- Publication number
- JPH0661266B2 JPH0661266B2 JP61070362A JP7036286A JPH0661266B2 JP H0661266 B2 JPH0661266 B2 JP H0661266B2 JP 61070362 A JP61070362 A JP 61070362A JP 7036286 A JP7036286 A JP 7036286A JP H0661266 B2 JPH0661266 B2 JP H0661266B2
- Authority
- JP
- Japan
- Prior art keywords
- thin film
- immobilized enzyme
- electrode
- enzyme
- glucose oxidase
- 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
Links
- 239000010409 thin film Substances 0.000 title claims description 18
- 108010093096 Immobilized Enzymes Proteins 0.000 title description 14
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 18
- 108010015776 Glucose oxidase Proteins 0.000 claims description 12
- 239000004366 Glucose oxidase Substances 0.000 claims description 12
- 229940116332 glucose oxidase Drugs 0.000 claims description 12
- 235000019420 glucose oxidase Nutrition 0.000 claims description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 229920000642 polymer Polymers 0.000 claims description 3
- 230000003100 immobilizing effect Effects 0.000 claims description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 14
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 8
- 239000010408 film Substances 0.000 description 8
- 229910052697 platinum Inorganic materials 0.000 description 7
- 238000006116 polymerization reaction Methods 0.000 description 7
- 229910021607 Silver chloride Inorganic materials 0.000 description 5
- 230000035699 permeability Effects 0.000 description 5
- 229920000767 polyaniline Polymers 0.000 description 5
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 5
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 102000003992 Peroxidases Human genes 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 229920000128 polypyrrole Polymers 0.000 description 3
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000008151 electrolyte solution Substances 0.000 description 2
- 229950006191 gluconic acid Drugs 0.000 description 2
- 235000012208 gluconic acid Nutrition 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 229910001369 Brass Inorganic materials 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000010951 brass Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 238000005868 electrolysis reaction Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
Landscapes
- 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。TECHNICAL FIELD The present invention relates to an immobilized enzyme thin film, and more specifically to an immobilized enzyme thin film made of a polymer of aniline retaining the enzymatic activity of 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、
共有結合法等が知られている。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。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。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。(Prior Art) Conventionally, entrapment method, cross-linking method,
The covalent bond method and the like are known. Further, recently, a glucose oxidase / polypyrrole conductive enzyme thin film has been produced as an immobilized enzyme thin film by, for example, an electrochemical polymerization method. However, such a polypyrrole enzyme thin film has a drawback that it has poor selectivity when used as an electrode coating film due to its conductivity. That is, there has been a problem that various molecules react on the surface of the polypyrrole thin film at the set potential for expressing the enzyme activity.
（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。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。(Object of the Invention) An object of the present invention is to provide a simple and robust immobilized enzyme thin film having excellent selectivity. Another object is to provide an immobilized enzyme thin film having excellent enzyme permeability.
（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。(Structure of the Invention) The object of the present invention is achieved by an immobilized enzyme thin film composed of a polymer of aniline on which glucose oxidase is immobilized.
以下詳細に本発明を説明する。The present invention will be described in detail below.
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 used in the present invention is a known enzyme that catalyzes a reaction that oxidizes glucose to produce D-gluconic acid.
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。The immobilized enzyme thin film of the present invention is produced by immobilizing aniline and glucose oxidase by a conventional method such as an electrochemical polymerization method.
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。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。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
〜５０mg/mlの範囲が適当である。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。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
以上であることが望ましい。また、重合温度は、例えば
通常５℃〜５０℃の間から選ばれる。That is, for example, aniline and glucose oxidase are added to an electrolytic solution, and it is carried out at a constant temperature by a constant potential or constant current electrochemical polymerization method in an electrolytic cell using an Ag / AgCl electrode as a reference electrode. There is no limitation on the electrolytic solution used here, and for example, sulfuric acid of 0.1 mM or more or a phosphate buffer solution of an appropriate concentration is selected, but as the acidity increases, the enzyme activity of the immobilized enzyme thin film tends to improve. is there. The concentrations of aniline and glucose oxidase are, for example, 25 mM to 1 M and 0.1 mg / ml, respectively.
A range of up to 50 mg / ml is suitable. Further, the electrolytic cell and the working electrode / counter electrode are not limited, and, for example, a glass cell and a platinum electrode can be used, respectively. When polymerizing at a constant potential, the set potential is 0.5 V vs Ag ・ AgCl to obtain a good quality film.
The above is desirable. The polymerization temperature is usually selected from the range of 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。The immobilized enzyme thin film thus obtained has a highly selective enzyme activity, and further, the enzyme film firmly adheres to the electrode surface and is not easily peeled off, and it has good 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) Examples are shown below to describe the immobilized enzyme thin film of the present invention in more detail, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded.
実施例１ 実験装置の概略を図１に示す。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０．１ＭH2SO4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。Example 1 An outline of an experimental apparatus is shown in FIG. Using a glass electrolytic cell having a platinum electrode as a working electrode and a counter electrode and an Ag / AgCl electrode as a reference electrode, in a 2 ml of 0.1 MH 2 SO 4 solution containing 0.1 M aniline and 10 mg of glucose oxidase, at a constant temperature, Electrochemical polymerization was carried out by applying a constant potential of 0.9 V (vs. Ag · AgCl) for 120 seconds.
この時、白金電極表面は薄縁色に変色した。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。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、
０．５mg/mlペルオキシダーゼ０．３mlおよび０．１Ｍ
リン酸緩衝液１．８mlの混合溶液中に浸漬した。約１５
分間放置した後、５０３nmの可視光スペクトルを測定し
た。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/cm2であつ
た。At this time, the platinum electrode surface was discolored to a light edge. After the completion, the peroxidase method was adopted to confirm that the enzyme-immobilized polyaniline film was formed on the platinum electrode surface and the enzyme activity was retained. That is, the electrode was washed and dried, and 1 M glucose 0.3 ml, 0.1 M phenol 0.3 ml, 0.1 M 4-aminoantipyrine 0.3 ml,
0.5 mg / ml peroxidase 0.3 ml and 0.1M
It was immersed in a mixed solution of 1.8 ml of phosphate buffer. About 15
After standing for a minute, a visible light spectrum at 503 nm was measured. The enzyme activity of glucose oxidase was determined from the absorbance of the visible light spectrum and found to be 276 ng / 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/AgCl2）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。In order to examine the oxygen permeability of the polyaniline film, a polyaniline film was formed on the surface of the platinum electrode using only aniline in the above-mentioned method, and the polyaniline film was added in a 0.1M phosphate buffer solution to 0 to -0.6V ( When a potential scan was performed in the potential range (against Ag / AgCl 2 ) in accordance with the usual method, as shown in Fig. 2, the reduction current was greatly increased by the aeration of oxygen gas, oxygen permeated through the membrane, and the platinum electrode It was found to be electrochemically reduced above.
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。この時、白金電
極表面は薄黄銅色となつた。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、
１１５ng/cm2であつた。Example 2 0.1 M aniline and 9.9 mg of glucose oxidase were dissolved in 2 ml of 0.1 M phosphate buffer, and electrochemical polymerization was carried out in the same manner as in Example 1. At this time, the platinum electrode surface became a light brass color. After the end, when the activity of glucose oxidase is determined by the peroxidase method,
It was 115 ng / 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。
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。(Effects of the Invention) According to the present invention, a strong immobilized enzyme thin film that retains enzyme activity, has high selectivity, and has good oxygen permeability can be obtained.
The immobilized enzyme thin film of the present invention can be applied to a biosensor, a glucose detection device for clinical tests, a bioreactor for producing gluconic acid, and the like.
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, when an electrochemical polymerization method or the like is used, an immobilized enzyme thin film can be easily prepared on the surface of a minute electrode, which can be an effective means for forming a biodevice 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。 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。 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FIG. 1 shows a schematic view of the electrolysis apparatus used in the examples. FIG. 2 shows the oxygen permeability of the polyaniline film produced in Example 1. Working electrode Counter electrode Reference electrode Salt bridge Glass electrolytic cell Electrolyte KCl saturated solution
Claims (1)
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. An immobilized oxygen thin film comprising a polymer of aniline obtained by immobilizing glucose oxidase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61070362A JPH0661266B2 (en) | 1986-03-28 | 1986-03-28 | Immobilized enzyme thin film |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61070362A JPH0661266B2 (en) | 1986-03-28 | 1986-03-28 | Immobilized enzyme thin film |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62228274A JPS62228274A (en) | 1987-10-07 |
| JPH0661266B2 true JPH0661266B2 (en) | 1994-08-17 |
Family
ID=13429245
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61070362A Expired - Lifetime JPH0661266B2 (en) | 1986-03-28 | 1986-03-28 | Immobilized enzyme thin film |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0661266B2 (en) |
Families Citing this family (6)
| 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 | Cell culture substrate, method for producing the same, and method for forming cell array |
| US6413410B1 (en) | 1996-06-19 | 2002-07-02 | Lifescan, Inc. | Electrochemical cell |
| AUPN661995A0 (en) * | 1995-11-16 | 1995-12-07 | Memtec America Corporation | Electrochemical cell 2 |
| US6863801B2 (en) | 1995-11-16 | 2005-03-08 | Lifescan, Inc. | Electrochemical cell |
| AU2002340079A1 (en) | 2001-10-10 | 2003-04-22 | Lifescan Inc. | Electrochemical cell |
-
1986
- 1986-03-28 JP JP61070362A patent/JPH0661266B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62228274A (en) | 1987-10-07 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EXPY | Cancellation because of completion of term |