JPH0786497B2 - Enzyme electrode using non-porous immobilized enzyme membrane - Google Patents
Enzyme electrode using non-porous immobilized enzyme membraneInfo
- Publication number
- JPH0786497B2 JPH0786497B2 JP1331059A JP33105989A JPH0786497B2 JP H0786497 B2 JPH0786497 B2 JP H0786497B2 JP 1331059 A JP1331059 A JP 1331059A JP 33105989 A JP33105989 A JP 33105989A JP H0786497 B2 JPH0786497 B2 JP H0786497B2
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- Prior art keywords
- membrane
- enzyme
- electrode
- glucose
- immobilized
- Prior art date
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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） 本発明の方法によれば常温、常圧の緩和な条件下で酵素
を固定化することができるので酵素活性の低下が少な
く、簡単に、大量に、安価に固定化酵素膜を作製するこ
とができ、その膜は弾力性があって強度も大きく、水溶
液中で膨潤することもなく、かつ本発明による酵素電極
に利用することができる。したがって産業上ではバイオ
センサーとして食品、酒類醸造及び医療関係等において
利用することができる。DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) According to the method of the present invention, an enzyme can be immobilized under mild conditions of room temperature and normal pressure, so that the enzyme activity is less likely to decrease, and An immobilized enzyme membrane can be produced in large quantities at low cost, the membrane has elasticity and high strength, does not swell in an aqueous solution, and can be used for the enzyme electrode according to the present invention. Therefore, in industry, it can be used as a biosensor in food, brewing alcohol, medical care, and the like.
（従来技術及び問題点） バイオセンサーに利用する固定化酵素膜の作製方法につ
いては共有結合法、架橋法または包括法等に関し多くの
特許及び研究報告があるが、その多くは固定化方法が複
雑で反応条件が厳しく、固定化操作により酵素活性が低
下する、膜の強度が低い、使用中に膜が膨潤する。酵素
活性の維持期間が短い、高価である等の問題点があり、
固定化酵素膜作製に高度の技術を要することもあって、
酵素の基質特異性に基づくバイオセンサーの優れた特性
は認められているものの広く産業界で実用される状況に
はない。(Prior art and problems) There are many patents and research reports on covalent bond method, cross-linking method, encapsulation method, etc. for the preparation method of immobilized enzyme membrane used for biosensor, but most of them have complicated immobilization method. The reaction conditions are severe, the enzyme activity is reduced by the immobilization operation, the strength of the membrane is low, and the membrane swells during use. There are problems such as short maintenance period of enzyme activity and high price,
Since it requires a high level of technology to prepare the immobilized enzyme membrane,
Although the excellent characteristics of the biosensor based on the substrate specificity of the enzyme have been recognized, they are not widely used in industry.
バイオセンサーとして最も多く用いられているグルコー
スセンサーにおいてもグルコースオキシダーゼ固定化膜
は１個１万円以上と高価であり、しかも使用できる期間
は30日間程度である。酵素活性を長期間持続することは
その性質上困難であっても、もう少し安価に作製するこ
とができれば使い捨てのような形により、産業界でさら
に広く利用されることが可能である。Even in the glucose sensor that is most often used as a biosensor, each glucose oxidase-immobilized membrane is expensive as 10,000 yen or more, and the usable period is about 30 days. Even if it is difficult to maintain the enzyme activity for a long period of time, it can be more widely used in the industrial field in a disposable form if it can be produced at a slightly lower cost.
（問題点を解決するための手段） 本発明者らはバイオセンサー用として緩和な条件で簡単
に酵素を固定化することができ、固定化膜が上部であっ
てしかも水中で膨潤しないような酵素の固定化方法につ
いて鋭意研究を進めたところ、塗膜形成能のある有機物
を水中に分散させた水系エマルジョンを利用するとそれ
らの目的が達せられ、かつ本発明の方法により、作製し
た膜が酵素電極に利用できることを知ったのである。(Means for Solving Problems) The inventors of the present invention can easily immobilize an enzyme under mild conditions for a biosensor, and the immobilization membrane is on the upper side and does not swell in water. As a result of intensive studies on the immobilization method, the use of an aqueous emulsion in which an organic substance capable of forming a coating film is dispersed in water can achieve those purposes, and the membrane prepared by the method of the present invention can be used as an enzyme electrode. I knew it could be used for.
まず、固定化酵素膜の製造方法について述べる。塗膜形
成能のある有機物を水中に分散させた水系エマルジョン
の製造方法としては、水に塗膜形成能のある有機物を混
和し、乳化剤を加えて強力に撹拌する方法、または反応
性のある種々のモノマーの乳化重合法等がある。実験室
内には市販の水系合成樹脂エマルジョン塗料を利用する
ことができる。市販の水性アクリル塗料は水にアクリル
樹脂を分散させたエマルジョン塗料であり、pH6.5程度
で多くの酵素はpHによって変性することはなく都合がよ
い。合成樹脂エマルジョンに酵素の水溶液を加え混合し
た後、平面上に薄く広げ水を蒸発させる方法、または引
き上げ法等により膜を作製し、必要に応じて連結乾燥を
行うと酵素は合成樹脂の網目構造によって固定化させ
る。ただし、この方法により作製した膜には光学顕微鏡
で認められる程度の細孔はほとんど存在せず、均質ない
わゆる非多孔膜である。酵素水溶液の調整には、蒸留水
または使用する酵素に適したpHの0.1M程度以下の濃度の
緩衝液を用いるのが望ましい。緩衝液濃度が高い場合に
はエマルジョンが破壊されて沈澱が生じ、樹脂膜が作製
できないことがある。液膜を形成後30〜60分程度室温に
放置して水を蒸発させ、樹脂を硬化させた後、凍結乾燥
を行うと膜の柔軟性が増加することがあるが、あまり早
い時期に凍結を行うと膜の強度が著しく低下する。First, a method for producing an immobilized enzyme membrane will be described. As a method for producing an aqueous emulsion in which an organic substance capable of forming a coating film is dispersed in water, a method of mixing the organic substance capable of forming a coating film with water, adding an emulsifier and stirring strongly, or various reactive substances is used. There is an emulsion polymerization method of the monomer. A commercially available water-based synthetic resin emulsion paint can be used in the laboratory. A commercially available water-based acrylic paint is an emulsion paint in which an acrylic resin is dispersed in water, and it is convenient that many enzymes do not denature with pH at about 6.5. After adding an aqueous solution of enzyme to a synthetic resin emulsion and mixing it, a membrane is prepared by a method of spreading it thinly on a flat surface and evaporating water, or by a method such as pulling up, and if necessary, drying by linking, the enzyme has a network structure of synthetic resin. Immobilize by. However, the film produced by this method has almost no pores as observed by an optical microscope, and is a homogeneous so-called non-porous film. For the preparation of the enzyme aqueous solution, it is desirable to use distilled water or a buffer solution having a pH of about 0.1 M or less suitable for the enzyme to be used. When the concentration of the buffer solution is high, the emulsion may be broken and precipitation may occur, and the resin film may not be produced. After forming the liquid film, leave it at room temperature for about 30 to 60 minutes to evaporate the water and harden the resin, then freeze-drying may increase the flexibility of the film, but freeze it too early. If this is done, the strength of the film will be significantly reduced.
次に非多孔固定化酵素膜を使用する酵素電極について説
明する。一般に固定化した酵素を使用して液体中で溶質
に化学反応を起こす場合、酵素の固定化には多孔質の物
質が用いられる。例えば、グルコースセンサーのように
酸素電極を利用する場合は、酸素が透過しやすいように
酵素の固定化に際してはポリアクリルアミドゲル等のゲ
ル状物質による包括固定化やガス透過性の多孔質テフロ
ン等への共有結合による固定化等が方法が用いられてい
る。したがって、細孔を有していない非多孔膜はガス検
出用電極を用いる酵素電極には用いられていない。しか
し、膜材と親和性のある低分子物質は非多孔膜に溶解
し、膜内を拡散により移動することが知られていること
から、低分子物質検出型の電極を使用する酵素電極にお
いては低分子物質が膜を透過すれば細孔のない非多孔膜
であっても使用できると考えられる。そこで前述の方法
によりグルコースオキシダーゼ固定化膜を作製し、酸素
電極の上に本発明の方法により作製した非多孔固定化酵
素膜を密着させ、その上にセルロース製透析膜を密着さ
せるとグルコースセンサーとして機能することを知った
のである。Next, an enzyme electrode using a non-porous immobilized enzyme membrane will be described. Generally, when the immobilized enzyme is used to cause a chemical reaction with a solute in a liquid, a porous substance is used for immobilizing the enzyme. For example, when using an oxygen electrode like a glucose sensor, when immobilizing an enzyme so that oxygen can easily permeate, entrapping immobilization with a gel-like substance such as polyacrylamide gel or using a gas-permeable porous Teflon, etc. The method is used such as immobilization by covalent bond. Therefore, the non-porous membrane having no pores is not used for the enzyme electrode using the gas detection electrode. However, it is known that low molecular weight substances that have an affinity for the membrane material dissolve in the non-porous membrane and move by diffusion in the membrane, so in enzyme electrodes using low molecular weight substance detection type electrodes, It is considered that a low-molecular substance can be used even if it is a non-porous membrane having no pores if it penetrates the membrane. Therefore, a glucose oxidase-immobilized membrane is prepared by the above-mentioned method, a non-porous immobilized enzyme membrane prepared by the method of the present invention is adhered on an oxygen electrode, and a cellulose dialysis membrane is adhered on it to give a glucose sensor. I knew it would work.
グルコースオキシダーゼ固定化膜を用いるグルコースセ
ンサーは、次の反応による溶液中の酸素濃度の低下を検
出し、反応したグルコール量を測定するものである。A glucose sensor using a glucose oxidase-immobilized membrane detects a decrease in oxygen concentration in a solution due to the following reaction and measures the amount of reacted glucose.
酸素電極に本発明の方法により作製したグルコースオキ
シターゼ固定化膜を密着させ、それを撹拌行っている0.
5％（w/v）グルコース水溶液中に挿入し５分間わたって
酸素濃度計の出力を測定したところ、その出力にはほと
んど変化はなかった。この結果は次のような現象による
ものと推察された。 The glucose oxidase-immobilized membrane prepared by the method of the present invention is closely attached to the oxygen electrode, and it is stirred.
When the output of the oximeter was measured by inserting it into a 5% (w / v) glucose aqueous solution for 5 minutes, there was almost no change in the output. This result was presumed to be due to the following phenomenon.
本固定化酵素膜は非多孔質であるため、固定化酵素膜の
内部でグルコースと酸素が反応することはなく、膜の表
面においてのみ反応する。したがって（１）式の反応に
関与するグルコースオキシターゼは固定化された酵素の
内、膜の表面に存在するものだけでその分子数はわずか
であると考えられ、（１）式によって消費される酸素量
も少ない。一方試料液体は撹拌により流動しているため
固定化酵素膜の表面には絶えず新しい液体が供給され
る。膜表面での反応量はわずかであるため試料液全体の
酸素濃度にはほとんど変化は生じない。その結果、５分
経過後の固定化酵素膜の表面の酸素濃度は初発の試料液
体本体の濃度とほとんど差はなかったものと推察され
た。したがって、このままではグルコースセンサーとし
ては使用できない。Since this immobilized enzyme membrane is non-porous, glucose and oxygen do not react inside the immobilized enzyme membrane, but only on the surface of the membrane. Therefore, the glucose oxidase involved in the reaction of the formula (1) is considered to be the only immobilized enzyme existing on the surface of the membrane and the number of molecules thereof is small, and the oxygen consumed by the formula (1) is considered to be small. The quantity is also small. On the other hand, since the sample liquid is flowing by stirring, new liquid is constantly supplied to the surface of the immobilized enzyme membrane. Since the reaction amount on the film surface is small, the oxygen concentration of the entire sample solution hardly changes. As a result, it was speculated that the oxygen concentration on the surface of the immobilized enzyme membrane after 5 minutes had almost no difference from the concentration of the initial sample liquid body. Therefore, it cannot be used as a glucose sensor as it is.
そこで第１図のように酸素電極（実験に使用したポーラ
ロ型酸素電極は白金電極に隔膜を密着させた形となって
いる。）に本発明の方法により作製した非多孔グルコー
スオキシダーゼ固定化膜を密着させ、さらにその上にセ
ルロース製透析膜を密着せて、0.5％（w/v）グルコース
を含むpH6.8リン酸緩衝液中に撹拌を行いつつ挿入した
ところ酸素濃度計の出力は初期値の7mVから30秒後には
約3mVまで低下し60秒後にはその出力は安定した。Therefore, as shown in FIG. 1, a non-porous glucose oxidase-immobilized membrane prepared by the method of the present invention is attached to an oxygen electrode (the polaro-type oxygen electrode used in the experiment has a platinum electrode and a diaphragm adhered thereto). The cellulose dialysis membrane was further adhered to it, and then it was inserted into a pH 6.8 phosphate buffer containing 0.5% (w / v) glucose while stirring, and the output of the oximeter was the initial value. After 7 mV, it decreased to about 3 mV after 30 seconds, and after 60 seconds the output became stable.
以上の結果は次の〜のような現象によるものと推察
された。It is speculated that the above results are due to the following phenomena.
水溶液中のグルコース（Ｇ）及び酸素（O2）が透析膜
（Ａ）の細孔中を拡散により固定化酵素膜（Ｃ）の方向
に移動した。Glucose (G) and oxygen (O 2 ) in the aqueous solution moved in the pores of the dialysis membrane (A) toward the immobilized enzyme membrane (C) by diffusion.
固定化酵素膜（Ｃ）表面に固定化されているグルコー
スオキシダーゼ（Ｅ）の接触作用により酸素（O2）とグ
ルコース（Ｇ）が反応し、透析膜と固定化酵素膜の間
（Ｂ）の酸素濃度が低下した。Oxygen (O 2 ) and glucose (G) react with each other by the contact action of glucose oxidase (E) immobilized on the surface of the immobilized enzyme membrane (C), and between the dialysis membrane and the immobilized enzyme membrane (B). Oxygen concentration decreased.
固定化酵素膜と隔膜の間（Ｄ）の酸素が固定化酵素膜
中を（Ｂ）方向へ移動し、（Ｄ）における酸素濃度が低
下した。Oxygen between the immobilized enzyme membrane and the membrane (D) moved in the immobilized enzyme membrane in the direction (B), and the oxygen concentration in (D) decreased.
電極（Ｐ）間の酸素が隔膜（Ｅ）中を拡散により
（Ｄ）方向へ移動し、その結果電極面の酸素濃度が低下
した。Oxygen between the electrodes (P) moved in the direction (D) due to diffusion in the diaphragm (E), and as a result, the oxygen concentration on the electrode surface decreased.
酸素濃度計の出力が低下した。The output of the oximeter decreased.
試料液体（Ｌ）は撹拌されているため透析膜の試料側の
表面には絶えず新しい液体が供給され、透析膜の表面の
酸素濃度はほとんど試料液体本体の酸素濃度と同一であ
る。しかし、透析膜と固定化酵素膜の間の（Ｂ）では酵
素膜表面で酸化反応により酸素が消費されるため、試料
液体中より酵素濃度が低く、透析膜中に酸素の濃度勾配
が生じる。溶液本体は撹拌により流動しているが透析膜
中の液体はその細孔に保持されているため流動せず、濃
度勾配は保護される。一定時間が経過すると酸素消費量
と拡散量とが平衡に達するものと推察された。Since the sample liquid (L) is agitated, a new liquid is constantly supplied to the sample-side surface of the dialysis membrane, and the oxygen concentration on the surface of the dialysis membrane is almost the same as the oxygen concentration of the sample liquid body. However, in (B) between the dialysis membrane and the immobilized enzyme membrane, oxygen is consumed by the oxidation reaction on the surface of the enzyme membrane, so the enzyme concentration is lower than in the sample liquid, and a concentration gradient of oxygen occurs in the dialysis membrane. The solution body flows by stirring, but the liquid in the dialysis membrane does not flow because it is held in the pores, and the concentration gradient is protected. It was assumed that the oxygen consumption and the diffusion amount reached equilibrium after a certain period of time.
次に本発明の実施例を示す。Next, examples of the present invention will be described.
実験例１ 本発明の方法により作製したグルコースオキ
シダーゼ固定化膜を装着したグルコースセンサーの応答
速度 （１）グルコースオキシダーゼ固定化膜の作製水0.4ml
にグルコースオキシダーゼを25.0mg溶解し、これを市販
の透明水性アクリルペイント（（株）アサヒペン製）0.
4mlに加え、よく混合した後ガラス板上に10×10cmに広
げ、120分間室温で風乾した。その後、−20℃に１時間
保って凍結し、−107℃で１時間凍結乾燥した後冷凍庫
に保管した。この膜のグルコースオキシダーゼ含有量は
0.25mg/cm2であり、膜の厚さは0.039mmであった。Experimental Example 1 Response speed of glucose sensor equipped with glucose oxidase-immobilized membrane prepared by the method of the present invention (1) Preparation of glucose oxidase-immobilized membrane 0.4 ml of water
Glucose oxidase (25.0 mg) was dissolved in a commercially available transparent water-based acrylic paint (manufactured by Asahipen Corporation).
The mixture was added to 4 ml, mixed well, spread on a glass plate to 10 × 10 cm, and air-dried at room temperature for 120 minutes. Then, it was frozen by keeping it at -20 ° C for 1 hour, freeze-dried at -107 ° C for 1 hour, and then stored in a freezer. The glucose oxidase content of this membrane is
It was 0.25 mg / cm 2 and the film thickness was 0.039 mm.
（２）グルコースセンサーの応答速度の測定ポーラロ式
酸素濃度計（エイブル（株）製AI−1007型）の白金電極
面上に前述の方法で作製したグルコースオキシダーゼ固
定化膜（作製後24時間冷凍庫に保管したもので以後作製
直後の膜という）、その上に透析用セルロース膜（VISK
ASE社製18/32）の順に密着させて固定し、それをグリコ
ース測定用のセルに設置した。セル内にはpH6.8のリン
酸緩衝液1.5mlを入れ30℃に保った。この状態でのセル
内のリン酸緩衝液は空気中の酸素と平衡であり酸素濃度
計の出力（E0）は7mVであるＱグルコース標準溶液とし
て0.05,0.1,0.2,0.3,0.4及び0.5％（w/v）のグルコース
を含む水溶液を調整し、それぞれの0.5mlをセルに注入
し、30〜120秒間の酸素濃度形の出力（EG）を測定し
た。測定結果を第２図に示した。試料を注入してから60
秒後には酸素濃度計の出力（EG）はほとんど安定した。
また、注入したグルコース溶液の濃度が高いほど初発の
酸素濃度計の出力（E0）との差が大きかった。したがっ
て第２図において注入した試料中のグルコース濃度と低
下した電圧E0−EGの関係をあらかじめ測定しておけば、
E0−EGを測定することにより溶液中のグルコース濃度を
求めることができる。(2) Measurement of response speed of glucose sensor Glucose oxidase-immobilized membrane prepared by the above-mentioned method on the platinum electrode surface of a polaro-type oxygen concentration meter (AI-1007 manufactured by Able Co., Ltd.) (in a freezer for 24 hours after preparation) The stored membrane is called the membrane immediately after production), and the cellulose membrane for dialysis (VISK)
18/32) manufactured by ASE Co., Ltd. was adhered and fixed in that order, and it was placed in a cell for measuring glucose. 1.5 ml of pH 6.8 phosphate buffer was placed in the cell and kept at 30 ° C. The phosphate buffer solution in the cell in this state is in equilibrium with oxygen in the air, and the output (E 0 ) of the oximeter is 7 mV. As a Q glucose standard solution 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5% adjust the aqueous solution containing (w / v) glucose was injected each 0.5ml into the cell and measuring the output of the oxygen concentration shaped 30-120 seconds (E G). The measurement results are shown in FIG. 60 after injection of sample
After a few seconds, the oximeter output (E G ) was almost stable.
Further, the higher the concentration of the injected glucose solution, the greater the difference from the output (E 0 ) of the initial oximeter. Therefore, in FIG. 2, if the relationship between the glucose concentration in the injected sample and the lowered voltage E 0 −E G is measured in advance,
The glucose concentration in the solution can be determined by measuring E 0 −E G.
アクリル樹脂エマルジョンからは弾力性のある厚さ10〜
100μｍ程度の薄膜が簡単に作製できるうえに、酸素の
透過性があり、膨潤しないため酸素検出型酵素電極用の
固定化酵素膜を作製するのに適している。From acrylic resin emulsion, elastic thickness 10 ~
Since a thin film of about 100 μm can be easily produced, it is permeable to oxygen and does not swell, so it is suitable for producing an immobilized enzyme membrane for an oxygen-sensing enzyme electrode.
実験例２ 本発明の方法により作製したグルコースオキ
シダーゼ固定化膜の耐久性 （１）作製直後の固定化酵素膜によるグルコース濃度と
酸素濃度計出力の関係 第２図から各グルコース標準溶液について120秒後の酸
素濃度計の出力を読み取り初発の出力（E0）との差を第
３図に示した（Ａ）。第３図の縦軸はグルコースによる
電圧低下E0−EG、横軸はグルコースの濃度（％,W/v）で
ある。Experimental Example 2 Durability of glucose oxidase-immobilized membrane prepared by the method of the present invention (1) Relationship between glucose concentration and oxygen analyzer output by immobilized enzyme membrane immediately after preparation 120 seconds after each glucose standard solution from FIG. The output of the oximeter was read and the difference from the initial output (E 0 ) is shown in FIG. 3 (A). In FIG. 3, the vertical axis represents the voltage drop E 0 -E G due to glucose, and the horizontal axis represents the glucose concentration (%, W / v).
（２）酵素電極を室温で７日間緩衝液中浸漬した後のグ
ルコース濃度と酸素濃度計出力の関係 実験例２の（１）に用いたもの同一の膜から切り取った
グルコースオキシダーゼ固定化膜を酸素電極に装着して
セルに固定し、セル内にpH6.8のリン酸緩衝液を満たし
て室温（約20℃）で７日間経過した後、実験１と同様に
各グルコース標準溶液について酸素濃度計の出力測定し
た。試料を注入してから120秒後の酸素濃度計の出力（E
0−EG）を第２図に示した（Ｂ）。第３図からこの酵素
膜は室温で１週間緩衝液中に浸漬するとわずかに活性が
低下するが酵素電極としては使用可能な程度の活性が残
存していると推察された。(2) Relationship between glucose concentration and oxygen concentration meter output after the enzyme electrode was immersed in a buffer solution at room temperature for 7 days. The same one used in (1) of Experimental Example 2 was used for the glucose oxidase-immobilized membrane cut from the same membrane. After attaching to the electrode and fixing it in the cell, filling the cell with phosphate buffer of pH 6.8 and allowing it to stand at room temperature (about 20 ° C) for 7 days, as in Experiment 1, use an oxygen concentration meter for each glucose standard solution. The output was measured. Output of the oximeter 120 seconds after the sample was injected (E
The 0 -E G) shown in FIG. 2 (B). From FIG. 3, it was inferred that the activity of this enzyme membrane slightly decreased when it was immersed in the buffer solution at room temperature for 1 week, but the activity remained enough to be used as an enzyme electrode.
実験例 ３ グルコースセンサーの感度に及ぼす固定化
酵素膜中の酵素剤量の影響 添加する酵素剤の量を変えて実施例１と同様の操作によ
りグルコースオキシダーゼ固定化膜を作製した。作製し
た膜のグルコースオキシダーゼ含有量は0.2mg/cm2及び
0.6mg/cm2であった。アクリル樹脂塗料0.4ml、水0.4ml
及び酵素剤を混和後5cm×5cmの広さに広げたので膜の厚
さはそれぞれ0.81mm及び0.89mmとなった。実験例２と同
様の条件で測定したグルコース濃度と酸素濃度計の出力
の関係を第４図に示した。同図は膜の酵素含有量が増加
するとグルコースセンサーとしての酸素濃度計の感度
（E0−Ｅ）が増加することを示している。Experimental Example 3 Effect of amount of enzyme agent in immobilized enzyme membrane on sensitivity of glucose sensor A glucose oxidase-immobilized membrane was prepared in the same manner as in Example 1 while changing the amount of enzyme agent added. The glucose oxidase content of the prepared membrane was 0.2 mg / cm 2 and
It was 0.6 mg / cm 2 . Acrylic resin paint 0.4 ml, water 0.4 ml
After the mixture and the enzyme preparation were mixed and spread to a width of 5 cm × 5 cm, the film thicknesses were 0.81 mm and 0.89 mm, respectively. The relationship between the glucose concentration measured under the same conditions as in Experimental Example 2 and the output of the oximeter is shown in FIG. The figure shows that the sensitivity (E 0 −E) of the oximeter as a glucose sensor increases as the enzyme content of the membrane increases.
第１図 非多孔膜を用いた酵素電極のモデル 第２図 グルコースセンサーの応答速度の測定結果 第３図 作製直後の固定化酵素膜及び７日間室温の緩衝
液中に浸漬した固定化酵素膜を装着した酵素電極による
グルコース濃度と酸素濃度計出力の関係 第４図 グルコースセンサーの感度に及ぼす固定化酵素
膜中の酵素剤量の影響Fig. 1 Model of enzyme electrode using non-porous membrane Fig. 2 Results of measurement of response speed of glucose sensor Fig. 3 Immobilized enzyme membrane immediately after preparation and immobilized enzyme membrane immersed in buffer solution for 7 days at room temperature Relationship between glucose concentration and oximeter output by the attached enzyme electrode Fig. 4 Effect of amount of enzyme in immobilized enzyme membrane on sensitivity of glucose sensor
フロントページの続き (72)発明者 大場 俊輝 東京都北区滝野川２丁目６番30号 国税庁 醸造試験所内 (56)参考文献 国際公開89／07139（ＷＯ，Ａ)Continuation of the front page (72) Inventor Toshiki Oba 2-6-30 Takinogawa, Kita-ku, Tokyo Inside the National Brewery Laboratory (56) References International Publication 89/07139 (WO, A)
Claims (1)
たエマルジョンまたは水系合成樹脂エマルジョン塗料に
酵素及び水または使用する酵素に適した緩衝液を加え混
合した後、薄く展開して水を蒸発させて製造した非多孔
固定化酵素膜を酸素電極等の低分子物質検出型電極に密
着させ、さらにその上に半透膜または多孔膜等の検出目
的成分透過性膜を密着させることを特徴とする酵素電極1. An emulsion in which an organic substance capable of forming a coating film is dispersed in water or a water-based synthetic resin emulsion paint, which is mixed with an enzyme and water or a buffer solution suitable for the enzyme to be used, and then thinly spread to remove water. Characterized by adhering a non-porous immobilized enzyme membrane produced by evaporation to a low molecular weight substance detection type electrode such as an oxygen electrode, and further adhering a semipermeable membrane or a permeation membrane of a target component to be detected such as a porous membrane thereon. Enzyme electrode
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1331059A JPH0786497B2 (en) | 1989-12-22 | 1989-12-22 | Enzyme electrode using non-porous immobilized enzyme membrane |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1331059A JPH0786497B2 (en) | 1989-12-22 | 1989-12-22 | Enzyme electrode using non-porous immobilized enzyme membrane |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03194457A JPH03194457A (en) | 1991-08-26 |
| JPH0786497B2 true JPH0786497B2 (en) | 1995-09-20 |
Family
ID=18239394
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1331059A Expired - Lifetime JPH0786497B2 (en) | 1989-12-22 | 1989-12-22 | Enzyme electrode using non-porous immobilized enzyme membrane |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0786497B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6343225B1 (en) * | 1999-09-14 | 2002-01-29 | Implanted Biosystems, Inc. | Implantable glucose sensor |
| AT509355B1 (en) * | 2010-02-10 | 2012-04-15 | Univ Graz Tech | TEST ARRANGEMENT |
-
1989
- 1989-12-22 JP JP1331059A patent/JPH0786497B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH03194457A (en) | 1991-08-26 |
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