JPH02236153A - Permselective membrane and electrode using the same - Google Patents
Permselective membrane and electrode using the sameInfo
- Publication number
- JPH02236153A JPH02236153A JP1057588A JP5758889A JPH02236153A JP H02236153 A JPH02236153 A JP H02236153A JP 1057588 A JP1057588 A JP 1057588A JP 5758889 A JP5758889 A JP 5758889A JP H02236153 A JPH02236153 A JP H02236153A
- Authority
- JP
- Japan
- Prior art keywords
- protein
- electrode
- crosslinking agent
- membrane
- immobilized
- 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.)
- Granted
Links
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
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Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明一は、たとえば電気化学的に検出可能な物質の生
成、または消費を検出する方式の固定化生体触媒電極な
どに好適に用いられる選択透過膜およびそれを用いる電
極に関する．
従来の技術
近年、生化学などの研究の発達によって従来未解明であ
った生体反応が解明されて化学工業の各分野に応用され
るようになってきた．
細胞、酵素などが触媒する生化学反応の応用範囲は広い
が、特に生化学反応を物質の検出手段に応用するいわゆ
るバイオセンシングは注目を集めている．このバイオセ
ンシングによって、従来の技術では測定不可能であった
物質あるいは測定に多大の時間と労力を費やした物質を
簡便に測定することができ、環境計測、食品製造、食品
分析、医療分析などの各分野において実用化が進んでい
る．
一般に、酵素などの生体触媒には、
■反応の特異性が高い，
■温和な条件下での測定が可能である．■高感度で微量
成分の測定が可能である．などの利点があるが、触媒と
して生体由来の物質を用いている故に、
■酵素などの生体触媒が高価である．
■温度、ｐＨなどの反応条件に制限がある．■様々な要
因によって酵素などの生体触媒の触媒能が失われる、い
わゆる失活が生じる．という欠点を併せ持っている．
このような問題点を解決するために、生体触媒を固定化
して用いることが考え出されており、多くの方法が提案
されている．そのうちで共有結合性の架橋剤を用いて生
体触媒を架橋反応によって固定化する方法は、その結合
が吸着やイオン結合などに比較して強固であるために、
実用化に適している．
このような固定化生体触媒を被検出物質の測定に応用す
る場合には、生体触媒とその生化学反応によって増減す
る物質を検出できる検出機構とを組合わせて使用する．
このとき検出機構としては、電気化学検出器、蛍光検出
器、熱検出器などが応用されている．特に、固定化酵素
による生化学反応における酸素の消費または過酸化水素
の生成を酸素電極または過酸化水素電極などを用いて電
気化学的に検出する方法は、装置構成の簡単さ、検出感
度の高さ、応答速度の速さなどの利点を有しており、最
もよく用いられている．
しかしながら、たとえば過酸化水素電極を用いた場合に
は、生化学反応によって生成された過酸化水素だけでな
く、測定試料に含まれるアスコルビン酸などの妨害物質
をも検出してしまい、測定の信頼性が低下してしまうこ
とが知られている．そこで固定化酵素膜と、酵素電極ま
たは過酸化水素電極との間に選択透過展を介在させる方
法が実用化されている．すなわち、酵素電極や過酸化水
素電極などの表面の近傍に緩衝液などを介して被検出物
質以外の物質の透過を制限する選択透過膜と、固定化酵
素膜とをこの順に配置するかあるいは電極表面に直接選
択透過膜と、固定化酵素膜とをこの順に形成する．これ
によって妨害物質の検出を防止するとともに、測定精度
を向上することができる．
従来では、このような選択透過膜として、たとえば特開
昭６０−５６２５４号に開示されているように、アセチ
ルセルロースなどの素材を用いた選択透過膜が使用され
ている．しかしながら、この選択透過膜を製造するため
には、取り扱いの不便な溶剤を使用しなければならない
という問題がある．また、所望の選択透過能を得るため
には、微妙に乾燥時間等を調節しなへければならず、多
大な時間と非常に面倒な操作とを要しなければ実用可能
な選択透過膜を得ることができない．また、本件発明者
等は、タンパク質と架橋剤とを用いて簡便に選択透過膜
を形成する方法を提案した（特開昭６３−１８２５５９
号）．シかしながら、選択透過膜に限らず、一般にタン
パク質と架橋剤とを用いて形成された固定化タンパク質
膜には、物理的強度が低いという問題があり、その取り
汲いが不便であり、耐久性に劣るという欠点がある．
このような固定化タンパク質膜の物理的強度の問題は、
たとえば物理的強度の大きい担体上にタンパク質を固定
化しても固定化タンパク質膜自体が脆くなり、部分的に
剥離することがあり、根本的に解決することはできない
．
したがって、上述したような選択透過膜に対する問題点
は、有効な解決策が提示されずに現在番こ至っている．
一般に、選択透過膜ではなく、酵素などの生体触媒であ
るタンパク質を固定化した固定化タンパク質膜の強度を
向上させる方法は、たとえば特公昭５８−４９８２１号
に示されてレ１る．この方法は、アミノおよびイミノ系
高分子とともに、生体触媒である酵素を固定化し、酵素
固定化膜の物理的強度を向上させるものである．しかし
ながら、このような方法を選択透過膜に応用する場合に
は、固定化されたタンパク質の物性が変化しやすいので
所望の選択透過能を得ること力《困難であるという問題
がある．
さらに、特開昭６２−３２３５２号に示されているよう
に、タンパク質である酵素をポリエステル布に固定化し
、酵素固定化膜の物理的強度の補強を図る方法も提案さ
れている．しかしながら，このような場合には、タンパ
ク質膜の膜厚が増大してしまい、選択透過膜の形成に応
用する場合番こは、固定化酵素電極の測定感度と測定速
度が悪化してしまうという問題がある．
発明が解決しようとする課題
したがって本発明の目的は上記技術的課題を解決し、所
望の選択透過能を有し、物理的強度に浸れ、なおかつｍ
便に製造することができる選択透過膜およびそれを用い
る電極を提供することである．
課題を解決するための手段
本発明は、タンパク質と、少なくとも１種類の架橋剤と
を含み、前記タンパク質以外に架橋剤と反応する高分子
物質が少なくとも１種類添加された混合溶液を膜状に展
開し、
架橋反応により前記タンパク質を固定化して成ることを
特徴とする選択透過．ｒｍである．また本発明は、前記
タンパク質が少なくとも１種類以上の球状タンパク質を
含む成分構成であり、前記少なくとも１種類の架橋剤が
アミノ基と架橋反応する架橋剤を含み、
前記高分子物質がアミノ基、アミン基の誘導体あるいは
その両方を有する直鎖状多糖類を含むことを特徴とする
前記選択透過膜である．前記タンパク質と直鎖状多糖類
との構成重量比は１０００：１から１０００：１０の範
囲であり、タンパク質と架橋剤との構成重量比は、１０
０：５から１００：５０の範囲であることが好ましい．
また前記直鎖状多糖類はキトサンであってもよい．さら
に本発明は、上述した選択透過膜を導電性基体近傍に設
けたことを特徴とする電極である．このような電極にお
いて前記選択透過膜の導電性基体と反対側表面上に、少
なくとも１種類のオキシダーゼを固定化した固定化酵素
膜を設けてもよい
作 用
導電性基体表面上にタンパク質を固定化し、これを選択
透過膜とし、さらに固定化酵素膜を形成する場合には、
この選択透過膜の物理強度が固定化酵素電極の寿命に大
きな影響を与える．一般に、固定化タンパク質膜の物理
的強度を向上させるためには、固定化タンパク質膜中に
おける架橋剤の濃度を高くすればよい．しかし、この固
定化タンパク質膜を選択透過膜として形成する場合には
、固定化タンパク質膜の物理的特性が架橋剤濃度によっ
て変化するので、選択透過能が悪化してしまうことがあ
り、望ましくない．また、固定化タンパク質膜中に架橋
剤と反応する補強物質を共存させて固定化タンパク質膜
の強度を向上させる方法もある．しかし、このような方
法では、補強物質が固定化タンパク質膜の物理的特性に
悪影響を与え、選択透過膜としての本来の機能、すなわ
ち選択性を失わせてしまう恐れがある．
本件発明者は、特にタンパク質と架橋剤とで構成される
選択透過膜中に架橋剤と反応する補強物質を共存させる
実験を行い、選択透過膜の物理的特性に悪影響を与えず
、なおかつ選択透過膜の物理的強度を向上させる補強物
質および、その製造条件を検討し、本発明を完成するに
至った．このような補強物質には、
■固定化されるタンパク質の溶液またはこのタンパク質
と架橋剤との混会溶液に溶解する物質であること．
■選択透過膜の物理的特性に悪影響を与えないために少
量で選択透過膜の物理的強度を向上させることができる
物質であること．
■架橋剤に対して、固定化すべきタンパク質と同程度の
反応性を有し、架橋反応によって不溶化する物質である
こと．
などの諸性質が要求される．
したがって本件発明者は、タンパク質と架橋剤との混合
溶液中に共存させることが可能で、架橋剤と架橋反応を
行い、かつ、選択透過膜の選択透過能に悪影響を与えな
い化ｉ！！ｉ物を探索した．たとえば、架橋剤としてグ
ルタルアルデヒド等を用い、溶媒として蒸留水または！
ｌ！胃液などを用いて一定濃度のタンパク質と架橋剤の
混合溶液を調製し、この温き溶液に各種化合物を共存さ
せてグルタルアルデヒド等と架橋反応を行わせ、固定化
タンパク質膜を作成した．作成された固定化タンパク質
膜の物理的強度および選択透過能について実験し、選択
透過膜の物理強度を向上させ、選択透過能に悪影響を与
えない化合物を探索することができる．
この結果、タンパク質を少なくとも１種顕以上の球状タ
ンパク質を含む成分構成にし、架橋剤としてアミン基と
架橋反応を行う化合物を用い、前記混き溶液に共存させ
る化合物としてアミン基、アミノ基の誘導体、あるいは
その両方を倉む直鎖状多糖類を使用すれば固定化タンパ
ク質膜の選択透過性に悪影響を与えることなく、なおか
つ固定化タンパク質膜の物理的強度を向上することがで
きることが見いだされた．
本発明によれば、容易に優れた選択透過膜を形成するこ
とが可能である．特に、導電性基体表面上に選択透過膜
を直接に設け，ることが容易に可能となり、さらに選択
透過膜上に固定化酵素膜を形成することにより、固定化
酵素電極を製造することができる．
本発明に使用される架橋剤としては、各種架橋剤を使用
することができるが、グルタルアルデヒド、ヘキサメチ
レンジイソシアネートなどの架嬌試薬は結き強度が大き
いので、好ましく用いられる．特にグルタルアルデヒド
はより好ましく用いられる．
タンパク質と架橋剤との横成重量比は、得られる選択透
過膜の選択透過性の点で、好ましくは１００：５〜１０
０：５０の範囲である．また本発明に従う選択透過膜に
使用されるタンパク質は、アルプミン、グロプリン、プ
ロラミンなどの球状タンパク質を少なくとも１種類以上
含む成分構成であれば、優れた選択透過能を有する選択
透過膜を製造することができる．
また、タンパク質と架碑剤との混合溶液に添加され架橋
剤と反応する高分子物質は、好ましくはアミノ基、アミ
ノ基誘導体あるいはその両方を有する直鎖状多糖類であ
り、具体的にはグルコサミン、ガラクトサミンなどのア
ミノ糖およびその誘導体を楕成単位とする多糖類を用い
ることが可能である．このようなものには、バクテリア
のべブナドグリカン、一部脱アセチル化したヒアルロン
酸や一部脱アセチル化したコンドロイナン硫酸、キトサ
ンが例示できる．容易に入手可能なキトサンは、このよ
うな高分子物質として好適に用いられる．
また本発明に従う選択透過膜においては、タンパク質と
前記直鎖状多糖類との構成重量比は、１０００：１から
１０００：１０の範囲にあることが好ましい．タンパク
質に対する直鎖状多糖類の構成重量比が１０００：１０
より大きくなると、選択透過膜の選択能が悪化してくる
．この原因については定かではないが、直鎖状多糖類の
タンパク質に対する構成重量比が大きくなると、相対的
にタンパク質に対する架橋剤濃度が低下し、タンパク質
に対する架橋反応が不充分になる．したがって，固定化
タンパク質膜が脆くなり、導電性基体表面との結合性、
密着性およびタンパク質問の結合性に影響し、膜の緻密
さが保たれなくなるためだと考えられる．また、上記構
成重量比が１０００＝１よりも小さくなると膜強度の向
上が不充分となる．
本発明に従えば、選択透過膜は先ず上述したタンパク質
と架橋剤とを蒸留水または＆！街液などに溶解し、さら
に上記高分子物質を添加し、たとえば導電性基体などの
表面上に塗布する．これによって高分子物質を添加した
混合溶液では、架橋反応が進行し、固定化タンパク質膜
−が形成される．このようにして、製造された選択透過
膜は、たとえば超音波処理などの物理的衝撃に対して非
常に耐久性に優れている．また高分子物質を添加したこ
とにより、選択透過能が悪化するといった事態を招来す
ることもない．
本発明に従う選択透過膜を導電性基体上に配置し、これ
によって過酸化水素測定用４Ｘ４極を構成することがで
きる．このとき、選択透過膜を［１液などを介して導電
性基体近傍に配置することも可能であるが、導電性基体
上に直接選択透過膜を形成してもよい．さらに、この選
択透過膜の導電性基体とは反対側に少なくとも１種類の
オキシダーゼ等の酵素を固定化することによって、固定
化酵素電極を製造することができる．このような固定化
酵素電極は物理的強度が大きく、耐久性および被検出物
質の，選択性に非常に優れている．また本発明に従う選
択透過膜は、バイオリアクターにおいて反応生成物、若
しくは原材料のみを選択的に分離するためなどにも使用
することが可能である．
以下に実施例を示し、本発明をより具体的に説明するが
、本発明はこれのみに限定されるものではない．なお、
％は重量％を表す．
実施例
測定装置
本実施例では、第１図に示されるフロー型測定装置を使
用した．第１図に示されるフロー型測定装置は、μｌオ
ーダの試料注入が可能な高速液体クロマトグラフイ用の
インジエクタ３と、本発明に従う選択透過膜を用いた電
極Ｅ１〜Ｅ４（Ｃｌ〜Ｃ５）および参照電極としてのＡ
ｇ／ＡｇＣｌ電極８が取付けられ、対極７としてステン
レス鋼製の管路が備えられた測定用セル５とを含んで構
成される．たとえば内径０．５ｍｍ、長さ１．５ｍのテ
フロン製の希釈用管路４は、インジエクタ３と、測定用
セル５との間に接続される．測定用セル５の内容積は４
０μｌであり、電極Ｅ１〜Ｅ４（Ｃ１〜Ｃ５）とＡｇ／
ＡｇＣｌ ＠極８とが、ｙＩＷＩ液の管路を介して対向
して配置される．電極Ｅ１〜Ｅ４（Ｃｌ〜Ｃ５）には、
ポテンシオスタット９によってＡｇ／ＡｇＣｌ電極８に
対して＋〇．６０Ｖの電圧が印加される．
このような構成は、恒温槽１２内に配宜され、恒温槽１
２内の温度は３７℃に保持される．緩衝液１の送液には
高速液体クロマトグラフイ用のボンプ２を用い、緩衝液
１としてｐＨ６．０の０．１Ｍリン酸ナトリウムＩ！衝
液が１．．Ｏｍ１／分の流量で送液される．測定を終え
た試料を含む緩衝液は廃液瓶１１にて捕捉される．なお
測定値は記録計１０によって記録される．
実施例１
タンパク質としてウシ血清アルプミン《フラクションＶ
，シグマ社製》を、架橋剤としてグルタルアルデヒドを
、タンパク質以外に架橋剤と反応する物質としてキトサ
ン（東京化成工業製）を用いた．
キトサンは１００ｍＭ塩酸水溶液に溶解して０．５％キ
トサン溶液とした．
ワツセルマン試験管中で
５％ウシ血清アルブミン水溶液 ４００μ！、２５％グ
ルタルアルデヒド水溶液 ２０μｌ、０．５％キトサ
ン溶液 ２０μｌ、蒸 留 水
５６０ μ！ 、を混合し
、
最終濃度 ２％ ウシ血清アルブミン、０．５％
グルタルアルデヒド、
０．０１％ キトサン
の混合溶液を調製した．
この混合溶液のタンパク質とキトサンの構成重量比は１
０００：５、タンパク質と架橋剤の構成重量比は１００
：２５である．
一方、一導電性基体である直径２ｍｍの白金線の側面を
熱収縮テフロンで被覆し、その断面を１６００番手のエ
メリー紙で研磨した．この研磨された表面に、マイクロ
シリンジで上記混合溶液５μｌを載せて４０℃で乾燥し
、白金電極上に直接タンパク質膜を形成した．このよう
にして、過酸化水素を検出するための電極Ｅ１を作成し
た．このタンパク質膜で被覆された白金電極から成る電
ｉｉＥ１を第１図に示すフロー型測定装置に組み込み、
１００ｍＭリン酸緩衝液１を流しながら、ｋｇ／ＡｇＣ
ｌ電極８に対してｏ．６ｖの電位を印加した．この状態
でインジェクタ３から５ｍＭ過酸化水素を注入したとこ
ろ、検出電流値は２１８ｎＡであった．次に５ｍＭアス
コルビン酸の溶液を注入したところ、検出電流値は２．
９ｎＡであった．したがって、第１表に示すようにアス
コルビン酸による検出電流値の同濃度の過酸化水素の検
出電流値に対する割合は１．３％であった．（以下余白
》
第 １
表
上述したように作成した電極Ｅ１を超音波処理装置で１
０分間処理した後、再び第１図に示すフロー型測定装置
に組み込んだ．５ｍＭ過酸化水素を注入したときの検出
電流値は２１８ｎＡであり、５ｍＭアスコルビン酸の溶
液を注入したときの検出電流値は２．９ｎＡであった．
この結果は、超音波処理を行う前の結果とともに第２表
に示されている．第２表から、超音波処理の前後におい
て、電極Ｅ１の過酸化水素およびアスコルビン酸に対す
る感度に変化はなかったことが判る．
第２表
実施ｒ！４２
タンパク質としてウシ血清アルブミン（フラクション■
，シグマ社製）を、架橋剤としてグルタルアルデヒドを
，タンパク質以外に架橋剤と反応する物質としてキトサ
ン（東京化成工業製》を用いた．
キトサンは１００ｍＭ酢酸水溶液に溶解して０．５％キ
トサン溶液とした．
ワツセルマン試験管中で
５％ウシ血清アルブミン水溶液 ４００μｌ、２５％グ
ルタルアルデヒド水溶液 ．２０μｌ、０．５％キトサ
ン溶液 ２０μｌ、蒸 留 水
５ ６ ０μｌ 、を混合
し、
最終濃度 ２％ ウシ血清アルブミン、０．５％
グルタルアルデヒド、
０．０１％ キトサン
の混合溶液を調製した．
この混合溶液のタンパク質とキトサンの楕成重量比は１
０００：５、タンパク質と架橋剤の楕成重量比は１００
：２５である．
一方、導電性基体である直径２ｍｍの白金線の側面を熱
収縮テフロンで被覆し、その断面を１６００番手のエメ
リー紙で研磨した．この研磨された表面に、マイクロシ
リンジで上記混合溶液５μｌを載せて４０℃で乾燥し、
白金電極上に直接タンパク質膜を形成した．このように
して、過酸化水素を検出するための電極Ｅ２を作成した
．このタンパク質膜で被覆された白金電極から成る電極
Ｅ２を図面に示すフロー型測定装置に組み込み、１００
ｍＭリン酸＆！衝液１を流しながら、Ａｇ／ＡｇＣ１電
極８に対して０．６Ｖｆ）電位を印加した．この状態で
インジェクタ３がら５ｍＭ過酸化水素を注入したところ
、検出電流値は２２０ｎＡであった．次に５ｍＭアスコ
ルビン酸の溶液を注入したところ、検出電流値は２．９
ｎＡであった．したがって、第１表に示すようにアスコ
ルビン酸による検出電流値の同濃度の過酸化水素の検出
電流値に対する割合は実施例１と同様に１．３％であっ
た．また、電極Ｅ２に超音波処理を行っても、過酸化水
素およびアスコルビン酸に対する感度に変化はなかった
．したがって、キトサンを溶解するための水溶液は、酸
性溶液であれば、塩酸であっても酢酸であってもよいこ
とが判った．実施例３
タンパク質としてウシ血清アルブミン（フラクション■
．シグマ社製）を、架橋剤としてグルタルアルデヒドを
、タンパク質以外に架橋剤と反応する物質としてキトサ
ン《東京化成工業製》を用いた．
キトサンは１００ｍＭ塩酸水溶液に溶解して０．５％キ
トサン溶液とした．
ワツセルマン試験管中で
５％ウシ血清アルブミン水溶液 ４００μ！２５％グル
タルアルデヒド水溶液 ２０μｌ、０．５％キトサン
溶液 ２００μｌ蒸 留 水
３ ８ ０μ！ 、を混合し、
最終濃度 ２％ ウシ血清アルブミン、０．５％
グルタルアルデヒド、
０．０５％ キトサン
の混合溶液を調製した．
この混合溶液のタンパク質とキトサンの構成重量比は１
０００：２５、タンパク質と架橋剤の構成重量比はＺｏ
ｏ：２５である．
一方、導電性基体である直径２ｍｍの白金線の側面を熱
収縮テフロンで被覆し、その断面を１６００番手のエメ
リー紙で研麿した．この研磨された表面に、マイクロシ
リンジで上記混合溶液５μｌを載せて４０℃で乾燥し、
白金電極上に直接タンパク質膜を形成した．このように
して、過酸化水素を検出するための電極Ｅ３を作成した
。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a selectively permeable membrane suitable for use in, for example, an immobilized biocatalyst electrode for detecting the production or consumption of electrochemically detectable substances. and the electrodes that use it. Conventional technology In recent years, with the development of research in biochemistry, biological reactions that were previously unexplained have been elucidated and are now being applied to various fields of the chemical industry. Biochemical reactions catalyzed by cells and enzymes have a wide range of applications, but so-called biosensing, which applies biochemical reactions as a means of detecting substances, is attracting particular attention. With this biosensing, it is possible to easily measure substances that could not be measured with conventional techniques or substances that require a great deal of time and effort to measure, and are useful in environmental measurement, food manufacturing, food analysis, medical analysis, etc. Practical use is progressing in various fields. In general, biocatalysts such as enzymes: 1) have high reaction specificity, and 2) can be measured under mild conditions. ■It is possible to measure trace components with high sensitivity. However, because biologically derived substances are used as catalysts, biocatalysts such as enzymes are expensive. ■There are restrictions on reaction conditions such as temperature and pH. ■Due to various factors, biocatalysts such as enzymes lose their catalytic ability, which is called deactivation. It also has the disadvantage of In order to solve these problems, the use of immobilized biocatalysts has been considered, and many methods have been proposed. Among these methods, the method of immobilizing the biocatalyst through a cross-linking reaction using a covalent cross-linking agent has a stronger bond than that of adsorption or ionic bonding.
Suitable for practical use. When applying such an immobilized biocatalyst to the measurement of a target substance, the biocatalyst is used in combination with a detection mechanism that can detect the substance that increases or decreases due to its biochemical reaction.
At this time, the detection mechanisms used include electrochemical detectors, fluorescence detectors, and thermal detectors. In particular, the method of electrochemically detecting the consumption of oxygen or the production of hydrogen peroxide in biochemical reactions by immobilized enzymes using an oxygen electrode or hydrogen peroxide electrode has a simple device configuration and a high detection sensitivity. It has advantages such as speed and response speed, and is the most commonly used. However, when a hydrogen peroxide electrode is used, for example, it detects not only hydrogen peroxide produced by biochemical reactions, but also interfering substances such as ascorbic acid contained in the measurement sample, resulting in poor measurement reliability. is known to decrease. Therefore, a method has been put into practical use that involves intervening selective permeation between the immobilized enzyme membrane and the enzyme electrode or hydrogen peroxide electrode. In other words, a selectively permeable membrane that restricts the permeation of substances other than the target substance through a buffer, etc., and an immobilized enzyme membrane are placed in this order near the surface of the enzyme electrode or hydrogen peroxide electrode, or the electrode A permselective membrane and an immobilized enzyme membrane are formed directly on the surface in this order. This prevents the detection of interfering substances and improves measurement accuracy. Conventionally, as such a selectively permeable membrane, a selectively permeable membrane using a material such as acetyl cellulose has been used, as disclosed in, for example, Japanese Patent Laid-Open No. 60-56254. However, in order to manufacture this selectively permeable membrane, there is a problem in that a solvent that is inconvenient to handle must be used. In addition, in order to obtain the desired permselective ability, it is necessary to delicately adjust the drying time, etc., and it is necessary to make a practical permselective membrane that does not require a large amount of time and extremely troublesome operations. I can't get it. In addition, the present inventors proposed a method for easily forming a selectively permeable membrane using a protein and a crosslinking agent (Japanese Patent Application Laid-Open No. 182559/1983).
issue). However, not only permselective membranes but also immobilized protein membranes formed using proteins and cross-linking agents in general have a problem of low physical strength, making it inconvenient to remove them. The drawback is that it is less durable. The problem of physical strength of such immobilized protein membranes is
For example, even if a protein is immobilized on a carrier with high physical strength, the immobilized protein membrane itself becomes brittle and may partially peel off, making it impossible to fundamentally solve the problem. Therefore, the above-mentioned problems with permselective membranes have not been proposed and effective solutions have not yet been proposed.
In general, a method for improving the strength of an immobilized protein membrane in which a protein such as a biocatalyst such as an enzyme is immobilized rather than a selectively permeable membrane is disclosed in, for example, Japanese Patent Publication No. 49821/1983. This method improves the physical strength of enzyme-immobilized membranes by immobilizing enzymes, which are biocatalysts, along with amino and imino polymers. However, when applying such a method to a permselective membrane, there is a problem in that it is difficult to obtain the desired permselective ability because the physical properties of the immobilized protein tend to change. Furthermore, as shown in JP-A No. 62-32352, a method has been proposed in which an enzyme, which is a protein, is immobilized on a polyester cloth to strengthen the physical strength of the enzyme-immobilized membrane. However, in such cases, the thickness of the protein membrane increases, and when applied to the formation of selectively permeable membranes, there is a problem that the measurement sensitivity and measurement speed of the immobilized enzyme electrode deteriorate. There is. Problems to be Solved by the Invention Therefore, an object of the present invention is to solve the above technical problems, to have a desired permselective ability, high physical strength, and m.
The object of the present invention is to provide a selectively permeable membrane that can be manufactured in a vacuum and an electrode using the same. Means for Solving the Problems The present invention develops into a film a mixed solution containing a protein and at least one type of crosslinking agent, to which at least one type of polymeric substance that reacts with the crosslinking agent is added in addition to the protein. A selective permselector, characterized in that the protein is immobilized by a cross-linking reaction. It is rm. Further, the present invention provides a component composition in which the protein includes at least one type of globular protein, the at least one type of crosslinking agent includes a crosslinking agent that crosslinks with an amino group, and the polymer substance includes an amino group, an amine. The permselective membrane described above is characterized in that it contains a linear polysaccharide having a derivative of the group or both. The composition weight ratio of the protein and the linear polysaccharide is in the range of 1000:1 to 1000:10, and the composition weight ratio of the protein and the crosslinking agent is 10.
It is preferably in the range of 0:5 to 100:50.
Further, the linear polysaccharide may be chitosan. Furthermore, the present invention is an electrode characterized in that the above-described permselective membrane is provided in the vicinity of a conductive substrate. In such an electrode, an immobilized enzyme membrane having at least one type of oxidase immobilized thereon may be provided on the surface of the permselective membrane opposite to the conductive substrate. , when using this as a selectively permeable membrane and further forming an immobilized enzyme membrane,
The physical strength of this selectively permeable membrane has a large effect on the lifespan of the immobilized enzyme electrode. Generally, in order to improve the physical strength of an immobilized protein membrane, it is sufficient to increase the concentration of a crosslinking agent in the immobilized protein membrane. However, when this immobilized protein membrane is formed as a permselective membrane, the physical properties of the immobilized protein membrane change depending on the crosslinking agent concentration, which is undesirable because the permselectivity ability may deteriorate. Another method is to improve the strength of an immobilized protein membrane by coexisting a reinforcing substance that reacts with a crosslinking agent in the immobilized protein membrane. However, in such a method, the reinforcing substance may adversely affect the physical properties of the immobilized protein membrane, causing it to lose its original function as a permselective membrane, that is, its selectivity. The inventor of the present invention conducted an experiment in which a reinforcing substance that reacts with the crosslinking agent coexists in a selectively permeable membrane composed of a protein and a crosslinking agent, and found that it does not adversely affect the physical properties of the selectively permeable membrane. The present invention was completed by studying reinforcing substances that improve the physical strength of membranes and their manufacturing conditions. Such reinforcing substances include: (1) A substance that is soluble in the solution of the protein to be immobilized or the mixed solution of this protein and a crosslinking agent. ■It must be a substance that can improve the physical strength of a selectively permeable membrane in small amounts without adversely affecting the physical properties of the selectively permeable membrane. ■It must be a substance that has the same level of reactivity with the crosslinking agent as the protein to be immobilized and that becomes insolubilized by the crosslinking reaction. Various properties such as these are required. Therefore, the inventor of the present invention has developed a protein that can coexist in a mixed solution of a protein and a crosslinking agent, performs a crosslinking reaction with the crosslinking agent, and does not adversely affect the permselective ability of the permselective membrane. ! I searched for i things. For example, use glutaraldehyde as a crosslinking agent, distilled water or! as a solvent!
l! A mixed solution of protein and a cross-linking agent at a certain concentration was prepared using gastric juice, etc., and various compounds were coexisted with this warm solution to perform a cross-linking reaction with glutaraldehyde, etc., to create an immobilized protein film. By conducting experiments on the physical strength and permselectivity of the created immobilized protein membrane, it is possible to search for compounds that improve the physical strength of the permselective membrane and do not adversely affect the permselectivity. As a result, the protein has a component composition containing at least one type of globular protein, a compound that performs a crosslinking reaction with an amine group is used as a crosslinking agent, and the compounds coexisting in the mixed solution include an amine group, an amino group derivative, It was discovered that by using a linear polysaccharide that carries both of these functions, the physical strength of the immobilized protein membrane can be improved without adversely affecting the permselectivity of the immobilized protein membrane. According to the present invention, it is possible to easily form an excellent permselective membrane. In particular, it is now possible to easily provide a selectively permeable membrane directly on the surface of a conductive substrate, and further, by forming an immobilized enzyme membrane on the selectively permeable membrane, an immobilized enzyme electrode can be manufactured. ．． As the crosslinking agent used in the present invention, various crosslinking agents can be used, but crosslinking reagents such as glutaraldehyde and hexamethylene diisocyanate have high binding strength and are therefore preferably used. In particular, glutaraldehyde is more preferably used. The weight ratio of protein and crosslinking agent is preferably 100:5 to 10 in terms of permselectivity of the resulting permselective membrane.
The range is 0:50. Furthermore, if the protein used in the permselective membrane according to the present invention has a composition containing at least one type of globular protein such as albumin, globulin, or prolamin, it is possible to produce a permselective membrane with excellent permselective ability. can. In addition, the polymeric substance that is added to the mixed solution of protein and crosslinking agent and reacts with the crosslinking agent is preferably a linear polysaccharide having an amino group, an amino group derivative, or both, and specifically, glucosamine It is possible to use polysaccharides whose oval units are amino sugars such as , galactosamine, and their derivatives. Examples of such substances include bacterial bevnadoglycan, partially deacetylated hyaluronic acid, partially deacetylated chondroinan sulfate, and chitosan. Chitosan, which is easily available, is suitably used as such a polymeric substance. In the permselective membrane according to the present invention, the weight ratio of the protein to the linear polysaccharide is preferably in the range of 1000:1 to 1000:10. Constituent weight ratio of linear polysaccharide to protein is 1000:10
As it becomes larger, the selectivity of the permselective membrane deteriorates. The reason for this is not clear, but as the weight ratio of linear polysaccharide to protein increases, the concentration of crosslinking agent relative to protein decreases, and the crosslinking reaction to protein becomes insufficient. Therefore, the immobilized protein film becomes brittle, and the bonding property with the conductive substrate surface decreases.
This is thought to be because it affects adhesion and protein-protein bonding, making it difficult to maintain the membrane's density. Furthermore, if the above-mentioned constituent weight ratio is smaller than 1000=1, the film strength will not be improved sufficiently. According to the present invention, the selectively permeable membrane first mixes the above-mentioned protein and crosslinking agent with distilled water or &! It is dissolved in street liquid, etc., the above-mentioned polymeric substance is added to it, and it is applied onto the surface of, for example, a conductive substrate. As a result, in the mixed solution to which the polymeric substance has been added, a crosslinking reaction proceeds and an immobilized protein film is formed. The selectively permeable membrane produced in this way has excellent durability against physical impacts such as ultrasonic treatment. In addition, the addition of polymeric substances does not cause a situation where the selective permeability is deteriorated. A selectively permeable membrane according to the present invention can be placed on a conductive substrate to constitute a 4×4 pole for measuring hydrogen peroxide. At this time, it is possible to place the selectively permeable membrane in the vicinity of the conductive substrate via a liquid or the like, but it is also possible to form the selectively permeable membrane directly on the conductive substrate. Furthermore, an immobilized enzyme electrode can be manufactured by immobilizing at least one type of enzyme such as oxidase on the side of the permselective membrane opposite to the conductive substrate. Such an immobilized enzyme electrode has great physical strength, durability, and excellent selectivity for the target substance. The selectively permeable membrane according to the present invention can also be used to selectively separate only reaction products or raw materials in bioreactors. The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto. In addition,
% represents weight%. Example Measuring Apparatus In this example, a flow type measuring apparatus shown in FIG. 1 was used. The flow-type measuring device shown in FIG. 1 includes an injector 3 for high-performance liquid chromatography capable of injecting a sample on the order of μl, electrodes E1 to E4 (Cl to C5) using a selectively permeable membrane according to the present invention, and A as a reference electrode
The measurement cell 5 is equipped with a g/AgCl electrode 8 and a stainless steel conduit as a counter electrode 7. For example, a dilution pipe 4 made of Teflon and having an inner diameter of 0.5 mm and a length of 1.5 m is connected between the injector 3 and the measurement cell 5. The internal volume of the measurement cell 5 is 4
0 μl, electrodes E1 to E4 (C1 to C5) and Ag/
AgCl @pole 8 are placed facing each other via the yIWI liquid conduit. Electrodes E1 to E4 (Cl to C5) include
+〇 relative to the Ag/AgCl electrode 8 by the potentiostat 9. A voltage of 60V is applied. Such a configuration is arranged in the thermostatic chamber 12, and the thermostatic chamber 1
The temperature inside 2 is maintained at 37°C. Bump 2 for high performance liquid chromatography was used to send buffer solution 1, and 0.1M sodium phosphate I! with a pH of 6.0 was used as buffer solution 1. The solution is 1. ．． The liquid is delivered at a flow rate of Om1/min. After the measurement, the buffer containing the sample is captured in the waste liquid bottle 11. Note that the measured values are recorded by a recorder 10. Example 1 Bovine serum albumin (fraction V) as a protein
, manufactured by Sigma Corporation,'' glutaraldehyde was used as a crosslinking agent, and chitosan (manufactured by Tokyo Kasei Kogyo) was used as a substance that reacts with the crosslinking agent in addition to proteins. Chitosan was dissolved in 100mM hydrochloric acid aqueous solution to make a 0.5% chitosan solution. 5% bovine serum albumin aqueous solution 400 μ in Wasselmann test tube! , 25% glutaraldehyde aqueous solution 20μl, 0.5% chitosan solution 20μl, distilled water
560μ! , final concentration 2% bovine serum albumin, 0.5%
A mixed solution of glutaraldehyde and 0.01% chitosan was prepared. The composition weight ratio of protein and chitosan in this mixed solution is 1
000:5, composition weight ratio of protein and crosslinking agent is 100
:25. On the other hand, the side surface of a 2 mm diameter platinum wire serving as a conductive substrate was covered with heat-shrinkable Teflon, and its cross section was polished with 1600-grit emery paper. 5 μl of the above mixed solution was placed on the polished surface using a microsyringe and dried at 40° C. to form a protein film directly on the platinum electrode. In this way, electrode E1 for detecting hydrogen peroxide was created. The electrode iiE1 consisting of a platinum electrode covered with this protein film was incorporated into the flow type measuring device shown in Fig. 1.
kg/AgC while flowing 100mM phosphate buffer 1.
l to the electrode 8. A potential of 6V was applied. When 5mM hydrogen peroxide was injected from injector 3 in this state, the detected current value was 218nA. Next, when a 5mM ascorbic acid solution was injected, the detected current value was 2.
It was 9nA. Therefore, as shown in Table 1, the ratio of the current value detected by ascorbic acid to the current value detected by hydrogen peroxide at the same concentration was 1.3%. (Left below) Table 1 Electrode E1 prepared as described above was processed using an ultrasonic processing device.
After processing for 0 minutes, it was again incorporated into the flow-type measuring device shown in Figure 1. The detected current value when 5mM hydrogen peroxide was injected was 218nA, and the detected current value when 5mM ascorbic acid solution was injected was 2.9nA. These results are shown in Table 2 along with the results before ultrasonication. From Table 2, it can be seen that there was no change in the sensitivity of electrode E1 to hydrogen peroxide and ascorbic acid before and after the ultrasonic treatment. Table 2 implementation r! 42 Bovine serum albumin as a protein (fraction ■
, manufactured by Sigma Corporation), glutaraldehyde was used as a cross-linking agent, and chitosan (manufactured by Tokyo Kasei Kogyo Co., Ltd.) was used as a substance other than protein that reacts with the cross-linking agent. Chitosan was dissolved in a 100 mM acetic acid aqueous solution and a 0.5% chitosan solution was used. In a Wasselmann test tube, 400 μl of 5% bovine serum albumin aqueous solution, 20 μl of 25% glutaraldehyde aqueous solution, 20 μl of 0.5% chitosan solution, and distilled water.
560 μl, final concentration 2% bovine serum albumin, 0.5%
A mixed solution of glutaraldehyde and 0.01% chitosan was prepared. The elliptical weight ratio of protein and chitosan in this mixed solution is 1
000:5, the elliptical weight ratio of protein and crosslinker is 100
:25. On the other hand, the side surface of the conductive substrate, a platinum wire with a diameter of 2 mm, was covered with heat-shrinkable Teflon, and its cross section was polished with 1600-grit emery paper. 5 μl of the above mixed solution was placed on this polished surface using a microsyringe and dried at 40°C.
A protein film was formed directly on the platinum electrode. In this way, electrode E2 for detecting hydrogen peroxide was created. Electrode E2 consisting of a platinum electrode coated with this protein film was incorporated into the flow type measuring device shown in the drawing, and
mM phosphoric acid &! A potential of 0.6 Vf) was applied to the Ag/AgC1 electrode 8 while flowing the buffer solution 1. When 5mM hydrogen peroxide was injected through injector 3 in this state, the detected current value was 220nA. Next, when a 5mM ascorbic acid solution was injected, the detected current value was 2.9.
It was nA. Therefore, as shown in Table 1, the ratio of the current value detected by ascorbic acid to the current value detected by hydrogen peroxide at the same concentration was 1.3%, as in Example 1. Furthermore, even when electrode E2 was subjected to ultrasonic treatment, there was no change in sensitivity to hydrogen peroxide and ascorbic acid. Therefore, it was found that the aqueous solution for dissolving chitosan may be either hydrochloric acid or acetic acid as long as it is an acidic solution. Example 3 Bovine serum albumin (fraction ■
．． (manufactured by Sigma), glutaraldehyde was used as a cross-linking agent, and chitosan (manufactured by Tokyo Kasei Kogyo) was used as a substance other than protein that reacts with the cross-linking agent. Chitosan was dissolved in 100mM hydrochloric acid aqueous solution to make a 0.5% chitosan solution. 5% bovine serum albumin aqueous solution 400 μ in Wasselmann test tube! 25% glutaraldehyde aqueous solution 20 μl, 0.5% chitosan solution 200 μl distilled water
3 8 0μ! , final concentration 2% bovine serum albumin, 0.5%
A mixed solution of glutaraldehyde and 0.05% chitosan was prepared. The composition weight ratio of protein and chitosan in this mixed solution is 1
000:25, the composition weight ratio of protein and crosslinking agent is Zo
o:25. On the other hand, the side surface of the conductive substrate, a platinum wire with a diameter of 2 mm, was covered with heat-shrinkable Teflon, and its cross section was polished with 1600-grit emery paper. 5 μl of the above mixed solution was placed on this polished surface using a microsyringe and dried at 40°C.
A protein film was formed directly on the platinum electrode. In this way, electrode E3 for detecting hydrogen peroxide was created.
このタンパク質膜で被覆された白金電極から成る電極Ｅ
３を第１図に示すフロー型測定装置に組み込み、１００
ｍＭリン酸ｗｉ衝液１を流しながら、Ａ ｇ ／ Ａ
ｇ Ｃ ｌ電極８に対して０．６Ｖの電位を印加した．
この状態でインジエクタ３から５ｍＭ過酸化水素を注入
したところ、検出電流値は２１９ｎＡであった．次に５
ｍＭアスコルビン酸の溶液を注入したところ、検出電流
値は６．８ｎＡであった．したがって、第１表に示すよ
うにアスコルビン酸による検出電流値の同濃度の過酸化
水素の検出電流値に対する割合は３．１％であった．実
施例１．２に比較してわずかに選択透過能に劣るが、充
分使用に耐える値である．また電極Ｅ３に超音波処理を
行っても、過酸化水素およびアスコルビン酸に対する感
度に変化はなかった．比較例１
直径２ｍｍの白金線の側面を熱収縮テフロンで被覆し、
その断面を１６００番手のエメリー紙で研磨し、白金電
極Ｃ１とした．
この白金電極Ｃ１を図面に示すフロー型測定装置に組み
込み、１００ｍＭリン酸緩衝液１を流しながら、Ａｇ／
ＡｇＣ１電極８に対して０．６Ｖの電位を印加し、５ｍ
Ｍ過酸化水素を注入したところ、検出電流値は５３０ｎ
Ａであり、次に５ｍＭアスコルビン酸の溶液を注入した
ところ、検出電流値は３３３ｎＡであった．したがって
、第１表に示すようにアスコルビン酸による検出電流値
の同濃度の過酸化水素の検出電流値に対する割合は６２
．９％であった．これによって選択透過膜を設けていな
い白金電極Ｃ１では、アスコルビン酸を含む試料を測定
するこはできないことが判った．
比較例２
タンパク質としてウシ血清アルブミン（フラクション■
，シグマ社製）を、架橋剤としてグルタルアルデヒドを
、タンパク質以外に架橋剤と反応する物質として１．４
−ジアミノブタンを用いた．ワツセルマン試験管中で
５％ウシ血清アルブミン水溶液 ４００μ！２５％グル
タルアルデヒド水溶液 ２０μＩＯ．５％１．４−ジ
アミノブタン溶液 ２０μｌ、蒸 留 水
５ ６ ０μ！ 、を混合し、
最終濃度 ２％ ウシ血清アルブミン、０．５％
グルタルアルデヒド、
０．０１％ １．４−ジアミノブタン
の混合溶液を調製した。Electrode E consisting of a platinum electrode covered with this protein film
3 was incorporated into the flow type measuring device shown in Fig. 1, and 100
While flowing 1 mM phosphoric acid solution, A g/A
A potential of 0.6 V was applied to the g Cl electrode 8.
When 5mM hydrogen peroxide was injected from injector 3 in this state, the detected current value was 219nA. Next 5
When a solution of mM ascorbic acid was injected, the detected current value was 6.8 nA. Therefore, as shown in Table 1, the ratio of the current value detected by ascorbic acid to the current value detected by hydrogen peroxide at the same concentration was 3.1%. Although the selective permeability is slightly inferior to that of Example 1.2, the value is sufficient for use. Furthermore, even when electrode E3 was subjected to ultrasonic treatment, there was no change in sensitivity to hydrogen peroxide and ascorbic acid. Comparative Example 1 The side surface of a platinum wire with a diameter of 2 mm was covered with heat-shrinkable Teflon,
The cross section was polished with 1600 grit emery paper to form a platinum electrode C1. This platinum electrode C1 was assembled into the flow type measuring device shown in the drawing, and while flowing 100mM phosphate buffer 1, Ag/
Apply a potential of 0.6 V to the AgC1 electrode 8, and
When M hydrogen peroxide was injected, the detected current value was 530n.
A, and when a 5mM ascorbic acid solution was then injected, the detected current value was 333nA. Therefore, as shown in Table 1, the ratio of the current value detected by ascorbic acid to the current value detected by hydrogen peroxide at the same concentration is 62
．． It was 9%. This revealed that it was not possible to measure samples containing ascorbic acid with the platinum electrode C1, which was not equipped with a selectively permeable membrane. Comparative Example 2 Bovine serum albumin (fraction ■
, manufactured by Sigma), glutaraldehyde as a cross-linking agent, and 1.4 as a substance that reacts with the cross-linking agent other than protein.
-Diaminobutane was used. 5% bovine serum albumin aqueous solution 400 μ in Wasselmann test tube! 25% glutaraldehyde aqueous solution 20μIO. 5% 1,4-diaminobutane solution 20μl, distilled water
5 6 0μ! , final concentration 2% bovine serum albumin, 0.5%
A mixed solution of glutaraldehyde and 0.01% 1,4-diaminobutane was prepared.
一方、導電性基体である直径２ｍｍの白金線の側面を熱
収縮テフロンで被覆し、その断面を１６００番手のエメ
リー紙で研磨した．この研磨された表面に、マイクロシ
リンジで上記混合溶液５μｌを載せて４０℃で乾燥し、
白金電極上に直接タンパク質膜を形成した．このように
して、過酸化水素を検出するための電極Ｃ２を作成した
．このタンパク質膜で被覆された白金電極から成る電極
Ｃ２を第１図に示すフロー型測定装置に組み込み、１０
０ｍＭリン酸緩衝液１を流しながら、Ａ ｇ ／ Ａ
ｇ Ｃ １電極８に対して０．６Ｖの電位を印加した．
この状態でインジエクタ３から５ｍＭ過酸化水素を注入
したところ、検出電流値は２９６ｎＡであった．次に５
ｍＭアスコルビン酸の溶液を注入したところ、検出電流
値は１４４ｎＡであった．したがって、第１表に示すよ
うにアスコルビン酸による検出電流値の同濃度の過酸化
水素の検出電流値に対する割きは４８．５％であった．
１，４−ジアミノブタンを添加して得られた選択透過膜
は選択透過能に劣り、電極Ｃ２では、妨害物質としてア
スコルビン酸を含む試料の測定を行うことができないこ
とが判った，また、超音波処理を行うとさらに選択性が
低下することから物理的強度にも劣ることが判った．
比較例３
タンパク質としてウシ血清アルプミン（フラクション■
．シグマ社製）を、架橋剤としてグルタルアルデヒドを
、タンパク質以外に架橋剤と反応する物質としてトリエ
チレンテトラアミンを用いた．
ワツセルマン試験管中で
５％ウシ血清アルブミン水溶液 ４００μ！、２５％グ
ルタルアルデヒド水溶液 ２０μ！、０．５％トリエ
チレンテトラアミン溶液２０μ！、蒸 留 水
５６０ μ！ 、を混合し
、
最終濃度 ２％ ウシ血清アルブミン、０．５％
グルタルアルデヒド、
０．０１％ トリエチレンテトラアミン、の混合溶液を
調製した．
一方、導電性基体である直径２ｍｍの白金線の側面を熱
収縮テフロンで被覆し、その断面を１６００番手のエメ
リー紙で研磨した．この研磨された表面に、マイクロシ
リンジで上記混合溶液５μｌを載せて４０℃で乾燥し、
白金電極上に直接タンパク質膜を形成した，このように
して、過酸化水素を検出するための電極Ｃ３を作成した
．このタンパク質膜で被覆された白金電極から成る電極
Ｃ３を図面に示すフロー型測定装置に組み込み、１００
ｍＭリン酸ｌ！衝液１を流しながら、Ａｇ／ＡｇＣ１
ｔｌｉ８に対して０．６Ｖの電位を印加した．この状態
でインジエクタ３から５ｍＭ過酸化水素を注入したとこ
ろ、検出電流値は４２６ｎＡであった．次に５ｍＭアス
コルビン酸の溶液を注入したところ、検出電流値は１０
４ｎＡであった．したがって、第１表に示すようにアス
コルビン酸による検出電流値の同濃度の過酸化水素の検
出電流値に対する割合は２４．４％であった。On the other hand, the side surface of the conductive substrate, a platinum wire with a diameter of 2 mm, was covered with heat-shrinkable Teflon, and its cross section was polished with 1600-grit emery paper. 5 μl of the above mixed solution was placed on this polished surface using a microsyringe and dried at 40°C.
A protein film was formed directly on the platinum electrode. In this way, electrode C2 for detecting hydrogen peroxide was created. Electrode C2 consisting of a platinum electrode coated with this protein film was incorporated into the flow type measuring device shown in FIG.
While flowing 0mM phosphate buffer 1, A g/A
A potential of 0.6 V was applied to g C 1 electrode 8.
When 5mM hydrogen peroxide was injected from injector 3 in this state, the detected current value was 296nA. Next 5
When a solution of mM ascorbic acid was injected, the detected current value was 144 nA. Therefore, as shown in Table 1, the ratio of the current value detected by ascorbic acid to the current value detected by hydrogen peroxide at the same concentration was 48.5%.
It was found that the permselective membrane obtained by adding 1,4-diaminobutane has poor permselective ability, and electrode C2 cannot measure samples containing ascorbic acid as an interfering substance. It was found that when sonicated, the selectivity further decreased and the physical strength was also inferior. Comparative Example 3 Bovine serum albumin (fraction ■
．． (manufactured by Sigma), glutaraldehyde was used as a cross-linking agent, and triethylenetetraamine was used as a substance other than protein that reacts with the cross-linking agent. 5% bovine serum albumin aqueous solution 400 μ in Wasselmann test tube! , 25% glutaraldehyde aqueous solution 20μ! , 0.5% triethylenetetraamine solution 20μ! ,Distilled water
560μ! , final concentration 2% bovine serum albumin, 0.5%
A mixed solution of glutaraldehyde and 0.01% triethylenetetraamine was prepared. On the other hand, the side surface of the conductive substrate, a platinum wire with a diameter of 2 mm, was covered with heat-shrinkable Teflon, and its cross section was polished with 1600-grit emery paper. 5 μl of the above mixed solution was placed on this polished surface using a microsyringe and dried at 40°C.
In this way, electrode C3 for detecting hydrogen peroxide was created by forming a protein film directly on the platinum electrode. Electrode C3 consisting of a platinum electrode covered with this protein film was incorporated into the flow type measuring device shown in the drawing, and
mM phosphate l! While flowing buffer solution 1, Ag/AgC1
A potential of 0.6V was applied to tli8. When 5mM hydrogen peroxide was injected from injector 3 in this state, the detected current value was 426nA. Next, when a solution of 5mM ascorbic acid was injected, the detected current value was 10
It was 4nA. Therefore, as shown in Table 1, the ratio of the current value detected by ascorbic acid to the current value detected by hydrogen peroxide at the same concentration was 24.4%.
トリエチレンテトラアミンを添加して得られた選択透過
膜は選択透過能に劣り、電極Ｃ３では妨害物質としてア
スコルピン酸を含む試料の測定を行うことができないこ
とが判った．また超音波処理を行うと、さらに選択性が
低下することから物理的強度にも劣ることが判った．
比較例４
タンパク質としてウシ血清アルブミン《フラクションＶ
．シグマ社製》を、架橋剤としてグルタルアルデヒドを
用いた．
ワツセルマン試験管中で
５％ウシ血清アルブミン水溶液 ４００μ！、２５％グ
ルタルアルデヒド水溶液 ２０μ！蒸 留 水
５ ８ ０μ！ 、を混
合し、
最終濃度 ２％ ウシ血清アルブミン、０．５％
グルタルアルデヒド、
の混合溶液を調製した．
一方、導電性基体である直径２ｍｍの白金線の側面を熱
収縮テフロンで被覆し、その断面を１６００番手のエメ
リー紙で研磨した．この研磨された表面に、マイクロシ
リンジで上記混合溶液５μｌを載せて４０℃で乾燥し、
白金電掻上に直接タンパク質膜を形成した．このように
して、過酸化水素を検出するための電極Ｃ４を作成した
．このタンパク質膜で被覆された白金電極から成る電極
Ｃ４を図面に示すフロー型測定装置に組み込み、１００
ｍＭリン酸緩衝液１を流しながら、Ａｇ／ＡｇＣＺ電極
８に対してｏ．６Ｖの電位を印加した．この状態でイン
ジエクタ３から５ｍＭ過酸化水素を注入したところ、検
出電流値は２１９ｎＡであった．次に５ｍＭアスコルビ
ン酸の溶液を注入したところ、検出電流値は２．６ｎＡ
であった．したがって、第１表に示すようにアスコルビ
ン酸による検出電流値の同濃度の過酸化水素の検出電流
値に対する割合は１．２％であった．このように、タン
パク質と架橋剤とからのみ成る選択透過膜は、その選択
透過能に優れている．しかし、この電極Ｃ４を超音波処
理装置で１０分間処理した後、再び第１図に示すフロー
型測定装置に組み込んだ，５ｍＭ過酸化水素を注入した
ときの検出電流値は２６２ｎＡであり、５ｍＭアスコル
ビン酸の溶液を注入したときの検出電流値は６．６ｒｔ
Ａであった．
この結果は、超音波処理を行う前の結果とともに第２表
に示されている．第２表から、超音波処理の後において
は、電ｆｉｃ４の過酸化水素およびアスコルビン酸に対
する感度が上昇し、また選択透過性が悪くなっている．
したがって、このような電極Ｃ４では、物理的強度が不
充分であり、その耐久性に劣ることが判った．
実施例４
直Ｐｉ２ ｍ ｍの白金線の側面を熱収縮テフロンで被
覆し、その断面を１６００番手のエメリー紙で研磨した
表面に実施例１と同様な材料および手順で選択透過膜を
形成する．この上に牛血清アルブミンをｌｍｇ／ｍｊ！
、グルコースオキシダーゼ（タイプ■、シグマ社製）を
１ｍｇ／ｍｌ、グルタルアルデヒドを０，２％になるよ
う１００ｍＭリン酸ナトリウム緩衝液（ｐＨ６．０）に
溶解した液をマイクロシリンジで３μｌ滴下し゛、４０
℃で１５分間加熱し、固定化酵素層を形成し、電極Ｅ４
とした．
この電極Ｅ４を図面に示すフロー型測定装置に組み込み
、１００ｍＭリン酸緩衝液１を流しながら、Ａｇ／Ａｇ
Ｃｌｉｌ極８に対して０．６Ｖの電圧を印加した．イン
ジエクタ３から３０ｍＭのグルコース水溶液５μｌを注
入したところ、検出電流値は３９３ｎＡであった．次に
、３０ｍＭのアスコルビン酸水溶液を同量注入したとこ
ろ、検出電流値は１１．２ｎＡであった．つまり、同濃
度のグルコースに対するアスコルビン酸の検出値は２．
８％であり、実用上全く無視できるものである．
さらに、この電極Ｅ４をフロー型測定装置から一旦取外
し、超音波処理装置で１０分間処理した後、再度フロー
型測定装置に取付けた．そして超音波処理前と同様に、
３０ｍＭのグルコースおよびアスコルビン酸水溶液を、
各々５μ！注入した．グルコースに対する検出電流値は
３９５ｎＡで、アスコルビン酸に対する検出電流値は１
１．１ｒ＋Ａであった．この結果は第３表に示されてい
る．このように、本電極Ｅ４は超音波処理による性能劣
化がないことが判った．
また、超音波処理した本電極を室温、Ｍ衝液中で保存し
たところ、３カ月後も感度低下や選択性の低下が認めら
れず優れた耐久性を示した．第３表
比較例５
選択透過腹作成時にキトサンを含まながった以外、実施
例４と同様に固定化酵素電極Ｃ５を作成した．
この電極Ｃ５を第１図に示すフロー型測定装置に組み込
み１００ｍＭリン酸緩衝液１を流しながら、Ａｇ／Ａｇ
Ｃｆ電極に対して０．６ｖの電圧を印加した，３０ｍＭ
のグルコース水溶液５μｌを注入したところ検出電流値
は３８５ｎＡであった．次に、３０ｍＭのアスコルビン
酸水溶液を同量注入したところ検出電流値は１０、３ｎ
Ａであった．
さらに、この電極Ｃ５をフロー型測定装置がら一旦取外
し、超音波処理装置で１０分間処理した．目視的には膜
に異常は認められなかったが、再度フロー型測定装置に
電極を取付け、超音波処理前と同様に、３０ｍＭのグル
コースおよびアスコルビン酸水溶液を、各々５μｌ注入
したところ、グルコースに対する検出電流値は４０２ｎ
Ａで、アスコルビン酸に対する検出電流値は１６．２ｎ
Ａであった．この結果は、実・施例４の結果とともに第
３表に示されている．このように、電極Ｃ５においては
、超音波処理によって膜透過性が変動し選択性の低下が
起きたことが判る．
また、超音波処理した本電極を室温・Ｈｉｌｔ液中で保
存したところ、２カ月後に目視的に膜の剥離が認められ
、超音波処理によって引き起こされた膜の部分的破損が
最終的に膜の剥離にまで進展したものと考えられる．
発明の効果
以上説明したように本発明によれば、タンパク質を用い
て選択透過能を有する機能性膜を構築する場合に、固定
化タンパク質膜の特性に悪影響を与えず、なおかっ、物
理的強度に優れた選択透過膜を容易に作成することがで
きる．It was found that the permselective membrane obtained by adding triethylenetetraamine had poor permselectivity, and electrode C3 was unable to measure samples containing ascorbic acid as an interfering substance. Furthermore, it was found that when ultrasonication was applied, the selectivity further decreased and the physical strength was also inferior. Comparative Example 4 Bovine serum albumin (fraction V) as protein
．． manufactured by Sigma Corporation, and glutaraldehyde was used as a crosslinking agent. 5% bovine serum albumin aqueous solution 400 μ in Wasselmann test tube! , 25% glutaraldehyde aqueous solution 20μ! Distilled water
5 8 0μ! , final concentration 2% bovine serum albumin, 0.5%
A mixed solution of glutaraldehyde was prepared. On the other hand, the side surface of the conductive substrate, a platinum wire with a diameter of 2 mm, was covered with heat-shrinkable Teflon, and its cross section was polished with 1600-grit emery paper. 5 μl of the above mixed solution was placed on this polished surface using a microsyringe and dried at 40°C.
A protein film was formed directly on the platinum electrode. In this way, electrode C4 for detecting hydrogen peroxide was created. Electrode C4 consisting of a platinum electrode covered with this protein film was incorporated into the flow type measuring device shown in the drawing, and
While flowing mM phosphate buffer 1, o.c. to Ag/AgCZ electrode 8. A potential of 6V was applied. When 5mM hydrogen peroxide was injected from injector 3 in this state, the detected current value was 219nA. Next, when a 5mM ascorbic acid solution was injected, the detected current value was 2.6nA.
Met. Therefore, as shown in Table 1, the ratio of the current value detected by ascorbic acid to the current value detected by hydrogen peroxide at the same concentration was 1.2%. As described above, a permselective membrane consisting only of proteins and a crosslinking agent has excellent permselectivity. However, after treating this electrode C4 with an ultrasonic treatment device for 10 minutes, the current value detected when 5mM hydrogen peroxide was injected into the flow-type measuring device shown in Figure 1 was 262nA, and 5mM ascorbic acid was detected. The detected current value when injecting acid solution is 6.6rt
It was A. These results are shown in Table 2 along with the results before ultrasonication. Table 2 shows that after ultrasonication, the sensitivity of electric fic4 to hydrogen peroxide and ascorbic acid increases, and the permselectivity deteriorates.
Therefore, it was found that such electrode C4 had insufficient physical strength and was inferior in durability. Example 4 The side surface of a platinum wire with a diameter of 2 mm is coated with heat-shrinkable Teflon, and the cross section thereof is polished with 1600-grit emery paper. A selectively permeable membrane is formed using the same materials and procedures as in Example 1. On top of this, add bovine serum albumin to lmg/mj!
Using a microsyringe, drop 3 μl of glucose oxidase (type ■, manufactured by Sigma) dissolved at 1 mg/ml and glutaraldehyde at 0.2% in 100 mM sodium phosphate buffer (pH 6.0).
℃ for 15 minutes to form an immobilized enzyme layer, and electrode E4
It was. This electrode E4 was assembled into the flow type measurement device shown in the drawing, and while flowing 100mM phosphate buffer 1, the Ag/Ag
A voltage of 0.6 V was applied to Cliil pole 8. When 5 μl of a 30 mM glucose aqueous solution was injected from Injector 3, the detected current value was 393 nA. Next, when the same amount of 30mM ascorbic acid aqueous solution was injected, the detected current value was 11.2nA. In other words, the detected value of ascorbic acid for the same concentration of glucose is 2.
8%, which can be completely ignored in practical terms. Furthermore, this electrode E4 was once removed from the flow-type measuring device, treated with an ultrasonic treatment device for 10 minutes, and then attached to the flow-type measuring device again. And as before sonication,
30mM glucose and ascorbic acid aqueous solution,
5μ each! Injected. The detection current value for glucose is 395 nA, and the detection current value for ascorbic acid is 1.
It was 1.1r+A. The results are shown in Table 3. Thus, it was found that the performance of electrode E4 did not deteriorate due to ultrasonic treatment. Furthermore, when this ultrasonicated electrode was stored in M solution at room temperature, no decrease in sensitivity or selectivity was observed even after 3 months, showing excellent durability. Table 3 Comparative Example 5 An immobilized enzyme electrode C5 was prepared in the same manner as in Example 4, except that chitosan was not included during the preparation of the selectively permeable electrode. This electrode C5 was installed in the flow type measuring device shown in Fig. 1, and while flowing 100mM phosphate buffer 1, Ag/Ag
A voltage of 0.6v was applied to the Cf electrode, 30mM
When 5 μl of glucose aqueous solution was injected, the detected current value was 385 nA. Next, when the same amount of 30mM ascorbic acid aqueous solution was injected, the detected current value was 10.3n.
It was A. Furthermore, this electrode C5 was once removed from the flow-type measurement device and treated with an ultrasonic treatment device for 10 minutes. Visually, no abnormality was observed in the membrane, but when the electrode was attached to the flow-type measurement device again and 5 μl each of 30 mM glucose and ascorbic acid aqueous solutions were injected, the detection of glucose was detected. The current value is 402n
At A, the detection current value for ascorbic acid is 16.2n
It was A. These results are shown in Table 3 along with the results of Example 4. Thus, it can be seen that in electrode C5, the membrane permeability changed due to ultrasonication, resulting in a decrease in selectivity. Furthermore, when this ultrasonicated electrode was stored in Hilt solution at room temperature, peeling of the membrane was visually observed after two months, and partial damage to the membrane caused by ultrasonication eventually caused the membrane to peel off. It is thought that this has progressed to the point of peeling. Effects of the Invention As explained above, according to the present invention, when constructing a functional membrane having selective permeation ability using proteins, the properties of the immobilized protein membrane are not adversely affected, and the physical strength is It is possible to easily create a selectively permeable membrane with excellent performance.
Claims (7)
み、前記タンパク質以外に架橋剤と反応する高分子物質
が少なくとも１種類添加された混合溶液を膜状に展開し
、 架橋反応により前記タンパク質を固定化して成ることを
特徴とする選択透過膜。(1) A mixed solution containing a protein and at least one type of crosslinking agent, to which at least one type of polymeric substance that reacts with the crosslinking agent is added in addition to the protein, is spread into a film shape, and the protein is removed by a crosslinking reaction. A selectively permeable membrane characterized by being formed by immobilization.
ンパク質を含む成分構成であり、 前記少なくとも１種類の架橋剤がアミノ基と架橋反応す
る架橋剤を含み、 前記高分子物質がアミノ基、アミノ基の誘導体あるいは
その両方を有する直鎖状多糖類を含むことを特徴とする
請求項第１項記載の選択透過膜。(2) The protein has a component composition including at least one type of globular protein, the at least one type of crosslinking agent includes a crosslinking agent that crosslinks with an amino group, and the polymer substance contains an amino group, an amino group, and a crosslinking agent. The selectively permeable membrane according to claim 1, characterized in that it contains a linear polysaccharide having one or both derivatives.
る請求項第２項記載の選択透過膜。(3) The permselective membrane according to claim 2, wherein the polymeric substance is chitosan.
１０００：１から１０００：１０の範囲であることを特
徴とする請求項第２項記載の選択透過膜。(4) The permselective membrane according to claim 2, wherein the weight ratio of the protein to the linear polysaccharide is in the range of 1000:1 to 1000:10.
：５から１００：５０の範囲であることを特徴とする請
求項第２項または第４項記載の選択透過膜。(5) The composition weight ratio of the protein and the crosslinking agent is 100
5. The permselective membrane according to claim 2 or 4, characterized in that the ratio is in the range of :5 to 100:50.
、導電性基体近傍に設けたことを特徴とする電極。(6) An electrode, characterized in that the permselective membrane according to any one of claims 1 to 5 is provided in the vicinity of a conductive substrate.
、少なくとも１種類のオキシダーゼを固定化した固定化
酵素膜を設けたことを特徴とする請求項第６項記載の電
極。(7) The electrode according to claim 6, characterized in that an immobilized enzyme membrane on which at least one type of oxidase is immobilized is provided on the surface of the permselective membrane opposite to the conductive substrate.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1057588A JP2648361B2 (en) | 1989-03-08 | 1989-03-08 | Permselective membrane and electrode using the same |
| US07/490,709 US5242793A (en) | 1989-03-08 | 1990-03-07 | Selective permeable membrane and electrode using the same |
| DE69012610T DE69012610T2 (en) | 1989-03-08 | 1990-03-08 | Selectively permeable membrane, its manufacturing process and its application to an electrode. |
| EP90104452A EP0386763B1 (en) | 1989-03-08 | 1990-03-08 | Selective permeable membrane, method of producing and electrode using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1057588A JP2648361B2 (en) | 1989-03-08 | 1989-03-08 | Permselective membrane and electrode using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02236153A true JPH02236153A (en) | 1990-09-19 |
| JP2648361B2 JP2648361B2 (en) | 1997-08-27 |
Family
ID=13060004
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1057588A Expired - Lifetime JP2648361B2 (en) | 1989-03-08 | 1989-03-08 | Permselective membrane and electrode using the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2648361B2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04173841A (en) * | 1990-11-05 | 1992-06-22 | Nec Corp | Method for partially forming polymer membrane having functional group |
| WO2009008556A1 (en) * | 2007-07-11 | 2009-01-15 | National Institute For Materials Science | Flexible and autonomous protein nanofilm, method of producing the same and application thereof |
| US7811829B2 (en) | 2006-06-08 | 2010-10-12 | Canon Kabushiki Kaisha | Measuring probe and production process thereof |
| JP2012075995A (en) * | 2010-09-30 | 2012-04-19 | Jnc Corp | Nanofiber-reinforced protein porous film |
| JP5598823B2 (en) * | 2009-08-03 | 2014-10-01 | 独立行政法人物質・材料研究機構 | Organic polymer separation method |
-
1989
- 1989-03-08 JP JP1057588A patent/JP2648361B2/en not_active Expired - Lifetime
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04173841A (en) * | 1990-11-05 | 1992-06-22 | Nec Corp | Method for partially forming polymer membrane having functional group |
| US7811829B2 (en) | 2006-06-08 | 2010-10-12 | Canon Kabushiki Kaisha | Measuring probe and production process thereof |
| WO2009008556A1 (en) * | 2007-07-11 | 2009-01-15 | National Institute For Materials Science | Flexible and autonomous protein nanofilm, method of producing the same and application thereof |
| JP2009131725A (en) * | 2007-07-11 | 2009-06-18 | National Institute For Materials Science | Soft protein nano-membrane having self-standing property and its manufacturing method and application |
| EP2179779A3 (en) * | 2007-07-11 | 2011-11-02 | National Institute for Materials Science | A flexible free-standing ultrathin or thin protein membrane, its fabrication method and application |
| US8741152B2 (en) | 2007-07-11 | 2014-06-03 | National Institute For Materials Science | Flexible free-standing ultrathin or thin protein membrane, its fabrication method and application |
| US8828239B2 (en) | 2007-07-11 | 2014-09-09 | National Institute For Materials Science | Flexible free-standing ultrathin or thin protein membrane, its fabrication method and application |
| JP5598823B2 (en) * | 2009-08-03 | 2014-10-01 | 独立行政法人物質・材料研究機構 | Organic polymer separation method |
| JP2012075995A (en) * | 2010-09-30 | 2012-04-19 | Jnc Corp | Nanofiber-reinforced protein porous film |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2648361B2 (en) | 1997-08-27 |
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