JP4695316B2 - Silver / silver chloride electrode and electrochemical biosensor using the same - Google Patents
Silver / silver chloride electrode and electrochemical biosensor using the same Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
Description
技術分野
本発明は酵素センサー、免疫センサー等のバイオセンサーに用いることのできる電極及び当該電極を用いた電気化学的バイオセンサーに関する。
背景技術
酵素センサー、免疫センサー等のバイオセンサーは、血液や尿等の体液中に含まれる微量成分を簡易迅速に検出・定量する手段として有用であり、特に在宅医療及び地域医療の充実に伴ない、家庭内や臨床検査の専門家がいない地域診療の場においての臨床診断が重要視されている今日においては、使用方法が簡単で且つ小型で使い捨てのできる簡易測定キットの開発が望まれている。
斯かる状況の下、電気化学的な測定法を用いたバイオセンサーにおいては、小型で使い捨て可能な電極の開発がなされ、絶縁体等にカーボン又はグラファイトの微粒子を含むゾル／ゲル状の材料（Ｗａｎｇ，Ｊ．，ｅｔ．ａｌ．，Ａｎａｌ．Ｃｈｅｍ．，１９９８，７０，１１７１−１１７５等）や市販のカーボンペーストインク等を印刷した印刷電極が開発されている。また、電気化学的バイオセンサーの参照電極としては、一般に銀・塩化銀電極が使用される場合が多く、斯かる電極を印刷により作製する方法としては、銀ペーストインクで印刷した後塩化銀層を塩酸中で電析する方法、銀の上に塩化第二鉄を反応させ塩化銀層を作製する方法、塩化銀を主体とする層を塗工により作製する方法（特開平８−９４５７３）、更には銀粉末と塩化銀末を予め混合しプレスして作製する方法（特開昭６２−４３５５６）或いは銀粉末と塩化銀末を高分子のバインダーで混合し塗布して作製する方法（特開平２−９００５２）等が知られている。
しかし、銀上に後から塩化銀層を塗布する方法は、操作が煩雑で塩化銀層が物理的に破損しやすい等の欠点があり、また、銀粉末と塩化銀末を予め混合する方法においては、電極の内部抵抗が大きくなったり、銀粉末と塩化銀末が均一でなかったり、これらの粒子径が大きいためスクリーンプリントで目詰まりを起す等の問題が生じていた。
発明の開示
本発明の目的は、極めて簡単に作製でき、且つ均質で電導率のよい銀・塩化銀電極及び当該電極を用いた電気化学的バイオセンサーを提供することにある。
本発明者らは、斯かる実状に鑑み、可溶化させた塩化銀を超音波照射下で析出させることにより得られる微細塩化銀粉末を用いて作製した銀・塩化銀電極が、優れた均質性及び電導性を有し、当該電極を用いることにより、小型で使い捨て可能なバイオセンサーが安価で容易に作製できることを見出し、本発明を完成するに至った。
即ち本発明は、塩化銀粉末を水系溶媒に溶解し、超音波照射下に有機溶媒を添加して析出させた微細塩化銀粉末と、銀ペーストを混練して得られた銀・塩化銀ペーストインクを用いて印刷することにより得られる銀・塩化銀電極を提供するものである。
また本発明は、当該銀・塩化銀電極を電極部に有する電気化学的バイオセンサーを提供するものである。
また本発明は、クロマトグラフィーマトリックス部分と電極部分から構成される検体溶液中の分析対象物質を検出及び定量するためのセンサーであって、そのクロマトグラフィーマトリックス部分が、（ａ）一個又は複数個の試料添加部位、（ｂ）試料添加部位の下流に接触して位置し、分析対象物と親和性を有する酵素標識された特異結合物質を移動可能に保持した特異反応部位、（ｃ）該特異反応部位の下流に接触して位置し、分析対象物質又は分析対象物質と同様な反応性を有する物質を固定化した競合反応部位、（ｄ）該競合反応部位の下流に接触して位置し、前記標識酵素に対する基質を移動可能に保持した酵素反応部位からなり、電極部分が、該酵素反応部位あるいはその下流に接触して位置し、作用電極、対電極及び参照電極が多孔質担体に印刷配置されたものであるシート状電気化学的バイオセンサーを提供するものである。
発明を実施するための最良の形態
本発明の銀・塩化銀電極は、塩化銀粉末を水系溶媒に溶解し、超音波照射下に有機溶媒を添加して析出させた微細塩化銀粉末と、銀ペーストを混練して得られた銀・塩化銀ペーストインクを用いて印刷することにより作製できる。以下（１）〜（４）にその詳細を説明する。
（１）水系溶媒への溶解
溶解は、塩化銀粉末を水系溶媒中に添加攪拌することにより行われる。
ここで、水系溶媒とは、塩化銀を溶解するものであれば特に限定されるものではないが、例えばアンモニア水、濃塩酸、アルカリ性シアン化物水溶液、チオ硫酸塩水溶液、炭酸アンモニウム水溶液等が挙げられ、好ましくは、アンモニア水である。
また、溶解は、通常３０〜５０℃で攪拌することで効率よく行うことができる。
（２）微細粉末化
微細粉末化は、超音波の照射下、塩化銀溶液に有機溶媒を添加することにより行なわれる。
超音波は、通常２０ＫＨｚ以上、特に塩化銀粉末の均質性の観点から、２８ＫＨｚ〜４２ＫＨｚで照射することが好ましい。また、超音波照射には、超音波を任意の容器に照射可能とする超音波浴、例えば超音波洗浄器等を用いることができる。
ここで用いられる有機溶媒としては、例えばメタノール、エタノール、メチルセロソルブ、ブチルセロソルブなど水系溶媒に溶解する有機溶媒ならいずれでも使用可能であるが特にメタノールが好ましい。
斯かる有機溶媒の添加量は、溶媒によって異なるが沈渣が有効に生成する量比の添加が必要で、例えば２倍〜１０倍程度が好ましい。添加はゆっくり行うことが好ましく、例えば全量に対して１秒あたりその１００分の１量を添加する程度の速度で行うことができる。
また、得られた沈渣は必要に応じて▲１▼静置、▲２▼上清除去、▲３▼有機溶媒添加の操作を繰り返して洗浄・溶媒転換を行い、懸濁液のまま銀ペーストインクに混練することができる。▲１▼静置の操作は、塩化銀微粉末が固化しない程度の強度で遠心分離で代替えすることができる。この場合の遠心力は５０×ｇ〜３００×ｇの範囲で設定可能だが１００×ｇ程度で数１０秒から１分程度の遠心操作が好ましい。▲３▼有機溶媒再添加の操作においては微細粉末化及び洗浄の効果向上の目的で超音波浴中での添加が望ましい。この有機溶媒添加操作の過程で微細粉末化に使用した溶媒から銀・塩化銀ペーストへの混合に都合の良い溶媒に転換することができる。例えばメタノールでの洗浄からペーストインクを構成する溶媒に転換して洗浄することができ、望ましくはメチルセルロソルブ、ブチルセロソルブ、酢酸メチルセロソルブ、酢酸ブチルセロソルブ、イソホロン、ブチルカルビトールアセテートなどを使用することができる。
また、▲１▼静置〜▲３▼有機溶媒添加の操作は、数回、例えば２〜８回、好ましくは５回程度繰り返し、▲２▼上清除去したものを使用するのが、溶媒を転換する上で好ましい。
（３）銀・塩化銀ペーストの製造
微細塩化銀粉末を、銀ペースト例えばポリエステル樹脂をバインダーとした一液性のポリマー型銀ペースト（例えば銀ペーストＬＳ−４１１（ＡＳＡＨＩ ＣＨＥＭＩＣＡＬ ＲＥＳＥＡＲＣＨ ＬＡＢＯＲＡＴＯＲＹ社製）等）中に混練し、印刷用の銀・塩化銀ペーストとすることができる。
微細塩化銀粉末の配合比は、重量比で銀・塩化銀ペーストの０．１％〜４０％であり、好ましくは０．２％〜２０％である。
得られた銀・塩化銀ペーストインクは、均質性がよく３００メッシュ程度のスクリーンを用いたスクリーンプリントでも目詰まりすることなく印刷することが可能となる。
（４）電極の印刷
銀・塩化銀ペーストインクを用いた電極の印刷は、電子工学分野においてよく知られている任意の方法を用いることができ、具体的には、ポリエステル繊維やステンレスのスクリーンメッシュに電極図形パターンを光感光技術で形成し、これを原版として印刷するスクリーン方式又はＸＹ２軸方向に可動可能なノズル若しくは印刷物固定台を利用し、電極図案パターンに沿ってペーストインクを空気圧等で吐出しながら印刷するインクジェット方式又はディスペンス方式等を利用することができる。
かくして得られた銀・塩化銀電極は、酵素センサー、免疫センサー等のバイオセンサーにおける電極部の電極、特に参照電極として使用することができる。例えば、分析対象物質を検体溶液から分離展開するクロマトグラフィーマトリックス部と電極部とから構成される免疫センサーに代表される電気化学的バイオセンサーの電極部に応用することが考えられ、装置全体の小型化を可能にする。
更に、本発明は、銀・塩化銀電極を用いた電気化学的バイオセンサーを提供するものである。以下にその内容を説明する。
即ち、本発明の電気化学的バイオセンサーは、検体溶液中の分析対象物質を検出及び定量するためのセンサーであって、そのクロマトグラフィーマトリックス部が、（ａ）一個又は複数個の試料添加部位、（ｂ）該試料添加部位の下流に接触して位置し、分析対象物質と親和性を有する酵素標識された特異結合物質を移動可能に保持した特異反応部位、（ｃ）該特異反応部位の下流に接触して位置し、分析対象物質又は分析対象物質と同様な反応性を有する物質を固定化した競合反応部位、（ｄ）該競合反応部位の下流に接触して位置し、前記標識酵素に対する基質を移動可能に保持した酵素反応部位からなり、電極部が、該酵素反応部位あるいはその下流に接触して位置し、印刷配置されたものである。
ここでいう分析対象物質とは、例えばペプチド、タンパク質、複合タンパク質、多糖類、脂質、複合脂質、ホルモン類、核酸類等の生体関連物質を意味し、更に具体的には、例えば卵胞刺激ホルモン、黄体刺激ホルモン、胎盤性ゴナドトロピン、甲状腺ホルモン、プロゲステロン、エストラジオール等のホルモン類、リポ蛋白、アポリポ蛋白、アルブミン、ヘモグロビン等の生体微量成分等が挙げられる。
検体溶液とは、斯かる生体関連物質を含む液体、主に血液、血漿、血清、唾液、尿等の生物由来の液体試料をいうが、粘液、体組織あるいは細胞等の固形を、緩衝液等の液体に懸濁もしくは溶解させたものであってもよい。
本発明の電気化学的バイオセンサーは、基本的に免疫反応を行うクロマトグラフィーマトリックス部である（ａ）〜（ｄ）と、電極部から構成されるものであるが、ここで、クロマトグラフィーマトリックス部は多孔質担体より構成される。
多孔質担体は、検体溶液を毛細管現象により移行させるための媒体として機能するものであれば特に限定されず、この様な素材としては、例えばセルロース膜（ろ紙）、ガラス繊維ろ紙（例えば、ＧＦ／Ａ，Ｗｈａｔｍａｎ製）、ニトロセルロース膜、ナイロン膜、ポリスチレン・ポリエステル等のプラスチック繊維による不織布やスポンジ様の多孔質素材等を用いることができる。多孔性部分の寸法は特に制限ないが、本センサーで１個の多孔性部分を使用する場合、幅２ｍｍ〜１０ｍｍ、長さ３０ｍｍ〜１５０ｍｍ、厚さ２０μｍ〜１０００μｍのものを用いることができる。
試料添加部位（ａ）は、短い多孔質部分を複数個用いて構成されていてもよく、この場合には検体溶液が毛細管現象により上流から下流に移動可能な様に互いに接触状態で配置すればよい（なお、「上流」及び「下流」とは、クロマトグラフィーマトリックスにおいて検体溶液が多孔性部分を移動する方向を意味する）。
また、試料添加部位（ａ）においては、必要に応じてＴｒｉｓ緩衝剤、ジエタノール緩衝剤、リン酸緩衝剤等のｐＨ緩衝剤又は非特異的結合を阻止する目的でゼラチン、牛血清アルブミン、カゼイン等の蛋白質や任意の界面活性剤を塗布することができる。
特異反応部位（ｂ）は、分析対象物質と親和性を有する酵素標識された特異結合物質を移動可能な状態で保持し、検体溶液中の分析対象物質と標識された特異結合物質との反応の場を提供する場である。
特異結合物質は、該分析対象物質と特異的に反応する物質であって、例えば、任意の抗原に対する抗体、任意の核酸に対する相補的な核酸、種々の生理活性物質に対するレセプター等を挙げることができるが、好ましくは任意の抗原に対する抗体である。
また、特異結合物質に導入する標識酵素としては、基質と組み合わせることにより電気化学的信号を発生できる酸化還元酵素であれば特に限定されず、例えばグルコースオキシダーゼ、ガラクトシダーゼ、アルカリフォスファターゼ、ホースデッシュパーオキシダーゼ等のオキシダーゼ類やデヒドロゲナーゼ類を用いることができる。また、これらの酵素を特異結合物質に導入する方法は汎用されている任意の方法を用いることができる。
当該酵素標識された特異結合物質は、該溶液を多孔質担体に含浸させ、凍結乾燥、陰圧乾燥、送風乾燥、自然乾燥等の任意の手段に付すことにより、移動可能な状態で保持することができる。
競合反応部位（ｃ）は、分析対象物質又は分析対象物質と同様な反応性を有する物質を固定化し、上流から移動してきた検体溶液中の分析対象物質と酵素標識された特異結合物質との反応混合物（分析対象物質と酵素標識された特異結合物質との反応生成物である複合体、及び未反応の酵素標識された特異結合物質）のうち、未反応の酵素標識された特異結合物質が捕獲、除去される。
ここで、分析対象物質と同様な反応性を有する物質とは、分析対象物質自体、特異結合物質と反応する部分を残してこれを断片化した物質、特異結合物質と結合可能な類似の構造を持つ物質、分析対象物質を熱・試薬・ｐＨなどの手段で変性させた物質、タンパク質・ペプチド・核酸などの場合その構成単位であるアミノ酸や核酸塩基の配列が近似したもの又は人工的に変化させた物質等が挙げられ、好ましくは分析対象物質である。
分析対象物質又は分析対象物質と同様な反応性を有する物質の固定化は、特に限定されないが、一般的には多孔性部分を分析対象物質又は分析対象物質と同様な反応性を有する物質の溶液に一定時間浸漬させた後、多量の緩衝液又は洗浄液、例えば一般的なｐＨ緩衝液又はこれらにＴｗｅｅｎ ２０等の界面活性剤を加えたものによって濯ぎ・洗浄し、乾燥することにより行うことができる。また、固定化する分析対象物質又は分析対象物質と同様な反応性を有する物質がタンパク質の場合は、脱離する可能性があるため共有結合により固定化するのが好ましい。その一例として、ジアゾ化ろ紙又はＩｍｍｕｎｏｄｙｎｅ ＡＢＣ（Ｐａｌｌ社製）等を用いることができる。
酵素反応部位（ｄ）は、前記標識用酵素と反応して信号を発生する基質を移動可能な状態で保持し、当該基質が上流から移動してきた分析対象物質と酵素標識された特異結合物質との複合体と反応し電気化学的信号発生物質を生成する。
基質としては、標識酵素により適宜選択され、例えばアルカリフォスファターゼ、ホースラディッシュパーオキシダーゼ、ガラクトシダーゼ、グルコースオキシダーゼを始めとするオキシダーゼ類、デヒドロゲナーゼ類を酵素として使用した場合、それぞれｐ−アミノフェニルフォスフェート、水素供与体及び過酸化水素、ｐ−アミノフェニル−β−Ｄ−ガラクトシド、還元体としてのグルコース等の基質及び酸素、水素供与体等の基質及びＮＡＤ＋を適宜組み合わせて利用することができる。
これらの基質の保持は酵素標識された特異結合物質の場合と同様に多孔性部分に基質溶液を含浸させ凍結乾燥、陰圧乾燥、送風乾燥等の手法で行うことができる。
電極部は、酵素反応部位（ｄ）で生成した電気化学的信号発生物質の信号を電気化学的に測定する部位であり、作用電極、対電極及び参照電極の三つの印刷電極よりなる。当該電極は、酵素反応部位（ｄ）或いはその下流に接触して位置し、多孔質担体に印刷配置され、毛細管現象によりクロマトグラフィーマトリックス内を浸透、移動してきた反応液と接触する。
電極は、作用電極及び対電極にはカーボンペーストインク、参照電極には銀・塩化銀ペーストインクが用いられ、印刷して作製される。ここで、用いられる銀・塩化銀電極としては、電導性がよく、参照電極として安定して使用できるもであれば特に限定されるものではないが、電導性及び簡便性等を考慮すれば、上述したような、塩化銀粉末を水系溶媒に溶解し、超音波照射下に有機溶媒を添加して析出させた微細塩化銀粉末と、銀ペーストを混練して得られた銀・塩化銀ペーストインクを用いて印刷されたものが好ましい。特に濾紙等の多孔質担体に銀・塩化銀電極を印刷配置する場合には、銀ペースト印刷後に塩化銀層を作製する方法は適用できず、予め銀・塩化銀粉末を混練したペーストを用いることが好ましい。また、多孔質担体に適合するように均質性及び電導性に優れた銀・塩化銀ペーストを用いることが好ましい。
尚、カーボンペーストインクはポリエステル樹脂をバインダーとした一液性のポリマー型カーボンペースト、例えばＦＴＵ−６０（ＡＳＡＨＩ ＣＨＥＭＩＣＡＬ ＲＥＳＥＡＲＣＨ ＬＡＢＯＲＡＴＯＲＹ社製）を使用することができる。
このように、多孔質担体上に電極部を配置することから、この領域に移動してきた検体溶液の下流への移動を止める絶縁部分を設けることが必要となる。ここでいう絶縁部分とは電気的に電導性がないものであって且つ水溶液の進入を防ぐものをいうが、当該絶縁部分は、コーティング剤、絶縁性塗料や接着剤等の各種プラスチックの有機溶媒溶液等を塗布浸透させ固化させることにより作製できる。
斯かる三つの電極は、外部と接触可能な状態にあり、電気化学測定用ポテンショスタット装置又はこれと同等の一定電圧を発生可能な定電圧装置で印加できるようにされ、発生する電流がアンペロメトリーによって測定される。
尚、当該電気化学的バイオセンサーは、蒸発防止、強度補強、遮光等の目的で、試料添加部位又はその一部及び測定装置との接続部分を残して透明ないしは不透明なプラスチックフィルム等でカバーすることができる。
実施例
以下、実施例により本発明をさらに具体的に説明する。
実施例１ 銀・塩化銀電極の作製と評価
（１）銀・塩化銀ペーストの作製
塩化銀（片山化学工業）１０ｇを濃アンモニア水（２８％、和光純薬）７０ｍＬに懸濁し３０℃湯浴中で撹拌して溶解した。塩化銀溶液を超音波槽に移し、約３０ＫＨｚの超音波照射下でメタノール１４０ｍＬを徐々に滴下して塩化銀の微細な粉末を析出させた。撹拌後５分静置し上清を吸引除去し沈渣にメタノール１５０ｍＬを加え沈渣を洗浄した。同様の操作を５回繰り返しアンモニアを除いた後ブチルセロソルブを用いてメタノールと同様に沈渣の洗浄操作を行った。最終的にブチルセロソルブ中の懸濁物として約６７０ｍｇの塩化銀微粉末を得た。この塩化銀微粉末懸濁液を銀ペーストインクＬＳ−４１１（アサヒ化学研究所）に対し２０から０．２５％の比率で混合し均一になるように練り合わせた。
（２）印刷参照電極の評価
前項で作製した銀・塩化銀参照電極を評価するために市販の参照電極と比較した。
（１）で作製した各混合比の電極用銀・塩化銀ペーストを用いて３０５メッシュ及び１２０メッシュのスクリーンを用いてスクリーン印刷して印刷参照電極を作製した。測定装置への接続部と電極部の間にフッ素樹脂のコーティング剤（ＣＴ−３００−２、アサヒ科学研究所）をライン状に塗布し、陰圧下で乾燥して絶縁部分を設けた。
基準電極とするＢＡＳ社製参照電極（ＲＥ−１Ｂ）と、銀・塩化銀ペーストのみを印刷した電極パッドを塩化銀粉末少量を飽和ＫＣｌ溶液に加えた試験電極液に浸漬し、ＲＥ−１Ｂを２６３Ａポテンショスタット装置（ＥＧ＆Ｇ）の参照電極側に、銀・塩化銀電極を作用電極側に接続して電極間の電位差を測定した。結果を表１及び２に示す。
（３）印刷電極での電気化学的測定による参照電極の評価
純セルロースクロマトグラフィペーパー３ＭＭ（Ｗｈａｔｍａｎ社）を印刷基板として用いた。カーボンペーストインクＦＴＵ−６０（アサヒ化学研究所）を用いて図１に示したように作用電極及び対電極をスクリーン印刷した。これを１２０℃、３０分間陰圧下で乾燥固化した。これに各混合比の銀・塩化銀ペーストインクを用いて図１に示すような配置で参照電極をスクリーン印刷した後同様に１２０℃、３０分間陰圧下で乾燥固化した。続いて、作用電極の先端から１０ｍｍの位置にフッ素樹脂のコーティング剤（ＣＴ−３００−２、アサヒ科学研究所）をライン状に塗布し陰圧下で乾燥して絶縁部分を設けた。最終的に印刷３電極を含む１０ｍｍ×３０ｍｍ部分を切り出し印刷電極とした。２６３Ａポテンショスタット装置（ＥＧ＆Ｇ）の測定端子に対応するそれぞれの電極をつなぎ、電気化学測定用ソフトウェアＭｏｄｅｌ ２７０／２５０を用いてＣｈｒｏｎｏａｍｐｅｒｏｍｅｔｒｙ（ＣＡ）、Ｃｙｃｌｉｃ Ｖｏｌｔａｍｍｅｔｒｙ（ＣＶ）によって電気化学測定を行った。電気化学的活性物質ｐ−アミノフェノール０．５ｍＭ、１ｍＭ塩化マグネシウム、０．１Ｍ塩化カリウムを含む０．１Ｍリン酸緩衝液（ｐＨ７．５）６０μＬを電極部に添加して電流値（μＡ）を計測した。ＣＡでは測定開始後６０秒の電流値、ＣＶでは０から３００ｍＶの電流値（μＣ）を測定した。結果を表３に示す。
実施例２
本発明による電気化学的バイオセンサーを作製し、それを用いて試料中のヒト血清アルブミンを測定した。
（１）試料添加部の作製
コンジュゲートパッド（５０×１００ｍｍ）を２％（ｗ／ｖ）ブロックエース（大日本製薬（株））を含むＰＢＳ溶液に浸漬し１０分間振盪処理した。これを０．０５％Ｔｗｅｅｎ２０を含むＰＢＳ溶液で５分間３回振盪洗浄し真空乾燥器中で乾燥した。最終的には１０ｍｍ×２０ｍｍの大きさになるように切断し絶縁体ベース上に配置して使用する。
（２）酵素標識抗体パッドの作製
酵素標識特異結合物質としてはβ−ガラクトシダーゼ標識した抗ヒト血清アルブミン抗体を用いた。標識用抗体はアメリカンコーレックス社のβ−ガラクトシダーゼ標識抗アルブミンヤギ抗体（０．２５ｍｇ／ｍＬ）を用いた。コンジュゲートパッド（５０×１００ｍｍ）を２％（ｗ／ｖ）ブロックエース（大日本製薬（株））を含むＰＢＳ溶液に浸漬し１０分間振盪処理した。これを０．０５％Ｔｗｅｅｎ２０を含むＰＢＳ溶液で５分間３回振盪洗浄し真空乾燥器中で乾燥した。このパッドを０．２％（ｗ／ｖ）ブロックエース１０μｇ／ｍＬの濃度に調製した標識抗体を含むＰＢＳ溶液４ｍＬ中に浸漬しこの液を保持したまま液体窒素に投入して急速冷凍した後凍結乾燥した。最終的には１０ｍｍ×２０ｍｍの大きさになるように切断し絶縁体ベース上試料添加部の下流側に配置して使用する。（３）ヒト血清アルブミン膜の作製
多孔性ナイロン膜Ｉｍｍｕｎｏｄｙｎｅ ＡＢＣ（Ｐａｌｌ社）（５０×１００ｍｍ）をヒト血清アルブミン（Ｃａｌｂｉｏｃｈｅｍ社）１ｍｇ／ｍＬを含むＰＢＳ溶液１０ｍＬ中で１時間振盪反応させた。これを０．０５％Ｔｗｅｅｎ２０を含むＰＢＳ溶液で５分間３回振盪洗浄し真空乾燥器中で乾燥した。さらに、２％（ｗ／ｖ）ブロックエース（大日本製薬（株））を含むＰＢＳ溶液に浸漬し１０分間振盪処理した後真空乾燥器中で乾燥した。最終的には１０ｍｍ×１０ｍｍの大きさになるように切断し絶縁体ベース上酵素標識抗体パッドの下流側に配置して使用する。
（４）基質パッドの作製
純セルロースクロマトィーペーパー３１ＥＴＣｈｒ（Ｗｈａｔｍａｎ社）（５０×１０ｍｍ）を５ｍＭｐ−アミノフェニル−β−Ｄ−ガラクトピラノシド、１ｍＭ塩化マグネシウムを含む０．１Ｍリン酸緩衝液（ｐＨ７．５）３ｍＬ中に浸漬しこの液を保持したまま液体窒素に投入して急速冷凍した後凍結乾燥した。最終的には１０ｍｍ×１０ｍｍの大きさになるように切断し絶縁体ベース上ヒト血清アルブミン膜の下流側に配置して使用する。
（５）参照電極用銀・塩化銀ペーストの作製
実施例１（１）と同様にして銀・塩化銀ペーストインクを作製した。
（６）電極パッドの作製
純セルロースクロマトグラフィーペーパー３１ＥＴＣｈｒ（Ｗｈａｔｍａｎ社）を印刷基板として用いた。カーボンペーストインクＦＴＵ−６０（アサヒ化学研究所）を用いて図１に示したように作用電極及び対電極をスクリーン印刷した。これを１２０℃、３０分間陰圧下で乾燥固化した。これに銀・塩化銀ペーストインクを用いて図１に示すような配置で参照電極をスクリーン印刷した後同様に１２０℃、３０分間陰圧下で乾燥固化した。続いて、作用電極の先端から１０ｍｍの位置にポリエステル樹脂のコーティング剤（ＣＴ−３００−２、アサヒ科学研究所）をライン状に塗布し陰圧下で乾燥して絶縁部分を設けた。最終的に印刷３電極を含む１０ｍｍ×３０ｍｍ部分を切り出し電極パッドとして基質パッドの下流側に配置して使用する。
（７）ヒト血清アルブミンの測定
上記のようにして作製した各パーツはそれぞれ互いにその１部を接触した形でプラスチック製絶縁体上に図１のように配置し接着した。電極パッドの接点部分を介してカーボン線の内作用電極を２６３Ａポテンショスタット装置（ＥＧ＆Ｇ）の作用極端子に、もう一方のカーボン線の対電極を対極端子に、銀・塩化銀電極参照電極側を参照極端子に接続した。さらに、この２６３Ａポテンショスタット装置からＧＰＩＢラインを通じてコンピュータに接続し電気化学測定用ソフトウェアＭｏｄｅｌ ２７０／２５０で計測およびデータ解析を行った。測定には作用電極の電位を参照電極に対して３００ｍＶに設定しクロノアンペロメトリーで測定した。
０．２％ブロックエースを含むＰＢＳ溶液中にヒト血清アルブミンを０〜２００μｇ／ｍＬの濃度で含む試料溶液３００μＬを試料添加部に添加して順次下流側に展開させ電流値の継時変化を測定し測定開始から１分後の電流値を記録した。結果を表４に示す。
産業上の利用可能性
本発明の銀・塩化銀電極は、均質性が高く電導率が優れるという性質を有し、簡易に且つスクリーン印刷法においても目詰まりなく作製することができる。また、当該銀・塩化銀電極を利用した本発明の電気化学的バイオセンサーは、分析対象物質を迅速且つ正確に検出及び定量することができると共に、その形態を小型で且つ使い捨て可能とすることができる。従って、簡易測定キットとして家庭内、緊急医療現場、ベッドサイド、地域診療等幅広い分野で使用できる。
【図面の簡単な説明】
図１は、本発明の電気化学的バイオセンサーの模式図である。図中の符号は次のとおりである。
１：試料添加部位
２：特異反応部位
３：競合反応部位
４：酵素反応部位
５：絶縁部分
６：測定装置との接続部
７：電極部
ａ：対電極
ｂ：作用電極
ｃ：参照電極Technical field
The present invention relates to an electrode that can be used in a biosensor such as an enzyme sensor or an immunosensor, and an electrochemical biosensor using the electrode.
Background art
Biosensors such as enzyme sensors and immunosensors are useful as simple and rapid means for detecting and quantifying trace components in body fluids such as blood and urine. Nowadays, where clinical diagnosis is important in a local medical field where there is no clinical laboratory specialist, it is desired to develop a simple measurement kit that is simple to use, small and disposable.
Under such circumstances, in biosensors using electrochemical measurement methods, small and disposable electrodes have been developed, and sol / gel-like materials (Wang that contain carbon or graphite fine particles in an insulator, etc.) J., et.al., Anal.Chem., 1998, 70, 1171-1175, etc.) and commercially available carbon paste inks have been developed. In general, a silver / silver chloride electrode is often used as a reference electrode for an electrochemical biosensor. As a method for producing such an electrode by printing, a silver chloride layer is formed after printing with a silver paste ink. A method of electrodepositing in hydrochloric acid, a method of producing a silver chloride layer by reacting ferric chloride on silver, a method of producing a layer mainly composed of silver chloride by coating (JP-A-8-94573), and Is a method in which silver powder and silver chloride powder are mixed and pressed in advance (Japanese Patent Laid-Open No. 62-43556) or a method in which silver powder and silver chloride powder are mixed and coated with a polymer binder (Japanese Patent Laid-Open No. 2). -90052) is known.
However, the method of coating a silver chloride layer on silver later has drawbacks such as complicated operation and the physical damage of the silver chloride layer, and in the method of previously mixing silver powder and silver chloride powder. However, problems such as increased internal resistance of the electrode, unevenness of silver powder and silver chloride powder, and clogging due to screen printing due to the large particle diameter of these particles occurred.
Disclosure of the invention
An object of the present invention is to provide a silver / silver chloride electrode which can be produced very easily and has a uniform and good conductivity, and an electrochemical biosensor using the electrode.
In view of such a situation, the inventors of the present invention have excellent homogeneity in the silver / silver chloride electrode produced using fine silver chloride powder obtained by precipitating solubilized silver chloride under ultrasonic irradiation. In addition, the present inventors have found that a small and disposable biosensor can be easily produced at low cost by using the electrode, which has electrical conductivity, and has completed the present invention.
That is, the present invention is a silver / silver chloride paste ink obtained by kneading a silver paste with a fine silver chloride powder obtained by dissolving silver chloride powder in an aqueous solvent and adding an organic solvent under ultrasonic irradiation. The present invention provides a silver / silver chloride electrode obtained by printing with the use of the material.
The present invention also provides an electrochemical biosensor having the silver / silver chloride electrode in the electrode part.
The present invention also provides a sensor for detecting and quantifying a substance to be analyzed in a sample solution composed of a chromatography matrix part and an electrode part, wherein the chromatography matrix part comprises (a) one or a plurality of substances. A sample addition site, (b) a specific reaction site located in contact with the downstream of the sample addition site and holding an enzyme-labeled specific binding substance having affinity for the analyte, and (c) the specific reaction A competitive reaction site, which is located in contact with the downstream of the site and immobilizes the analyte or a substance having the same reactivity as the analyte, (d) is located in contact with the downstream of the competitive reaction site, It consists of an enzyme reaction site that holds the substrate for the labeled enzyme so that it can move, and the electrode part is located in contact with the enzyme reaction site or downstream thereof, working electrode, counter electrode and reference electrode There is provided a sheet-like electrochemical biosensor in which printed placed on a porous support.
BEST MODE FOR CARRYING OUT THE INVENTION
The silver / silver chloride electrode of the present invention is obtained by kneading a silver paste with fine silver chloride powder obtained by dissolving silver chloride powder in an aqueous solvent and adding an organic solvent under ultrasonic irradiation. -It can produce by printing using a silver chloride paste ink. Details will be described below in (1) to (4).
(1) Dissolution in aqueous solvent
Dissolution is performed by adding and stirring silver chloride powder in an aqueous solvent.
Here, the aqueous solvent is not particularly limited as long as it dissolves silver chloride, and examples thereof include aqueous ammonia, concentrated hydrochloric acid, an aqueous alkaline cyanide solution, an aqueous thiosulfate solution, and an aqueous ammonium carbonate solution. Preferably, it is ammonia water.
Moreover, melt | dissolution can be efficiently performed by stirring at 30-50 degreeC normally.
(2) Fine powder
Fine pulverization is performed by adding an organic solvent to the silver chloride solution under irradiation of ultrasonic waves.
It is preferable to irradiate the ultrasonic waves at 20 KHz or higher, and particularly 28 KHz to 42 KHz from the viewpoint of the homogeneity of the silver chloride powder. For ultrasonic irradiation, an ultrasonic bath capable of irradiating an ultrasonic wave to an arbitrary container, such as an ultrasonic cleaner, can be used.
As the organic solvent used here, any organic solvent that can be dissolved in an aqueous solvent such as methanol, ethanol, methyl cellosolve, butyl cellosolve can be used, but methanol is particularly preferable.
The amount of the organic solvent to be added varies depending on the solvent, but it is necessary to add an amount ratio that effectively produces sediment, and for example, about 2 to 10 times is preferable. The addition is preferably performed slowly. For example, the addition can be performed at a rate of adding 1/100 of the amount per second to the total amount.
In addition, the obtained sediment is washed and solvent-converted by repeating the steps of (1) standing, (2) removing the supernatant, and (3) adding an organic solvent as necessary, and the silver paste ink remains as a suspension. Can be kneaded. (1) The standing operation can be replaced by centrifugation at such a strength that the silver chloride fine powder does not solidify. The centrifugal force in this case can be set in the range of 50 × g to 300 × g, but a centrifugal operation of about several hundred seconds to about one minute is preferable at about 100 × g. (3) In the organic solvent re-addition operation, addition in an ultrasonic bath is desirable for the purpose of making fine powder and improving the cleaning effect. In the course of this organic solvent addition operation, the solvent used for fine powdering can be converted into a solvent convenient for mixing into the silver / silver chloride paste. For example, it can be washed by changing from a washing with methanol to a solvent constituting paste ink, and preferably methyl cellosolve, butyl cellosolve, methyl cellosolve acetate, butyl cellosolve, isophorone, butyl carbitol acetate, etc. can be used. .
The operation of (1) standing to (3) adding an organic solvent is repeated several times, for example, 2 to 8 times, preferably about 5 times, and (2) the supernatant is removed. Preferred for conversion.
(3) Manufacture of silver / silver chloride paste
Fine silver chloride powder is kneaded in a silver paste, for example, a one-part polymer type silver paste (for example, silver paste LS-411 (manufactured by ASAHI CHEMICAL RESEARCH LABORATORY), etc.) using a polyester resin as a binder. It can be a silver chloride paste.
The mixing ratio of the fine silver chloride powder is 0.1% to 40%, preferably 0.2% to 20% of the silver / silver chloride paste by weight.
The obtained silver / silver chloride paste ink has good homogeneity and can be printed without clogging even by screen printing using a screen of about 300 mesh.
(4) Electrode printing
For electrode printing using silver / silver chloride paste ink, any method well known in the field of electronics can be used. Specifically, an electrode figure pattern is applied to polyester fiber or stainless steel screen mesh. Forming with photosensitive technology and printing using this as the original plate or inkjet method using a nozzle or print fixing base movable in the XY biaxial direction and printing while discharging paste ink with air pressure etc. along the electrode design pattern Alternatively, a dispensing method or the like can be used.
The thus obtained silver / silver chloride electrode can be used as an electrode of an electrode part in a biosensor such as an enzyme sensor or an immunosensor, particularly as a reference electrode. For example, it can be applied to the electrode part of an electrochemical biosensor represented by an immunosensor composed of a chromatography matrix part that separates and develops a substance to be analyzed from a sample solution and an electrode part. Make it possible.
Furthermore, the present invention provides an electrochemical biosensor using a silver / silver chloride electrode. The contents will be described below.
That is, the electrochemical biosensor of the present invention is a sensor for detecting and quantifying a substance to be analyzed in a sample solution, and the chromatography matrix part thereof is (a) one or a plurality of sample addition sites, (B) a specific reaction site that is located in contact with the downstream of the sample addition site and holds an enzyme-labeled specific binding substance having affinity for the analyte, and (c) downstream of the specific reaction site A competitive reaction site that is immobilized in contact with the analyte or a substance having the same reactivity as the analyte, (d) located in contact with the downstream of the competitive reaction site, and It consists of an enzyme reaction site holding a substrate so as to be movable, and the electrode part is located in contact with the enzyme reaction site or downstream thereof, and is printed and arranged.
The substance to be analyzed herein means biologically related substances such as peptides, proteins, complex proteins, polysaccharides, lipids, complex lipids, hormones, nucleic acids, and more specifically, for example, follicle stimulating hormone, Examples include luteinizing hormone, placental gonadotropin, thyroid hormone, progesterone, estradiol and other hormones, and lipoproteins, apolipoproteins, albumin, hemoglobin, and other biological trace components.
The sample solution refers to a liquid containing such a biological substance, mainly a liquid sample derived from organisms such as blood, plasma, serum, saliva, urine, etc., but solids such as mucus, body tissue or cells are used as a buffer solution, etc. It may be suspended or dissolved in the liquid.
The electrochemical biosensor of the present invention basically comprises a chromatography matrix part (a) to (d) that performs an immune reaction and an electrode part. Here, the chromatography matrix part Is composed of a porous carrier.
The porous carrier is not particularly limited as long as it functions as a medium for transferring the sample solution by capillary action. Examples of such a material include cellulose membrane (filter paper) and glass fiber filter paper (for example, GF / A, manufactured by Whatman), nitrocellulose membrane, nylon membrane, non-woven fabric made of plastic fibers such as polystyrene / polyester, sponge-like porous material, and the like. The size of the porous portion is not particularly limited, but when one porous portion is used in the present sensor, one having a width of 2 mm to 10 mm, a length of 30 mm to 150 mm, and a thickness of 20 μm to 1000 μm can be used.
The sample addition site (a) may be composed of a plurality of short porous portions. In this case, if the sample solution is arranged in contact with each other so that the sample solution can move from upstream to downstream by capillary action. Good (“upstream” and “downstream” mean the direction in which the analyte solution moves through the porous portion in the chromatography matrix).
In addition, at the sample addition site (a), pH buffer such as Tris buffer, diethanol buffer, phosphate buffer or gelatin, bovine serum albumin, casein, etc. for the purpose of preventing non-specific binding as necessary. Or any surfactant can be applied.
The specific reaction site (b) holds an enzyme-labeled specific binding substance having an affinity for the analysis target substance in a movable state, and allows the reaction between the analysis target substance in the sample solution and the labeled specific binding substance. It is a place that provides a place.
The specific binding substance is a substance that specifically reacts with the substance to be analyzed. Examples thereof include an antibody against an arbitrary antigen, a complementary nucleic acid against an arbitrary nucleic acid, and a receptor for various physiologically active substances. Are preferably antibodies against any antigen.
The labeling enzyme introduced into the specific binding substance is not particularly limited as long as it is an oxidoreductase that can generate an electrochemical signal when combined with a substrate. For example, glucose oxidase, galactosidase, alkaline phosphatase, horse dish peroxidase, etc. Oxidases and dehydrogenases can be used. Moreover, the method of introduce | transducing these enzymes into a specific binding substance can use the arbitrary methods generally used.
The enzyme-labeled specific binding substance is held in a movable state by impregnating the solution into a porous carrier and subjecting the solution to any means such as freeze drying, negative pressure drying, air drying, and natural drying. Can do.
The competitive reaction site (c) immobilizes an analysis target substance or a substance having the same reactivity as the analysis target substance, and reacts the analysis target substance in the sample solution that has moved from the upstream with the enzyme-labeled specific binding substance. Unreacted enzyme-labeled specific binding substance is captured in the mixture (complex that is the reaction product of the analyte and enzyme-labeled specific binding substance and unreacted enzyme-labeled specific binding substance) Removed.
Here, the substance having the same reactivity as the analysis target substance refers to the analysis target substance itself, a substance obtained by fragmenting the substance that reacts with the specific binding substance, and a similar structure that can bind to the specific binding substance. In the case of substances, analytes, denatured substances by means of heat, reagents, pH, etc., proteins, peptides, nucleic acids, etc. The substance to be analyzed is preferable.
The immobilization of the analysis target substance or the substance having the same reactivity as the analysis target substance is not particularly limited, but in general, the porous portion is a solution of the analysis target substance or the substance having the same reactivity as the analysis target substance. After being soaked for a certain period of time, it can be performed by rinsing, washing with a large amount of buffer solution or washing solution, for example, a general pH buffer solution or a surfactant added with a surfactant such as Tween 20, and drying. . In addition, when a protein to be immobilized or a substance having the same reactivity as the substance to be analyzed is a protein, it is preferably immobilized by a covalent bond because it may be detached. As an example, diazotized filter paper or Immunodyne ABC (manufactured by Pall) can be used.
The enzyme reaction site (d) holds a substrate that reacts with the labeling enzyme and generates a signal in a movable state, and the analyte to which the substrate has moved from the upstream and the enzyme-labeled specific binding substance Reacts with the complex to produce an electrochemical signal generator.
The substrate is appropriately selected according to the labeling enzyme. For example, when alkaline phosphatase, horseradish peroxidase, galactosidase, oxidase such as glucose oxidase, or dehydrogenase is used as the enzyme, p-aminophenyl phosphate, hydrogen donation, respectively. And hydrogen peroxide, p-aminophenyl-β-D-galactoside, substrate such as glucose as reductant and oxygen, substrate such as hydrogen donor and NAD + Can be used in appropriate combinations.
The substrate can be retained by a technique such as freeze drying, negative pressure drying, and air drying by impregnating a porous portion with a substrate solution, as in the case of an enzyme-labeled specific binding substance.
The electrode part is a part for electrochemically measuring the signal of the electrochemical signal generating substance generated at the enzyme reaction part (d), and is composed of three printed electrodes, a working electrode, a counter electrode and a reference electrode. The electrode is located in contact with the enzyme reaction site (d) or downstream thereof, printed on a porous carrier, and in contact with the reaction solution that has permeated and moved through the chromatography matrix by capillary action.
The electrodes are produced by printing using carbon paste ink for the working electrode and counter electrode, and silver / silver chloride paste ink for the reference electrode. Here, the silver / silver chloride electrode used is not particularly limited as long as it has good conductivity and can be stably used as a reference electrode, but considering conductivity and simplicity, Silver / silver chloride paste ink obtained by kneading a silver paste with a fine silver chloride powder prepared by dissolving silver chloride powder in an aqueous solvent and adding an organic solvent under ultrasonic irradiation as described above What was printed using is preferable. In particular, when a silver / silver chloride electrode is printed and arranged on a porous carrier such as filter paper, a method of preparing a silver chloride layer after silver paste printing cannot be applied, and a paste previously kneaded with silver / silver chloride powder should be used. Is preferred. Further, it is preferable to use a silver / silver chloride paste excellent in homogeneity and conductivity so as to be compatible with the porous carrier.
As the carbon paste ink, a one-component polymer type carbon paste using a polyester resin as a binder, for example, FTU-60 (manufactured by ASAHI CHEMICAL RESEARCH LABORATORY) can be used.
As described above, since the electrode portion is disposed on the porous carrier, it is necessary to provide an insulating portion that stops the movement of the sample solution that has moved to this region to the downstream. The insulating part here is one that is not electrically conductive and prevents the ingress of aqueous solution. The insulating part is an organic solvent for various plastics such as coating agents, insulating paints and adhesives. It can be prepared by coating and infiltrating a solution or the like.
These three electrodes are in contact with the outside and can be applied by a potentiostat device for electrochemical measurement or a constant voltage device capable of generating a constant voltage equivalent thereto. Measured by a meter.
The electrochemical biosensor should be covered with a transparent or opaque plastic film, etc. for the purpose of preventing evaporation, reinforcing the strength, shielding light, etc., leaving the sample addition site or part of it and the connection with the measuring device. Can do.
Example
Hereinafter, the present invention will be described more specifically with reference to examples.
Example 1 Preparation and evaluation of silver / silver chloride electrode
(1) Preparation of silver / silver chloride paste
10 g of silver chloride (Katayama Chemical Co., Ltd.) was suspended in 70 mL of concentrated aqueous ammonia (28%, Wako Pure Chemical Industries) and dissolved by stirring in a 30 ° C. hot water bath. The silver chloride solution was transferred to an ultrasonic bath, and 140 mL of methanol was gradually added dropwise under ultrasonic irradiation of about 30 KHz to precipitate a fine silver chloride powder. After stirring, the mixture was allowed to stand for 5 minutes, the supernatant was removed by suction, and 150 mL of methanol was added to the precipitate to wash the precipitate. The same operation was repeated 5 times, ammonia was removed, and the sediment was washed with butyl cellosolve in the same manner as methanol. Finally, about 670 mg of silver chloride fine powder was obtained as a suspension in butyl cellosolve. This silver chloride fine powder suspension was mixed with silver paste ink LS-411 (Asahi Chemical Research Laboratories) at a ratio of 20 to 0.25% and kneaded to be uniform.
(2) Evaluation of printed reference electrode
In order to evaluate the silver / silver chloride reference electrode prepared in the previous section, it was compared with a commercially available reference electrode.
Using the silver / silver chloride paste for electrodes of each mixing ratio prepared in (1), screen printing was performed using a screen of 305 mesh and 120 mesh to prepare a printed reference electrode. A fluororesin coating agent (CT-300-2, Asahi Scientific Research Laboratories) was applied in a line between the connecting part to the measuring device and the electrode part, and dried under negative pressure to provide an insulating part.
A reference electrode (RE-1B) manufactured by BAS as a reference electrode and an electrode pad on which only a silver / silver chloride paste is printed are immersed in a test electrode solution in which a small amount of silver chloride powder is added to a saturated KCl solution. A silver / silver chloride electrode was connected to the working electrode side on the reference electrode side of the 263A potentiostat device (EG & G), and the potential difference between the electrodes was measured. The results are shown in Tables 1 and 2.
(3) Evaluation of reference electrode by electrochemical measurement on printed electrode
Pure cellulose chromatography paper 3MM (Whatman) was used as a printed substrate. Using carbon paste ink FTU-60 (Asahi Chemical Research Laboratory), the working electrode and the counter electrode were screen-printed as shown in FIG. This was dried and solidified at 120 ° C. under a negative pressure for 30 minutes. A reference electrode was screen-printed with the silver / silver chloride paste ink of each mixing ratio in the arrangement as shown in FIG. 1 and then dried and solidified at 120 ° C. under a negative pressure for 30 minutes. Subsequently, a fluororesin coating agent (CT-300-2, Asahi Scientific Research Laboratories) was applied in a line at a position 10 mm from the tip of the working electrode, and dried under negative pressure to provide an insulating portion. Finally, a 10 mm × 30 mm portion including the printed 3 electrodes was cut out and used as a printed electrode. Each electrode corresponding to the measurement terminal of the H.263A potentiostat device (EG & G) was connected, and electrochemical measurement was performed by Chronoamperometry (CA) and Cyclic Voltammetry (CV) using the electrochemical measurement software Model 270/250. Electrochemically active substance p-aminophenol 0.5 mM, 1 mM magnesium chloride, 0.1 M phosphate buffer solution (pH 7.5) containing 0.1 M potassium chloride was added to the electrode part, and the current value (μA) was determined. Measured. For CA, a current value 60 seconds after the start of measurement was measured, and for CV, a current value (μC) of 0 to 300 mV was measured. The results are shown in Table 3.
Example 2
An electrochemical biosensor according to the present invention was prepared and used to measure human serum albumin in a sample.
(1) Preparation of sample addition part
The conjugate pad (50 × 100 mm) was immersed in a PBS solution containing 2% (w / v) Block Ace (Dainippon Pharmaceutical Co., Ltd.) and shaken for 10 minutes. This was washed with a PBS solution containing 0.05% Tween 20 for 3 minutes by shaking and dried in a vacuum dryer. Finally, it is cut to a size of 10 mm × 20 mm and placed on an insulator base for use.
(2) Preparation of enzyme-labeled antibody pad
An anti-human serum albumin antibody labeled with β-galactosidase was used as an enzyme-labeled specific binding substance. The antibody for labeling was β-galactosidase labeled anti-albumin goat antibody (0.25 mg / mL) manufactured by American Corex. The conjugate pad (50 × 100 mm) was immersed in a PBS solution containing 2% (w / v) Block Ace (Dainippon Pharmaceutical Co., Ltd.) and shaken for 10 minutes. This was washed with a PBS solution containing 0.05% Tween 20 for 3 minutes by shaking and dried in a vacuum dryer. This pad is immersed in 4 mL of a PBS solution containing a labeled antibody prepared to a concentration of 0.2% (w / v) Block Ace 10 μg / mL, and this solution is retained and placed in liquid nitrogen for quick freezing and freezing. Dried. Finally, it is cut so as to have a size of 10 mm × 20 mm and disposed on the downstream side of the sample addition portion on the insulator base. (3) Preparation of human serum albumin membrane
Porous nylon membrane Immunodyne ABC (Pall) (50 × 100 mm) was shaken for 1 hour in 10 mL of PBS solution containing 1 mg / mL human serum albumin (Calbiochem). This was washed with a PBS solution containing 0.05% Tween 20 for 3 minutes by shaking and dried in a vacuum dryer. Furthermore, it was immersed in a PBS solution containing 2% (w / v) Block Ace (Dainippon Pharmaceutical Co., Ltd.), shaken for 10 minutes, and then dried in a vacuum dryer. Finally, it is cut to a size of 10 mm × 10 mm and placed on the downstream side of the enzyme-labeled antibody pad on the insulator base.
(4) Preparation of substrate pad
Pure cellulose chromatography paper 31ETChr (Whatman) (50 × 10 mm) in 3 mL of 0.1 M phosphate buffer (pH 7.5) containing 5 mM p-aminophenyl-β-D-galactopyranoside and 1 mM magnesium chloride It was immersed, and this liquid was retained, poured into liquid nitrogen, rapidly frozen, and then lyophilized. Finally, it is cut to a size of 10 mm × 10 mm and used on the insulator base on the downstream side of the human serum albumin membrane.
(5) Preparation of silver / silver chloride paste for reference electrode
A silver / silver chloride paste ink was prepared in the same manner as in Example 1 (1).
(6) Preparation of electrode pad
Pure cellulose chromatography paper 31ETChhr (Whatman) was used as a printed substrate. Using carbon paste ink FTU-60 (Asahi Chemical Research Laboratory), the working electrode and the counter electrode were screen-printed as shown in FIG. This was dried and solidified at 120 ° C. under a negative pressure for 30 minutes. A silver / silver chloride paste ink was used to screen-print the reference electrode in the arrangement as shown in FIG. 1, and then dried and solidified at 120 ° C. under a negative pressure for 30 minutes. Subsequently, a polyester resin coating agent (CT-300-2, Asahi Scientific Research Laboratories) was applied in a line at a position 10 mm from the tip of the working electrode and dried under negative pressure to provide an insulating portion. Finally, a 10 mm × 30 mm portion including the printed three electrodes is cut out and arranged on the downstream side of the substrate pad for use.
(7) Measurement of human serum albumin
Each part produced as described above was arranged and adhered on a plastic insulator as shown in FIG. The inner working electrode of the carbon wire is connected to the working electrode terminal of the 263A potentiostat device (EG & G) through the contact portion of the electrode pad, the counter electrode of the other carbon wire is used as the counter electrode terminal, and the silver / silver chloride electrode reference electrode side is Connected to the reference terminal. Further, this 263A potentiostat device was connected to a computer through a GPIB line, and measurement and data analysis were performed using electrochemical measurement software Model 270/250. For measurement, the potential of the working electrode was set to 300 mV with respect to the reference electrode, and measurement was performed by chronoamperometry.
300 μL of a sample solution containing human serum albumin at a concentration of 0 to 200 μg / mL in a PBS solution containing 0.2% Block Ace is added to the sample addition section and sequentially developed downstream to measure the change in current value over time. The current value 1 minute after the start of measurement was recorded. The results are shown in Table 4.
Industrial applicability
The silver / silver chloride electrode of the present invention has a property of high homogeneity and excellent electrical conductivity, and can be produced easily and without clogging even in a screen printing method. In addition, the electrochemical biosensor of the present invention using the silver / silver chloride electrode can detect and quantify a substance to be analyzed quickly and accurately, and can be compact and disposable in form. it can. Therefore, it can be used as a simple measurement kit in a wide range of fields such as home, emergency medical treatment, bedside, and regional medical care.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of an electrochemical biosensor of the present invention. The symbols in the figure are as follows.
1: Sample addition site
2: Specific reaction site
3: Competitive reaction site
4: Enzyme reaction site
5: Insulation part
6: Connection with measuring device
7: Electrode part
a: Counter electrode
b: Working electrode
c: Reference electrode
Claims (6)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| JP2001509974A JP4695316B2 (en) | 1999-07-13 | 2000-07-12 | Silver / silver chloride electrode and electrochemical biosensor using the same |
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| Application Number | Priority Date | Filing Date | Title |
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| JP1999198689 | 1999-07-13 | ||
| JP19868999 | 1999-07-13 | ||
| JP2001509974A JP4695316B2 (en) | 1999-07-13 | 2000-07-12 | Silver / silver chloride electrode and electrochemical biosensor using the same |
| PCT/JP2000/004660 WO2001004614A1 (en) | 1999-07-13 | 2000-07-12 | Electrochemical biosensor using silver silver chloride electrode and chromatography matrix |
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| JP4695316B2 true JP4695316B2 (en) | 2011-06-08 |
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| WO (1) | WO2001004614A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2386331A (en) * | 2002-03-07 | 2003-09-17 | Surescreen Diagnostics Ltd | Deposition of a chemical solution on a test device using a printer |
| EP2280284A4 (en) * | 2008-05-26 | 2014-10-29 | Panasonic Healthcare Co Ltd | Biosensor |
| CN101858918B (en) * | 2010-06-09 | 2013-03-20 | 长沙市食品质量安全监督检测中心 | Method for detecting ractopamine in animal-derived food by microgap array electrode-based electrochemical immunosensor |
| JP6785856B2 (en) | 2016-06-30 | 2020-11-18 | タツタ電線株式会社 | Biological electrodes and methods for forming bioelectrodes |
| JP6653843B2 (en) | 2016-06-30 | 2020-02-26 | タツタ電線株式会社 | Electrode material |
| JP6612984B2 (en) * | 2016-06-30 | 2019-11-27 | タツタ電線株式会社 | Silver chloride paste |
| US11199519B2 (en) * | 2018-06-14 | 2021-12-14 | Nok Corporation | Method for producing silver-silver chloride electrode |
| CN111707723A (en) * | 2020-06-09 | 2020-09-25 | 上海三屹电子科技有限公司 | Silver/silver chloride slurry with high signal-to-noise ratio for biological detection electrode and preparation method thereof |
| KR102650140B1 (en) * | 2021-05-06 | 2024-03-21 | 주식회사 아이센스 | Paste for reference electrode, reference electrode, and biosensor including the same |
| CN114496345A (en) * | 2021-12-30 | 2022-05-13 | 上海宝银电子材料有限公司 | Silver chloride conductive silver paste and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4981785A (en) * | 1988-06-06 | 1991-01-01 | Ventrex Laboratories, Inc. | Apparatus and method for performing immunoassays |
| JPH06317553A (en) * | 1993-05-06 | 1994-11-15 | Nakagawa Boshoku Kogyo Kk | Reference electrode for concrete burying |
| JPH07198669A (en) * | 1993-12-28 | 1995-08-01 | Taiyo Yuden Co Ltd | Electrode for chemical sensor, manufacture thereof and chemical sensor plate |
| WO1997001761A1 (en) * | 1995-06-27 | 1997-01-16 | Dainabot Co., Ltd. | Assay method and assay kit |
-
2000
- 2000-07-12 WO PCT/JP2000/004660 patent/WO2001004614A1/en active Application Filing
- 2000-07-12 JP JP2001509974A patent/JP4695316B2/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4981785A (en) * | 1988-06-06 | 1991-01-01 | Ventrex Laboratories, Inc. | Apparatus and method for performing immunoassays |
| JPH06317553A (en) * | 1993-05-06 | 1994-11-15 | Nakagawa Boshoku Kogyo Kk | Reference electrode for concrete burying |
| JPH07198669A (en) * | 1993-12-28 | 1995-08-01 | Taiyo Yuden Co Ltd | Electrode for chemical sensor, manufacture thereof and chemical sensor plate |
| WO1997001761A1 (en) * | 1995-06-27 | 1997-01-16 | Dainabot Co., Ltd. | Assay method and assay kit |
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