JP4700687B2 - バイオセンサ - Google Patents
バイオセンサ Download PDFInfo
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- JP4700687B2 JP4700687B2 JP2007510476A JP2007510476A JP4700687B2 JP 4700687 B2 JP4700687 B2 JP 4700687B2 JP 2007510476 A JP2007510476 A JP 2007510476A JP 2007510476 A JP2007510476 A JP 2007510476A JP 4700687 B2 JP4700687 B2 JP 4700687B2
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- biosensor
- reaction layer
- electrode
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Description
20mMリン酸緩衝液(pH7)1mLに大豆油エマルジョン2mLを添加し、十分に撹拌した後、リポプロテインリパーゼ含有溶液を1mL添加し、37℃にて20分間振盪させる。振盪後、反応停止液を10mL添加し、さらにn−ヘプタン6mLおよびイオン交換水4mLを添加し、十分に撹拌する。なお、「大豆油エマルジョン」とは、20mMリン酸緩衝液(pH7)で調製した10%ウシ血清アルブミン溶液16mLに大豆油24mLを添加し、十分に撹拌したものである。また、「反応停止液」とは、n−ヘプタン/2−プロパノール/2N硫酸(10/40/1、w/w/w)混合液である。
50μM DCIP、0.2mM PMS、400mM グリセロールを含んだ0.2%トリトンX−100を含む10mMリン酸緩衝液(pH7.0)中に、PQQ依存性グリセロールデヒドロゲナーゼを溶解してPQQ依存性グリセロールデヒドロゲナーゼ溶液を調製した。該溶液に含まれる酵素と基質との反応をDCIPの600nmの吸光度変化によって追跡し、その吸光度の減少速度を酵素の反応速度とした。1分間に1μmolのDCIPが還元される酵素活性を1単位(U)とした。なお、DCIPのpH7.0におけるモル吸光係数は16.3mM−1とした。
ソルビトール(2質量%)、酵母エキス(0.3質量%)、肉エキス(0.3質量%)、コーン・スティープ・リカー(0.3質量%)、ポリペプトン(1質量%)、尿素(0.1質量%)、KH2PO4(0.1質量%)、MgSO4・7H2O(0.02質量%)、CaCl2(0.1質量%)、pH7.0よりなる培地400mLを500mL容坂口フラスコに一本あたり100mLずつ移し、121℃、20分間でオートクレーブを行った。
電極系が形成されたセンサ基板として、市販のセンサ電極(BVT社製;AC1.W5.R1)を準備した。このセンサ基板において、電極系は直径約6mmの略円形状である。さらに、上記で準備した電極系の上部に、直径6mmの孔を開けた厚さ0.2mmのPETシートを貼り合わせ、スペーサを形成した。電極系上のスペーサの孔の内部に、上記の参考例1で得たGlyDH(20U/mL)と、リポプロテインリパーゼ(天野エンザイム製、10,000U/mL)と、電子受容体として1−メトキシ−5−メチルフェナジニウムメチルサルフェート(同人化学研究所製:10mmol/L;以下、「m−PMS」とも称する)とを混合したリン酸緩衝生理食塩水を10μL滴下し、室温で乾燥させて反応層を形成した。
実施例1−1で使用した電極系上に、0.5%(w/v)のカルボキシメチルセルロースナトリウム(和光純薬製;以下、「CMC」とも称する)水溶液を滴下乾燥後、実施例1と同様にして得られたGlyDH(20U/mL)、リポプロテインリパーゼ(10,000u/mL)およびm−PMS(10mmmol/L)を含む0.5%CMC溶液を滴下し、乾燥させて反応層を形成した。
電極系が形成されたセンサ基板として、市販のセンサ電極(BVT社製;AC1.W5.R1)を準備した。このセンサ基板において、電極系は直径約6mmの略円形状である。さらに、上記で準備した電極の上部に、直径6mmの孔を開けた厚さ0.2mmのPETシートを貼り合わせ、スペーサを形成した。
フは、その傾きが大きいほどセンサの測定感度が高いことを意味する。本実施例においては、図6に示すような傾きの比較的大きいグラフが得られる。従って、本実施例によれば、測定感度に優れるセンサが提供されることがわかる。
反応層形成用組成物を調製する際に、界面活性剤であるトリトンX−100を添加しなかったこと以外は、上記の実施例2−1と同様の手法により、センサ基板上にスペーサおよび反応層を形成し、試料溶液の添加後の酸化電流(応答電流)値の測定を行った。得られた結果をプロットしたグラフを図6に示す。本実施例においては、図6に示すように、得られるグラフの傾きが小さいが、ある程度の線形性は認められる。
反応層形成用組成物を調製する際に、リン酸緩衝生理食塩水に代えて蒸留水を用いたこと以外は、上記の実施例2−1と同様の手法により、センサ基板上にスペーサおよび反応層を形成し、試料溶液の添加後の酸化電流(応答電流)値の測定を行った。得られた結果をプロットしたグラフを図6に示す。本実施例においては、図6に示すように、得られるグラフの傾きは小さいが、ある程度の線形性は認められる。
Claims (15)
- 絶縁性基板と、
前記絶縁性基板上に形成された、作用極および対極を含む電極系と、
前記電極系の上部または近傍に形成された、リポプロテインリパーゼ、補酵素依存型グリセロールデヒドロゲナーゼおよび電子受容体を含む反応層と、
を備える、前記電極系に流れる電流値に基づいて中性脂肪の濃度を測定するためのバイオセンサであって、
前記補酵素がピロロキノリンキノンまたはフラビンアデニンジヌクレオチドである、バイオセンサ。 - 前記電極系が、参照極をさらに含む、請求項1に記載のバイオセンサ。
- 前記電子受容体が、フェリシアンイオン、p−ベンゾキノン、p−ベンゾキノン誘導体、フェナジンメトサルフェート、フェナジンメトサルフェート誘導体、メチレンブルー、チオニン、インジゴカーミン、ガロシアニン、サフラニン、フェロセンおよびその誘導体、α−ナフトキノンおよびα−ナフトキノン誘導体からなる群から選択される1種または2種以上である、請求項1または2のいずれか1項に記載のバイオセンサ。
- 前記反応層が、界面活性剤およびpH緩衝剤をさらに含む、請求項1〜3のいずれか1項に記載のバイオセンサ。
- 前記界面活性剤が非イオン性界面活性剤である、請求項4に記載のバイオセンサ。
- 前記pH緩衝剤のpHが6〜9である、請求項4または5に記載のバイオセンサ。
- 前記反応層が、親水性高分子をさらに含む、請求項1〜6のいずれか1項に記載のバイオセンサ。
- 前記親水性高分子が、タンニン酸、ペクチン、カゼイン、カラギナン、ファーセレラン、プルラン、コラーゲン、キチン、キトサン、コンドロイチン硫酸ナトリウム、リグニンスルホン酸、およびこれらの誘導体からなる群から選択される1種または2種以上である、請求項7に記載のバイオセンサ。
- 前記反応層が、
前記電子受容体および前記親水性高分子を含み、前記グリセロールデヒドロゲナーゼを実質的に含まない、第1反応層と、
前記第1反応層の上層に形成された、前記グリセロールデヒドロゲナーゼおよび前記親水性高分子を含み、前記電子受容体を実質的に含まない、第2反応層と、からなる、請求項7または8に記載のバイオセンサ。 - 前記第1反応層と前記第2反応層との間に、実質的に前記親水性高分子からなる第3反応層が形成された、請求項9に記載のバイオセンサ。
- 前記電極系の上層に、略矩形の開口部を有する絶縁層が形成され、前記対極の前記開口部からの露出面積が、前記作用極の前記開口部からの露出面積の1.2〜3.0倍であることを特徴とする、請求項1〜10のいずれか1項に記載のバイオセンサ。
- 前記電極系をバイオセンサ外部へと電気的に導通させるためのリード部およびコネクタ部をさらに備え、
前記電極系を構成する材料が、10μmの厚さにおいて100Ω/□以下の表面抵抗値を有するカーボンを主成分とする材料であり、前記リード部および前記コネクタ部を構成する材料が、10μmの厚さにおいて50mΩ/□以下の表面抵抗値を有する金属を主成分とする材料である、請求項1〜11のいずれか1項に記載のバイオセンサ。 - 前記金属が、銀、金、白金、およびパラジウムからなる群から選択される1種または2種以上の金属である、請求項12に記載のバイオセンサ。
- 前記グリセロールデヒドロゲナーゼが、界面活性剤の存在下、2価性架橋試薬で化学修飾されてなる修飾グリセロールデヒドロゲナーゼである、請求項1〜13のいずれか1項に記載のバイオセンサ。
- 前記グリセロールデヒドロゲナーゼが、グルコノバクター属に属する細菌由来である、請求項14に記載のバイオセンサ。
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GB0204232D0 (en) * | 2002-02-22 | 2002-04-10 | Isis Innovation | Assay |
CN100339702C (zh) * | 2002-03-01 | 2007-09-26 | 松下电器产业株式会社 | 生物传感器 |
JP2004271746A (ja) * | 2003-03-06 | 2004-09-30 | Fuji Xerox Co Ltd | 画像形成装置 |
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KR101368597B1 (ko) * | 2009-03-31 | 2014-02-27 | 코오롱인더스트리 주식회사 | 투명전극, 전도성 적층체 및 전도성 수지막 |
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2006
- 2006-03-27 KR KR1020077021705A patent/KR101226264B1/ko active IP Right Grant
- 2006-03-27 ES ES06730046T patent/ES2331533T3/es active Active
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- 2006-03-27 CA CA2603123A patent/CA2603123C/en not_active Expired - Fee Related
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- 2006-03-27 EP EP06730046A patent/EP1873516B1/en not_active Not-in-force
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US20170101661A1 (en) | 2017-04-13 |
US20090236222A1 (en) | 2009-09-24 |
ES2331533T3 (es) | 2010-01-07 |
EP1873516A4 (en) | 2008-05-07 |
CA2603123C (en) | 2014-01-28 |
US9957542B2 (en) | 2018-05-01 |
DE602006010048D1 (en) | 2009-12-10 |
WO2006104077A1 (ja) | 2006-10-05 |
CN101151526B (zh) | 2012-05-30 |
CN101151526A (zh) | 2008-03-26 |
KR101226264B1 (ko) | 2013-01-25 |
KR20070116608A (ko) | 2007-12-10 |
JPWO2006104077A1 (ja) | 2008-09-04 |
EP1873516B1 (en) | 2009-10-28 |
EP1873516A1 (en) | 2008-01-02 |
CA2603123A1 (en) | 2006-10-05 |
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