JP5022285B2 - 中性脂肪測定用バイオセンサ - Google Patents
中性脂肪測定用バイオセンサ Download PDFInfo
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- JP5022285B2 JP5022285B2 JP2008089443A JP2008089443A JP5022285B2 JP 5022285 B2 JP5022285 B2 JP 5022285B2 JP 2008089443 A JP2008089443 A JP 2008089443A JP 2008089443 A JP2008089443 A JP 2008089443A JP 5022285 B2 JP5022285 B2 JP 5022285B2
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Description
能を有するバイオセンサの開発が進められている。
の濃度を測定するための装置である。以下、バイオセンサ10を構成する各部材について
詳細に説明する。
(1)不織布に中性脂肪分解酵素を含む溶液(以下、「中性脂肪分解酵素溶液」とも称する)を含浸させる工程
(2)上記工程(1)で形成された中性脂肪分解酵素溶液を含浸した不織布を乾燥する工程
上記工程(1)は、中性脂肪分解酵素溶液を不織布に含浸させることができればよい。具体的には、中性脂肪分解酵素溶液を不織布に滴下して含浸させる方法、不織布を中性脂肪分解酵素溶液に浸漬させて含浸させる方法などがあり、いずれの方法も好適に使用されうる。中でも、適量、好ましくは0.1〜10μlの量の中性脂肪分解酵素溶液を、不織布に直接滴下する方法が使用できる。
37℃で5分間予備加温したオリーブオイルエマルジョン溶液 2mlに酵素溶液 0.2mlを添加し、15分間反応させた。その後、0.2M TCA溶液 2mlを添加して反応を停止させ、グリセロールおよび遊離脂肪酸を生成させた。なお、「オリーブオイルエマルジョン溶液」とは、オリーブオイル 5gおよび5% Triton(登録商標)X−100 5mlの混合溶液を10分間超音波処理し、この混合溶液に4% BSA溶液 25mlおよび0.1M リン酸緩衝液 pH 7.0 15mlを添加し、十分に撹拌したものである。
50μM DCIP(2,6−ジクロロインドフェノール)、0.2mM PMS(1−メトキシ−5−メチルフェナジニウムメチルサルフェイト)、および450mM グリセロールを含む0.1% Triton(登録商標)X−100を含む10mM リン酸緩衝液(pH7.0)中に、酵素溶液を加えた。この溶液中の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。ここで、1分間に1μmolのDCIPを還元するグリセロールデヒドロゲナーゼの量を1活性単位(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。
本発明のバイオセンサの一例として、以下の手法により、図1および図2に示す形態の中性脂肪センサを作製した。
電極系が形成されたセンサ基板として、ディスポーザブル印刷電極 DEP Chip EP−N(有限会社バイオデバイステクノロジー製)を使用した。DEP Chip EP−Nは、絶縁性基板20の上に、それぞれカーボンからなる作用極30、参照極40、対極50が形成され、絶縁層70を挟んで、カーボンからなる作用極作用部分30a、銀/塩化銀からなる参照極作用部分40a、カーボンからなる対極作用部分50aが形成されている。
試料溶液としては、中性脂肪含有標準血清を用いた。
試料溶液としては、グリセロール標準液を用いた。
図3に示される結果より、本発明のバイオセンサにおいては、試料溶液として中性脂肪含有標準血清を用いた場合の応答電流値と、グリセロール標準液を用いた場合の応答電流値とはほとんど同じ値を示し、近似直線もほぼ一致した。
20 絶縁性基板、
30 作用極、
30a 作用極作用部分、
40 参照極、
40a 参照極作用部分、
50 対極、
50a 対極作用部分、
60 電極系、
70 絶縁層、
80 ポリマー層、
90 不織布層。
Claims (7)
- 絶縁性基板と、
前記絶縁性基板上に形成された、少なくとも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、グリセロールデヒドロゲナーゼ、電子受容体、および親水性ポリマーを含むポリマー層と、
前記ポリマー層上に形成された、不織布に中性脂肪分解酵素が担持されてなる不織布層と、
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。 - 前記グリセロールデヒドロゲナーゼは、ピロロキノリンキノン(PQQ)、フラビンモノヌクレオチド(FMN)、フラビンアデニンジヌクレオチド(FAD)、ニコチンアミドアデニンジヌクレオチド(NAD)、およびニコチンアミドアデニンジヌクレオチドリン酸(NADP)からなる群より選択される少なくとも1種の補酵素が結合した補酵素結合型グリセロールデヒドロゲナーゼであることを特徴とする、請求項1項に記載のバイオセンサ。
- 前記電子受容体は、フェリシアン化カリウムなどのシアン化合物、フェロセン、フェロセンカルボン酸などのフェロセン誘導体、p−ベンゾキノン、α−ナフトキノン、p−ベンゾキノン誘導体およびα−ナフトキノン誘導体などのキノン誘導体、フェナジンメトサルフェート(PMS)、フェナジンメトサルフェート誘導体、2,6−ジクロロインドフェノールナトリウムなどのフェノール誘導体、ルテニウム錯体およびオスミウム錯体などの金属錯体、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、ならびにこれらの混合物からなる群より選択される1種であることを特徴とする、請求項1または2に記載のバイオセンサ。
- 前記親水性ポリマーは、カルボキシメチルセルロースなどのセルロース系化合物、ポリビニルアルコールなどのポリビニル系化合物、ポリアクリル酸ナトリウムなどのポリアクリル酸系化合物、ポリメタクリル酸ナトリウムなどのポリメタクリル酸系化合物、ポリスチレンスルホン酸ナトリウムなどのポリスチレンスルホン酸系化合物、カルボキシメチルスターチなどのスターチ系化合物、ポリアクリルアミドなどのポリアクリルアミド系化合物、ポリビニルピロリドンなどのポリビニルピロリドン系化合物、ポリ酢酸ビニルなどのポリ酢酸ビニル系化合物、ポリグルタミン酸、ポリアスパラギン酸、ゼラチン、デンプン、ポリエチレンオキシド、デキストラン、ポリエチレングリコール、プルラン、およびこれらの誘導体からなる群より選択される少なくとも1種からなることを特徴とする、請求項1〜3のいずれか1項に記載のバイオセンサ。
- 前記中性脂肪分解酵素は、リポプロテインリパーゼであることを特徴とする、請求項1〜4のいずれか1項に記載のバイオセンサ。
- 前記不織布は、スパンボンド不織布、ニードルパンチ不織布、サーマルボンド不織布、ケミカルボンド不織布、ステッチボンド不織布、およびこれらの混合物からなる群より選択される少なくとも1種であることを特徴とする、請求項1〜5のいずれか1項に記載のバイオセンサ。
- 前記不織布の構成材料は、ポリアミド繊維、ポリビニルアルコール繊維、ポリエチレン繊維、ポリスルホン繊維、ポリエーテルスルホン繊維、ポリカーボネート繊維、ポリウレタン繊維、ポリエステル繊維、ポリプロピレン繊維、ポリオレフィン繊維、アラミド繊維、セルロース繊維、再生セルロース繊維、レーヨン繊維、ビニロン繊維、アクリル繊維、リヨセル繊維、絹繊維、麻繊維、ガラス繊維、キチン・キトサン繊維、炭素繊維、これらの誘導体、およびこれらの混合体からなる群より選択される少なくとも1種であることを特徴とする、請求項1〜6のいずれか1項に記載のバイオセンサ。
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