JP3871967B2

JP3871967B2 - Sensing element using photoresponsive DNA thin film

Info

Publication number: JP3871967B2
Application number: JP2002154388A
Authority: JP
Inventors: 利彦長村
Original assignee: Japan Science and Technology Agency; National Institute of Japan Science and Technology Agency
Current assignee: Japan Science and Technology Agency; National Institute of Japan Science and Technology Agency
Priority date: 2002-05-28
Filing date: 2002-05-28
Publication date: 2007-01-24
Anticipated expiration: 2022-05-28
Also published as: JP2003344286A

Description

【０００１】
【産業上の利用分野】
本発明は、表面プラズモン共鳴励起蛍光法により気体，液体を高感度，高応答速度で測定する光応答性ＤＮＡ薄膜を利用したセンシング素子に関する。
【０００２】
【従来技術及び問題点】
ＮＯ₂ガス等の検出には、有機又は無機半導体薄膜を用いた電気的方法，多孔質ガラスの有機化合物を担持させた系で発生する蛍光又は吸収変化，色素会合体累積膜から発生する蛍光，表面プラズモンの共鳴角変化に応じた反射率変化等を利用した光学的方法等が採用されている。これらの方法によるとき、ｐｐｍ以下からｐｐｂオーダの感度で目的物が検出される。しかし、数分〜数時間と遅い応答速度のため測定に時間がかかり、濃度，物性等が短時間で変化する目的物の検出には適していない。
【０００３】
応答性は、検出対象の吸着・拡散・脱着を容易にするため膜厚を可能な限り薄くすることにより改善される。しかし、薄膜化に応じて感度が低下するので、感度低下を補う必要がある。そこで、本発明者等は、表面プラズモン共鳴又は導波モードで増強された光電場により光応答性薄膜から発した蛍光を気体，液体等の測定対象流体に透過させ、蛍光強度又は蛍光スペクトルを測定するセンシング素子を開発した（特願２００１−０５６４１５号）。この方法では、合成高分子に蛍光色素を分散させた薄膜を金属薄膜に積層し、金属薄膜との界面で発生する表面プラズモン共鳴又は導波モードで増強された光電場による色素の励起を利用して感度を上げている。該センシング素子を使用すると、高速応答性は勿論、ｐｐｂオーダの高感度検出も可能になる。
表面プラズモン共鳴又は導波モード励起蛍光法の採用で応答性，感度が改善されるものの、ＮＯ₂ガスに応答する高分子に加わる制約が大きく、蛍光強度に依存する応答性の向上にも限度がある。
【０００４】
【課題を解決するための手段】
本発明は、先願で提案したセンシング素子の感度，応答速度を更に改善すべく案出されたものであり、二重螺旋部に蛍光分子を取り込んだ光応答性ＤＮＡ薄膜を使用することにより蛍光強度を増加させ、或いはＤＮＡ自体の光励起でＤＮＡのクロモフォア間から蛍光分子に効率的にエネルギー移動させると共に、薄膜化により応答性を一段と改善したセンシング素子を提供することを目的とする。
【０００５】
本発明のセンシング素子は、その目的を達成するため、マッチング媒体を介して励起光入射側表面にプリズムが接合された光透過性固体基板と、光透過性固体基板の他面に金属薄膜を介して堆積された光応答性ＤＮＡ薄膜と、光応答薄膜の上方に透光板で閉じられ、測定対象流体Ｓを流動させ又は収容する測定空間と、全反射角を超えた入射角で励起光がプリズムの一面に入射するように光応答性固体基板を搭載し、回転量が調節可能な回転テーブルとを備え、表面プラズモン共鳴又は導波モード条件を満足する入射角の入射光を閉じ込めることにより励起された光応答性ＤＮＡ薄膜から発する蛍光が測定空間の測定対象流体を透過して測定系に送られることを特徴とする。
【０００６】
光源一体型測定セルにセンシング素子を組み込むこともできる。この場合、光透過性固体基板の一面に金属薄膜を介して光応答性ＤＮＡ薄膜を堆積し、他面に集光レンズを装着する。測定対象流体を流動させ又は収容する測定空間を光透過性固体基板の光応答性ＤＮＡ薄膜側に設け、励起光源を内蔵した透明媒質を光応答性ＤＮＡ薄膜の上方に配置している。
【０００７】
光応答性ＤＮＡ薄膜は、二重螺旋部に蛍光分子をインターカレート又はグルーブに取り込んだＤＮＡの水溶液又は疎水処理ＤＮＡの有機溶液を用い、スピンコート法，溶媒蒸発法，ＬＢ法，交互吸着法等で成膜される。ＤＮＡには、天然ＤＮＡ，合成ＤＮＡ，合成ＲＮＡ，ＤＮＡ／ＲＮＡ混合系等がある。ナトリウムイオン（Ｎａ⁺）を長鎖アルキルトリメチルアンモニウムイオン又は長鎖ジアルキルジメチルアンモニウムイオンで置換した疎水処理ＤＮＡも使用可能である。疎水処理ＤＮＡでは、長鎖アルキルの外周部に蛍光分子を取り込んでも良い。
【０００８】
蛍光分子としては、本発明に制約を加えるものではないが一つ又は複数の置換基Ｒ（Ｒ：アミノ基，モノアルキルアミノ基，ジアルキルアミノ基，モノヒドロキシアルキルアミノ基，ジヒドロキシアルキルアミノ基，ピリジル基，キノリル基，イソキノリル基，アクリジニル基，ヒドロキシル基，ピリジニウム基，キノリニウム基，イソキノリニウム基，アクリジニウム基，スルホニウム基）をもつ脂肪族，芳香族，複素環族の有機化合物又はその分子集合体等、既存の蛍光物質，色素が使用される。具体的には、ローダミンＢ，エチジウム色素，クマリン６，ルブレン誘導体，ペリレン誘導体，フルオロセイン，アクリフラビン，プロフラビン，アクリジンオレンジ，フェノサフラニン，ナイルブルー，クレシルブルー，メチレンブルー，ピレン誘導体，アントラセン誘導体，フルオレノン誘導体，フルオレン誘導体，チオフェン誘導体，ビチオフェン誘導体，バイラニン，ポルフィリン，アミノアクリジン，ルテニウムトリスビピリジン錯体等がある。
【０００９】
【作用及び実施の形態】
適当な濃度のＤＮＡ水溶液及び色素（蛍光分子）の水溶液を混ぜ合わせると、ＤＮＡの塩基対と色素の比や色素の構造等に依存して、ＤＮＡの二重螺旋部にインターカレート又はグルーブに色素（蛍光分子）が取り込まれる。色素取込み反応は、ＤＮＡと色素との組合せにもよるが、直ちに或いは数時間程度で完了する。疎水処理ＤＮＡを使用する場合、2-メトキシエタノール，エタノール等の有機溶媒中で疎水処理ＤＮＡ及び蛍光分子を溶解混合すること又は疎水処理ＤＮＡ，色素それぞれを溶解した有機溶媒を混合することによって色素取込み反応が進行する。疎水処理ＤＮＡでは、疎水外周部に色素が取り込まれることもある。
【００１０】
蛍光分子を取り込んだＤＮＡ又は疎水処理ＤＮＡは、溶液中に比べて蛍光分子の運動を強く抑制し、蛍光分子周囲の微視的環境を変える。分子運動の抑制は蛍光強度の増加をもたらす。微視的環境の変化は、吸収スペクトルを変化させ、蛍光強度を増加させる原因となる。ＤＮＡ水溶液又は疎水処理ＤＮＡ有機溶液からスピンコート法，溶媒蒸発法，ＬＢ法等で成膜すると、運動が抑制され周囲の微視的環境が変化した状態のままで蛍光分子が薄膜に固定された光応答性ＤＮＡ薄膜又は光応答性疎水処理ＤＮＡ薄膜（以下、適宜「光応答性ＤＮＡ薄膜」で総称する）が作製される。
【００１１】
光応答性ＤＮＡ薄膜を励起光で照射すると、蛍光分子が励起され蛍光が発生する。発生した蛍光は、ＤＮＡ又は疎水処理ＤＮＡに蛍光分子を取り込むことにより分子運動が抑制されること、蛍光分子間やＤＮＡ又は疎水処理ＤＮＡ自体の発色団と蛍光分子の間に生じるエネルギー移動等が応答速度，感度の双方を向上させ、結果としてセンシング素子の繰返し耐久性，ガス応答性，検出限界が改善される。
【００１２】
光応答性ＤＮＡ薄膜の励起には、ＤＮＡ，疎水処理ＤＮＡ又は蛍光分子の吸収波長に当る波長の励起光Ｌ₁が使用される。励起光Ｌ₁の照射に際し、酸素（Ｏ₂），二酸化窒素（ＮＯ₂），酸化窒素（ＮＯ），笑気ガス（Ｎ₂Ｏ），二酸化硫黄（ＳＯ₂），硫化水素（Ｈ₂Ｓ），一酸化炭素（ＣＯ），二酸化炭素（ＣＯ₂），水（Ｈ₂Ｏ），アンモニア（ＮＨ₃），メタノール（ＣＨ₃ＯＨ），エタノール（Ｃ₂Ｈ₅ＯＨ），アミン類，アルデヒド類，各種炭化水素，メルカプタン類，光化学オキシダント，酸化性化合物、還元性化合物，酸化還元酵素，ハロゲン誘導体麻酔ガス等を含むガス又は液体が光応答性ＤＮＡ薄膜に吸収され或いは薄膜中を拡散すると、蛍光分子が測定対象流体と相互作用し、特定波長での蛍光強度変化，スペクトル変化又は励起光の反射強度変化として測定対象流体が検出される。
【００１３】
光応答性ＤＮＡ薄膜は、表面プラズモン共鳴励起蛍光測定システム（図１）の測定セル２０に組み込まれる。
この表面プラズモン共鳴励起蛍光測定システムは、励起光源１１で発生した励起光Ｌ₁を偏光板１２，半波長板１３，偏光板１４，チョッパー１５，ミラー１６，１７を経て測定セル２０に導く光学系１０を備えている。励起光Ｌ₁には、連続発振レーザ光，パルス発振レーザ光又は分光された定常光が使用される。
測定セル２０では、励起光Ｌ₁の入射側にプリズム２１を接触させた固体基板２２が回転テーブル２３に搭載されている。固体基板２２には、光透過性に優れたガラス，石英，サファイア，透明プラスチック等が使用される。
【００１４】
測定系３０では、測定セル２０から発する蛍光Ｌ₂を集光レンズ３１で集光し、光電子増倍管３２に入力する。光電子増倍管３２で電気変換・増幅された蛍光Ｌ₂は、蛍光分光光度計３３で蛍光強度が測定され、或いはスペクトル分析される。蛍光Ｌ₂の一部は、光電子増倍管３２で電気信号に変換・増幅され、デジタルメモリ３４に送られ、時間変化が記録される。
【００１５】
プリズム２１の一面に対する励起光Ｌ₁の入射角θは、全反射条件を超える入射角θにおいて表面プラズモン共鳴を満足して反射率が最小となるように回転テーブル２３の回転量で調整される。回転テーブル２３は、コンピュータ３７で駆動制御されるステージコントローラ３６により回転量が制御される。励起光Ｌ₁の反射光Ｌ₃はフォトダイオード３８によって検出・電気変換され、チョッパー１５に同期した信号がロックイン増幅器３５で増幅され、デジタルメモリ３４で記録される。
【００１６】
固体基板２２には、Ａｇ，Ａｕ，Ａｌ等の金属薄膜２４を介して光応答性ＤＮＡ薄膜２５が堆積されている。固体基板２２は、プリズム２１，金属薄膜２４，光応答性ＤＮＡ薄膜２５を除く表面部がシリコンシート２６で覆われ、プリズム２１との間にマッチング媒体２７が充填されている。マッチング媒体２７は、プリズム２１の屈折率に固体基板２２の屈折率を一致させるために設けられる層であり、市販の屈折液等が使用される。
【００１７】
固体基板２２を保持するホルダー２８には、ガラス板２９で閉じられた測定空間Ｖにガス，液体等の測定対象流体Ｓを導く流入管２８in及び測定空間Ｖから測定対象流体Ｓを送り出す排出管２８outが形成されている。
測定対象流体Ｓには、酸素，二酸化窒素，酸化窒素，笑気ガス，二酸化硫黄，硫化水素，一酸化炭素，二酸化炭素，水，アンモニア，メタノール，エタノール，アミン類，アルデヒド類，各種炭化水素，メルカプタン類，光化学オキシダント，酸化性化合物，還元性加工物，酸化還元酵素，ハロゲン誘導体麻酔ガス等を含む気体又は液体が使用される。また、流入管２８in及び排出管２８outを介して循環させることなく、測定空間Ｖに測定対象流体Ｓを静置させた状態での測定も可能である。
【００１８】
測定セルとしては、光源一体型のセル（図３）も使用可能である。光源一体型測定セル４０は、固体基板４１の一面に金属薄膜４２及び光応答性ＤＮＡ薄膜４３を堆積しており、固体基板４１の他面に集光レンズ４８が配置されている。励起光Ｌ₁の反射光Ｌ₃は、フォトダイオード４９で検出され、電気信号に変換される。金属薄膜４２及び光応答性ＤＮＡ薄膜４３がシリコンシート４４で取り囲まれ、プラスチック等の透明媒質４５で覆われている。透明媒質４５に励起光源４６及び偏光板４７が配置されている。固体基板４１の内部にある測定空間Ｖに測定対象流体Ｓを供給しながら、励起光源４６から励起光Ｌ₁が光応答薄膜４３に向けて出射される。
【００１９】
光応答性ＤＮＡ薄膜２５，４３が励起光Ｌ₁によって励起されると、光応答性ＤＮＡ薄膜２５，４３から蛍光Ｌ₂が発せられる。蛍光Ｌ₂は、測定空間Ｖにある測定対象流体Ｓを透過した後、測定系３０の集光レンズ３１で集光され、光電子増倍管３２に入力される。光電子増倍管３２で電気変換・増幅された蛍光Ｌ₂は、蛍光分光光度計３３で蛍光強度が測定され、或いはスペクトル分析される。光応答性ＤＮＡ薄膜２５，４３に測定対象流体Ｓが吸着又は拡散されることにより光応答性ＤＮＡ薄膜２５，４３の蛍光強度を高速且つ敏感に変化する。また、測定対象流体Ｓの吸着又は拡散で光応答性ＤＮＡ薄膜２５，４３の屈折率が変わり表面プラズモン共鳴角度がシフトすることにより、励起光Ｌ₁の反射光Ｌ₃が強度変化することもある。したがって、蛍光Ｌ₂又は反射光Ｌ₃の強度は、測定対象流体Ｓに応じた変化を取り込んだ信号として扱われる。
【００２０】
たとえば、プリズム２１を介し金属薄膜２４，４２にＰ偏光を入射すると、ある角度で金属薄膜２４，４２中にある金属の自由電子のプラズマ振動とＰ偏光が結合し、ほとんど反射光Ｌ₃のない表面プラズモン共鳴が生じる。また、金属薄膜２４，４２上に膜厚が約１００ｎｍ以下の光応答性ＤＮＡ薄膜２５，４３を配置すると、光応答性ＤＮＡ薄膜２５，４３の膜厚に応じて表面プラズモン共鳴が生じる入射角θが次第に広角度に移っていく。更に、薄膜２５，４３の膜厚を光の波長の半分程度以上にすると、Ｐ偏光，Ｓ偏光の何れにおいても膜厚に応じて厚み方向に一つ又は複数のノードをもつ定在波ができ、反射光Ｌ₃がほとんどでなくなる導波モードとなる。
【００２１】
ＤＮＡ又は疎水処理ＤＮＡに取り込まれた蛍光性色素は、二重螺旋部にインターカレート又はグルーブに、或いは長鎖アルキル基の疎水外周部に捕捉される。そのため、励起波長及び蛍光分子を適切に選択すると、ＤＮＡ自体の光励起によってＤＮＡのクロモファ間から蛍光分子に効率の良いエネルギー移動が生じる。蛍光分子へのエネルギー移動は、センシングの観点から検出対象流体に対する応答分子の著しい増加に対応する。更に、表面プラズモン共鳴により反射率が極小になる入射角では、金属薄膜２４，４２で電場増強され薄膜２５，４３に閉じこまれた光によって蛍光分子中の色素が非常に強く励起される。その結果、１００ｎｍ以下の超薄膜においても高感度蛍光測定が可能になり、気体，液体等の測定対象流体の吸着，拡散，脱着が厚膜に比較して一層高速化される。
【００２２】
この点、特願２００１−０５６４１５号で提案したセンシング素子では，通常の高分子に色素を無秩序分散させているので，高分子のクロモファ間又は高分子と色素間の効率的なエネルギー移動がほとんど期待できない。そのため、実質的には表面プラズモン共鳴条件より数倍厚い数百ｎｍ程度の薄膜で生じる導波モードに依らざるを得ない。これに対し、光応答性ＤＮＡ薄膜２５，４３を使用したセンシング素子は、繰返し応答性，応答速度等が格段に改善される。また、表面プラズモン共鳴センサーで一般に実施されている測定対象流体の吸着，拡散，脱着に伴う屈折率変化による反射光の強度変化を蛍光測定に併用できることも利点である。
【００２３】
表面プラズモン共鳴を満足する入射角θでは、入射光（励起光Ｌ₁）が光応答性ＤＮＡ薄膜２５，４３に閉じ込められた状態にあるので、光応答性ＤＮＡ薄膜２５，４３に含まれている蛍光性分子が効率よく励起される。光応答性ＤＮＡ薄膜２５，４３の内部に拡散し、或いは表面に吸着した気体，液体等の測定対象流体Ｓと蛍光性分子との相互作用の影響を受け、励起で生じる蛍光Ｌ₂の強度が敏感に変化する。しかも、光応答性ＤＮＡ薄膜２５，４３が薄いため、光応答性ＤＮＡ薄膜２５，４３に対する測定対象流体Ｓの吸着又は拡散が迅速に進行し、相互作用の影響が高応答速度で蛍光強度の変化に反映される。したがって、蛍光Ｌ₂の蛍光強度又はスペクトル分析結果から、或いは反射光強度の変化から測定対象流体Ｓが高感度且つ高応答速度で検出又は測定される。
【００２４】
【実施例】
ＤＮＡ：８１.３ｍｇ及びエチジウムブロミド：０.９９ｍｇをそれぞれ純水：５ｍｌに溶かした水溶液を用意した。水溶液を相互に混合した後でエチジウムブロミドの吸収スペクトルを測定したところ、吸収スペクトルが長波長側にシフトしており、図４の蛍光スペクトル（ａ，ｂ）にみられるように蛍光強度も増加していた。この測定結果は、エチジウムブロミドがＤＮＡに取り込まれていることを示す。また、エチジウムブロミド単独系（ｄ）に比較してＤＮＡとの混合系水溶液の蛍光励起スペクトル（ｃ）がＤＮＡの吸収波長帯域で著しく増大している結果から、ＤＮＡからエチジウムブロミドへのエネルギー移動が読み取れる。疎水処理ＤＮＡ：５８.７ｍｇ及びローダミンＢ：０.４８ｍｇを2-メトキシエタノール：１０ｍｌに溶解した有機溶液について吸収スペクトルを測定したところ、疎水処理ＤＮＡがない場合に比較してローダミンＢの蛍光強度が増加しており、ローダミンＢが疎水処理ＤＮＡに取り込まれていることを確認できた。更に、円偏光二色性スペクトル測定からも、発光色素のＤＮＡ又は疎水処理ＤＮＡへの取込みが確認された。
【００２５】
固体基板２２にスライドガラスを使用し、固体基板２２上に膜厚５０ｎｍのＡｇ薄膜（金属薄膜２４）を真空蒸着法で設けた後、疎水処理ＤＮＡ，ローダミンＢの有機溶液を膜厚５０ｎｍの銀蒸着膜にスピンコート法で塗布し、膜厚２０ｎｍの光応答性ＤＮＡ又は疎水処理ＤＮＡ薄膜を積層した。プリズムを介し波長５４３.５ｎｍのＨｅ−Ｎｅレーザ光を入射させたところ、入射角５５度近傍で表面プラズモン共鳴に起因する反射率の極小域（ディップ）が検出された。反射率が極小となる５５度の入射角で蛍光スペクトルを測定した結果、直接励起に比較して２桁も大きな蛍光強度が得られ（図５）、表面プラズモン共鳴励起の効果が確認された。。
【００２６】
固体基板２２を測定セル２０に組み込み、０.５ｐｐｍの二酸化窒素ガス（ＮＯ₂）を測定空間Ｖに送り込みながら入射角θ＝５５度で励起光Ｌ₁をプリズム２１の一面に入射させた。
励起光Ｌ₁で誘起された表面プラズモン励起により、光応答性ＤＮＡ薄膜２５から蛍光Ｌ₂が発した。蛍光は１分間で約４０％減少し、二酸化窒素ガス（ＮＯ₂）の供給を止めて排気すると１分以内に元の状態に完全復帰した。短時間での完全復帰は、高速応答性に優れた光応答性ＤＮＡ薄膜２５であることを示す。検出精度は約１０ｐｐｂ程度であり、高濃度側では約１００ｐｐｍまで同様な応答性で検出できた。
【００２７】
そこで、二酸化窒素ガス（ＮＯ₂）の給気，排気を繰り返しながら蛍光Ｌ₂の強度変化を調査した。図６の調査結果にみられるように、二酸化窒素ガス（ＮＯ₂）の給気，排気サイクルに高い一致性で蛍光強度が周期的に変化し、ローダミンＢを含む疎水処理ＤＮＡ薄膜２５の優れた高速応答性，高感度，繰返し耐久性が確認された。
ＤＮＡ，エチジウムブロミドの水溶液から作製された光応答性ＤＮＡ薄膜も、同様に高速応答性，高感度，繰返し耐久性に優れ、二酸化窒素ガスの高性能測定が可能であった。
【００２８】
【発明の効果】
以上に説明したように、本発明のセンシング素子では、金属により電場増強された表面プラズモン共鳴で光応答性ＤＮＡ薄膜を励起し、光応答性ＤＮＡ薄膜から発した蛍光を気体，液体等の測定対象流体に透過させ、蛍光強度又は蛍光スペクトルを高感度測定している。光応答性ＤＮＡ薄膜の使用により、高速応答性で測定対象流体を高感度測定できる。薄膜化した光応答性ＤＮＡ薄膜の表面プラズモン共鳴励起蛍光が短時間で大幅に減少するため、短時間で物性や濃度が変化する測定対象流体に対しても適用でき、オンラインの環境ガスモニタ，呼吸器ガスモニタ，医療用ガスモニタ，光化学オキシダントセンサ等、広範な分野で使用される。
【図面の簡単な説明】
【図１】本発明に従った表面プラズモン共鳴励起蛍光測定システムの一例
【図２】測定セルの詳細
【図３】光源一体型測定セルの側面図（ａ）及び平面図（ｂ）
【図４】エチジウムブロミドとＤＮＡとの混合系及びエチジウムブロミド単独水溶液の蛍光スペクトル（ａ，ｂ）及び蛍光励起スペクトル（ｃ，ｄ）を示すグラフ
【図５】ローダミンＢを含む疎水処理ＤＮＡ薄膜の表面プラズモン共鳴励起及び直接励起で発生した蛍光スペクトルを示すグラフ
【図６】ローダミンＢを含む疎水処理ＤＮＡ薄膜の表面プラズモン励起蛍光が二酸化窒素ガスの給気，排気に応じて強度変化することを示したグラフ
【符号の説明】
１０：光学系２０：測定セル２１：プリズム２２，４１：固体基板２３：回転テーブル２４，４２：金属薄膜２５，４３：光応答性ＤＮＡ薄膜２７：マッチング媒体２８：ホルダー２９：ガラス板
３０：測定系４０：光源一体型測定セル４５：透明媒質
Ｌ₁：励起光Ｌ₂：蛍光 θ：入射角
Ｓ：測定対象流体Ｖ：測定空間[0001]
[Industrial application fields]
The present invention relates to a sensing element using a photoresponsive DNA thin film that measures gas and liquid with high sensitivity and high response speed by surface plasmon resonance excitation fluorescence.
[0002]
[Prior art and problems]
For detection of NO ₂ gas, etc., an electrical method using an organic or inorganic semiconductor thin film, a fluorescence or absorption change generated in a system supporting an organic compound of porous glass, a fluorescence generated from a dye aggregate accumulation film, An optical method using a change in reflectance according to a change in the resonance angle of surface plasmons is employed. When these methods are used, the target is detected with a sensitivity of the order of ppb from ppm or less. However, it takes a long time to measure due to a slow response speed of several minutes to several hours, and is not suitable for detecting a target object whose concentration, physical properties, etc. change in a short time.
[0003]
Responsiveness is improved by making the film thickness as thin as possible in order to facilitate adsorption, diffusion, and desorption of the detection target. However, since the sensitivity decreases as the film thickness is reduced, it is necessary to compensate for the decrease in sensitivity. Therefore, the present inventors transmit fluorescence emitted from the photoresponsive thin film to a measurement target fluid such as gas or liquid by a photoelectric field enhanced by surface plasmon resonance or waveguide mode, and measure fluorescence intensity or fluorescence spectrum. Has been developed (Japanese Patent Application No. 2001-056415). In this method, a thin film in which a fluorescent dye is dispersed in a synthetic polymer is laminated on a metal thin film, and the excitation of the dye by a surface plasmon resonance generated at the interface with the metal thin film or a photoelectric field enhanced by a guided mode is used. To increase sensitivity. When the sensing element is used, not only high-speed response but also high-sensitivity detection on the order of ppb becomes possible.
Although responsiveness and sensitivity are improved by adopting surface plasmon resonance or guided mode excitation fluorescence method, there are significant restrictions on the polymer that responds to NO ₂ gas, and there is a limit to improvement of responsiveness depending on fluorescence intensity. is there.
[0004]
[Means for Solving the Problems]
The present invention has been devised in order to further improve the sensitivity and response speed of the sensing element proposed in the prior application. By using a photoresponsive DNA thin film in which fluorescent molecules are incorporated in the double helix portion, the present invention has been proposed. It is an object of the present invention to provide a sensing element that increases the intensity or efficiently transfers energy from between chromophores of DNA to fluorescent molecules by photoexcitation of the DNA itself and further improves the responsiveness by thinning.
[0005]
In order to achieve the object, the sensing element of the present invention has a light-transmitting solid substrate in which a prism is bonded to the excitation light incident side surface via a matching medium, and a metal thin film on the other surface of the light-transmitting solid substrate. The light-responsive DNA thin film deposited in this manner, a measurement space that is closed by a light-transmitting plate above the light-responsive thin film and flows or contains the fluid S to be measured, and excitation light at an incident angle that exceeds the total reflection angle Equipped with a light-responsive solid substrate so as to be incident on one surface of the prism, and a rotary table with adjustable rotation amount, excited by confining incident light at an incident angle that satisfies surface plasmon resonance or waveguide mode conditions The fluorescence emitted from the photoresponsive DNA thin film is transmitted to the measurement target fluid in the measurement space and sent to the measurement system.
[0006]
A sensing element can also be incorporated in the light source integrated measurement cell. In this case, a photoresponsive DNA thin film is deposited on one surface of a light transmissive solid substrate via a metal thin film, and a condensing lens is mounted on the other surface. A measurement space for flowing or containing a fluid to be measured is provided on the light-responsive DNA thin film side of the light-transmissive solid substrate, and a transparent medium containing an excitation light source is disposed above the light-responsive DNA thin film.
[0007]
The photoresponsive DNA thin film uses an aqueous solution of DNA in which fluorescent molecules are intercalated or grooved in a double helix or an organic solution of hydrophobically treated DNA, spin coating method, solvent evaporation method, LB method, alternating adsorption method Etc. are formed. Examples of DNA include natural DNA, synthetic DNA, synthetic RNA, and a DNA / RNA mixed system. Hydrophobized DNA in which sodium ions (Na ⁺ ) are replaced with long-chain alkyltrimethylammonium ions or long-chain dialkyldimethylammonium ions can also be used. In the hydrophobically treated DNA, fluorescent molecules may be incorporated into the outer periphery of the long chain alkyl.
[0008]
As the fluorescent molecule, one or a plurality of substituents R (R: amino group, monoalkylamino group, dialkylamino group, monohydroxyalkylamino group, dihydroxyalkylamino group, pyridyl is not limited to the present invention. Group, quinolyl group, isoquinolyl group, acridinyl group, hydroxyl group, pyridinium group, quinolinium group, isoquinolinium group, acridinium group, sulfonium group), aliphatic, aromatic, heterocyclic organic compounds or molecular aggregates thereof, etc. Existing fluorescent materials and dyes are used. Specifically, rhodamine B, ethidium dye, coumarin 6, rubrene derivative, perylene derivative, fluorescein, acriflavine, proflavine, acridine orange, phenosafranine, nile blue, cresyl blue, methylene blue, pyrene derivative, anthracene derivative, fluorenone derivative , Fluorene derivatives, thiophene derivatives, bithiophene derivatives, bilanine, porphyrin, aminoacridine, ruthenium trisbipyridine complex, and the like.
[0009]
[Operation and embodiment]
When an aqueous DNA solution and a dye (fluorescent molecule) solution of appropriate concentration are mixed, depending on the DNA base pair to dye ratio, dye structure, etc., the DNA double helix is intercalated or grooved. A dye (fluorescent molecule) is taken up. Although the dye uptake reaction depends on the combination of DNA and dye, it is completed immediately or in about several hours. When hydrophobically treated DNA is used, the dye is incorporated by dissolving and mixing the hydrophobically treated DNA and fluorescent molecules in an organic solvent such as 2-methoxyethanol or ethanol, or by mixing the hydrophobicly treated DNA and the organic solvent in which each dye is dissolved. The reaction proceeds. In the case of hydrophobically treated DNA, a dye may be incorporated into the hydrophobic outer periphery.
[0010]
The DNA incorporating the fluorescent molecule or the hydrophobically treated DNA strongly suppresses the movement of the fluorescent molecule as compared with the solution, and changes the microscopic environment around the fluorescent molecule. Inhibition of molecular motion results in an increase in fluorescence intensity. Changes in the microscopic environment change the absorption spectrum and increase the fluorescence intensity. When a film is formed from a DNA aqueous solution or hydrophobically treated DNA organic solution by spin coating, solvent evaporation, LB method, etc., the movement is suppressed and the fluorescent molecules are fixed to the thin film while the surrounding microscopic environment is changed. A photoresponsive DNA thin film or a photoresponsive hydrophobically treated DNA thin film (hereinafter collectively referred to as “photoresponsive DNA thin film” as appropriate) is prepared.
[0011]
When the photoresponsive DNA thin film is irradiated with excitation light, the fluorescent molecules are excited to generate fluorescence. The generated fluorescence is suppressed by the movement of molecules by incorporating fluorescent molecules into DNA or hydrophobically treated DNA, and the energy transfer that occurs between fluorescent molecules or between the chromophores of the DNA or hydrophobically treated DNA itself and the fluorescent molecules. Both speed and sensitivity are improved. As a result, the repeated durability, gas responsiveness, and detection limit of the sensing element are improved.
[0012]
For excitation of the photoresponsive DNA thin film, excitation light L ₁ having a wavelength corresponding to the absorption wavelength of DNA, hydrophobically treated DNA or fluorescent molecules is used. Upon irradiation with the excitation light L ₁ , oxygen (O ₂ ), nitrogen dioxide (NO ₂ ), nitrogen oxide (NO), laughing gas (N ₂ O), sulfur dioxide (SO ₂ ), hydrogen sulfide (H ₂ S) , Carbon monoxide (CO), carbon dioxide (CO ₂ ), water (H ₂ O), ammonia (NH ₃ ), methanol (CH ₃ OH), ethanol (C ₂ H ₅ OH), amines, aldehydes, When a gas or liquid containing various hydrocarbons, mercaptans, photochemical oxidants, oxidizing compounds, reducing compounds, oxidoreductases, halogen derivative anesthetic gases, etc. is absorbed or diffuses in the photoresponsive DNA thin film, fluorescent molecules Interacts with the fluid to be measured, and the fluid to be measured is detected as a change in fluorescence intensity at a specific wavelength, a change in spectrum, or a change in reflection intensity of excitation light.
[0013]
The photoresponsive DNA thin film is incorporated in the measurement cell 20 of the surface plasmon resonance excitation fluorescence measurement system (FIG. 1).
This surface plasmon resonance excitation fluorescence measurement system is an optical system that guides excitation light L ₁ generated by an excitation light source 11 to a measurement cell 20 through a polarizing plate 12, a half-wave plate 13, a polarizing plate 14, a chopper 15, and mirrors 16 and 17. 10 is provided. As the excitation light L ₁ , continuous wave laser light, pulsed laser light or spectrally steady light is used.
In the measurement cell 20, a solid substrate 22 having a prism 21 in contact with the incident side of the excitation light L ₁ is mounted on the rotary table 23. For the solid substrate 22, glass, quartz, sapphire, transparent plastic or the like excellent in light transmittance is used.
[0014]
In the measurement system 30, the fluorescence L ₂ emitted from the measurement cell 20 is collected by the condenser lens 31 and input to the photomultiplier tube 32. The fluorescence L ₂ electrically converted and amplified by the photomultiplier tube 32 is measured for fluorescence intensity by the fluorescence spectrophotometer 33 or subjected to spectrum analysis. A part of the fluorescence L ₂ is converted / amplified into an electric signal by the photomultiplier tube 32 and sent to the digital memory 34 to record a change with time.
[0015]
The incident angle θ of the excitation light L ₁ with respect to one surface of the prism 21 is adjusted by the amount of rotation of the turntable 23 so that the surface plasmon resonance is satisfied and the reflectance is minimized at the incident angle θ exceeding the total reflection condition. The rotation amount of the rotary table 23 is controlled by a stage controller 36 that is driven and controlled by a computer 37. The reflected light L ₃ of the excitation light L ₁ is detected and electrically converted by the photodiode 38, and a signal synchronized with the chopper 15 is amplified by the lock-in amplifier 35 and recorded in the digital memory 34.
[0016]
A photoresponsive DNA thin film 25 is deposited on the solid substrate 22 via a metal thin film 24 of Ag, Au, Al or the like. The solid substrate 22 is covered with a silicon sheet 26 except for the prism 21, the metal thin film 24, and the photoresponsive DNA thin film 25, and a matching medium 27 is filled between the prism 21. The matching medium 27 is a layer provided to match the refractive index of the solid substrate 22 with the refractive index of the prism 21, and a commercially available refractive liquid or the like is used.
[0017]
The holder 28 for holding the solid substrate 22 includes an inflow pipe 28in for introducing the measurement target fluid S such as gas and liquid into the measurement space V closed by the glass plate 29, and a discharge pipe 28out for sending the measurement target fluid S from the measurement space V. Is formed.
The fluid S to be measured includes oxygen, nitrogen dioxide, nitric oxide, laughing gas, sulfur dioxide, hydrogen sulfide, carbon monoxide, carbon dioxide, water, ammonia, methanol, ethanol, amines, aldehydes, various hydrocarbons, Gases or liquids containing mercaptans, photochemical oxidants, oxidizing compounds, reducing products, oxidoreductases, halogen derivative anesthetic gases and the like are used. Further, it is possible to perform measurement in a state where the measurement target fluid S is allowed to stand in the measurement space V without being circulated through the inflow pipe 28in and the discharge pipe 28out.
[0018]
A light source integrated cell (FIG. 3) can also be used as the measurement cell. In the light source integrated measurement cell 40, a metal thin film 42 and a photoresponsive DNA thin film 43 are deposited on one surface of a solid substrate 41, and a condensing lens 48 is disposed on the other surface of the solid substrate 41. The reflected light L ₃ of the excitation light L ₁ is detected by the photodiode 49 and converted into an electric signal. The metal thin film 42 and the photoresponsive DNA thin film 43 are surrounded by a silicon sheet 44 and covered with a transparent medium 45 such as plastic. An excitation light source 46 and a polarizing plate 47 are disposed on the transparent medium 45. The excitation light L ₁ is emitted from the excitation light source 46 toward the photoresponsive thin film 43 while supplying the measurement target fluid S to the measurement space V inside the solid substrate 41.
[0019]
When the photoresponsive DNA thin films 25 and 43 are excited by the excitation light L ₁ , fluorescence L ₂ is emitted from the photoresponsive DNA thin films 25 and 43. After passing through the measurement target fluid S in the measurement space V, the fluorescence L ₂ is collected by the condenser lens 31 of the measurement system 30 and input to the photomultiplier tube 32. The fluorescence L ₂ electrically converted and amplified by the photomultiplier tube 32 is measured for fluorescence intensity by the fluorescence spectrophotometer 33 or subjected to spectrum analysis. As the measurement target fluid S is adsorbed or diffused on the photoresponsive DNA thin films 25 and 43, the fluorescence intensity of the photoresponsive DNA thin films 25 and 43 is rapidly and sensitively changed. Further, the reflected light L ₃ of the excitation light L ₁ may change in intensity due to the refractive index of the photoresponsive DNA thin films 25 and 43 changing due to the adsorption or diffusion of the fluid S to be measured and the surface plasmon resonance angle shifting. . Therefore, the intensity of the fluorescence L ₂ or the reflected light L ₃ is treated as a signal incorporating a change corresponding to the measurement target fluid S.
[0020]
For example, when the incident P-polarized light to a metal thin film 24, 42 via the prism 21, an angle in free electron plasma oscillation and the P-polarized light is coupled metal present in the metal thin film 24 and 42, almost no reflection light L ₃ Surface plasmon resonance occurs. Further, when the photoresponsive DNA thin films 25 and 43 having a film thickness of about 100 nm or less are disposed on the metal thin films 24 and 42, the incident angle θ at which surface plasmon resonance occurs according to the film thickness of the photoresponsive DNA thin films 25 and 43. Will gradually move to a wider angle. Furthermore, when the film thickness of the thin films 25 and 43 is set to about half or more of the wavelength of light, a standing wave having one or a plurality of nodes in the thickness direction can be formed in both P-polarized light and S-polarized light depending on the film thickness. The waveguide mode is such that almost no reflected light L ₃ is present.
[0021]
The fluorescent dye incorporated into the DNA or the hydrophobically treated DNA is captured by the double helix in an intercalation or groove, or in the hydrophobic outer periphery of the long chain alkyl group. Therefore, when the excitation wavelength and the fluorescent molecule are appropriately selected, efficient energy transfer occurs between the chromophores of the DNA to the fluorescent molecule by photoexcitation of the DNA itself. Energy transfer to fluorescent molecules corresponds to a significant increase in response molecules to the fluid to be detected from a sensing perspective. Further, at an incident angle at which the reflectivity is minimized by surface plasmon resonance, the electric field is enhanced by the metal thin films 24 and 42 and the light in the fluorescent molecules is excited very strongly by the light confined in the thin films 25 and 43. As a result, high-sensitivity fluorescence measurement is possible even for ultra-thin films of 100 nm or less, and adsorption, diffusion, and desorption of a measurement target fluid such as gas and liquid are further accelerated as compared with a thick film.
[0022]
In this respect, in the sensing element proposed in Japanese Patent Application No. 2001-056415, since the dye is randomly dispersed in a normal polymer, efficient energy transfer between the polymer chromophore or between the polymer and the dye is almost expected. Can not. For this reason, it is inevitable to rely on a guided mode generated in a thin film of about several hundreds of nanometers that is several times thicker than the surface plasmon resonance condition. On the other hand, the sensing element using the photoresponsive DNA thin films 25 and 43 is remarkably improved in repetitive response, response speed, and the like. Further, it is also advantageous that the fluorescence measurement can be used in combination with a change in the intensity of reflected light due to a change in refractive index accompanying the adsorption, diffusion, and desorption of a measurement target fluid, which is generally performed with a surface plasmon resonance sensor.
[0023]
At an incident angle θ satisfying the surface plasmon resonance, incident light (excitation light L ₁ ) is confined in the photoresponsive DNA thin films 25 and 43, and thus is included in the photoresponsive DNA thin films 25 and 43. The fluorescent molecule is efficiently excited. The intensity of the fluorescence L ₂ generated by excitation is affected by the interaction between the fluorescent substance and the fluid S to be measured such as gas or liquid diffused inside the photoresponsive DNA thin film 25 or 43 or adsorbed on the surface. It changes sensitively. In addition, since the photoresponsive DNA thin films 25 and 43 are thin, the adsorption or diffusion of the measurement target fluid S on the photoresponsive DNA thin films 25 and 43 proceeds rapidly, and the influence of the interaction changes the fluorescence intensity at a high response speed. It is reflected in. Therefore, the fluid S to be measured is detected or measured with high sensitivity and high response speed from the fluorescence intensity or spectral analysis result of the fluorescence L ₂ or from the change in reflected light intensity.
[0024]
【Example】
Aqueous solutions prepared by dissolving 81.3 mg of DNA and 0.99 mg of ethidium bromide in 5 ml of pure water were prepared. When the absorption spectrum of ethidium bromide was measured after mixing the aqueous solutions with each other, the absorption spectrum was shifted to the longer wavelength side, and the fluorescence intensity increased as seen in the fluorescence spectrum (a, b) of FIG. It was. This measurement result shows that ethidium bromide is incorporated into DNA. In addition, compared to the ethidium bromide single system (d), the fluorescence excitation spectrum (c) of the mixed aqueous solution with DNA remarkably increases in the absorption wavelength band of DNA, indicating that energy transfer from DNA to ethidium bromide I can read. When the absorption spectrum of an organic solution in which 58.7 mg of hydrophobically treated DNA and 0.48 mg of rhodamine B were dissolved in 10 ml of 2-methoxyethanol was measured, the fluorescence intensity of rhodamine B was higher than when no hydrophobically treated DNA was present. It was confirmed that rhodamine B was incorporated into the hydrophobically treated DNA. Furthermore, uptake of the luminescent dye into DNA or hydrophobically treated DNA was also confirmed from circular dichroism spectrum measurement.
[0025]
A glass slide is used for the solid substrate 22, an Ag thin film (metal thin film 24) having a thickness of 50 nm is provided on the solid substrate 22 by vacuum deposition, and then an organic solution of hydrophobically treated DNA and rhodamine B is silver having a thickness of 50 nm. The deposited film was applied by spin coating, and a 20 nm-thick photoresponsive DNA or hydrophobically treated DNA thin film was laminated. When a He—Ne laser beam having a wavelength of 543.5 nm was made incident through the prism, a minimum region (dip) of reflectance caused by surface plasmon resonance was detected at an incident angle of about 55 degrees. As a result of measuring the fluorescence spectrum at an incident angle of 55 degrees at which the reflectance is minimized, a fluorescence intensity that is two orders of magnitude greater than that in direct excitation was obtained (FIG. 5), and the effect of surface plasmon resonance excitation was confirmed. .
[0026]
The solid substrate 22 was incorporated into the measurement cell 20, and the excitation light L ₁ was incident on one surface of the prism 21 at an incident angle θ = 55 degrees while sending 0.5 ppm of nitrogen dioxide gas (NO ₂ ) into the measurement space V.
Fluorescence L ₂ was emitted from the photoresponsive DNA thin film 25 by surface plasmon excitation induced by the excitation light L ₁ . The fluorescence decreased by about 40% in 1 minute, and when the supply of nitrogen dioxide gas (NO ₂ ) was stopped and exhausted, the original state was completely restored within 1 minute. Complete recovery in a short time indicates that the photoresponsive DNA thin film 25 is excellent in high-speed response. The detection accuracy was about 10 ppb, and detection was possible with the same responsiveness up to about 100 ppm on the high concentration side.
[0027]
Therefore, the intensity change of the fluorescence L ₂ was investigated while repeating the supply and exhaust of nitrogen dioxide gas (NO ₂ ). As can be seen from the investigation results in FIG. 6, the fluorescence intensity periodically changes with high consistency with the supply and exhaust cycles of nitrogen dioxide gas (NO ₂ ), and the hydrophobic treated DNA thin film 25 containing rhodamine B is excellent. High-speed response, high sensitivity, and repeated durability were confirmed.
The photoresponsive DNA thin film prepared from an aqueous solution of DNA and ethidium bromide was also excellent in high-speed response, high sensitivity, and repeated durability, and was capable of high-performance measurement of nitrogen dioxide gas.
[0028]
【The invention's effect】
As described above, in the sensing element of the present invention, the photoresponsive DNA thin film is excited by surface plasmon resonance enhanced by an electric field by a metal, and the fluorescence emitted from the photoresponsive DNA thin film is measured as a gas, liquid, or the like. The fluorescence intensity or fluorescence spectrum is measured with high sensitivity through the fluid. By using the photoresponsive DNA thin film, the measurement target fluid can be measured with high sensitivity and high sensitivity. Since the surface plasmon resonance excitation fluorescence of the thinned photoresponsive DNA thin film is greatly reduced in a short time, it can be applied to a measurement target fluid whose physical properties and concentration change in a short time. Used in a wide range of fields such as gas monitors, medical gas monitors, and photochemical oxidant sensors.
[Brief description of the drawings]
FIG. 1 shows an example of a surface plasmon resonance excitation fluorescence measurement system according to the present invention. FIG. 2 shows details of a measurement cell. FIG. 3 shows a side view (a) and a plan view (b) of a light source integrated measurement cell.
FIG. 4 is a graph showing fluorescence spectra (a, b) and fluorescence excitation spectra (c, d) of a mixed system of ethidium bromide and DNA and an aqueous solution of ethidium bromide alone. FIG. 5 is a graph of a hydrophobically treated DNA thin film containing rhodamine B. Graph showing fluorescence spectrum generated by surface plasmon resonance excitation and direct excitation FIG. 6 shows that the surface plasmon excitation fluorescence of a hydrophobically treated DNA thin film containing rhodamine B changes in intensity according to supply and exhaust of nitrogen dioxide gas. Graph 【Explanation of symbols】
10: Optical system 20: Measurement cell 21: Prism 22, 41: Solid substrate 23: Rotary table 24, 42: Metal thin film 25, 43: Photoresponsive DNA thin film 27: Matching medium 28: Holder 29: Glass plate 30: Measurement System 40: Light source integrated measurement cell 45: Transparent medium L ₁ : Excitation light L ₂ : Fluorescence θ: Incident angle S: Fluid to be measured V: Measurement space

Claims

A light-transmitting solid substrate in which a prism is bonded to the excitation light incident side surface through a matching medium; a photoresponsive DNA thin film deposited on the other surface of the light-transmitting solid substrate via a metal thin film; A light-responsive solid substrate so that excitation light is incident on one surface of the prism at an incident angle exceeding the total reflection angle, and a measurement space that is closed by a translucent plate above and flows or contains the fluid S to be measured. Equipped with a rotating table with adjustable rotation amount, and fluorescence emitted from a photoresponsive DNA thin film excited by confining incident light at an incident angle satisfying surface plasmon resonance or waveguide mode conditions is measured in the measurement space. A sensing element using a photoresponsive DNA thin film, which is transmitted to a measurement system through a fluid to be measured.

A photo-responsive DNA thin film is deposited on one side with a metal thin film and a condensing lens is mounted on the other side. Incident light having an incident angle that satisfies a surface plasmon resonance or waveguide mode condition, comprising a measurement space in which the target fluid flows or accommodates, and a transparent medium that is disposed above the photoresponsive DNA thin film and contains an excitation light source A sensing element using a photoresponsive DNA thin film characterized in that fluorescence emitted from a photoresponsive DNA thin film excited by confining a light is transmitted through a fluid to be measured and a condenser lens and sent to a measurement system.

The sensing element according to claim 1 or 2, wherein a photoresponsive DNA thin film is formed from an aqueous solution of DNA in which fluorescent molecules are intercalated or incorporated into grooves in the double helix.

Fluorescent molecules are intercalated, grooved, or peripheries of long-chain alkyl in the double helix of hydrophobically treated DNA in which sodium ion (Na ⁺ ) as a counter cation is replaced with long-chain alkyltrimethylammonium ion or long-chain dialkyldimethylammonium ion The sensing element according to claim 1, wherein a photoresponsive DNA thin film is formed from an organic solution of hydrophobically treated DNA incorporated in the membrane.

The fluorescent molecule has one or more substituents R (R: amino group, monoalkylamino group, dialkylamino group, monohydroxyalkylamino group, dihydroxyalkylamino group, pyridyl group, quinolyl group, isoquinolyl group, acridinyl group, hydroxyl group The sensing element according to claim 3 or 4, which is an aliphatic, aromatic, or heterocyclic organic compound having a group, pyridinium group, quinolinium group, isoquinolinium group, acridinium group or sulfonium group, or a molecular assembly thereof.