JP5757535B2 - 走査光源による導波管に基づく検出システム - Google Patents
走査光源による導波管に基づく検出システム Download PDFInfo
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Description
本明細書に記載される全ての出版物及び特許出願は、参照として組み込まれるものとして各個別の出版物又は特許出願が具体的かつ個別に示されているように、同じ範囲の参照により本明細書に組み込まれる。
本発明は、簡単で費用効率の良いやり方で1つ以上の光学的導波管内に光1つ以上の波長の光を結合するための方法及び装置を提供する。走査光源、検出器、基板、及び複数の導波管及び光学感知部位を含む検出システムを用いた光学的検出のための器具、方法、及びキットもまた供給される。本システムの1つの基板は、複数の実質的に平行な励起導波管及び複数の実質的に平行な集光導波管を含む。励起導波管及び集光導波管は、交差して交差領域及び2次元のアレイを形成する。本システムの他の基板は、複数の実質的に平行な導波管、及び複数の感知部位を含む。光学感知部位はセンサーを含み、1つ以上の導波管と光通信する。様々な環境及び生体試料の検出は、本明細書に記載される器具、方法及びキットを用いて達成することができる。光波誘導及びエバネッセント場蛍光励起の一般的な理論的原理は本明細書に開示される実施形態に当てはまる。
「生物活性のある検体」という用語は、本明細書で用いられるときには、ウイルス、細菌、菌類、植物、動物及び人間を含むがそれらに制限されるものではない生物有機体の何らかの物理的又は生化学的特性に影響を与えることがある任意の物質を意味する。特に本明細書で用いられる場合、本発明による生物活性のある検体は、発現タンパク質、細胞指標、抗体、血清タンパク質、コレステロール、多糖類、核酸、生物学的検体、遺伝子、タンパク質又はホルモン、もしくはそれらの任意の組み合わせなどのような薬、プロドラッグ、医薬品、薬物代謝産物、生体指標を含むが、それらに制限されるものではない。生物活性のある検体はさらに、気体、化学物質もしくは汚染物質、又はそれらの組み合わせ(例えば、環境源からの)を含むがそれらに制限されるものではない天然又は人工の物質を含むことができる。分子レベルにおいて、生物活性のある検体は、ポリペプチド、糖タンパク質、多糖、脂質、核酸又はそれらの組み合わせとすることができる。
蒸着手順中に、良く制御された厚さを有する良く定義された材料の層が全ウエハにわたり蒸着される。導波管層蒸着に用いられる最も一般的な材料は、ガラスとしても知られるシリカ(SiO2)、及び窒化珪素(Si3N4)である。シリカの光学特性(主にその屈折率)は、蒸着中に導入されるドーピング(Ge、P、及びB等)の量により制御される。シリコン、ガラス、エポキシ、ニオブ酸リチウム、リン化インジウム及びSiON(酸窒化珪素)及びその誘導体などのような他の材料も用いられる。クラッディング層に対しては、材料はシリコン、シリカ(SiO2)、ガラス、エポキシ、ニオブ酸リチウム及びリン化インジウムを含むことができるがそれらに制限されるものではない。
蒸着に続いて、エッチング手順の前に、削り取られないように領域をマスキングすることにより、蒸着されたウエハにPLC装置の所望の2次元構造が転写される。マスキングは、ウエハを感光性材料で覆うこと、これをリソグラフマスクを通して光にさらすこと、及びマスクを低位置に残したまま、露光された材料を取り除くことを含むいくつかの手順で行われる。こうした手順の結果は、マスク1725が、基板1704のコア1723層の頂部に示される図17Bに示される。
エッチング手順において、マスクされない領域の材料は、基板(図17C参照)の頂部コア1723層から除去される。エッチング速度は、既知のパラメータであり、それゆえエッチング深さは時間によって制御可能することができる。エッチングの2つの最も一般的な技術は、ウェットエッチング、及び反応性イオンエッチング(RIO)である。図17Cは、導波管1727をもたらすエッチング手順の結果を示す。
本発明のセンサーの1つの特徴は、少数の生物活性のある検体分子を検出する能力である。次の実験は、この能力を示す。センサーの検出限界(LOD)は、溶液中の蛍光標識されたオボアルブミンの測定により判定された。使用された染料はAlexa Fluor 660であった。これは658nmで励起され、発光は690nmで測定された。全ての測定は室温(25℃)で行われた。LODは、2つの異なる方法を用いて測定された。すなわち、1)95%を超える信頼度の上記のバッファバックグラウンドにおける、検出可能な標識化されたオボアルブミンの最低濃度の判定(P<0.05、スチューデントt−検定)、及び2)センサー応答対濃度の標準曲線対濃度からの分析感度の判定(MDL = 2*SD/傾斜、ここでSDはバッファーバックグラウンド測定の標準偏差であり、傾斜は標準曲線の初期傾斜)である。こうした方法は、両者がLODに対して95%信頼水準を特定する点で比較可能である。3つの異なる手順の等温線からのLODの値が表1に示される。
本発明は、タンパク質間相互作用の測定に有用である。この実施例において、オボアルブミンの抗オボアルブミン抗体への結合を測定するために、導波管に基づく光学的検出技術が用いられた。実験が行われる前、52.5×4,500μm感知素子付きチップは、抗オボアルブミン抗体で浸漬被覆された。この処置は、いくつかの手順を伴った。第1にチップが洗浄された。チップをウエハの切断中に保護するために、チップ製造者は、チップの活性表面を薄いポリマー層で被膜した。ポリマー層は、チップをアセトンに5分間、続いてイソプロパノールに5分間浸漬し、第2のイソプロパノールすすぎを追加で1分間行うことにより除去された。その後、チップは、脱イオン水中で3回洗われ、減圧デシケーター内で乾燥された。次に、表面が活性化された。導波管に捕捉分子を固定するために、二酸化珪素の薄い層(〜10nm)が、ピラニア処理によってチップ上に形成された。チップは、9%(v/v)H2O2及び66.5%(v/v)H2SO4を包含するピラニア溶液に45分間攪拌して浸漬された。この手順は、有機汚染物質を除去し、抗体結合に用いられた反応性シラノール基の薄い(1〜2ナノメートル)層を生成した。その後、チップは、4回脱イオン水中ですすがれ、続いて再蒸留水中で最終すすぎされた。
導波管に基づく光学的検出システムは、プライマ伸長測定でのDNAの重合の定量化に用いることができる。プライマ伸長反応において、mRNA標的はDNAプライマと雑種を作り、逆転写酵素はmRNA標的をデオキシヌクレオチド(dATP、 dTTP、 dGTP、 dCTP)をDNAプライマーの3’末端に付加するためのテンプレートとして用いる。
転写産物の定量リアルタイムPCR検出は、例えば、bcr/abl融合転写産物は、Kreuzer等(上記参照)に記載されたものから修正されたPCR測定法プロトコルを用いて、本発明の検出システムを用いて達成することができる。システムは、励起導波管付き基板チップ、集光導波管、及び交差領域を含む。励起導波管及び集光導波管が交差する交差領域は、定量リアルタイムPCRを行うための感知ウェル付き光学感知部位を含む。検出システムは、走査経路に沿ったある点で、基板の励起導波管と結合しかつ、光通信する走査光源をさらに含む。システムはさらに光学的検出器を含む。
複数の被験者のHIV+状態の蛍光免疫法に基づく検出は、図7Aに示される実施例のような検出システムを用いて達成することができる。システムは、励起導波管付き基板チップ、集光導波管、及び交差領域を含む。励起導波管及び集光導波管が交差する交差領域は、蛍光免疫法を行うための感知ウェル付き光学感知部位を含む。検出システムは、走査経路に沿ったある点で基板と結合し、かつ光通信する走査光源、及び励起導波管をさらに含む。システムはさらに光学的検出器を含む。
2部位サンドイッチ免疫測定法を、本発明の検出システム(例えば、図7A〜図7Eに示されるシステム)を用いた測定法に使用することができる。抗体は、かかる測定法において2つの異なる役割を果たし、それにより、固体化抗体が検体を捕捉し、一方で、可溶性の、蛍光標識された抗体が検体結合を検出する、すなわち「トレースする」。競合的結合を防ぐために、捕捉及びトレーサー抗体は、検体分子上の異なる部位に結合しなければならない。反復エピトープを伴う大きな検体(例えば、ウイルス及び細菌)において、単一の抗体(反復エピトープに特異的)は、捕捉及びトレーサーの役割の両方に通常用いることができる。複数の、固有のエピトープを表すより小さい検体(例えば、タンパク質及び多糖)は、各々固有のエピトープに特有の2つの異なる抗体を必要とする。
Claims (13)
- 生物活性検体分子を検出するための検出システムであって、
走査光源と、
複数の導波管、及び基板の1つ以上の導波管と光通信する複数の光学感知部位を備える基板と;
前記基板と結合し、かつ光通信する検出器と;
複数の光線のそれぞれが走査経路に沿ってある点で前記基板の前記導波管の1つと結合され、かつ光通信するように、前記基板に対して前記走査光源から発せられた前記複数の光線を空間的に平行移動させ、各導波管内を進む光パルスを生成するための手段とを備える、システム。 - 前記基板が、複数の実質的に平行な励起導波管、及び複数の実質的に平行な集光導波管であって、前記励起導波管と集光導波管とが交差して交差領域の2次元配列を形成し、励起導波管と集光導波管とが交差し、各交差で前記交差領域と光通信を提供する導波管と、交差領域と各々光通信する複数の光学感知部位とを備え、
走査光源が、走査経路に沿ったある点で、前記基板の第1の端部で1つ以上の前記励起導波管と結合し、かつ光学的に通信し、
前記検出器が前記基板の第2の端部で1つ以上の前記集光導波管と結合し、かつ光通信する、請求項1のシステム。 - 前記基板が複数のインカップリング導波管及び複数のアウトカップリング導波管と、各々インカップリング導波管及びアウトカップリング導波管と光通信する複数の光学感知部位と、を備え、前記走査光源が、走査経路に沿ったある点で、かつ、前記基板の第1の端部で、1つ以上のインカップリング導波管に結合され、かつ光通信し、前記検出器が前記基板の1つ以上の前記アウトカップリング導波管と結合され、かつ光通信する、請求項1のシステム。
- 前記走査光源が検出器をさらに備え、前記光源が前記光学感知部位と光通信する複数の導波管の1つ以上と結合され、かつ光通信する点で、検出器も前記1つ以上の導波管と結合され、かつ光通信する、請求項1のシステム。
- 前記基板が複数のインカップリング導波管及び複数のアウトカップリング導波管を備え、前記光源が1つ以上の前記インカップリング導波管と結合され、かつ光通信する点で、前記検出器が1つ以上の前記アウトカップリング導波管と結合され、かつ光通信する、請求項4のシステム。
- 前記走査光源全体又はその任意の部分が移動可能で、前記光線の前記空間的な平行移動が前記走査光源全体又はその任意の部分の移動によって実行される、請求項1のシステム。
- 前記基板が移動可能で、前記光線の前記空間的な平行移動が前記基板の移動によって実行される、請求項1のシステム。
- 検出方法であって、
検出すべき生物活性のある検体分子を包含する疑いのある試料を検出システムの基板上の光学感知部位へ送達するステップであって、前記基板は複数の導波管を備える、ステップと、
走査光源を空間的に平行移動させ、当該走査光源により生成された複数の光線を空間的に平行移動させるステップであって、各光線が走査経路に沿ってある点で前記導波管の1つに結合され、かつ光通信するように、各光線が前記基板の導波管の1つの中を進む第1の光波のパルスを生成し、前記導波管が前記光学感知部位と光通信し、前記第1の光波が前記光学感知部位に組み込まれたセンサーによって第2の光波に変換可能である、ステップと、
前記基板と光通信する検出器を用いた前記第2の光波の測定可能な変化を検出することであって、前記センサーが前記生物活性のある検体分子と相互作用するときに前記第2の光波の測定可能な変化が起きる、検出するステップと、を備える検出方法。 - 前記基板が前記光学感知部位と光通信する複数の実質的に平行な励起導波管を備え、前記第1の光波は、前記光学感知部位に関連するセンサーによって、前記光学感知部位と光学的に通信があり、前記励起導波管と交差する複数の実質平行な集光導波管のうちの1つ以上で伝えられる第2の光波に変換可能であり、
前記第2の光波の測定可能な変化が前記集光導波管と光通信する検出器を用いて検出され、前記センサーが前記生物活性のある検体分子と相互作用するときに前記第2の光波の測定可能な変化が起きる、請求項8の方法。 - 前記走査光源が検出器をさらに備え、前記光源が前記光学感知部位と光通信する前記複数の導波管と結合され、かつ光通信する点で、前記検出器も前記複数の導波管と結合され、かつ光通信する、請求項8の方法。
- 前記基板が複数のインカップリング導波管、及び前記光学感知部位と光通信する複数のアウトカップリング導波管を備え、前記光源が1つ以上の前記インカップリング導波管と結合され、かつ光通信する点で、前記検出器が1つ以上のアウトカップリング導波管と結合され、かつ光通信する、請求項8の方法。
- 前記生物活性のある検体が、核酸、タンパク質、抗原、抗体、微生物、気体、化学物質及び汚染物質からなる群から選択される、請求項8の方法。
- 前記センサーが免疫測定法に対応するように適合していて、前記生物活性のある検体と相互作用する前記センサーが免疫測定法の結果を備える、請求項8の方法。
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KR20120035912A (ko) | 2012-04-16 |
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JP2012525595A (ja) | 2012-10-22 |
IL215898A0 (en) | 2012-01-31 |
EP2425286A1 (en) | 2012-03-07 |
CA2759396A1 (en) | 2010-11-04 |
US20130215425A9 (en) | 2013-08-22 |
WO2010127001A1 (en) | 2010-11-04 |
AU2010241641B2 (en) | 2015-05-14 |
EP2425286B1 (en) | 2020-06-24 |
US20100302544A1 (en) | 2010-12-02 |
CN102460254A (zh) | 2012-05-16 |
IL215898A (en) | 2016-08-31 |
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Free format text: JAPANESE INTERMEDIATE CODE: R250 |