単分子計数と強度校正による光測定のダイナミックレンジの拡大
Lucas Smith, Manish Kohli1, Andrew M Smith
1Department of Oncology , Mayo Clinic , Rochester , Minnesota 55905 , United States.
Journal of the American Chemical Society
|September 15, 2018
まとめ
この研究は,画像と分子数計を使用して,光測定のダイナミック範囲を拡張します. 癌バイオマーカーのフェムトモラ検出限界は,従来の光測定の限界を克服することによって達成されます.
科学分野:
- 生物化学
- 分析化学
- バイオテクノロジー
背景:
- 伝統的な光測定は,限られたダイナミックレンジ (1000倍) と感度 (亜ナノモラー) を有する.
- 生物学的分子は,フェムトモラーレベルまで,膨大な多様性 (1,000,000倍) を表しています.
- 既存の方法は,このような広い濃度範囲で分析物を正確に定量化するのに苦労しています.
研究 の 目的:
- 光ベースの測定のダイナミックレンジと感度を拡大する.
- 集合強度のための分子数と単一分子校正を組み合わせた方法を開発する.
- 低濃度分析剤を検出するための現在の表面捕獲アッセイの限界を克服する.
主な方法:
- 集合強度の単一分子校正と分子数計を組み合わせたイメージングを用いた.
- 表面捕捉された光ラベルの光学限界を評価し,光素のクラスを比較した.
- 幅広い光照射範囲をカバーするために検出器の取得パラメータを使用します.
- 癌に関連したmiRNAバイオマーカーの密集ラベリングのためにローリングサークル増幅を使用した.
主要な成果:
- ファイコエリトリンは,強烈で均質な単一フッ素光度により,優れたフッ素素である.
- 単一分子検出とカリブレーションを ほぼ5度の濃度範囲で達成しました.
- miRNAバイオマーカーの検出限界は29フェムトモラー (fM) であった.
- 非特異的に結合した光物質による制限を克服した.
結論:
- 光測定の性能を大幅に向上させるためのイメージングと分子数算の戦略を開発した.
- 表面光検査の検出限界とダイナミックレンジを拡大するための実用的なアプローチを示した.
- この方法は,様々な生物学的サンプルにおける敏感なバイオマーカーの検出に広く適用可能であることが示されています.
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