Single-Molecule Detection and Identification of Multiple Species by Multiparameter Fluorescence Detection

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• 作者：Jerker Widengren ; Volodymyr Kudryavtsev ; Matthew Antonik ; Sylvia Berger ; Margarita Gerken ; and Claus A. M. Seidel
• 刊名：Analytical Chemistry
• 出版年：2006
• 出版时间：March 15, 2006
• 年：2006
• 卷：78
• 期：6
• 页码：2039 - 2050
• 全文大小：519K
• 年卷期：v.78,no.6(March 15, 2006)
• ISSN：1520-6882

文摘

Two general strategies are introduced to identify andquantify single molecules in dilute solutions by employinga spectroscopic method for data registration and specificburst analysis, denoted multiparameter fluorescence detection (MFD). MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the eight-dimensional fluorescenceinformation (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum,fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time and allows forselection of specific events for subsequent analysis. Usingthe multiple fluorescence dimensions, we demonstrate adye labeling scheme of oligonucleotides, by which it ispossible to identify and separate 16 different compoundsin the mixture via their characteristic pattern by MFD.Such identification procedures and multiplex assays withsingle-molecule sensitivity may have a great impact onscreening of species and events that do not lend themselves so easily to amplification, such as disease-specificproteins and their interactions.