Widengren Jerker, Kudryavtsev Volodymyr, Antonik Matthew, Berger Sylvia, Gerken Margarita, Seidel Claus A M
Department of Applied Physics, Royal Institute of Technology, Albanova University Center, 10691 Stockholm, Sweden.
Anal Chem. 2006 Mar 15;78(6):2039-50. doi: 10.1021/ac0522759.
Two general strategies are introduced to identify and quantify single molecules in dilute solutions by employing a spectroscopic method for data registration and specific burst 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 fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time and allows for selection of specific events for subsequent analysis. Using the multiple fluorescence dimensions, we demonstrate a dye labeling scheme of oligonucleotides, by which it is possible to identify and separate 16 different compounds in the mixture via their characteristic pattern by MFD. Such identification procedures and multiplex assays with single-molecule sensitivity may have a great impact on screening of species and events that do not lend themselves so easily to amplification, such as disease-specific proteins and their interactions.
介绍了两种通用策略，通过采用光谱方法进行数据记录和特定猝发分析来识别和量化稀溶液中的单分子，这种方法称为多参数荧光检测（MFD）。MFD使用脉冲激发和时间相关单光子计数来实时同时监测八维荧光信息（基本各向异性、荧光寿命、荧光强度、时间、激发光谱、荧光光谱、荧光量子产率、荧光团之间的距离）的变化，并允许选择特定事件进行后续分析。利用多个荧光维度，我们展示了一种寡核苷酸的染料标记方案，通过该方案可以通过MFD根据其特征模式识别和分离混合物中的16种不同化合物。这种具有单分子灵敏度的识别程序和多重分析可能会对筛选不易扩增的物种和事件（如疾病特异性蛋白质及其相互作用）产生重大影响。
