Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA.
Bioconjug Chem. 2013 Feb 20;24(2):215-23. doi: 10.1021/bc3005073. Epub 2013 Jan 16.
Fluorescent biosensors based on environmentally sensitive dyes enable visualization and quantification of endogenous protein activation within living cells. Merocyanine dyes are especially useful for live cell imaging applications, as they are extraordinarily bright, have long wavelengths of excitation and emission, and can exhibit readily detectable fluorescence changes in response to environment. We sought to systematically examine the effects of structural features on key photophysical properties, including dye brightness, environmental responsiveness, and photostability, through the synthesis of a library of 25 merocyanine dyes, derived from combinatorial reaction of 5 donor and 5 acceptor heterocycles. Four of these dyes showed optimal properties for specific imaging applications and were subsequently prepared with reactive side chains and enhanced aqueous solubility using a one-pot synthetic method. The new dyes were then applied within a biosensor design for Cdc42 activation, where dye mero60 showed a remarkable 1470% increase in fluorescence intensity on binding activated Cdc42 in vitro. The dye-based biosensors were used to report activation of endogenous Cdc42 in living cells.
基于环境敏感染料的荧光生物传感器可实现活细胞内内源性蛋白质激活的可视化和定量。甲川花菁染料特别适用于活细胞成像应用，因为它们异常明亮，激发和发射波长较长，并且可以在响应环境时表现出可检测的荧光变化。我们通过组合合成了 25 种甲川花菁染料库，从 5 个供体和 5 个受体杂环的组合反应中，系统地研究了结构特征对关键光物理性质的影响，包括染料的亮度、环境响应性和光稳定性。这四种染料在特定的成像应用中表现出最佳的特性，随后使用一锅合成法制备了带有反应性侧链和增强的水溶性的染料。然后将这些新染料应用于 Cdc42 激活的生物传感器设计中，其中染料 mero60 在体外与激活的 Cdc42 结合时，荧光强度显著增加了 1470%。基于染料的生物传感器用于报告活细胞内内源性 Cdc42 的激活。
