Spectroscopic Study and Evaluation of Red-Absorbing Fluorescent Dyes

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• 作者：Volker Buschmann ; Kenneth D. Weston ; and Markus Sauer
• 刊名：Bioconjugate Chemistry
• 出版年：2003
• 出版时间：January 2003
• 年：2003
• 卷：14
• 期：1
• 页码：195 - 204
• 全文大小：180K
• 年卷期：v.14,no.1(January 2003)
• ISSN：1520-4812

文摘

The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commerciallyavailable red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes includedin this study are Alexa647, ATTO655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635,DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes willfacilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescenceassays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on thespectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilizedto assess the photophysical properties of the dyes under high excitation conditions. The dyes can beclassified into groups based on the results presented. For example, while the fluorescence quantumyield of ATTO655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surroundingmedium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced bythe viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin andsubsequent addition of streptavidin results in reversible fluorescence quenching or changes in therelaxation time of other photophysical processes of some dyes, most likely due to interactions withtryptophan residues in the streptavin binding site.