Yin Ruwen, Fang Yuan, Zhou Xinqi, Stains Cliff I
Department of Chemistry, University of Virginia, Charlottesville, VA, United States.
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, United States.
Methods Enzymol. 2020;639:23-36. doi: 10.1016/bs.mie.2020.04.007. Epub 2020 Apr 29.
Molecular imaging of biological analytes provides detailed insights into signaling processes. Ratiometric probes are particularly attractive due to the ability to quantify analyte production. However, design strategies for ratiometric probes can be hindered by spectral overlap of the product and reactant species. In this chapter, we provide protocols for the synthesis and application of RF, a ratiometric probe for HO displaying dramatic changes in both excitation and emission wavelengths, designed using an approach we term chemoselective alteration of fluorophore scaffolds. The probe contains a chemoselective functional group within a fluorescent xanthene scaffold, resulting in the in situ synthesis of a new fluorophore upon reaction with HO. Under physiological conditions, RF exhibits far-red to near-infrared excitation and emission, and upon reaction with HO, RF is chemically converted into tetramethylrhodamine, producing a significant (~66nm) blue-shift in excitation and emission. RF can be used for ratiometric, molecular imaging of endogenous HO production in living cells.
生物分析物的分子成像为信号传导过程提供了详细的见解。由于能够量化分析物的产生，比率探针特别具有吸引力。然而，产品和反应物物种的光谱重叠可能会阻碍比率探针的设计策略。在本章中，我们提供了RF的合成和应用方案，RF是一种用于HO的比率探针，在激发和发射波长上都有显著变化，它是使用我们称为荧光团支架的化学选择性改变的方法设计的。该探针在荧光呫吨支架内含有一个化学选择性官能团，与HO反应后会原位合成一种新的荧光团。在生理条件下，RF表现出远红到近红外的激发和发射，与HO反应后，RF会化学转化为四甲基罗丹明，在激发和发射上产生显著的（约66nm）蓝移。RF可用于活细胞内源性HO产生的比率分子成像。
