The Synthesis And Spectral Properties Of FRET-based Dual-excitation PH Fluorescence Ratiometric Probes

With the development of science and technology and social progress, thefluorescence ratio technology has become one of the most important techniques influorescence analysis. The fluorescence ratio technology is to detect the materialsby recording the ratio of two fluorescence emission peaks. Compared withconventional fluorescent probes, the most obvious advantage of this probe is that itcan eliminate the errors caused by the concentrations of probe molecules and theirsurrounding environment, and other factors. Meanwhile, the probes largelyincrease the scope of responses and provide more precise experimental results,which can bring great convenience to the study of properties of materials forpeople.Therefore, the fluorescence ratio probes have been greatly developed.Dual-excitation ratiometric fluorescent probes belongs to the ratiometric offluorescence probes. Dual-excitation ratiometric fluorescent probes allow themeasurement of fluorescence intensities at two excitation wavelengths, whichshould provide a built-in correction for environmental effects. However, most ofthe reported small-molecule dual-excitation ratiometric probes have rather limitedexcitation wavelength separations (20-70nm) and/or very small molar absorptioncoefficient at one excitation wavelength. These shortcomings can lead to seriouscross-excitation and thus errors in the measurement of fluorescence intensities andratios. Herein, we have presented a FRET-based molecular strategy forconstruction of small-molecule dual-excitation ratiometric probes with the donorand acceptor excitation bands exhibiting large excitation separations andcomparable excitation intensities, which is highly desirable for determining thefluorescence intensities and signal ratios with high accuracy and for enhancementof the dynamic range of signal ratios. Based on this strategy, we initially created acoumarin-rhodamine FRET platform, which was then employed to develop the firstclass of FRET-based dual-excitation ratiometric pH probes, having twowell-separated excitation bands (excitation separations>160nm) and comparableexcitation intensities. In addition, these pH probes may be considered as in a kindof secured ratioing mode. As a further application of these newly constructed pHprobes, the dual-excitation ratiometric pH probes were transformed into the firsttype of photocaged dual-excitation ratiometric pH probes, for improvement of spatiotemporal resolution. It is expected that the modular nature of theFRET-based molecular strategy should render it applicable to other small-moleculedual-dye energy transfer systems based on diverse fluorescent dyes for thedevelopment of a wide variety of dual-excitation ratiometric probes withoutstanding spectral features including large excitation separations and comparableexcitation intensities.