Design, Synthesis, And Applications Of A Series Of Ratiometric Organic Fluorescent Probes

Fluorescent probes can selectively detect a special analyte, which results the changes of fluorescence emission signals. It has attracted great notices of scientists, because there are some advantages of fluorescence analysis, such as high sensitivity, good selectivity, low cost, and easy operation. Moreover, fluorescence imaging has the favourable performance, for example, in situ real time and visual monitoring, noninvasive dectection for biosamples, and good repeatability. So, fluorescent probes have been widely used in many fields, containing theragnostics, pharmacy, pathology and biology, as an essential detection tool. However, the emission intensity of fluorescence varies with probe concentration, probe environment, and the quantitative measurements, which will reduce the accuracy of detection results. In contrast, the ratiometric fluorescent probes can eliminate this shortcoming by self-calibration of two emission bands and improve the detection accuracy.In this thesis, we focus on improving the detection efficiency of fluorescence analysis, and constructing a serial of ratiometric flu orescent probes based on internal charge transfer(ICT) and F?rster resonance energy transfer(FRET) processes. Ratiometric fluorescent probes for p H, hydrogen sulfide, and cyanide anions have been designed and synthesised using p yrrole-based cyanine, coumarin, naphthalimide, and rhodamine as fluorophores, respectively. The concrete contents are as follows:(1) We have introduced a new strategy to construct a unique class of pyrrole-based cyanine dyes, Py Cy fluorophores, by attaching two indolium moieties at the α-positions of the pyrrole core. Under acid conditions, the nitrogen atom of the pyrrole core is protonated and reducing the ability of electron-donating, Py Cy shows much weaker fluorescence. Under basic conditions, the deprotonated Py Cy enhances the ICT character, and induce a redshift of their absorption and emission spectra. So, Py Cy can sensing a wide range of p H and exhibits dual-mode fluorescence signals at different p H regions. We further demonstrated that these novel dye s are suitable for monitoring mitochondrial p H variations in living cells and real-time sensing of minor p H changes induced by alkaline phosphatase.(2) We have introduced a new strategy, for controlling FRET process through tuning the overlap level of the donor emission spectr um with the acceptor absorption via modulation of the acceptor fluorophore absorption wavelength. Based on this strategy, we constructing a ratiometric fluorescent H2 S sensor(CN-N3) based on coumarin–naphthalimide dyad, the ratio emission signal is modulated by the FRET process, which works in coordination with the ICT mechanism. CN-N3 has a sensitivity and selectivity for H2 S, which can induce a 8.7-fold increase of fluorescence intensity ratio at 534 nm and 474 nm. What ’s more, CN-N3 was applied to visualize both the intracellular exogenous and endogenous H2 S through blue and green emission channels.(3) We have designed and synthesized a FRET-based ratiometric probe for H2S(CP-H2S), the coumarin-pyronine dyad is a new FRET platform for constructing fluorescent probe. CP-H2 S has a sensitivity and selectivity for H2 S, and the detection limit was calculated to be 2.2×10-7 M. Addition of H2 S to CP-H2 S can inhibit the FRET process and induce a 256.1-fold increase of fluorescence intensity ratio at coumarin and pyronine, the changes of fluorescent emission from red to blue was observed by naked eyes. What’s more, CP-H2 S was successfully applied to monitor H2 S in living cells.(4) We have constructed a ratiometric probe for sensing cyanide anions based on coumarin-rhodamine FRET dyad, and studied the spectra prperties and the detailed responding mechanism. When CR reacts with cyanide anions, the FRET process will be destroyed and the ratio values of cuomarin to rhodamine( I486/I587) increases by 43.1-fold. The changes of fluorescent emission from red to blue cound be observed by naked eyes. The probe shows an excellent linear relationship between the emission ratios and the concentrations of CN- from 0 to 13 equivalents, suggesting that the chemodosimeter is potentially useful for quantitative determination of CN-. We further demonstrated that the probe well responds to CNat around physiological p H and has a good cell membrane permeability, so CR is suitable for detecting CN- in living cells.