Construction And Biosensing Study Of Fluorescent Probes Based On Coumarin And Xanthene Derivatives

In the life system,some biological small molecules,anions and cations involve in a large number of physiological and pathological processes,which play important roles in the human life and health.In order to better understand their function and influence and further elaborate their production,transportation and downstream functions,the development of high selectivity and high sensitivity detection methods of these molecules or ions has been a hot topic in biological detection field.Fluorescence analysis method has lots of advantages,such as high sensitivity,high selectivity,no damage to sample preparation,rapid real-time in-situ detection,multi-signal output mode and so on,thus fluorescent probes are considered to be an important tool for the study and detection of physiological processes of the active species in living animals and living cells,and also have been widely used in the detection of biologically active molecules,anion and cation.In this thesis,we designed and synthesized a series of fluorescent probes based on coumarin dye,hemicyanine-xanthene dye with near-infrared emission,carboxylic acid-functionalized coumarin-?,?-unsaturated malononitrile dye and Rhodol-HBT dye with aggregation-induced emission.These probes have been successfully applied to detect and sense biological gas transmitter H₂ S,food additive SO₃^2-,biothiols Cys and GSH as well as toxic Hg²⁺ and Ag⁺ in vitro and in cell.The main contents of this thesis are as follows:1.A novel colorimetric and fluorescent dual signal probe DNPOCA for sensing H₂ S by introducing 2,4-dinitrophenyl ether as the recognition group to 7-hydroxycoumarin-4-acetic acid methyl ester as fluorophore based on ICT mechanism was designed and synthesized.Upon the addition of H₂ S to the solution of DNPOCA in PBS?20 mM,pH 7.4,10% CH3 CN,v/v?,7-hydroxycoumarin-4-methyl acetate was released due to thiolysis of 2,4-dinitrophenyl ether.A remarkable red-shifted from 318 nm to 495 nm in the UV-vis spectrum and an obvious enhancement of the emission intensity at 463 nm in the fluorescence spectra were observed.The solution color change from colorless to pale yellow under visible light and dark to blue under an UV light,which indicated the detection of H₂ S could be realized by naked-eye and fluorescence spectrometer.The H₂ S probe exhibited excellent selectivity and high sensitivity with the detection limit as low as 49.7 nM.Furthermore,the probe could be successfully applied for H₂ S imaging in living cells with low cytotoxicity and good cell permeability,demonstrating the potential utility of the probe.2.We designed and synthesized NIR-DNP as a new dual functional probe for detecting H₂ S and SO₃^2-based on hemicyanine-xanthene as NIR fluorophore.In PBS?10 mM,pH 7.4,10% DMSO,v/v?,probe NIR-DNP was non-fluorescent due to efficient photo-induced electron transfer?d-PET?from the excitation of the hemicyanine-xanthene to the 2,4-dinitrobenzene group.Upon the addition of H₂ S,hemicyanine-xanthene was released due to thiolysis of 2,4-dinitrophenyl ether.The maximum absorption wavelength at 595 nm in the UV-visible absorption spectrum was red shifted to 685 nm with solution color changing from purple to blue,and an obvious enhancement of the emission intensity at 707 nm was observed.Upon the addition of SO₃^2-,SO₃^2-reacted with the unsaturated double bonds and destroyed the large conjugate system of the probe,a remarkable blue-shifted from 595 nm to 375 nm in the UV-vis spectrum was observed with solution color changing from purple to colorless.The probe showed good selectivity and sensitivity,and the detection limit of probe for H₂ S and SO₃^2-in aqueous solutions is 36.3 nM and 33.3 nM,respectively.Further investigation showed that probe NIR-DNP could be used to develop an easy-to-prepare and easy-to-detect paper-based test strip for cheap and effective detection of SO₃^2-.Also,probe NIR-DNP has the potential to image exogenous and endogenous H₂ S in living cells.3.Two novel probes CR1 and CR2 with three potential reaction sites for sensing Cys and GSH were designed and synthesized by introducing p-methylphenyl thioester or p-nitrophenyl thioester to the carboxylic acid-functionalized coumarin-?,?-unsaturated malononitrile.The Cys-induced transthioesterifcation-rearrangement-cyclization cascade reaction,GSH-induced transthioesterifcation-cyclizatiom cascade reaction,and Hcy-induced transthioesterifcation reaction lead to the corresponding blue-green fluorescence product CR-Cys,yellow-green fluorescence product CR-GSH,and non-fluorescent product CR-Hcy.In PBS?10 mM,pH 7.4,30% CH3 CN,v/v?,upon the addition of Cys,a remarkable blue-shifted from 510 nm to 417 nm in the UV-vis spectrum and an obvious enhancement of the emission intensity at 493 nm in the fluorescence spectra were observed.Upon treatment with GSH,a remarkable blue-shifted from 510 nm to 450 nm in the UV-vis spectrum and an obvious enhancement of the emission intensity at 503 nm in the fluorescence spectra were observed.The probe showed good selectivity and sensitivity,and the detection limit of probe for Cys and GSH in aqueous solutions is 29 nM and 371 nM,respectively.Meanwhile,we successfully applied probe CR1 for imaging Cys and GSH in living cells,demonstrating the potential utility of the probe.4.Two new AIE compoundsRBTP and RBTH,which is an intergrant of rhodol and HBT chromophores produced by introducing a benzothiazole unit to the ortho-position of the phenolic hydroxy of rhodol dyes,was designed and synthesized.Among them,RBTP owing to containing thiosemicarbazidescould be used as a new “turn-on”dual-mode probe for discrimination and detection of Hg²⁺ and Ag⁺ in aqueous solution.First,the analysis of photophysical properties and single crystals structures of RBTP and RBTHshowed that the enhanced emission of both compounds in the aggregates can be explained by the synergistic effect of the restriction of intramolecular rotations and vibrations and the ESIPT process.More importantly,in HEPES?10 mM,pH 7.4,40% THF,v/v?,upon the addition of Hg²⁺,a significant red fluorescence enhancement at 595 nm and a color change from colorless to red were observed.Upon treatment with Ag⁺,a visible green fluorescence enhancement at 520 nm was observed.Further studies indicated different sensing mechanism for Hg²⁺ and Ag⁺ were,respectively,based on an irreversible Hg²⁺-promoted oxadiazole forming reaction and the special molecular packing of the [RBTP-Ag]+ complex in aqueous solution.RBTP as a probe could sense Hg²⁺ and Ag⁺ with high selectivity and sensitivity by two different approaches,and the detection limit of probe for Hg²⁺ and Ag⁺ in aqueous solutions is 0.27 ?M and 0.45 ?M,respectively.Furthermore,its potential as practical biological imaging agents was further confirmed in HepG2 cells.