Coumarin-based Fluorescent Probe For Detecting H₂S /Thiols From Dual Emission And Application

Hydrogen sulfide, cysteine, homocysteine and glutathione, play important roles in some physiological processes in living organisms. It is practical significance that the rapid detection of small sulfur molecules in the organism. Fluorescent probes for detecting sulfur molecules have been widely researched because of its selectivity, sensitivity, ease of operation, economy and real-time detection. A novel discrimination strategy for biothiols was presented based on the potential reaction sites of a coumarin- derivative, which was modified by chlorine, methylenemalononitrile. It would be possible to distinguish sulfide and thiols for generating different derivatives to emit different fluorescence.In order to introduce the reactive groups to coumarin, 4-chloro-3-formyl-7-(N,Ndiethylamino)coumarin was synthesized by Pechmann condensation and Vilsmeier-Haack reactions from 2-N,N-diethyl phenol. Then the probe consisting chloro and cyanide ethylidene groups, 3-methylenemalononitrile-4-chloro-7-(N,N-diethylamino)coumarin was synthesized from 4-chloro-3-formyl-7-(N,N-diethylamino)coumarin by condensation with malononitrile, and verified by NMR, HRMS and IR. The mechanism for detecting H2 S was investigated by using 1H RMS and titration-NMR. The selectivity and the influence of hydrogen sulfide or biothiols concentration, reaction time and the p H of buffer solution on ultraviolet and fluorescence were investigated by spectrum method. The cytotoxicity of probe was tested by MTT assay. The endogenous hydrogen sulfide and thiols were simultaneously monitored in MCF-7 cells by multicolor imaging. The results showed that the probe exhibited high sensitivity to hydrogen sulfide and thiols. The fluorescence intensity at 564, 471 and 492 nm increases with increasing the concentration of hydrogen sulfide, Cys or glutathione. The action time of hydrogen sulfide, cysteine or glutathione with probe was less than 10 min. The detection limits(S/N=3) of hydrogen sulfide, cysteine and glutathione were calculated to be 41.7, 68.8 and 248 nmol/L. The probe can simultaneously detect hydrogen sulfide and thiols from different emission. The probe exhibited low cytotoxicity, and could simultaneously monitor endogenous hydrogen sulfide and thiols in MCF-7 cells using multicolor imaging.