The Recognition Of Biothiols By Fluorescent Probes Based On Fluorescein Skeleton

Fluorescent probes have been favored by scientists because of their simple application,low detection limit,good sensitivity,and easy imaging in biological cells.With the development of cross-discipline,fluorescent probes have been widely used in many fields such as chemistry,biology,environmental science,physiology and other fields in the past decade.In this thesis,we designed and synthesized three fluorescent probes based on fluorescein scaffold to detect three biothiols:Cys,Hcy and GSH.The main works are as follows:(1)In the first chapter,the recognition methods of biothiols by fluorescence probes are introduced,including Michael addition reaction,aldehyde cyclization reaction,conjugate addition cyclization reaction,nucleophilic substitution reaction,metal complexes approach,cut off of sulfonyl group reaction and so on.(2)In the second chapter,2,4-dinitrobenzenesulfonyl group was used as a switch to modify the fluorescence of fluorescein scaffold to synthesize the probe DMB-COA.DMB-COA can be used to recognize biothiols under physiological condition(PBS,pH=7.40).The detection limit is up to 0.286?M(Cys),and the fluorescence can be increased by 65 times(Cys).At the same time,the reaction and luminescence mechanism of the probe were studied by density functional theory(DFT)and ~1H NMR titration.(3)In the third chapter,the fluorescence probe DMB-COB was obtained by using fluorescein monoaldehydes as the framework.Similarly,2,4-dinitrobenzenesulfonyl group was used as fluorescence quenching group.DMB-COB has lower detection limit than probe DMB-COA under physiological conditions.The lowest detection limit can reach 0.052?M(Cys),and the highest fluorescence enhancement factor can reach 46 times(Cys).The mechanism of biothiols recognition by probe DMB-COB was studied by means of ~1H NMR titration,mass spectrometry,and Gaussian calculation.Furthermore,biological imaging was performed in HeLa cells successfully.(4)In the fourth chapter,we change the fluorescein monoaldehyde group into a cyano group and modify the 2,4-dinitrobenzenesulfonyl group to obtain the fluorescent probe DMB-CN.In the PBS buffer solution of pH 7.40,the detection limit of the probe is lower than that of the probe DMB-COB.the lowest detection limit can reach 0.016?M(GSH),and the fluorescence enhancement ratio can be up to 43 times(Cys).Similarly,we used density functional theory(DFT)and ~1H NMR titration to study the mechanism of the recognion,and performed biological imaging in HeLa cells.