Construction And Biological Imaging Of Fluorescent Probes For Biothiol And Hydrogen Peroxide

In recent years,organic small molecule fluorescent probes have attracted extensive attention from researchers because of their good selectivity,low detection limit,simple operation,and real-time detection.The relative balance of intracellular redox states is primarily achieved by controlling the relative amounts of reducing substances and reactive oxygen species?ROS?in the cells.As the most important reducing substance,biothiol plays an important role in the regulation of cell function,metabolism,and signal transduction.Studies have found that biothiols such as cysteine?Cys?,homocysteine?Hcy?and glutathione?GSH?are important targets for chemical,medical and biological research.It plays an important role in many physiological and biological processes such as protein synthesis,cell growth and metabolism.As an important reactive oxygen species,hydrogen peroxide is extremely important in the activities of living organisms and the external environment.Researches have shown that hydrogen peroxide participates in physiological and pathological regulation processes,and is closely related to cell proliferation and differentiation and migration processes.But many diseases are also caused by excessive hydrogen peroxide,such as cancer,alzheimer's disease and other diseases.Therefore,the dynamic changes of the redox state of the cell can provide a large number of effective physiological and pathological information for fields such as life medicine.In the first part of the thesis,our team developed a deep-red emission fluorescent probe?DRP-Cys?for Cys based on a hemicyanine scaffold.The electrophilic reagent malononitrile group could chromatically shift excitation/emission wavelengths to avoid the limitations such as photo-damage or auto-fluorescence in biological samples.Compared with other amino acids,Cys could trigger the Michael addition reaction to release a dramatic enhancement in fluorescence centering at around 645 nm in DMSO aqueous solution under physiological conditions with an ultrafast response time of about 1 min and a low limit of detection of 10nM which significantly benefited from the introduction of the malononitrile moiety.The probe was also successfully applied for visualizing endogenous Cys in living HeLa cells and mice with low cytotoxicity and good cell-membrane permeability.In the second part of the thesis,a two-separated-emission fluorescent probe for biothiols was developed based on the combination of nitrobenzofurazan?NBD?and phenanthroimidazole fluorophores linked by a facile ether bond.In the presence of Cys and Hcy,the probe in DMF-H₂O solution two separated fluorescence emissions at 480 and 550nm upon excitation of two independent wavelengths.However,addition of GSH to the probe can only lead to blue fluorescence at 480 nm.This difference is reasonablely attributed to that NBD-GSH intermediate other than NBD-Cys/Hcy could not undergo an intramolecular cyclization-rearrangement reaction.The probe exhibits a rapid response with low limits of detection?14.7 nM for Cys,14.4 nM for Hcy,and 13.4 nM for GSH?with the large concentration range of 0-100?M for Cys/Hcy and 0-200?M for GSH.Furthermore,the probe is successfully applied for simultaneously discriminating endogenous Cys,Hcy,and GSH in living HeLa cells and zebra-fish models.In the third part of the thesis,our team designed a fluorescence-enhanced two-photon hydrogen peroxide fluorescence spectrometer by ligating the fluorescent dye6-benzothiazole-2-hydroxyquinoline?BQ-OH?with a large two-photon cross section and4-carbobenzyl bromide.The probe is capable of specifically recognizing hydrogen peroxide,while other reactive oxygen species do not cause significant fluorescence changes.In the presence of hydrogen peroxide,the probe has a fluorescence enhancement of about 56 times,a fast response?20 minutes?,high sensitivity,and low detection limit.After testing,the detection limit is 14 nM.