Studies On The Development And Application Of Novel Cyanine Fluorescence Probes

Objective:To develop novel fluorescence probes based on cyanine dye and apply them for the fluorescence imaging analysis of intracellular ATP levels or viscosity changes.Methods:This thesis includes the below two parts.1.Development of near-infrared（NIR）ATP fluorescence probe NIR-ATP and its application in fluorescence imaging analysis during ferroptosis.CS-2 cyanine dye was choosed as fluorescence moiety and diethylenetriamine was selected as ATP recognition group to develop NIR fluorescence probe NIR-ATP for detecting ATP.The structure of NIR-ATP was confirmed by ¹H-NMR,¹³C-NMR and HRMS.Then the analysis performances of NIR-ATP to ATP were sequentially studied,such as linear range,detection limit and selectivity,by UV-vis spectrophotometer and fluorescence spectrophotometer.The biocompatibility of NIR-ATP was investigated by MTT assay at cell level.NIR-ATP was applied to the fluorescence imaging performances for intracellular ATP by inverted fluorescence microscope,and was applied to the fluorescence imaging analysis of ATP level changes during ferroptosis.2.Development and application of mitochondria-targeting viscosity responsive fluorescence probes N-1 and N-2Benzothiazole cyanine dye was used as fluorescence moiety,and combined with the“molecule rotor”strategy to develop mitochondria-targeting fluorescence probes N-1 and N-2 for detection of microenvironmental viscosity changes.The structures of N-1 and N-2was confirmed by ¹H-NMR,¹³C-NMR and HRMS.The fluorescence response properties of N-1 and N-2 to microenvironmental viscosity was investigated,such as linear range and anti-interference ability,by UV-visible spectrophotometer and fluorescence spectrophotometer.Investigated the biocompatibility for cells of N-1 and N-2 by MTT assay.The mitochondria-targeting performances of N-1 and N-2 was studied by confocal laser fluorescence microscopy,and N-1 and N-2 were applied to detect the mitochondrial viscosity changes by fluorescence imaging.Results:The target probe molecules were confirmed by ¹H-NMR,¹³C-NMR and HRMS.Studies on fluorescence response analysis and cell fluorescence imaging analysis showed that:（1）After response to ATP,the fluorescence intensity of NIR-ATP at 745 nm showed selective and sensitive enhancement.NIR-ATP also exhibited good fluorescence imaging performance for the change of ATP level in cells,it has been used to detect the change of ATP level in the process of cell ferroptosis.（2）N-1 and N-2 have weak fluorescence at low viscosity environment respectively.However,with the increase of viscosity in solvent environment,the fluorescence intensity of both of them showed a significant enhanced response.Meanwhile,the change of environmental p H and polarity didn’t cause obvious fluorescence response of N-1 and N-2,which means that N-1 and N-2 can be used for selective environmental viscosity detection.Furthermore,colocation imaging showed that N-1 and N-2 have good mitochondria-targeting ability.N-1 and N-2 have been used to monitor microenvironmental viscosity changes within mitochondria.Conclusion:Two types of fluorescence probes for detecting ATP or viscosity changes were obtained using novel cyanine dyes as fluorescence moiety and were successfully applied in cell fluorescence imaging.These probes provide not only new analytical tools for monitoring intracellular ATP levels or mitochondrial viscosity changes,but also noval strategy for design of new cyanine-based probes.