Construction And Application Of Fluorescent Probes For Monitoring Mitochondrial Membrane Potential And Autophagy

Cell viability is an important indicator reflecting the normal state of cells.In biological research,changes in many factors will affect cell activity,such as depolarization of the mitochondrial membrane potential（MMP）,abnormality of lysosomal pH values,and decrease of esterase activity.Mitochondria are the power plants of cells,which provide energy for various life activities of organisms.Mitochondrial membrane potential MMP is an important parameter reflecting the function of mitochondria.The formation of up to-180mv MMP is related to the proton excretion behavior of the inner mitochondrial membrane.Abnormal mitochondrial membrane potential will cause mitochondrial function to be blocked with cell activity to decrease.Lysosomes are the organelles responsible for cell breakdown and can break down macromolecular substances,such as proteins,polysaccharides and necrotic cells.The physiological pH of lysosomes（4.5-5.5）is related to the presence of various acid hydrolases in lysosomes.Abnormal pH value will directly affect the enzyme activity,resulting in the destruction of cell dynamic balance,and affecting cell activity.Compared with other techniques,the application of fluorescent probe technology in biology and pathology has become more and more mature.In this paper,Based on the study of mitochondria and lysosome targeted fluorescent probes,two fluorescent probes for detecting mitochondrial membrane potential MMP and a ratio type pH fluorescent probe for detecting lysosome were successfully designed.The spectral properties and biological applications of the three probes were studied in detail.（Ⅰ）A novel fluorescent probe REP based on organic cation group was designed.In living cells with high level of MMP,REP could target mitochondria and deliver a strong fluorescent signal.After the decrease of MMP,the probe would be release from mitochondria and the emission was significantly quenched.The MMP decrease caused by oxidative damage was also successfully visualized with the probe.（Ⅱ）A double targeted fluorescent probe DPEI with double cation groups was synthesized.The ligand with quinolinium moiety and strong electronic donor displays red fluorescence peaked at 630 nm.DPEI was highly enriched in mitochondria of living cells,and re-localizes into nucleolus upon mitochondrial depolarization owing to the affinity to RNA.With the introduction of double cation groups,DPEI has achieved the goal of high efficiency and rapid response to analytes.Cell damage induced by H₂O₂ was also successfully observed with the probe.（Ⅲ）A proportional pH fluorescent probe LEP-6 was developed to detect lysosome and autophagy.Pyridine is used as a pH responsive site and lysosomal targeting site.Under alkaline conditions,the probe emits blue fluorescence at 460nm;under acidic conditions,Protonation of pyridine enhances the intramolecular push-pull electrons,resulting in green fluorescence at 560 nm.In cell imaging study,the probe can be excited at 405nm,and the fluorescence signals of green channel and blue channel were from lysosomes.In the detection of autophagy,the probe successfully detected the decrease of lysosomal pH.