Construction And Application Of Hypochlorous Acid Fluorescent Probe In Mitochondrial Environment

Hypochlorous acid(HOCl),as a kind of reactive oxygen species(ROS)plays a pivotal role in immunoregulation of antibody,aging,apoptosis and defense against pathogen invasion.Research shows that abnormal concentration of hypochlorous acid will result in many diseases which include Alzheimer's disease,cancer,liver injury,ischemic stroke,rheumatoid arthritis and lymphadenitis.In addition,mitochondria server as the energy supply site not only provide energy for cells and involved in cell signal transduction while also the important place where produce hypochlorous acid.Therefore real-time monitoring of hypochlorous acid fluctuation in mitochondria has a warning effect in early stage to mitochondrial dysfunction,tissue damage and various diseases.It's extremely difficult to detect hypochlorous acid in vivo own to its high activity,live-half and it is necessary to develop a kind of real-time,rapid and efficient method for hypochlorous acid detection.Fluorometry possesses Simple preparation,high tempo-spatial resolution,high sensitivity and non-destructive imaging which promopted it widely used in the detection and imaging of biological molecules.Reasonable construction of fluorescent probe is the key of the application of fluorometry.Based on the above,to develop a fluorescent probe with excellent performance for detecting hypochlorous acid in mitochondria is the exciting point of research.In this thesis,we focus on rational construction of high-performance fluorescent probe initiated the following contents:1.Using indole cation as mitochondrial targeting group,a near-infrared fluorescent probe DMI for hypochlorous acid detection was synthesized based on Konevenagel reaction.The C=C bond as the reaction site could broken and formed corresponding aldehyde compound upon addition of hypochlorous acid to the probe,which would further lead to fluorescence quenched.This process can be identified with “naked eye”.The probe possesses excellent performance with response rapid(within 4 s),low detection limit(0.05?M),low cytotoxicity and high selectivity.Furthermore,the probe can target mitochondria and successfully realizing the detection of HOCl in mitochondria.2.Considering that the fluorescence quenched probe is easily disturbed by other factors and resulting in low sensitivity of probe.In this part,we designed and synthesized an “off-on” probe CPD for HOCl detection based on coumarin derviatives.The coumarin derviativies as molecular parent and pyridine cations were introduced as mitochondrial targeting group through Knoevenagel one-step reaction and the C=C bond was used as the reaction site.When the probe reacts with HOCl,the generated oxygen anions would interact with the adjacent hydroxyl group of the parent which inititate the excited state intramolecular proton transfer(ESIPT)and realized dual-channel detection.An obvious fluorescence enhancement was observed when added HOCl,which greatly weakened the interference caused by environmental factors.The results exhibite excellent optical properties with low detection limit(0.012 ?M),high selectivity and fast response time(within 10 s).It has been successfully used indetection of HOCl in mitochondria and zebrafish.3.Traditional fluorescent probe molecules tend to aggregate on the surface of biomolecules or form clusters in the hydrophobic cavity of biomolecules,and would occur aggregation caused quenching at a certain concentration,which seriously limits the application of probes in living organisms.To solve this problem,two kinds of aggregation induced emission(AIE)fluorescent probe BTD-1 and BTD-2 were synthesized usigng benzothiazole derivatives choosed as fluorescent matrix.The results showed that both probe BTD-1 and BTD-2 had obvious AIE phenomenon and could form spheres with a particle size of about 100 nm in 80% aqueous solution.With the C=N bond as reaction site,the aldehyde group compounds with significantly enhanced fluorescence were produced after the reaction with HOCl.Probe BTD-1 and BTD-2 can detect HOCl in mitochondria and same results were obtained in live zabrafish experiment.