Design And Synthesis Based On NBD Parent Fluorescent Probe And Its Application In Cells And In Vivo

In the living body,there exists various endogenously generated active species as well as potential tumor markers.The type and content of these substances are different between normal and abnormal cells.Recently,researchers have developed many detection technology for bioanalysis.However,due to the diversity and complexity of the environment in vivo,the sensitivity is low and the selectivity is unsatisfactory.Therefore,it is necessary to develop the analytical methods with high selectivity and sensitivity.Fluorescent probe can selectively convert the chemical information of the analyte to the fluorescence signal.When the surrounding environment changes,the fluorescent emission wavelength and intensity will change,which will reflect the characteristics of the environment.With these optical information,the sensitive detection and visualization of the analyte in the organism can be achieved.As one of the most important tools,small molecular fluorescent probe possesses the advantages of rational design,high selectivity,good accuracy and rapid response,thereby playing the increasiongly important role in bioanalysis inside the cell in vivo.In this paper,NBD-Cl was selected as the parent fluorophore.Several probes were designed and applied to detect and monitor the signal molecules and tumor markers.The definite works are as follows:Chapter one: A simple introduction about the progress of the application of fluorescence probe,selected topic significance and the main research content.Chapter two: The introduction of a probe for detecting ketoglutarate acid,the design,synthesis and further practical application of organisms.Probe for ?-ketoglutaric acid??-KA?usually provides low sensitivity,invisible colour change and com-plex procedure with surfacants,which limits the accurate,sensitive quantification and mornitoring of ?-KA in biological samples.Essentially different from the routine sensing mechanism,a special mech-anism of photo-induced electron transfer?PET?combining with large absorption red-shift?rather than emission?has been designed to develop a facile yet multifunctional probe for the simultaneous fluorimet-ric,absorption-ratiometric and colorimetric detections of ?-KA in biological samples.Effective inhibitionof PET ensured the intense fluorescence turn-on?60 fold?and qutie low detection limit?0.9 mM?for ?-KA without need of any surfacants.Dramatical colour change caused by the large absorption red shift?100 nm?from UV–vis region enabled the colormetric analysis of ?-KA by naked-eye.Meanwhile,the largeabsorption red shift also contributed to the ratiometric detection,allowing for the accurate quantificationof ?-KA in complex biological sample.With this probe,ratiometric detection,naked eye mornitoring andimaging of ?-KA in serum,living cells and tissues are realized for the first time.Chapter three: The introduction of a probe for detecting Oxidative stress?hypochlorite and hydrogen sulfide?,the design,synthesis and further practical application of organisms.Homeostasis of ClO-/H₂ S plays crucial role in the damage and repair of gastric tissue,but has been rarely investigated due to the challenge of in situ analysis in the highly acidic gastric environment.Herein,we design a new H+-activated optical mechanism including the controllable photo-induced electron transfer?PET?and the switch of electron push-pull?SEPP?to develop the simple yet multifunctional probe?Z?-4-?2-benzylidenehydrazinyl?-7-nitrobenzo[c][1,2,5] oxadiazole?BNBD?.First,probe BNBD?Off?is protonated by the highly acidic media to trigger the strong fluorescence?On?.Then the analyte ClO-or H₂ S reacts with the protonated BNBD,leading to the ultrasensitive?ClO-: 2.7 n M;H₂S: 6.9 nM?fluorescence quenching via the rapid oxidation of C=N?50 s?and nitro reduction?10 s?,respectively.With the logical discrimination by absorbance/colour?ClO-: 300 nm/colorless;H₂S: 400 nm/orange?,a strategy for in situ quantifying ClO-/H₂ S in gastric mucosa and juice is developed.For the first time,the in situ quantitative monitoring of the endogenous H₂ S,the ClO-/H₂ S homeostasis,as well as the pathologic manifestation in gastric mucosa are realize,overcoming the challenge of ClO-/H₂ S analysis in highly acidic condition and enabling the in situ tissue quantification of ClO-/H₂ S.In conjugation with the assessment of mucosal damage,this work confirms the injurious/rehabilitative effects of ClO-/H₂ S on gastric mucosa?at 50-90 mm depth?,which may facilitate the auxiliary diagnosis of stomach diseases induced by oxidative stress.Chapter four: The introduction of a probe for detecting Hydrogen proton,the design,synthesis and further practical application of organisms.The cells control their pH change in a very accurate range.pH plays important roles in cell autophagy and apoptosis.Evidences imply that the internal milieu of tumor is acidified.Although the acidification in cells is investigated,the biological effects from multiple stimulating factors under the complex intracellular environment have not been thoroughly elaborated yet.Hitherto,there is few pH probe performs in wide acidity range,which needs to be urgently developed.Herein,we report a new fluorescent probe?CM-BHNBD?for the real-time detection of mitochondrial and intramucosal acidification.The probe respond to pH via an H+-driven PET-TICT?photoinduced electron transfer-twist intramolecular charge transfer?mechanism,and can linearly report pH within a wide acidity units:7.00-2.00 following ¹40-fold fluorescence increase.The probe also possesses excellent membrane permeability,good photostability,and negligible cytotoxicity.The probe is successfully applied for quantifying the acidification in HeLa cells under the simultaneous stimulation of nutrient deprivation and oxidative stress.Our results demonstrate that the mitochondrial pH is in a dynamic fluctuating state during the acidification process,which suggests a potential cross-talk effect between cell autophagy and apoptosis.We also use the probe for quantifying the intramucosal pH variation in stomach and esophagus via manipulating cellular proton pump.The development of our probe is potentially expected to be used to monitor the intracellular/intramucosal acidification for biomedical research.Chapter five: Conclusion and future prospect.