The Novel Fluorescent Probe Based On Aggregation-induced Emission For Fluorescence Detection Of Bioactive Molecules

Biological signaling molecules directly or indirectly participate in cell signaling,immunity and metabolism,which in turn affect the homeostasis of the cellular microenvironment.The abnormal expression of related biological signaling molecules like phosphatase(ALP)and azoreductase is causing a variety of major diseases.Therefore,various means need to be developed to monitor biological signaling molecules in cells,providing an important means for disease analysis and drug discovery.At present,with the specific and highly sensitive,based on small organic molecules of fluorescent probes have been widely used for visual monitoring of biologically active molecules.Aggregation-Induced Emission(AIE)-based fluorescent probes can effectively avoid Aggregate Caused Quenching(ACQ)and exhibit better bioimaging capabilities.Accordingly,we designed and synthesized two types of AIE-based fluorescent probes,respectively monitor the activities of ALP and azoreductase in the cellular environment.In first chapter,we introduced the research background and the luminescence mechanism of fluorescent probe,the research progress of fluorescent probe for detecting ALP and azoreductase,as well as the research content of this thesis.In second chapter,an AIE fluorescent probe AE-Phos detecting alkaline phosphatase based on the activity was presented,distinguishing alkaline phosphatase activity in different cell lines.The probe AE-Phos was obtained by phosphorylation on the fluorophore SA.Combining the advantages of the characteristics of AIE and ESIPT,the introduction of a phosphate group quenched the fluorescence of salicylaldehyde azine(SA),prevented the formation of ESIPT and AIE,and made the probe AE-Phos with excellent water solubility.After dephosphorylation by ALP,the phosphate groups of AE-Phos were removed,releasing the hydroxyl group.The free hydroxyl groups can form intramolecular hydrogen bonds with nitrogen atoms,which ensure intramolecular rotation allowed only for the N-N bond,and then SA show the AIE characteristics of J aggregation in water,accompanied by strong fluorescence emission.At the same time,the ESITP property of salicylaldehyde-like structure provides a large Stokes shift.The data shows that the probe has great selectivity for ALP with a low detection limit(0.012 U/L).Compared with traditional ALP probes,AE-Phos combines the properties of"AIE+ESIPT"with a large Stokes-shift,showing extremely low background fluorescence and high fluorescence enhancement ratio.The reaction mechanism of the probe AE-Phos with ALP has been reliably verified by different methods such as fluorimeter,reverse phase Ultra-Performance Liquid Chromatography(UPLC)and dynamic light scattering(DLS).Furthermore,when detecting cellular endogenous ALP,AE-Phos was able to effectively differentiate and visualize endogenous ALP activity in different cell lines such as human osteoblasts,murine melanoma cells,and macrophages.In third chapter,an AIE fluorescent probe AIE-Cl for specific recognition of azoreductase was presented,integrating diagnostic and therapeutic.The probe AIE-Cl consists of a novel AIE fluorophore AIE-NH₂ and a chemical drug nitrogen mustard for tumor treatment,which are linked by an azo bond.The data show that when the azo bond is not reduced,the probe basically does not emit fluorescence in the buffer solution,which proves that the azo bond effectively quenches the fluorescence of AIE-NH₂;by using sodium hydrosulfite to chemically mimic azoreductase,azo bond is reduced to an amino group,the fluorophore AIE-NH₂ is released which exhibits excellent fluorescence emission in aqueous environment due to its AIE property.By studying its fluorescence emission at different p H and viscosity,the results show that the fluorophore can releases stronger fluorescence in high-viscosity environment of cells,which shows that the probe has application value in biological imaging.