Preparation And Application Of Fluorescent Probes For Bio-Imaging

Fluorescent probe is a class of fluorescent chemicals that its fluorescence properties (such as fluorescence intensity, lifetime, excitation wavelength and emission wavelength) will be influenced by the surrounding environment, and treatment with analytes, which will give out signals of color, fluorescence or UV-absorption spectra changes. Fluorescent probes contain organic dyes, luminescent nanomaterials and fluorescent protein. Molecular imaging based on fluorescent probes has been an essential tool for sensing toxins in environment and bioactive molecules in biomedical fields, tumor imaging and cellular imaging. Sulfide hydrogen, due to its active chemical properties, such as reductive properties, nucleophilicity, and forming complexes with metal ions, is found to be emerging signaling agent implicated in various pathological and physiological processes. The development of sensitive and selective methods for the sensing of sulfide hydrogen is therefore very important.Nanocarrier has been proven to be an excellent tool for gene delivery. However evaluation of gene therapy efficacy by nanocarriers based on cationic liposomes or polymers is difficult. Luminescent nanocarriers are increasingly employed to track the gene delivery. In this thesis, based on the review of their research status (Chapter 1), synthesis of functional fluorescent probes and their applications for fluorescent imaging aqueous sulfides and in vivo fluorescent tracking gene release were explored as following:In chapter 2, by using an easily prepared organic fluorescent moleculel-oxo-1H-phenalene-2,3-dicarbonitrile (OPD), we report its undiscovered application as high sensitive and selective fluorescent probe towards to aqueous sulfides and was further exploited for the fluorescence sensing of sulfides in HeLa cells. A novel reaction mechanism was proposed which is totally different from reported mechanisms. The sensitive detection of sulfides carries great significance for chemical industry and biological analysis. As-synthesized OPD compound provides promising fluorescent properties and unique reactive properties toward aqueous sulfides. In the absence of sulfide, OPD fluorescent probe was weakly fluorescent, but upon treated with sulfide, it was found that the sulfide-adduct emitted strong fluorescence at 576 nm. OPD fluorescent probe showed high selectivity and sensitivity towards Na2S over thiols and other inorganic sulfur compounds through a sulfide-involved reaction which was confirmed by high resolution mass spectroscopy (HRMS) and nuclear magnetic resonance (NMR) results. It was proposed that there were sulfide addition, structure rearrangement, oxidized reaction, hydrolysis to OPD fluorescent probe. The fluorescence intensity increases linearly with sulflde concentration in the range of 1.0-30 uM with a limit of detection of 52 nM.In chapter 3, we present a facile strategy to construct a smart nonviral nanocarrier to specifically monitor the release of plasmid DNA in HeLa cells. Accurately monitoring the gene release by luminescence imaging is highly desirable for evaluating the role of gene in the treatment process, while advances in the assay methods have enabled more reliable images for evaluating gene therapy efficacy. The luminescent nanocarrier was constructed by modifying uniform LaF3:Ce3+-Tb3+nanocrystals with functional amphiphilic polymer poly-(ethylene glycol)-poly-(lactic-co-glycolic acid) (PEG-PLGA) with enhanced effect and positively charged polymer poly-(methyl methacrylate)-(l-allyl-3-methylimidazolium chloride) (MMA-AMIM). The functionalized LaF3:Ce3+-Tb3+@ MMA-AMIM/PEG-PLGA nanospheres have been excellent candidates for loading plasmid DNA and indicating intracellular gene release. Luminescence of LaF3: Ce3+-Tb3+@MMA-AMIM/PEG-PLGA nanospheres and RFP was monitored for intracellular localization. This new imaging mode has shown promise and will bring new sight into gene release and for the further application in analytical chemistry and biomedical fields.