Novel Fluorescent PH Probes And Related Intracellular PH Imaging

Proto nation and deprotonation of biomolecules are frequently involved in many cellular events, such as cell growth, endocytosis, calcium regulation and other cellular processes. Moreover, abnormal pH; values are often associated with cell dysfunction and diseases such as cancer and Alzheimer’s disease. Therefore, precise and rapid detection of pH values is of great importance in cellular analysis or diagnosis. Different methods have been developed to monitor pH value in cells, including electrochemistry, nuclear magnetic resonance (NMR), absorbance spectroscopy and fluorescence probe analysis. Among all of these methods, fluorescent probe analysis has been extensively studied and applied due to the advantages of excellent sensitivity and specificity, noninvasiveness and high signal-to-noise ratio. In fact, the fluorescence imaging technology has also become the theme of pHi monitoring, since it can offer the real-time temporal/spatial information. Thus developing the pH probes of improved sensitivity, selectivity and reversibility is of great importance.In the first part of this thesis, three fluorescent probes (NBD-OH, NBD-en and NBD-Py) based on ICT and PET mechanisms were synthesized. In these probes, NBD-NH2fluorophore of favorable biocompatibility, visible light-excitability and large Stokes shift was utilized as the fluorophore, simple amino or hydroxyl groups act as the proton receptors. Their absorption and fluorescence properties were investigated at different pH values. These aqueous soluble probes display high sensitivity and photostability. Among these, NBD-Py is sensitive to pH at pH range of 4.5-9.0with a pKa of6.48, which is valuable for intracellular pH; study. The presence of metal cations such as Na+, K+, Ca2+, Mg2+and other transition metal cations doesn’t interfere with its fluorescent pH response. Fluorescent imaging in HeLa cells with this probe revealed that it can be an effective intracellular pH imaging agent and has fine lysosomal targeting ability, which provides a new and reliable tool for the research of protonation/deprotonation process in lysosomes.Since the ratiometric fluorescent pH probe can offer the quantitative information of analyte due to internal caliberation effect between two excitation/emission bands, in the second part of this thesis, a FRET-based ratiometric pH probe (NBDPEAC) was developed by integrating NBDPZ pH probe as FRET acceptor with a pH-insensitive coumarin fluorophore as the donor. This probe shows two emission bands centered respectively at544and470nm. Its emission behavior at different pH values shows the emission intensity at544nm is largely decreased while the intensity at470nm is slightly increased along with pH increasing from3.5to9.0. By fitting linearly the pH fluorescence titration profile of NBDPEAC, the pH application range of NBDPEAC can be found to be from4.2to6.2. The pH imaging ability of NBDPEAC has been confirmed in MCF-7cell, which displays its fine biocompatibility and quantitative pH imaging ability.