Design And Application Of Novel Fluorescent Probes For Fe³⁺ And Zn²⁺

Many metal cations are involved in living systems and play vital roles in different physiological processes and pathology. As the most abundant transitional metal in human body, iron is the essential component of variable proteins and enzymes and is associated with a variety of biological functions. As to zinc, it is the second most abundant transitional metal in human body and plays also many indispensable physiological roles. Metabolism disorder of zinc has been found to be tightly associated with many neurodegenerative diseases. Therefore, the in situ detection of iron and zinc is of great importance to understand their essential roles in life. Since the fluorescence imaging displays the high sensitivity/selectivity in detecting bio-species in non-invasive manner, the development of fluorescent probes for Fe3+ and Zn2+is now attracting much attention currently.Due to the emission quenching nature of paramagnetic Fe3+and Fe2+, most pf the reported fluorescent probes for Fe3+ display the Fe3+-induced emission decrement, and the development of Fe3+ fluorescent probes of turn-on response is of great significance and challenging. As to the fluorescent Zn2+ probes, two kinds of probes including the turn-on probes and ratiometric probes have been frequently reported. However, the most of these fluorescent probes showed the shorter absorption and emission wavelengths, and the imaging application will be disturbed by the autofluorescence of biomolecules. Therefore, it is of great importance to explore novel rationales and platform to develop fluorescent Zn2+ probes of longer excitation and emission wavelength. In addition, integrating of other functions into Zn2+ fluorescent probes should be helpful to extend the application of novel fluorescent Zn2+ probes.In this thesis, two new turn-on fluorescent probes for Fe3+, L1 and L2, were constructed by integrating N-hydroxyethyldiethylenetriamine (HEDTA) motif as Fe3+ chelator respectively with fluorophore anthracene or rhodamine B. The hydroxyl group of their HEDTA, as a hard base, is essential for the enhancement their affinity for Fe3+ over Fe2+. L1 is a PET sensor displays the reversible turn-on response specifically to Fe3+. Spectroscopic study disclosed that L1 is able to sense Fe3+ in Tris-HCl buffer containing 50% methanol via displaying the Fe3+-specific emission enhancement over other metal cations. The intracellular Fe3+ imaging has been realized in SKOV-3 and HeLa cells via L1 staining, which displayed not only the reversible Fe3+ imaging ability but also the ability of discriminating Fe3+ from Fe2+ inside the cells. Since the non-emissive spirolactam form of rhodamine can be converted into the fluorescent quinone form in suitable conditions, we developed a new Fe3+ fluorescent L2, in which one HEDTA motif was coupled with two rhodamine B motifs in spirolactam form. L2 exhibits the distinct fluorescence enhancement (enhancement factor is about 90) specifically upon the presence of Fe3+ due to the Fe+-induced ring opening of lactame to form the emissive quinone form of rhodamine B, and the obvious color change to pink can be observed by naked eyes. The intracellular imaging in cells stained by L2 disclosed that L2 is preferential accumulated in mitochondria and is able to imaging effectively the Fe3+ enhancement in mitochondria. Althouth L2 is a chemodosimeter, it displays still the reversible response to Fe3+, showing its advantage over other reported irreversible chemodosimeter of turn-on Fe3+ response. In fact, L2 is one of the very few fluorescent Fe3+ chemodosimeter displaying the reversible turn-on fluorescent response to Fe3+ in aqueous medium. This study provides also a new clue to develop turn-on fluorescent probe for Fe3+. The detection limit of L1 and L2 for Fe3+ is 5.80 x 10-7 and 1.07× 10-6 M, respectively, which were lower than the permitted iron level in drinking water (300 ug/L).The short excitation and emission wavelengths of many turn-on Zn2+fluorescent probe molecules make them not suitable for biological imaging studies. In this study, a new fluorescent Zn2+ probe BDP-m-BPA was developed by modifying BODIPY with a (4-hydroxyl)styryl group and a Zn+ chelator respectively at α- and meso-positions. In this way, the ICT in BODIPY was enhanced by the integrated (4-hydroxyl)styryl group in its a-position. Compared with the normal BODIPY fluorophore, the emission maxima of this probe was red shifted and close close to the near-infrared region. In the meantime, the the Stokes shift is also increased. BDP-m-BPA displays dual emission bands at 575 nm (intrinsic band) and 660 nm (excimer band). The emission of this probe demonstrates a distinct emission enhancement specifically upon Zn2+ addition due to the Zn2+ coordination to BPA blocking the PET process from BPA to BODIPY fluorophore. The Zn2+-induced emission enhancement factor is about 9.5 according to the emission at 575 nm after 6 equiv Zn2+ was added. Different from the distinct emission enhancement for intrinsic band, the excimer band displays not obviously enhancement upon Zn2+ addition, although this band is somehow overlapped by the Zn2+-enhanced intrinsic emission band. Therefore, BDP-m-BPA is not only a new turn-on Zn2+ probe but also a ratiometric one. The intracellular Zn2+imaging in live cells showed that the BDP-m-BPA is cell membrane permeable and is able to imaging intracellular Zn2+ in the reversible manner via both the mono-channel imaging and dual channel imaging. This study suggested that modifying the a-position of BODIPY dyes could be a reliable approach to enhance the excitation or emission wavelengths of BODIPY fluorophore to improve the imaging ability of BODIPY-based probe.The attractive development of nanotechnology is now encouraging scientists to explore the MRI/fluorescence bimodal probes for metal cations based on the magnetic nanoparticles.. In the latter of this thesis, we modified the paramagnetic Fe3O4 nanoparticles with an 8-aminoquinoline derived Zn2+ fluorescent sensor QTEPA on the surface of this particle with purpose to construct fluorescence/MRI probe for Zn2+..The resulted Fe3O4@SiO2-QTEPA magnetic nanoparticles have fine biological compatibility and retain the superparamagnetic nature of Fe3O4 nanoparticle. In addition, this nanopartilecs displays the distinct emission enhancement in Tris-HCl buffer specifically upon Zn2+addition, and the enhancement factor is about 22-fold. This nanoparticle demonstrates not only the reversible intracellular Zn2+ imaging ability but also the T2-weighted magnetic resonance imaging ability in both live cells and mice. The current results demonstrates the possibility of Zn2+ imaging in the specific region discriminated by MRI with the same agents.