| Zinc is one of the most important transition metal cation in human body. Although most of Zn2+ is bound by biomolecules such as proteins acting as metal center and structure factor, there is still many "free" or "chelatable" Zn2+ in nerve systems functioning as conventional synaptic neurotransmitter. In addition some of Zn2+ is closely associated with signal transduction as second messenger to regulate the cell growth, proliferation and apoptosis. Moreover, Zn2+ metabolism is still correlated to insulin secretion, neurodegenerative disorders and develop retardation of children. It is obvious that clarifying the process of zinc uptake, transportation, reservation and release is essential for elucidating the vital roles of Zn2+ and helpful to develop techniques for the diagnoses and therapy of the related diseases. In fact, developing techniques for spatial and temporal Zn2+ tracking in biological systems is currently one of the most attractive fields. Zn2+ fluorescent probing becomes one of the most important techniques due to its higher sensitivity and selectivity, and developing Zn2+ fluorescent sensors of longer excitation wavelength and special in vitro and in vivo distribution pattern should not only favor to overcome the disadvantages such as the photo-toxicity, interference of excitation but also satisfy the requirement to determine Zn2+ in specific organs or organelles.Due to the lower coordination number of classic Zn2+ fluorescent sensor TSQ, the Zn2+ imaging with TSQ may suffer from the interference of partially bound Zn2+ in living systems. In the first part of this study, a new Zn2+ fluorescent sensors, BPSQ, was prepared by modifying TSQ with the synergic Zn2+ coordinating motif Bis(2-pridylmetyl)amine (BPA). Crystal structural resolution of its zinc complex discloses a Zn2+ binding stoichiometry of 1:1, which favors to reduce the interference of partially coordinated Zn2+ in labile intracellular Zn2+ detection. This sensor exhibits two emission bands in aqueous medium, one centered at 413 nm and another is composed by two mutually overlaid bands centered at 496 and 529 nm. Zn2+ addition leads to～8-fold emission enhancement for the latter band with the former one being almost intact. In addition, such an emissive response is specific for Zn2+. All these suggests that BPSQ is not only intensity-based sensor but also a ratiometric sensor for Zn2+ In acetonitrile, the free sensor displays only the emission band of lower wavelengths due to the blockage of photo-induced proton transfer to solvent, and ratiometric Zn2+ sensing behavior become even distinct due to the Zn2+-induced emission shift to band of higher wavelength. The current intracellular Zn2+ imaging confirms the fine intracellular Zn2+ imaging ability and cell membrane permeability of BPSQ. The ratiometric Zn2+ imaging via BPSQ-staining is still undergoing.The second part of this study focuses on the biological Zn2+ imaging ability of a new visible light excitable Zn2+ fluorescent sensor, NBD-TPEA. This sensor was constructed based on the fluorophore 4-amino-7-nitro-2,1,3-benzoxadiazole, and the excitation and emission maxima are 466 and 544 nm, respectively. In addition, this sensor exhibits a specific Zn2+-amplified fluorescence. Its intracellular Zn2+ imaging ability has been verified on several cell lines by screening the staining conditions, and its cell membrane permeability is fine. Moreover, both the excitations at 458 and 488 nm are effective for intracellular Zn2+ imaging. The in vitro imaging also confirms the preferential affinity of NBD-TPEA to Golgi and lysosomes, and this sensor is qualified as the fluorescent tracker to monitor the fluctuated Zn2+ level in living PC 12 cells. As the visible light excitable sensor, the in vivo Zn2+ imaging via NBD-TPEA saining has also been realized on zebrafish larva. This is the first example of in vivo Zn2+ imaging. This experiment still disclosed two zygomorphic luminescent areas (ZLAs) on the chest of larva as the "Zn2+-riched regions". The confocal fluorescent imaging of the head of Zn2+-fed zebrafish larva still displayed Zn2+-riched dots which may be related to the neuromasts. All these suggest that NBD-TPEA is a practical sensor of fine in vitro and in vivo Zn2+ imaging ability, and should have potential applications in zinc neurobioinorganic chemistry and development biology.Finally, this study focuses on the imaging ability and distribution pattern of a group of novel Zn2+ sensors based on 1,8-naphthaliimide fluorophore, NBPEA-I, ⅡandⅢ. All the three sensors exhibit the similar excitation and emission maxima at～450 and 550 nm, respectively. Moreover, their Zn2+-amplified fluorescence is specific. The in vitro Zn2+ imaging on HeLa cells confirmed the intracellular Zn2+ imaging ability and cell membrane permeability of these sensors. Although NBPEA-I exhibits the preferential affinity to Golgi and lysosomes, this sensor still distributed uniformly in cytoplasm. More important is that this sensor is able to enter into the nucleolus. The tail-modified NBPEA-II and III display the similar in vitro Zn2+ imaging ability. However, a new Zn2+ sensor of same Zn2+ binding motif based on 4-amino-7-nitro-2,1,3-benzoxadiazole fluorophore cannot penetrate into the nucleus. This result infers that the different fluorophore parent should be essential for the intracellular distribution pattern of Zn2+ sensors. In vivo imaging on zebrafish larva via NBPEA-I-staining discovered the similar ZLAs. However, brighter fluorescence was observed on zebrafish yolk than the case of NBD-TPEA. This imaging still displays some bright spots on the head and neck, similar to the case of NBD-TPEA. However, the in vivo imaging with NBPEA-I still exhibits the bright line on the tail notochord of Zn2+-fed zebrafish, which is also different from the case of NBD-TPEA.All the current study on the biological Zn2+ imaging ability of the above novel Zn2+ sensors should not only favor to their potential application but also give some clues for developing novel Zn2+ sensor of special in vitro and in vivo distribution patterns. |
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