Novel Fluorescent Probes For Bio-inorganic Species:Design And Imaging Studies

Endogenous bio-inorganic species maintain complex and sophisticated homeostasis in biological system, involving in the regulation of various physiological processes, play essential roles in keeping the health of the living system. Furthermore, exogenous toxic species including heavy metal ions and CN-, will accumulated through food chain and ultimately enter human body, causing serious damage either directly or indirectly, on the various organs and physiological functions of the human body. Therefore, the development of the detection methods of these species is in highly demand for the research of their physiological functions and pathological effects. Fluorescent imaging technique is highly sensitive, none invasive to biological samples, capable of providing temporal and spatial information of the guest species, which make it one of the most widely used methods for the detection of bio-inorganic species. A number of fluorescent probes for the detection and imaging of these bio-inorganic species have been developed. However, researchers still have to face some difficulties and challenges.This thesis systemically summarizes the classic design princeples of fluorescent probes and the current studies of the fluorescent probes for bio-inorganic species. We have conducted the following works with regard to some of the difficulties and challenges in this field:Firstly, H2S is a reactive small molecule with very short half-life (minutes), however, most of the current fluorescent probes are not capable for the rapid detection of H2S. A series of coumarin-merocyanine hybrid dyes (CMCs) are designed and constructed for ratiometric detection of H2S, taking advantage of nucleophilicity of H2S. The structure-function relationship of these probes is preliminarily studied. CouMC exhibits rapid ratiometric H2S detection ability within few seconds, which is one of the fastest fluorescent probes for H2S. In addition, the probe also shows mitochondrial targeting capabilities, and the fast imaging of mitochondria H2S is achieved in MCF-7cells. Furthermore, we develop a novel and universal FRET-based strategy for designing ratiometric H2S fluorescent probes, and three new probes are constructed based on this strategy. One of these probes exhibits endogenous H2S imaging ability in HepG-2cells, while another probe with red and near infrared emission shows H2S imaging ability in nude mice.Secondly, ratiometric probes for monitoring mitochondria pH are challenging, although highly demanded. A pH-sensitive fluorophore FITC is integrated with a pH-insensitive merocyanine dye, and a ratiometric probe (Mito-pH) for pH measurement in mitochondria is constructed, since the merocyanine dyes are reported to have mitochondrial targeting ability. This probe shows rapid, reversible pH detection ability. Cell imaging experiments confirms that Mito-pH is selectively accumulated in mitochondria. Furthermore, this probe is also capable of monitoring the fluctuation of mitochondira pH triggered by redox stimuli in MCF-7cells.Thirdly, Cu2+tends to quenching fluorescence, and the current reaction-based Cu2+probes are usually irreversible and time-consuming. To solve this problem, a coumarin fluorophore is selected as energy donor, sulfonamide substituted benzoxadiazole (SBD) fluorophore is introduced as receptor, bis-picolyl amine (BPA) group is chosen as recognition site, and FRET-based ratiometric probe (CSBPA) for Cu2+is rationally designed and synthesized. Cu2+chelation interrupts the FRET efficiency in the probe system, and the rapid and reversible ratiometric Cu2+detection ability is achieved. CSBPA is successfully applied to the ratiometric imaging of Cu2+in living cells.At last, most of the reported Hg2+fluorescent probes suffer form low sensitivity and slow response to Hg2+. An amino-substituted benzoxadiazole (ABD) is selected as fluorophore, mercaptal (1,3-dithiane, DT) group is designed for Hg2+recognition, and a Hg2+fluorescent probe (DT-ABD) with fluorescence enhancement response is constructed. Specific Hg2+induced deprotection reaction changes ICT process of the probe system and a fast fluorescence enhancement is achieved. This probe overcomes the heavy-atom quenching effect of Hg2+. In additon, by introducing coumarin derivatives, based on similar response mechanism, a novel ratiometric fluorescent Hg2+probe (CDTT) is constructed. Both of the two fluorescent probes are capable for in vivo Hg2+imaging in zebrafish larvae, and the distribution and toxicity of Hg2+in zebrafish are preliminarily studied.