| Numerous free radicals are generated in the course of biology metabolism. Superoxide anion radicals (O2-·), hydroxyl radicals (HO·), ROO·and NO·are some typical ones. They have important relation with aging and many diseases. It is reported that appropriate level of reactive oxygen species (ROS) could help to build biological immune system, while excess ROS have damages on organisms. Recent studies demonstrated that ROS can cause many diseases such as heart disease, cancer, malignancy and so on. There are a lot of methods reported to detect ROS, among which using fluorescent probes is an ideal method because of its obvious advantages like high sensitivity, good selectivity and low detection. Thus, studies on fluorescent probes for detecting cellular ROS have greatly developed.Besides, various bio-active molecules are indispensable component of organism. They also take part in various life activities. Thus, studies on bio-active molecules including nucleic acid, protein, peptide, and other bio-active ingredients play an important role in life science. Recent years, more and more studies focus on detecting bio-active molecules. Among these methods, using fluorescent probes has many advantages such as high sensitivity, high selectivity and easy to operate. Besides, what is more attractive is that fluorescent probes can penetrate into cells without any damage, and combine with bio-active molecules to form strongly fluorescent substance. With the help of confocal microscopic imaging, visualizing and real-time detecting of bio-active molecules can be achieved. Recent years, imaging of living cells has made great progress, and fluorescent probe has become the most important tool because of its advantages.Based on the changes in spectrum characters of the fluorescent probes reacting with reactive oxygen species or bio-active molecules, we have achieved the detection of these ingredients. The designed fluorescent probes were also applied to confocal imaging of several kinds of cells. We carried out two aspects of investigation:First, based on Ebselen, the well known mimetic of GPx, which exhibits its catalytic GPx activity, we designd to introduce the selenium-nitrogen bond which reacts with thiols to fluorescence dyes. We designed and synthesized a novel rhodamine-based fluorescent probe containing a Se-N bond for detecting thiols. Due to the strong nucleophilicity of the sulfhydryl, the Se-N bond of the probe was cleaved, resulting in the formation of the strongly fluorescent dye rhodamine 6G. The spectral properties of the probe were tested in simulated physiological condition. And we achieved the detection of thiols by detecting the fluorescence changes. The fluorescence increase is proportional to the concentration of thiol in the range of 3.0×10-9 -1.2×10-7 M. The limit of detection is 1.4 nM. Confocal microscopic imaging of HL-7702 and HepG2 cells reveals that the probe is cell-permeable and intracellular thiol-responsive.Second, we designed and synthized a novel fluorescent probe 3,3'-Bis (trifluoromethylbenzeneseleninyl) fluorescein for lipid peroxide by two kinds of synthesis method. The products were well characterized by 1H NMR, 13C NMR, 77Se NMR, IR and MS. The reaction mechnism of fluorescent probe and lipid peroxide was studied. Encountering lipid peroxide in organic system, the weakly fluoresced probe showed strong fluorescent response due to the reaction between lipid perxide and the selenium-oxygen bond of the probe. The spectra properties of the probe were studied and the maximum exitation and emmision lied at 515/535 nm respectivly. The kinectic scan showed the good light stability of the probe and the probe could completely trap cumene hydroperoxide in 20 min. The selectivity of the probe was tested and the result showed that the probe showed good selectivity toward CuOOH than other ROS. Finally, to demonstrate the practicability of the probe, it was applied to detect the lipid peroxide of different kinds of rats including normal ones, rats with bleomycin-induced pulmonary fibrosis and others cured by Chinese traditional drugs and western drugs. The results showed that different fluorescent responses were detected in different kinds of rats, which was in accordance with their various extents of lipid peroxidation. |
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