Used In The Research And Application Of New Fluorescent Probes For The Detection Of Bioactive Molecules Within The Cell Selenium

The progress of life is a total result of many relative chemical reactions which a number of biological active substances take part in. Many active substances can combine with amino acid, protein or other biological molecules, and play important biological action, special physiological function and high biochemical effects.Nowadays, 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, easy to operate, and the availability of a wide range of indicator dyes. 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. Recently, 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 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 have carried out three aspects of investigation:First, this paper presented the design and synthesis of organoselenium fluorescent probe for thiols.Inspired by Ebselen, the well known mimetic of GPx, we designd to introduce the selenium-nitrogen bond which reacts with thiols to fluorescence dyes. A novel rhodamine 110-based fluorescent probe containing a Se-N bond for detecting thiols was designed and synthesized, and characterized by IR, 1H-NMR, 13C-NMR, 77Se-NMR and MS. The probe was synthesized in high yield and was stable over 3 months when kept in a refrigerator at -4 oC.Second, this paper presented the analysis and application of organoselenium fluorescent probe in vitro and in vivo.The spectral properties of the probe were tested in simulated physiological condition. The probe was nonfluorescent in the form of rhodamine 110 spirolactam. Upon selective reaction with thiols, Se-N bond is cleaved and strong fluorescence generates, accompanying a fluorescence intensity enhancement higher than 170-fold (λ_ex/λ_em = 499/522 nm). The probe features excellent immunity to interferences, including reactive oxygen species (ROS), metal ions, bioamines and biological antioxidants. There was a good linearity between fluorescence intensity and GSH concentrations in the range of 0.015 to 1.0μM. The detection limit of 144 pM was achieved. Confocal microscopy experiments in both HL-7702 cells and HepG2 cells show that Rh-Se-2 can visualize difference and changes in thiols concentrations in normal and aberrant cell types, allowing a simple and reliable fluorometric system for intracellular thiols analysis.Third, this paper presented the design and synthesis of an organoselenium fluorescenct probe for peroxynirte.