| Fluorescence imaging, a kind of molecular imaging technology, with high sensitivity and good resolution, could detect substrate fast in real-time and in-situ with nondestructive, exhibiting high value in applications of clinical medicine and the research of basic life science. The high selective recognition and fluorescence imaging of biomolecules by labeling the models of enzyme acitivity centers could reflect the real existence and concentration distribution of the biomolecular and metal ion in organism, and were benefited to the research of the physiological reactions and pathogenesis, becoming one of the hot issues recently. In this paper, a series of labeled models of enzyme activity center, with polyamine (strong chelator of metal ions) as ligand and traditional fluorescence dyes as indicators, were designed and synthesized. Its applications in detection and fluorescence imaging of the biomolecular and metal ion were then investigated.1. A series of models of copper enzyme activity center, labeling with the traditional fluorescence dyes were assembled, and its applications in detection and fluorescence imaging of nitric oxide (NO) were studied. By incorporating a Rhodamine B moiety as the fluorescent indicator, CuFL2(with tis(2-aminoethyl)amine (Tren) as the chelator) and CuFL3(with1,4,7,10-Tetraazacyclododecane (cyclen) as the chelator) was obtained as models of the copper enzyme activity center. They both gave a rapid (the fluorescence enhanced within30s) and high selective response toward NO through the ring-open of the rhodamine B moiety, causing by the reaction of NO and Cu(Ⅱ). Especially, CuFL2showed a700-fold fluorescence enhancement to NO (quantum yield0.13) with the detection limit of1nM, which is the best of the NO fluorescence sensors reported. And the quantum yield of CuFL3was0.16(detection limit1μM). Furthermore, they both could be applied to the NO imaging in vitro. By introducing1,8-naphthaleneimide as the luminescence unit, CuFL5with ethanediamine was synthesized as another model of copper enzyme activity center. The fluorescence was restoring by the reduction of Cu(Ⅱ) to Cu(Ⅰ), and enhanced by the generation of FL5-NO. CuFL5also exhibited a rapid (the fluorescence enhanced within30s), high selective and high sensitive (detection limit1nM) response to NO, and could also be applied to the NO imaging in vitro. Compared to the enhanced response, the ratio of two signals had more extensive value of application. Therefore, CuFL6(with Tren as the chelator) with both rhodamine and coumarin as the fluorescence unit was obtained as another model of copper enzyme activity center. It gave a fluorescence ratio response toward NO, which was specific. The cell imaging experiment proved that CuFL6could image NO through the fluorescence ratio changes.2. The models of the activity center of glucose tolerance factor (GTF) and CuZnSOD, with two different fluorescent indicators, were constructed based on the FRET mechanism. The detection system FL7:FL8(1:2) of Cr3+was comprised by two parts:a coumarin-labeled glutathione as the model of GTF activity center (FL7), and a rhodamine-labeled glucose as the substrate (FL8). According to the reaction of GTF and glucose in organism, FL7and FL8could be connected together through Cr3+, leading to a ratio response toward Cr3+. It improved the selectivity toward Cr3+with the detection limit of0.1ppm, and be used in cell imaging of Cr3+. In this paper, two different Cu-Cu homonuclear complexes, CuFL1-im-CuFL9and CuFL2-im-CuFL9, and a Cu-Zn heteronuclear complex, CuFL1-im-ZnFL9, as the models of the CuZnSOD activity center with different labels was constructed.It provides a new platform for the research of super oxygen anion detection and imaging. |
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