| More and more researchers pay attention to fluorescence analytical methods for its high sensitivity, selectivity,low detection limits other advantages. Among these methods,the hybridization technique of fluorescent probes has more applications in monitoring pathogen microorganisms.In this dissertation the authors synthesized a novel fluorescent probe of rare earth complexes. We studied the fluorescence time of the new complexes. They were applied in the detection of pathogenic microorganisms of the environment. The details are summarized as follows:Four kinds of rare earth europium complexes were synthesized, including complex Eu（TTA）3（5-NH2-phen）, Eu（TTA）3phen, Eu（DBM）3phen and complex Eu（ADBM）3phen. The Eu（TTA）3（5-NH2-phen） complex （ETN） was synthesized using TTA and 5-NH2-phen as the ligands and the Eu（TTA）3phen complex using with TTA and phen instead. The complex Eu（DBM）3phen was synthesized using DBM and phen and the complex Eu（ADBM）3phen was synthesized with ADBM and phen. We did scientific studies on the fluorescence intensity and life time of the four rare earth europium complexes, and then chose the long-lived luminescent europium complex ETN as the biomarker of the probe. The complex ETN had stable light and high fluorescence quantum yield of 0.62. It had active radical as well as long life time （0.68 ms）. It was proved as a kind of fluorescence dye with super luminescence property.（2） A two-probe tandem DNA sandwich hybridization assay including capture DNA1, probe DNA2, and target DNA3 was prepared. The long-lived luminescent europium complex ETN was used as the biomarker. The replace of amino in the particle ETN were easy to label oligonucleotide for time-resolved fluorescence assays. The sequences of Enterobacter cloacae and staphylococcus epidermidis genes were designed using software Primer Premier 5.0 according to the literature. Amino-modified capture DNA1 was covalently immobilized on the glutaraldehyde treated glass slides. The detection was done by monitoring the time-resolved fluorescence intensity from the glass surface after the hybridization reaction with the ETN labeled probe DNA2 and complementary target DNA3. The detection limit of the Enterobacter cloacae DNA was low of 5×10-11 mol L-1, and the detection limit of the of pure staphylococcus epidermidis DNA was low of 8.0×10-10 mol L-1. The results of the present work proved that this new approach was easy to operate with high sensitivity and specificity. It could be conducted as a powerful tool for the detection of pathogen microorganisms in the environment.（3）The original extracted DNA samples from Enterobacter cloacae and staphylococcus epidermidis without any purification and amplification process were directly used in the hybridization assay. The ETN labeled probes were employed for the specific detection of original extracted DNA from Enterobacter cloacae and staphylococcus epidermidis strain. Furthermore, The DNA concentration of Enterobacter cloacae was detected in the total extracted DNA from environment. This novel method was verified forward easier to operate with high selectivity and sensitivity. It is promised to be an important player in the future diagnosis of pathogen microorganisms. |
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