| Objective:The novel nano-flouresent materials, named solid-state fluorescence carbon nanodots (SCDs), were combined with immunochromatography technology and bio-imaging technology to achieve rapid and specific detection of microorganism and monitoring of total bacteria under field conditions, to provide new selected technical means for microbial detection/monitoring.Methods:1. Synthesis and optical properties analysis of SCDs:SCDs were synthesized by microwave method, the macro and micro structure of SCDs were characterized and influence of the spectral characteristics, fluorescence stability, photo bleaching solution pH, organic solvents and ions to the fluorescence quantum efficiency of SCDs were researched. The feasibility of SCDs is applied in the biological field as a fluorescent probe was explored.2. Research of SCDs applied in Dry rapid detection:with the SCDs as fluorescent markers and specific antibodies of Yersinia pestis as a biological probe, the principle of double-antibody sandwich immunochromatography mode based Y. pestis SCDs immunochromatographic test strips were prepared and its detection performance was evaluated systematically.3. Research of SCDs applied research in the liquid fast Monitoring:with the SCDs as the new non-toxic fluorescent dyes, rapid staining for all types of bacteria and HeLa cells was set up and the staining mechanism of SCDs was investigated, the staining performance had been compared with a commercial dye SYTO9. Then, SCDs rapid staining technology and flow cytometry were combined for rapid monitoring and systematically evaluation of total bacteria in the sample.Results:1. The SCDs which can emit fluorescence in both liquid and solid state was synthesized. Its high quantum yield was 37% and 28% in solid and liquid respectively. It has strong fluorescence stability, resistance against photo bleaching and can be stored for a long time in dark; the pH value from 3 to 11,the organic reagent including 10mg/ml PEG20000, 10mg/ml SDS,10mg/ml BSA,1% Triton X-100, 1% Tween20 and ions Na+, K+, Cl-,PO43- of concentration 0.3mol/L-1mol/L have no significant effect on fluorescence of SCDs (P> 0.05). However, Zn2+, Cd2+, Mg2+, Ca2+, SO42-, Fe3+, Fe2+, Hg2+, NO3- have different degrees of inhibition on fluorescence intensity of SCDs (P<0.05).2. In the dry-fast detection, the SCDs-based plague immunochromatographic test strip has been successfully prepared by optimizing labeling conditions and sample handling liquid. It can achieve specific detection of Y. pestis. The visually positive test with brownish yellow change, the strip sensitivity is up to 105CFU/ml.3. In the liquid fast monitoring, SCDs could quickly and efficiently stain all kinds of bacteria undifferentiated without binding to biological impurities. Penetration of SCDs to different bacteria are not identical, the lmin penetration of Staphylococcus aureus, Escherichia coli, Bacillus anthracis spores were 6%,6.7%,5%, respectively; Nucleic acid had no effect on the fluorescence intensity of SCDs (P>0.05), while fluorescence intensity of SYTO9 would increase after nucleic acid binding (P<0.05).Staining effects of the two dyes are good enough. Their fluorescence intensity of stained bacteria was not weakened either after soaking 2hours or saving from light 30 days. Coupled with flow cytometry, bacteria stained with SCDs can be distinguished with unstained bacteria and other impurities by the fluorescence intensity. The number of bacteria stained with SCDs that counted by cytometry and pure stained bacteria are linearly correlated(r=0.995, P<0.05).Conclusion:This study we synthesized a solid-state light emission SCDs having good optical properties. Such that make the novel detection and monitoring technology based on SCDs as fluorescent markers and fluorescent dye can obtain excellent microbiological properties, so as to provide an alternative fluorescent nanophobes for developing a novel type of biological detection technique. |
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