Cdte Quantum Dot Based Detection Method For Pathogenic Bacteria And Macrophages

Since food-borne diseases caused by food-borne pathogen is one of the biggest dangers to food safety which has been concerned by all the countries, and each reported detection methods for food-borne pathogen has its shortcomings, developing a higher sensitivity, wider applicability, more rapid and convenient new detection method is very meaningful in order to meet the need of detection. The application of semiconductor quantum dots in life science has caused extensive concern at home and abroad, especially in the the application research as fluorescent probe, because of its good biocompatibility, strong light stability and high yield. However, sensitivity of single quantum dot labeling technology in many application studies is not ideal, and there is little report about the quantum dot assembly technic which can significantly improve the detection sensitivity. Based on this, we used nano-probe technology and fluorescence immunoassay in DNA hybridization analysis, and made sequence-specific oligonucleotides of Salmonella, Staphylococcus aureus and Listeria monocytogenes as the model analytes to establish new ultra-trace amount analysis system. Meanwhile, we used CdTe QDs as fluorescent marker to analyse the images of different physiological status of macrophages from mouse, and further broaden the application of quantum dots.Firstly, we synthesized CdTe QDs in an aqueous solution and characterized by TEM and fluorescence spectra. Then one nanogold was connected with hundreds of quantum dots to construct signal amplified probes using short DNA chain. Combining with the Staphylococcus aureus specific sequences DNA capture probes, we captured specific sequencestarget DNA by hybridization to form a "sandwich" composites which were fixed on the micro well plate and detected the concentration of target DNA which was quantified by the fluorescent intensity of the signal probes. Under the optimized conditions, the fluorescent intensity is proportional to the concentration of Staphylococcus aureus target DNA over the range of 10~1000 fmol/L with a detection limit of 10 fmol/L, and the relative standard deviation is 3.0% (500 fmol/L, n=11). At the same time, the fluorescent intensity is proportional to the Staphylococcus aureus amounts which were calculated by standard flat counting in the same concentration over the range of 20~7000 cfu/mL, and the detection limit is 20 cfu/mL. At last, the method was assessed by analyzing the fluorescent intensity of Staphylococcus aureus in milk samples. Results showed that the new established detection method has good accuracy and can be extended to the real sample testing.Secondly, a reverse microemulsion method was used to prepare good disperse, uniform amino-silica nanoparticles which were characterized by TEM. Using short DNA chain as the medium, a silica nanoparticle could be connected with hundreds of CdTe QDs as signal amplification probes. Then we synthesized amino-functionalized magnetic nanoparticles though hydrothermal method which were combined with corresponding capture DNA as capture probes. We simultaneously detected Salmonella, Listeria monocytogenes and Staphylococcus aureus through the similar DNA hybridization model. Under the optimized conditions, the detection limit of Salmonella target DNA is 20 fmol/L, the detection limit of Listeria monocytogenes target DNA is 25 fmol/L, the detection limit of Staphylococcus aureus target DNA is 22 fmol/L. Meanwhile, the detection limit of Salmonella colony number is 30 cfu/mL, the detection limit of Listeria monocytogenes colony number is 20 cfu/mL, the detection limit of Staphylococcus colony number is 20 cfu/mL.Finally, quantum dots with different colors emission were used as cell imaging luminescent markers to label macrophage-specific antibodies. A successful test for mouse macrophages of different physiological status was carried out using antibody functionalized QDs with green and orange colours. Combining with the capture characteristic of magnetic nanoparticles, we simplified the traditional purification steps for macrophages and successfully observed the macrophages of two status under the fluorescent microscope at the same time. A new method for monitoring immune stress changes of body has been eatablished.