Fluorescent Silica Nanoparticles Labeling Based Detection Method Of Food-borne Pathogen

Food safety is the serious public health problems over the world, and food-borne disease caused by pathogenic microorganisms is one of the biggest dangers to food safety. So the fast and sensitive detection of food-borne pathogens is of great importance for food safety. At present, various detection methods have been reported for food-borne pathogen, but each has their advantages and disadvantages, which urged researchers to find more effective analytical methods with wide applicability, high sensitivity, and simple operation. The 21st century's two leading technology, nanotechnology and biotechnology meet people's needs. Nanomaterials are revolutionizing the development of bioprobes and biosensors. The use of nanomaterials to construct nanoparticle probes and sensors, can improve the analytical sensitivity and stability. And the new proposed analytical methods therefore have much better and more widely practical applications. In the present work, we studied the applications of dye-doped silica nanoparticles in immunoassay and DNA hybridization analysis by coulping with high sensitive fluorescence detection method. We used human IgG and sequence-specific oligonucleotides of Salmonella, Staphylococcus aureus and Listeria monocytogenes as the model analytes to establish new analysis method system, and achieved ultratrace detection with satisfactory results.Firstly, a reverse microemulsion method was successfully used to prepare good disperse, uniform Ru(bpy)32+-encapsulated fluorescent silica nanoparticles. And TEM and spectrofluorometric measurements were used to characterize the nanoparticles. Then the as-prepared nanoparticles were used as label for immunoassay of human IgG. Under the optimized conditions, the calibration graph for IgG was linear over the range 1~100 ng/mL with a detection limit of 0.3 ng/mL(3σ). The RSD for 11 parallel measurements of 30 ng/mL IgG was 2.2%. This method has been successfully applied to detect IgG in human serum samples.Secondly, the prepared Ru(bpy)32+-encapsulated fluorescent silica nanoparticles was modified with avidin on the surface. Then the biotinylated Salmonella sequence-specific indicator DNA was effectively bound on the nanoparticle surface to construct DNA-nanoparticles probes. Subsequently, a sandwich-type DNA hybridization analysis was conducted, in which the Salmonella target DNA were first hybridized to the Salmonella captured oligonucleotide probes immobilized on polystyrene microwells, and then the fluorescent nanoparticles modified indicator oligonucleotides were assembled through DNA hybridization and used as probes to monitor the presence of the sequence-specific Salmonella target DNA. The content of Salmonella target DNA was quanitied by the final fluorescent intensity. Under the selected conditions, it was found that the fluorescent intensity is proportional to the concentration of Salmonella target oligonucleotides over the range of 10~1000 fmol/L with a detection limit of 3 fmol/L (3S/N), and the relative standard deviation is 3.3% (300 fmol/L, n=11.). In the same method, we also developed a novel fluorescent bioconjugated nano-probes based detecttion method for Staphylococcus aureus sequence-specific DNA. It was found that the fluorescence intensity is proportional with the concentration of Staphylococcus target DNA over the range of 3~1000 fmol/L with a detection limit of 1 fmol/L. The proposed methods have been successfully applied to detect Salmonella and Staphylococcus aureus in real bacteria samples, respectively.Finally, based on molecular beacon and fluorescence nanoparticles labelling techniques, we constructed a novel sensitive detection method for Listeria monocytogenes target DNA in homogeneous system. In this experiment the FITC-IgG complex performs as nuclear materials, and core/shell type FITC fluorescence silica nanoparticles were successfully synthesized and effectively prevented the disclosure of FITC with a traditional method. Then FITC-IgG-encapsulated fluorescent silica nanoparticles was labeled to 5'end and Au nanoparticles was labeled to 3'end of Listeria monocytogenes sequence-specific molecular beacon probe to constructed molecular beacon fluorescent nanoprobe for the detection of Listeria monocytogenes DNA in homogeneous solution. Under the selected conditions, it was found that the fluorescent intensity is proportional to the concentration of Listeria monocytogenes target DNA over the range of 1~200 nmol/L with a detection limit of 0.3 nmol/L (3S/N), and the relative standard deviation is 2.6% (50 nmol/L, n=11.).