Study On Rapid Detection Of Salmonella In Milk By Biofunctionalised Magnetic Nanoparticle Sensor Based On Nuclear Magnetic Resonance

Salmonella is a common food-borne pathogens,which is harmful to human health seriously,but the traditional cultural methods,immunoassays and molecular biology methods could not meet the demend of rapid detection of food-pathogens in food,so it is necessary to establish an efficient and fast detection methods to control it.When the magnetic nanoparitcles are chose to water proton,the local magneic field of the water proton could be effectively changed,and the transverse relaxtion time?T₂?of the water proton is reduced.Under the effection of coupling agent?EDC/NHSS?,the Salmonella antibody was conjugated with nanoparticles duo to its abundant functional groups on the surface to provide targeting.In this paper,the magnetic nanoparticles modified with antibody were used as probe,and then a NMR biosensor was successful developed for the rapid detection Salmonella in the milk.In order to control the temperature accurately and serve for the rapid detection,the temperature control equipment was redesigned and equiped with internet technology to make it intelligent,accurate and simple.Each chapter are described as follows:The first chapter summarized the research progross of the rapid and sensitive detection of target by NMR biosensor,and the application of the progress of the NMR corlllary equipment in different areas.In the second chapter,preparation and characterization of different kinds of magnetic nanoprobes.In order to compare the relaxation signal output of nanoprobes prepared by different modification methods on the NMR biosensor,Fe₃O₄MNP-Antibody,Fe₃O₄ MNP-SA and Fe₃O₄ MNPC-SA were synthesized.Compared wih mother magnetic nanoparitcles,the hydrodynamic size of the Fe₃O₄MNP-antibody and Fe₃O₄ MNP-SA were increased by 29 nm and 12 nm respectively.Therefore,the increment in hydrodynamic size indicated that streptavidin and antibody modified successfully via the amidation reaction.Nanoparticle clusters were prepared by PLL and the optimal amount for synthesis of Fe₃O₄ MNPC was 8?g.After conjugation with streptavidin,the hydrodynamic size of Fe₃O₄ MNPC-SA was increased by 118 nm and the zeta potential of Fe₃O₄ MNPC surface changed from-30.57 mV to-25.63 mV.In comparison with Fe₃O₄ MNPC,the dydrodynamic size and zeta potential of Fe₃O₄ MNPC-SA increased significantly,indicating the successful modification with streptavidin.Through the relaxation analysis of nanoparticles,there was a“diffusion limited case”that would reduce the effect of transverse relaxation time of the water protons.In the third chapter,NMR biosensor for rapid detection based on nuclear magnetic resonance of nanoparticles was developed.Combination with 96 plate and double sandwich to immobilize the targer pathogens and then nanoprobe immobilized on the 96-well microplates were eluted,thereby reducing the transverse relaxation time?T₂?of neighbouring water molecules.The optimal experimental parameters included the following:0.5?g/mLcapture antibody and 0.5?g/mL biotin-antibody were added to the 96-well microplates and 10?g nanoprobe were used to lable the target pathogen and incubated for 30 min,then nanoprobes were eluted by glycine-Hcl?0.05 mol/L,pH=2.6?.Under the optimal parameters,the NMR biosensor would take 150 min to complete all the tests and the method detected Salmonella at 10⁵CFU/mL in milk,which also showed high selectivity over other non-target bacteria.The proposed biosensor could be a potential tool for the sensitive and rapid detection of foodborne pathogens in food.