Rapid Detection Of Salmonella By NMR Based On Superparamagnetic Nanoprobe

Rapid and accurate detection of Salmonella in food is an effective means to prevent and control Salmonella infection.Rapid food detection technology provides key technical support for food safety problems caused by microbial contamination.Salmonella not only poses a major threat to human health,but also causes huge losses to the global food industry.The traditional culture method,immunology method and molecular biology method can not meet people's demand for rapid detection of pathogenic bacteria in food,so it is of great significance to establish efficient and rapid detection methods.This study established a new method for the detection of Salmonella in food with high specificity,high sensitivity,rapid and versatility.In order to improve the sensitivity of the NMR sensor,the streptavidin-biotin system signal amplification method was introduced to enhance the binding amount and binding force to the target.On the basis that the size of target bacteria is much larger than that of probes,the difficulty of separating target signal probe and free probe in homogeneous system is overcome by the membrane filtration technology.Combined with LF-NMR technology,the target bacteria in food samples can be detected quickly,sensitively and specifically.The research contents of each section are as follows:In the first chapter,the research progress of rapid detection of Salmonella and Salmonella was summarized.In addition,the paper briefly introduces MRI contrast agent,magnetic nanoparticles,rapid detection technology of MRI,research status and development trend at home and abroad.The second chapter introduced the preparation and characterization of NMR nanosensors.In order to compare the relaxation properties of the nanoprobe before and after modification with the Antibody and SA,we modified the SMN with antibodies and streptavidin respectively.The zeta data shows that when SA is combined with magnetic beads,the surface negative potential increases from-11 mV to-7 mV,and the hydration particle size increases by 20 nm.The changes of hydrodynamic size and zeta potential indicate that SA successfully binds to SMN surface.FT-IR showed that the characteristic bands of SA protein appeared at 1717 cm-1(indicating the presence of C=O),1583 cm-1 and 1440 cm-1(indicating the presence of N-H and C-N),which further proved that the modification of SA on the surface of SMN was successful.The relaxation properties of the probes were analyzed by NMR,and the relaxation properties of SMN,SMN-SA and SMN-Antibody were weakened accordingly.Finally,PES filter membrane with the highest pass rate of the probe and the best filtration batch was selected as the filter membrane in this experiment.In the third chapter,the rapid detection of Salmonella by NMR based on magnetic signal of SMN-SA-Bt-PcAb nanoprobe was introduced.Rapid detection of Salmonella has important public health significance.In order to effectively monitor food safety,it is necessary to establish portable,fast and effective detection technology.In this paper,a nuclear magnetic resonance(NMR)biosensor based on superparamagnetic nanoparticle(SMN)with ultrasmall particle size is introduced for the detection of Salmonella in milk samples.The free biotinylated antibody specifically binds to the Salmonella in the homogeneous system through the antigen-antibody interaction,and the biotinylated antibody-Salmonella is captured by streptomycin on the SA-modified NMP by specific binding to biotin.The probes that are not attached to the target bacteria are separated by membrane filtration.Finally,the transverse magnetization time(T2)of the filtrate was determined by NMR technique.The optimal parameters were obtained by optimization:the concentration of biotinylated polyclonal antibody and SMN-SA was 1.0 ?g/mL,50?g/mL,and the incubation time with the bacteria was 45 min and 60 min respectively.Under optimal conditions,the whole process can accurately detect Salmonella at levels as low as 10-4 cfu/mL in less than 2 hours.In summary,the NMR biosensor combined membrane filtration technology has the advantages of fast speed,high sensitivity,strong specificity and good anti-interference,and it is a promising platform for the detection of biological macromolecular antigen.In the fourth chapter,a biosensor based on superparamagnetic nanoparticles combined with membrane filtration and low field NMR for rapid detection of Salmonella is introduced.First,the free biotinylated capture antibody and biotinylated detection antibody were specifically bound to different targets of Salmonella in milk samples,using the modified by chain mildew avidin NMP-SA on the chain mildew avidin biotin with specific antibodies to capture the interaction between biotin.Finally,PES membrane filtration was performed on the final reaction system to remove unbound probes,and then the filtrate was analyzed by NMR to indirectly reflect the content of the target substance retained on the filter membrane.The optimal parameters were obtained through optimization:The concentration of biotinylated capture and detection antibody was 0.4 ?g/mL,and the incubation time with bacteria was 45 min and 60 min respectively.The concentration of SMN-SA was 50?g/mL,and the immune reaction time was 30 min at room temperature.This method shows high specificity and anti-interference to Salmonella,and the limit of detection(LOD)in pure culture and real samples was 103 cfu/mL.This method has great application potential for realize rapid and sensitive detection of food-borne pathogenic bacteria in complex actual samples.