| Listeria monocytogenes(L.monocytogenes)is a common foodborne pathogen that can cause symptoms such as sepsis and meningitis in humans after infection,and can even lead to death in severe cases.Consumption of food contaminated with bacteria is the main source of human infection with L.monocytogenes,especially the food which is not sufficiently heated such as fresh fruits,vegetables,meat,and raw milk.Therefore,it is important to establish a rapid,accurate and sensitive detection method for the monitoring and prevention of food poisoning caused by L.monocytogenes.The magnetic separation technology based on magnetic nanoparticles(MNPs)is an efficient target separation and enrichment technique,by modifying different biorecognition molecules to realize the separation of specific targets.Meanwhile,the magnetic separation platform can also be used as a dispersed solid-phase extraction substrate for the construction of biosensor,combined with various signal markers to achieve signal conversion and analysis,and further realize high-sensitivity detection of targets.In this study,a series of detection methods for L.monocytogenes in food were established based on the novel cefepime magnetic separation platform combined with enzyme-catalyzed signal amplification methods.The contents of each chapter are described as follows:Chapter one:The research progress of magnetic separation technology and detection methods of foodborne pathogens based on magnetic separation platform is reviewed.Chapter two:A magnetic separation strategy for L.monocytogenes based on Cefe-affinity recognition was established,and a colorimetric assay based on horseradish peroxidase(HRP)-labeled catalysis was constructed for specific detection of L.monocytogenes.Firstly,the preparation of Cefe-MNPs capture probes was verified through various characterization methods,and the magnetic separation parameters were optimized,and the magnetic capture capacity of Cefe-MNPs on L.monocytogenes was evaluated.Moreover,monoclonal antibody(m Ab)was used as the second recognition molecule in this study,and an enzymatic signal amplification colorimetry for the specific detection of L.monocytogenes was established.The detection conditions of the proposed method were optimized,and the specificity and sensitivity of the method were evaluated.Under the optimal conditions,the limit of detection(LOD)of this method for L.monocytogenes in PBS and spiked lettuce sample were both 3.1×10~2 CFU/m L,with good specificity.Chapter three:A“flexible”magnetic separation strategy mediated by polyethylene glycol(PEG)“molecular arms”was established,and the Cefe-PEG-MNPs capture probes were prepared for the separation and enrichment of L.monocytogenes.In addition,gold nanoparticles(Au NPs)were used as the carrier to simultaneously load m Ab and HRP,so as to provide specific recognition ability and increase the labeling amount of HRP.An enzyme-catalyzed signal amplification colorimetric method was finally constructed based on a sandwich strategy to detect L.monocytogenes.Firstly,PEG and Cefe were coupled successively on the surface of MNPs,and the preparation of Cefe-PEG-MNPs was verified by a variety of characterization methods,and the magnetic separation parameters were optimized,and the magnetic capture capacity of Cefe-PEG-MNPs on L.monocytogenes was evaluated.Moreover,the synthesis conditions of Au NPs-HRP/m Ab were optimized,the preparation of Au NPs-HRP/m Ab was characterized,and the specificity and sensitivity of enzyme-catalyzed signal amplification colorimetry were evaluated.Under the optimal conditions,the LODs of L.monocytogenes in PBS and spiked lettuce sample were 3.1×10~1 CFU/m L and3.1×10~2 CFU/m L,respectively,with good specificity.Chapter four:A“brush-like”magnetic separation strategy mediated by polyglutamic acid(PGA)was established,and Cefe-PGA-MNPs capture probes were prepared for the separation of L.monocytogenes in food.The m Ab-labeled mesoporous palladium-platinum(Pd Pt)nanozyme was prepared,and a nanozyme-catalyzed signal amplification colorimetric method was constructed based on a sandwich strategy for the detection of L.monocytogenes.Firstly,the preparation of Cefe-PGA-MNPs and Pd Pt-m Ab were verified by various characterization methods,the magnetic separation parameters were optimized,and the magnetic capture performance of Cefe-PGA-MNPs on L.monocytogenes was evaluated.Moreover,the catalytic performance of Pd Pt nanozyme was explored,and a signal acquisition and analysis platform based on smartphone was established,and the detection conditions of the nanozyme-catalyzed colorimetry were optimized.Under optimal conditions,the LODs of L.monocytogenes in PBS,spiked lettuce sample and watermelon juice sample were 3.1×10~1 CFU/m L,3.1×10~1 CFU/m L,and 3.1×10~2 CFU/m L,respectively,with good specificity.In addition,the results of standard recovery experiments show that the recovery of this method was between 96.5%and 116.4%,which has excellent reliability.In summary,this study established three detection methods for L.monocytogenes based on cefepime magnetic separation platform combined with enzyme-catalyzed signal amplification colorimetry,and the detection performance in food samples was evaluated.The results showed that the established methods had good specificity,accuracy and sensitivity,which provide theoretical reference and techniques for the detection of food safety in our country. |
