Construction Of A Rapid Detection Method For Common Foodborne Pathogens

Foodborne disease is a major threat to public health safety in the worldwide scale.Salmonella has been a major cause in a large number of outbreaks of foodborne illness.Therefore,the establishment of a rapid,accurate and sensitive pathogen detection method has become an inevitable requirement,so as to more effectively protect the supply of food chain and ensure the safety of public health.The fluorescence biosensing has become a research central issue.Relevant research have shown that the fluorescence sensing platform based on nanomaterials can be used for the rapid detection of small molecules(ATP,DNA,micro RNA).A group reported a metal organic framework material(MOF-La)based on lanthanide element,which can be used to construct a fluorescence sensing platform for nucleic acid.The novelty of MOF-La is that the fluorescence change of the fluorescence nanoprobe based on the MOF-La is dependent on the charge(positive charge or negative charge)of the labeled fluorescence molecule on the aptamer.Therefore,in our project,we expected that a new material can be synthesized,which is similar to MOF-La for the rapid detection of Salmonella detection platform.Zeolitic Imidazolate Frameworks(ZIFs)is a large family of Metal-Organic Frameworks(MOFs),which is a coordination polymer constructed from Zn²⁺and imidazole ligands.As a classic ZIFs material,ZIF-8 has been successfully applied in many fields.However,the fluorescence quenching ability of ZIF-8 is worse than that of traditional nanomaterials(for example,gold nanoparticles,graphene oxide,quantum dot).Therefore,the application of ZIF-8 in fluorescence biosensing was limited.Therefore,in our project,ZIF-8 could be modified by two different methods and its fluorescence quenching performance was changed,so we could obtain a new type of nanomaterial with unique fluorescence quenching performance,which could be used to construct fluorescence nanoprobe.In the synthesis process of ZIF-8 material,the reaction system was composed of the different organic solvents respectively.The morphology and structure of them was characterized by TEM,SEM,XRD and other characterization methods.We selected the methanol as the organic solvent for the ideal reaction system.The response effect of FAM-P1 and ZIF-8 assembled fluorescent nanoprobe to target chain P1'was investigated.The results showed that the trend of the fluorescent molecule FAM was decreased in the process of the self-assembly of ZIF-8/FAM-P1and then increased in the response to the target chain P1'.In the modification process of ZIF-8,one method was to directly exchange Zn²⁺of ZIF-8 with lanthanide ions ZIF-8,generating three different lanthanide doped materials(La-ZIF-8,Nd-ZIF-8 and Ce-ZIF-8),and they were characterized by TEM.The results showed that the three lanthanide doped materials could not maintain the original the morphology.The response effects of three fluorescence nanoprobes based on La-ZIF-8,Nd-ZIF-8,Ce-ZIF-8 and FAM-P1 to target chain P1'were investigated.The results showed that the trend of the fluorescent molecule FAM was decreased in the self-assembly of La-ZIF-8/FAM-P1 or Nd-ZIF-8/FAM-P1 or Ce-ZIF-8/FAM-P1and then increased in the response to the target chain P1'.Another method was to indirectly modify the surface of ZIF-8 with La³⁺and square acid.The morphology and structure of the generated material La-sq-ZIF-8 was characterized by TEM,TEM-EDS-mapping,XRD,FT-IR,etc.The results showed that the framework of ZIF-8 was still existing,and La³⁺was evenly distributed on the surface of the material.The response effects of two fluorescence nanoprobes based on La-sq-ZIF-8,FAM-P1 and TAMRA-P1 to target chain P1'were investigated respectively.The results showed that the fluorescent molecule FAM was decreased in the process of the self-assembly of La-sq-ZIF-8/FAM-P1 and then decreased in the response to the target chain P1'.The fluorescent molecule TAMRA was decreased in the process of the self-assembly of La-sq-ZIF-8/TAMRA-P1 and then increased in the response to the target chain P1'.The results showed that the nanomaterial La-sq-ZIF-8 was synthesed by modifed the surface of ZIF-8 with La³⁺and square acid,which be a new material with unique fluorescence quenching performance.The aptamers P1 for Salmonella typhimurium was be modified by the FAM(FAM with negative charge)or TAMRA(TAMRA with positive charge),forming the fluorescence probe which can combine with La-sq-ZIF-8.In the assembly of two fluorescence nanoprobes,La-sq-ZIF-8/FAM-P1 and La-sq-ZIF-8/TAMRA-P1,the fluorescence of FAM and TAMRA was partially quenched by La-sq-ZIF-8.When the target DNA P1'was add to the reaction system,the fluorescence phenomenon was completely opposite.The fluorescence intensity of FAM was further decreased and the fluorescence intensity of TAMRA was restored.The detection sensitivity of two fluorescence nanoprobes for target chain P1'with different concentrations was investigated,and the results showed that the detection limit could be as low as 1 n M.In the vitro detection,the two fluorescent nanoprobes showed good detection ability between the cell concentrations of Salmonella typhimurium 10²-10⁹ cfu/m L.We concluded that two fluorescent nanoprobes can be used for Salmonella typhimurium in the vitro detection,with the high sensitivity and high accuracy.