The Study On The Detection Methods For Salmonella Typhimurium And Staphylococcus Aureus Based On Time-resolved Fluorescence Nanoparticles

Foodborne pathogens are the main factors that cause food safety accidents.Although conventional foodborne pathogens detection methods are quite reliable,the preparation work is laborious,time consuming and unable to meet the rapid detection needs.Time-resolved fluorescence technique can distinguish the fluorescence lifetime of the fluorescent substance contained in the sample,and thus eliminates the background fluorescence and offers a signal with remarkably high signal-to-noise ratio.It has been widely used in clinical medicine,however,the application in food detection fields is rare.In this study,we synthesized two kinds of time resolved nanoparticles,combining with magnetic separation,aptamer recognition and time-resolved fluorescence resonance energy transfer method?TR-FRET?,a series of rapid,accurate and sensitive methods for the detection of foodborne pathogens were developed.Firstly,Na YF₄:Ce/Tb nanoparticles were synthesized through facile one pot solvothermal route,combining with the reported Salmonella typhimurium?S.typhimurium?aptamer,a highly sensitive and selective TR-FRET aptasensor was developed for the identification of S.typhimurium.Under the optimal conditions,the detection linear range of S.typhimurium covered from 10² to 10⁶ cfu/m L?y=0.796-0.096x,R²=0.9932?with a limit of detection as low as 25 cfu/m L.The application of this bioassay in real sample was also investigated,and the results were consistent with those of plate-counting method.Therefore,this simple and rapid method owns a great potential in the application of food safety.Secondly,Fe₃O₄ magnetic nanoparticles?MNPs?and another time-resolved fluorescence nanoparticle?TRNPs?were synthesized.Both of these two TRNPs?Na YF₄:Ce/Tb and Na Gd F₄:Eu?can be excited at the same wavelength and the emission wavelength do not overlap.Na YF₄:Ce/Tb and Na Gd F₄:Eu TRNPs modified with S.typhimurium and Staphylococcus aureus?S.aureus?aptamers respectively were used as signal probes.The MNPs immobilized with S.typhimurium and S.aureus aptamers separately were used as capture probes.We established a dual-color time-resolved fluorescence aptasensor for simultaneous detection of S.aureus and S.typhimurium.Under the optimal conditions,the detection linear range of S.typhimurium covered from 10² to 10⁵ cfu/m L?y=10,213x-12,208.92,R²=0.9922?with a limit detection of 15 cfu/m L and the detection linear range of S.aureus covered from 10² to 10⁵ cfu/m L?y=4803.20x-1933.87,R²=0.9982?with a limit detection of 20 cfu/m L.The application of this bioassay in real sample was also investigated,and results were consistent with those of plate-counting method.Therefore,this simple and rapid method owns a great potential in the application for the multiplex analysis in food safety.Finally,by employing Na YF₄:Ce/Tb,Na Gd F₄:Eu TRNPs and high quenching efficiency of tungsten disulfide?WS₂?,a dual-color time-resolved fluorescence resonance energy transfer based assay was established for simultaneous determination of S.typhimurium and S.aureus.Under the optimal conditions,the detection linear range of S.typhimurium covered from 10² to 10⁶ cfu/m L?y=8670.59x-11372.41,R²=0.9905?with a limit detection of20 cfu/m L and the detection linear range of S.aureus covered from 10² to 10⁶ cfu/m L?y=4613.55x-5332.39,R²=0.9962?with a limit detection of 33 cfu/m L.The bioassay was successfully applied to the determination of S.typhimurium and S.aureus in real sample and results were consistent with those of plate-counting method.Therefore,this simple and rapid method owns a great potential in the application for the multiplex analysis in food safety.