Establishment Of Two Novel Method For The Detection Of Four Foodborne Pathogens By SERS Immunochromatography

Bacterial contamination of food and water has long been a major public health issue and one of the major threats to food safety and agricultural and livestock production.Common foodborne pathogens include Salmonella typhimurium（S.typhi）,which contaminates food,Staphylococcus aureus,S.aureus),Escherichia coli（E.coil）O157:H7 and Pseudomonas aeruginosa（P.aeruginosa）,which contaminates water supplies,cause millions of infections globally each year and lead to a high number of deaths in cases in less developed countries.Early,rapid and accurate identification of food-borne pathogens is of great significance to human public health,food safety and the development of animal husbandry.However,the existing detection methods for foodborne pathogens,such as bacterial culture and PCR technology,have the problems of a long time,low sensitivity and high cost.Therefore,this study proposed two new immunochromatography technologies based on Surface-Enhanced Raman Scattering（SERS）,aiming at the difficulties and pain points in the detection of food-borne pathogens with low sensitivity,and low detection flux.It is used for rapid,high-sensitivity,and low-cost detection of four foodborne pathogens in food.The main research contents and conclusions are as follows:1.Preparation of two new nanomaterials Mag@Au and MoS₂-Au@Ag.Firstly,new magnetic nanospheres Mag@Au and two-dimensional MoS₂-Au@Ag nanosheets were synthesized by the PEI-mediated seed growth method and PEI-based layer-upon-layer self-assembly method.Mag@Au and MoS₂-Au@Ag were characterized by TEM and SEM.The results of TEM and SEM showed the morphologies of the products at each stage of the synthesis of the two novel nanomaterials,indicating the successful synthesis of the materials.The enhancement factors of Mag@Au and MoS₂-Au@Ag were2.49×10⁷ and 1.68×10⁸,respectively,indicating that the prepared two novel nanomaterials had high SERS enhancement ability.The material stability of Mag@Au and MoS₂-Au@Ag is verified.The results show that these two materials have strong batch stability and salt stability.These results indicated that Mag@Au and MoS₂-Au@Ag,two new nanomaterials,could be introduced into the immune chromatography system as excellent SERS labels.2.Establishment of magnetic SERS immunochromatography method for detection of S.aureus and S.typhi based on Mag@Au.The surface biofunctionalization of Mag@Au-modified Raman molecules was performed,and the trapping efficiency of the Mag@Au tag was verified.The results showed that the maximum trapping efficiency（>80%）of the two bacteria could be achieved by modifying the Mag@Au label of the corresponding antibody in 15 min.The visual detection line constructed by mixing 20 nm Au@BSA with detection antibody does not affect the detection effect of the immunochromatographic system,and can accurately locate the detection line.Through condition optimization,it was found that the best detection effect was achieved when the antibody membrane concentration of S.aureus and S.typhi were1.0 and 0.8 mg/m L,10μL Mag@Au label was used,and PBS solution containing 1%Tween 20 and 10%FBS was used.Under optimal conditions,the established magnetic SERS immunochromatographic system can achieve rapid（30 min）and highly sensitive quantitative detection of S.aureus and S.typhi（detection limit of S.typhi is 12 cells/m L,and detection limit of S.aureus is 9 cells/m L）.The established magnetic SERS immunochromatography showed good specificity and did not react to the other 6 non-target bacteria.It also has good repeatability（RSD<8.03%）and high detection accuracy in complex food samples.The average recoveries of S.typhi and S.aureus are between 84.33%and 112.16%,and the relative standard deviation is less than 12.91%.3.Establishment of a one-line SERS immunochromatography method for the detection of E.coil O157:H7,S.typhi,and P.aeruginosa based on MoS₂-DAu@Ag.Three kinds of Raman molecules with non-interfering main peaks,DTNB,MBA,and 2-MPY,were screened out,which laid a foundation for the construction of SERS immunochromatography which could detect three target bacteria on one detection line.According to SEM and TEM images,the MoS₂-DAu@Ag label modified with the corresponding antibody had a strong binding force to the three target bacteria.By optimizing the conditions,it was found that the antibody membranes of E.coil O157:H7,S.typhi,and P.aeruginosa were 1.0,0.8,and 0.8 mg/m L,respectively,and were reactive in PBS solution containing 1%Tween 20 and 10%FBS for 20 min.The detection effect is the best.Under optimal conditions,the established one-line SERS immunochromatography could achieve rapid（20 min）and highly sensitive quantitative detection of the three target bacteria（E.coil O157:H7,P.aeruginosa,and S.typhi detection limits were 41,26 and 34cells/m L,respectively）.The established one-line SERS immunochromatography showed good specificity and did not react to the other 5 non-target bacteria.It also had good repeatability（RSD<11.56%）and high accuracy in complex food samples.The average recoveries of E.coil O157:H7,P.aeruginosa,and S.typhi ranged from 83.38%to 118.81%.The relative standard deviation is less than 9.40%.Two new immunochromatographic methods were successfully constructed to achieve rapid and sensitive quantitative detection of 4 foodborne pathogens,which has a strong application value in the field and early diagnosis of foodborne pathogens.