Study On Detection Method Of Foodborne Pathogenic Bacteria Based On Au@Ag NPs/solid-phase SERS Substrates

At present,the main problem in the field of food-borne pathogen detection is that the rapid detection of pathogenic bacteria and the distinction between live and dead bacteria cannot be achieved.Therefore,the research on rapid,sensitive,cheap and convenient detection methods for food-borne pathogenic bacteria is of great significance to ensure food safety and human health.Surface-enhanced Raman scattering(SERS)technology can obtain a complete microbial fingerprint.The SERS-based pathogen detection method has the advantages of high sensitivity,high selectivity,fast reaction time,and simple sample preparation.In this study,two SERS substrates with filter paper and slides as solid substrates were constructed to identify foodborne pathogens and specific quantitative detection based on aptamers.The main research work includes:Firstly,a method based on Au@Ag NPs/filter paper substrate for the detection of five food-borne pathogens(Salmonella typhimurium,Staphylococcus aureus,Escherichia coli,Shigella flexneri,Listeria monocytogenes)was constructed for the identification and differentiation of different bacterial species.The good water absorption of the filter paper fixes the bacteria on the substrate,increases the contact between the bacteria and the substrate,and enhances the bacterial SERS signal.The results showed that the positions of the main peaks of the five bacterial SERS spectra were close,but the relative intensities of the vibration peaks were significantly different,and different bacterial species had unique Raman peaks.Combining the principal component analysis(PCA)method with the bacterial SERS spectrum can effectively classify and identify five bacteria.At the same time,this method was used to quantitatively detect Staphylococcus aureus and Salmonella typhimurium,and the minimum detection concentration was 10~5 cfu/m L.Secondly,a"sandwich"method for detecting Staphylococcus aureus based on aptamer functionalized Au@Ag NPs/slide substrate was constructed to achieve specific and quantitative detection of bacteria.Based on the specific binding of aptamer and target,the sandwich structure of the"capturing base-target-signal molecular probe"was constructed.At the same time,the Au@Ag NPs solution was added dropwise on the substrate to capture the bacteria to further cover the surface of the bacteria to build more hot spots and improve detection sensitivity.Thus,SERS detection of Staphylococcus aureus was realized.Under the optimal experimental conditions,the logarithmic value of the intensity of ROX SERS and the number of Staphylococcus aureus colonies showed a good linear relationship in the concentration range of 10~2 cfu/m L?10~7 cfu/m L(y=422.181x+125.389,R~2=0.9926)with a detection limit of 34cfu/m L.The specificity analysis revealed that the method was higher specificity towards the corresponding target.The results of the standard recovery of milk samples were similar to those of the plate counting method,and the standard recovery was 90.73%?102.55%,indicating the accuracy and reliability of the developed method.Finally,an aptamer-based method for the detection of Staphylococcus aureus without immobilization of Au@Ag NPs/slide substrate was constructed.This method eliminates the need for bacterial immobilization and improves the convenience and efficiency of detection.The substrate that binds to the ROX-aptamer through electrostatic interaction has a certain SERS signal,and then based on the specific binding of the aptamer to the target bacteria,the ROX-aptamer falls off the surface of the substrate,resulting in a decrease in the signal intensity of the substrate,thus achieving the detection of Staphylococcus aureus.Under the optimal experimental conditions,a good linear relationship was found between ROX relative SERS intensity and the number of staphylococcus aureus colonies within the concentration range of10~2 cfu/mL?10~7 cfu/m L(y=795.527x-401.229,R~2=0.9926)with a detection limit of 31cfu/m L.The specificity analysis revealed that the method was higher specificity towards the corresponding target.The results of the standard recovery of milk samples were similar to those of the plate counting method,and the standard recovery was 90.60%?107.26%,indicating the accuracy and reliability of the developed method.