Comparative Study Of Three Methods For Detection Of Foodborne Pathogens Based On Two Kinds Of SERS Labeled Immunoassay

The number of foodborne diseases caused by foodborne pathogens is increasing,which poses a serious threat to global public health and food safety.Traditional pathogen detection methods are time-consuming and laborious,and require strict experimental operating environment.Therefore,it is very important to develop a highly sensitive and accurate pathogen detection technology to ensure food safety and human health.Surface enhanced Raman scattering(SERS)is a kind of highly sensitive inelastic scattering.This technique can measure the sample adsorbed on the surface of rough metal particles by ordinary Raman spectroscopy,and the Raman intensity of the sample is increased by 10³-10⁶ times.Surface enhanced Raman scattering has been widely used in environmental analysis,food safety,biomedical analysis and other fields due to its high sensitivity and fast detection speed.The purpose of this paper is to design two kinds of SERS tags for the rapid detection ofEscherichia coli O157:H7,Listeria monocytogenes and Salmonella typhimurium,and optimize the detection scheme to provide a theoretical basis for the rapid detection of pathogenic bacteria in food.The main research contents are as follows:1.Establishment of SERS labeled immunoassaySERS labeled immunoassay is a method that combines surface enhanced Raman scattering with labeled immunoassay.It uses the high accuracy and spectral selectivity of surface enhanced Raman spectroscopy,combined with immune reaction,to label the target nano immunoassay technology.Firstly,the bacterial aptamers were prepared Fe₃O₄@Au as the magnetic capture tool and SERS substrate of target bacteria,the target bacteria can be enriched and separated from the sample solution,which can be verified by electron microscopy and capture efficiency experiments.Secondly,gold nanoparticles(Au NPs)need to be prepared as auxiliary enhancement particles,modified with Raman molecule DTNB to provide SERS signal,and then labeled with the specific recognition of the target bacteria(such as antibodies).The obtained nanomaterials were used as SERS tags.By optimizing the size of gold nanoparticles,the modification amount of Raman molecule DTNB and the modification time,the optimal size of gold nanoparticles was 50 nm,and the optimal modification amount of Raman molecule DTNB was 20?M and the best modification time was 4 h.Finally,based on the establishment of SERS labeled immunoassay,two kinds of SERS tags were prepared for the detection of Escherichia coli O157:H7,Listeria monocytogenes and Salmonella typhimurium.2.The SERS label based on antibody functionalization is used for the detection of three food-borne pathogensBased on the establishment of SERS labeled immunoassay,this study designed a kind of SERS tag functionalized with bacterial antibody to detect three kinds of foodborne pathogens in food samples.After the prepared Au NPs were modified with Raman molecule DTNB,they were coupled with antibodies against Escherichia coli O157:H7,Salmonella typhimurium and Listeria monocytogenes,respectively,as SERS tags.Using bacterial aptamer-modified gold-shell magnetic beads as the capture substrate,after completing the capture of Escherichia coli O157:H7,Listeria monocytogenes and Salmonella typhimurium,the antibody functionalized SERS tag is added.The optimal parameters are 2?L of aptamer gold-shell magnetic beads,45 min for capturing bacteria,10?g of bacterial antibody modification,and30 min for antibody functionalized SERS tags.The Raman test results of the three bacteria showed that the detection limits of Escherichia coli ATCC 35150,Listeria monocytogenes ATCC 19115 and Salmonella typhimurium ATCC 14028 were all 100 cells/mL under the optimal detection conditions.Finally,the specificity and repeatability experiments were carried out in biological samples to verify the feasibility of the method.3.A functionalized SERS tag based on Staphylococcus aureus protein A is used for the detection of three food-borne pathogensOn the basis of the establishment of the SERS labeled immunoassay,a kind of SERS tag functionalized with Staphylococcus aureus protein A was designed to detect three kinds of food borne pathogens in food samples.The prepared Au NPs were modified with Raman molecule DTNB and coupled with Staphylococcus aureus protein A as a universal SERS tag.Using bacterial aptamer-modified gold-shell magnetic beads as the capture substrate,after the capture of Escherichia coli,Salmonella typhimurium and Listeria monocytogenes were completed,the SERS tag functionalized with Staphylococcus aureus protein A was added.The optimal parameters are that the optimal modification amount of Staphylococcus aureus protein A is 15?g,the amount of antibody added is 0.8?g,the antibody incubation time is 20 min,and the optimal reaction time of the general SERS label is 20 min.The results of the Raman test of the three bacteria showed that under the optimal detection conditions,the detection limit of Escherichia coli ATCC 35150 was 10 cells/mL,the detection limit of Listeria monocytogenes ATCC 19115 was 10 cells/mL and the detection limit of Salmonella typhimurium ATCC 14028 was 25 cells/mL.Finally,the specificity and repeatability of the method were tested in biological samples,and compared with the traditional colloidal gold strip method to verify the feasibility of the method.Conclusion:On this basis,we established the SERS tag based on antibody modification and the SERS tag based on Staphylococcus aureus protein A modification,and completed the detection of three kinds of foodborne pathogens in food samples.Compared with the antibody used to modify the SERS labels,the detection limit was 100 cells/mL.The established SERS tags modified by Staphylococcus aureus protein A for the detection of three food-borne pathogenic bacteria markers improved the detection sensitivity.The limits of detection for Escherichia coli ATCC 35150,Listeria monocytogenes ATCC 19115 and Salmonella typhimurium ATCC 14028 were 10 cells/mL,10 cells/mL and 25 cells/mL,respectively,and Staphylococcus aureus protein A modified SERS labels used as a general-purpose SERS labels,in food safety detection and diagnosis of infectious diseases has great potential.