| Food safety has a great impact on people's health,and its strict supervision and control is one of the important tasks of public health.In recent years,food safety problems caused by foodborne pathogens occur frequently,so the detection of foodborne pathogens is very important.Salmonella typhimurium(S.typhimurium)and Escherichia O157:H7(E.coli O157:H7)are the two most common foodborne pathogens reported around the world.The traditional culture method,molecular biology method and immunology are the main technology methods used for the detection of foodborne pathogens,however,the above methods have some shortcomings,especially not suitable for rapid,sensitive and accurate on-site real-time detection.Therefore,it is very important to establish a detection method of foodborne pathogens by using modern analytical technology.Lateral flow immunoassay(LFIA)is a new immunoassay method which combines chromatography with immunoassay technology.LFIA is simple,rapid,sensitive and almost does not need professional equipment,so it is suitable for on-site testing.Antibody and label are the most importment factors to the sensitivity of LFIA.Due to its good chemical stability,light stability and high luminescence,Quantum dots(QDs)is a new type of fluorescent label which can improve the sensitivity and quantitative ability of LFIA.However,there are some problems to need to be solved for QDs,such as poor biocompatibility,instability in complex environment,easy to agglomerate and so on.Core-shell nanomaterials have unique physical and chemical properties such as antiaggregating,super hydrophilicity and antireflection,and can overcome the disadvantages of the single metal nanoparticles(gold or silver)such as uniform particle size and easy to agglomerate,so core-shell nanomaterials have become a hot topic in the field of LFIA in recent years.A new high-performance Si O2-core quantum dots(QDs)–shell nanocomposites(Si O2@PEI-QDs)was synthesized using the polyethyleneimine(PEI)-mediated adsorption method in this study,and served as high-performance fluorescent labels for lateral flow immunoassay.We established a high sensitivity and rapid detection of Salmonella typhimurium via LFIA based on Si O2@PEI-QDs.Secondly,multiplex detection is one of the directions of foodborne pathogen detection technology.In view of the fact that vegetables,fruit juice,milk and other foods may be contaminated by S.typhimurium and E.coli O157:H7 simultaneously,the multiplex LFIA detection methods were further explored.A high-sensitivity and dual-channel detection of E.coli O157:H7 and S.typhimurium was established using the Si O2@PEI-QD-based LFIA strip with specific antibody.The results showed that the two kinds of foodborne pathogens could be detected by this sensitive and rapid double channel LFIA method.Compared with antibodies,polypeptides could be synthesized with low cost,so polypeptides were used as bacterial identification element instead of specific antibody,a dual-channel detection of E.coli O157:H7 and S.typhimurium was established.The main research contents were as follows:1.Preparation and characterization of Si O2@PEI-QDs nanocomposite.First,Si O2 NPs with dimensions of 150 nm were synthesized according to a modified Stfiber method and acted as the supporting core.Second,PEI-coated Si O2 NPs(Si O2@PEI)were prepared under the sonication,during which the cationic polymer PEI quickly self-assembled on the negatively charged Si O2surface.Finally,Si O2@PEI-QDs was formed via electro-static interaction under the sonication.The result of transmission electron microscopy(TEM),the energy dispersive spectroscopy(EDS),Zeta potential and fluorescence properties showed that the particle size of the nanocomposite microspheres was 182 nm(8nm in the middle layer of PEI),and the prepared Si O2@PEI-QDs had excellent stability and high luminescence,could be served as a new high-performance fluorescent label for LFIA.2.A Si O2@PEI-QDs-based LFIA strip for the detection of S.typhimuriumFirst,the egg yolk antibody of S.typhimurium for LFIA was prepared,then the prepared egg yolk antibody and the commercial antibody of S.typhimurium were compared through the capture experiment and the capture antibody-detection antibody paired screening experiment to find the antibody with high capture efficiency,easy availability and low price.The screening results showed that when the commercial S.typhimurium antibody was used for both capture antibody and detection antibody,it had higher specificity and better detection effect.Therefore,it was selected as the capture antibody and detection antibody of LFIA method.Second,the antibody of S.typhimurium was conjugated with the prepared Si O2@PEI-QDs serving as fluorescent labels,then Si O2@PEI-QDs-based LFIA strip for the detection of S.typhimurium was established.The results of method validation showed that the fluorescence immunoassay based on Si O2@PEI-QDs was simple,specific and sensitive.The running time for detection was within 15 min.This method had good specificity and could identify S.typhimurium specifically,whereas all nontarget bacteria group such as Listeria monocytogenes,Acinetobacter baumannii and Staphylococcus aureus showed no fluorescene signal on the test line.The results of food simulated samples of tap water and milk showed that the sensitivity of the Si O2@PEI-QD-based LFIA strip for the detection of S.typhimurium was as low as 5×102 CFU·m L-1 in milk sample.The accuracy and reproducibility of the method were good,and the detection results were consistent with the traditional plate culture method.In addition,the sensitivity of Si O2@PEI-QDs strip was 20 times higher than that of the commercial QDs nanocomposite-based LFIA strip.The high-performance Si O2@PEI-QDs nanocomposites was used as fluorescent label for LFIA-based detection of S.typhimurium for the first time.3.Dual-channel detection of S.typhimurium and E.coli O157:H7using the Si O2@PEI-QDs-based LFIA strip with antibody as the recognition element.In order to realize multiplex detection of foodborne pathogens,a dual-channel detection of S.typhimurium and E.coli O157:H7 was established using the Si O2@PEI-QDs-based LFIA strip and the antibody was used as the recognition element.The conditions of the LFA detection system such as the nitrocellulose membrane,buffer solution,detection time and detector antibody concentration were optimized.The specificity,sensitivity and reproducibility of the established method were evaluated.Besides,the sensitivity of the established method was compared with traditional colloidal gold immunochromatography.Under the optimized conditions,the results showed that dual-channel detection based on the Si O2@PEI-QDs was sensitive and specific.The visualization limit of the fluorescence signal of the Si O2@PEI-QDs-based strip was 1×104CFU·m L-1for S.typhimurium and E.coli O157:H7.The limit of detection(LOD)of the Si O2@PEI-QD-based strip was 1×103 CFU·m L-1 for S.typhimurium and E.coli O157:H7.The sensitivity of immunochromatography based on the Si O2@PEI-QDs in this study was100 times higher than that of traditional colloidal gold immunochromatography.In the detection of food simulated samples such as vegetables,fruit juice and milk,the recovery rate of the method was85%?123%,and the reproducibility was good.The results were consistent with that of the traditional plate culture method.Besides,the method was simple and could detect two kinds of foodborne pathogenic target bacteria within 15 minutes simultaneously,which improved the detection efficiency,reduced the cost and had a good practical application prospect.4.Dual-channel detection of S.typhimurium and E.coli O157:H7using the Si O2@PEI-QDs-based LFIA strip with polypeptides as the recognition element.Polypeptide is a new type of bacterial recognition element which can be chemical synthesized and had good stability.the polypeptides-modified Si O2@PEI-QDs composite were prepared by streptavidin-biotin interaction.The surface of Si O2@PEI-QDs was modified by polypeptide of the target bacteria,and served as a new fluorescent label of LFIA.A dual-channel decetion of S.typhimurium and E.coli O157:H7 was established using the Si O2@PEI-QDs-based LFIA strip using polypeptide as the recognition element of the bacteria.The results showed that dual-channel detection method based on the Si O2@PEI-QDs and polypeptide was sensitive and specific.The visualization limit of detetion was 1×104CFU·m L-1both for S.typhimurium and E.coli O157:H7.The limit of detection of the fluorescence signal of the Si O2@PEI-QD-based strip was 1×103CFU·m L-1both for S.typhimurium and E.coli O157:H7.The sensitivity of LFIA based on polypeptide recognition was similar to that of LFIA based on antibody,while polypeptides could be synthesized and had good stability.Therefore,in the absence of high-performance antibody,polypeptides could be served as an alternative bacterial specific recognition element.In conclusion,A LFIA for the detection of foodborne pathogens based on high-performance Si O2@PEI-QDs fluorescent label had been established for the first time,and the sensivity of which was better than those of commercial QDs nanocomposite-based LFIA strip and traditional colloidal gold immunochromatography.By using spetific antibody and polypeptide respectively,a dual channel detection of S.typhimurium and E.coli O157:H7 was established.The methods had the advantages of simple operation,high sensitivity,multi-channel and quantitative detection ability.It had good application in food simulated samples such as vegetables,fruit juice and milk,could meet the needs of immediate detection of foodborne pathogens,and had a good practical application prospect.This method could also be extended for the detection of other foodborne pathogens,providing a new technology and method for on-site real-time detection of foodborne pathogens. |
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