| Salmonella is a food-borne pathogen that poses great threat to human health and is also one of the common food-borne pathogens in dairy products.Its impact on human health and safety is greater than other food-borne pathogens.Due to the characteristics of high infectivity and high pathogenic rate of Salmonella,as well as the shortcomings of the existing Salmonella detection methods,such as time-consuming and laborious,cumbersome operation and high detection cost,it is necessary to establish a new,rapid and convenient method for the detection of foodborne pathogens to ensure food safety.In this paper,a novel Salmonella detection method based on quantum dots(CdSe/ZnS QD)was developed.A multiplex fluorescent biosensor based on streptavidin(SA)biotin(Bio)system and linear long-chain polymer poly-l-lysine(PLL)was developed for the detection of Salmonella in milk.The main research contents are as follows:Firstly,the preparation and characterization of fluorescent probe in fluorescence immunosensor were studied.This paper focuses on the synthesis of the sensor probe as a signal source with high stability(SA modified CdSe/ZnS QD nanoparticles).The hydration particle size data showed that the original hydration particle size of CdSe/ZnS QD was 15nm,and the hydration particle size of CdSe/ZnS QD-SA was increased to 25nm after modification of CdSe/ZnS QD by EDC/NHSS.TEM showed that the particle size distribution of CdSe/ZnS QD-SA was uniform without agglomeration.In addition,fluorescence spectra showed that the optical properties of CdSe/ZnS QD-SA did not change significantly compared with the original CdSe/ZnS QD.At the same time,agarose gel electrophoresis showed that the migration distance and migration speed of CdSe/ZnS QD-SA were significantly changed compared with that of unmodified CdSe/ZnS QD.These characterization results indicated that SA was successfully coupled to the quantum dots,indicating the successful synthesis of fluorescent probes.Secondly,a synthetic probe was used to construct the detection system,and a multiplex fluorescence biosensor method based on streptavidin(SA)biotin(Bio)system and linear polymer PLL was established to detect Salmonella in milk,and the detection methodology was evaluated.First,Salmonella was captured on a96-well black plate to form a double-antibody sandwich structure with paired Salmonella monoclonal antibody.Secondly,SA was fixed to biotinylated antibody(BT-m Ab)using SA-Bio specificity.Then,biotin-modified poly-l-lysine(BT-PLL)binds SA specifically again through SA-Bio system to achieve signal amplification.Finally,the water-soluble CdSe/ZnS QD labeled SA was added to the black 96-well plate and covalently coupled with BT-PLL.The fluorescence signal of the fluorescence immune complex in the 96-well plate was detected by the layer superposition of SA with biotin and the covalent coupling of biotinylated PLL.By optimizing the main experimental parameters of the detection system,the detection system process achieves the best effect.The optimal process parameters are as follows:The synthesis mole ratio of CdSe/ZnS QD-SA is 1:20.The dosage of biotinylated antibody was 2.5μg/m L,the dosage of SA was 5μg/m L,and the dosage of BT-PLL was 17.5μg/m L.The probe incubation time was 30 min.Under the optimal experimental parameters,the detection limit of Salmonella in PBS buffer was 4.9×10~3CFU/m L.The detection limit in milk was 4.9×10~4CFU/m L,and the recovery were 84%-114%with RSD of 6.55%-9.28%.Therefore,the fluorescence sensor has good applicability for the detection of Salmonella in milk samples.In addition,the sensor has short detection time and good specificity,and can complete the detection of Salmonella in actual samples within 1.5 h.Therefore,the new detection platform established in this study can be applied to the rapid detection of foodborne pathogens in actual samples. |
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