Rapid Detection Of Salmonella By LF-NMR Based On PAMAM-mediated Functionalized Nanoparticle Probe

Salmonella is one of the main causes of food-borne infectious diseases,which is a serious threat to public health.It can enter the food supply chain at all stages of production,processing and sales.Therefore,the high prevalence of Salmonella requires reliable and efficient detection methods.Traditional detection methods based on plate counting and real-time polymerase chain reaction are time-consuming and laborious,and there is an urgent need to establish a new rapid and accurate detection method.Magnetic nanoparticles combined with low-field NMR technology is an effective method for rapid detection of food-borne pathogens.Immunomagnetic nanoparticles directly capture Salmonella antibodies with high dosage and low sensitivity.In this study,in order to reduce the cost and improve the sensitivity,a functionalized modified superparamagnetic nanoparticle(SPIONs)probe based on polyamidoamine(PAMAM)mediated detection of Salmonella in milk combined with low-field nuclear magnetic resonance.The main research contents are as follows:1?Preparation and characterization of low-field nuclear magnetic resonance biosensor probes.In the preparation process of the biosensor probe,it is necessary to synthesize the signal source biotin-modified bovine serum albumin nanoparticles(BT-BSA-SPIONS)and the signal amplifier streptavidin-modified dendrimer-amine(SA-PAMAM).The optimal process conditions for probe synthesis were determined by single factor experiments.The structure and morphology of the prepared BT-BSA-SPIONs were analyzed by means of zeta potential,fourier transform infrared spectroscopy(FT-IR)and scanning electron microscopy(SEM).It was found that the prepared BT-BSA-SPIONs were successfully modified and had good dispersion properties.The SA-PAMAM was characterized by zeta potential and fourier transform infrared spectroscopy,and it was found that SA was successfully modified on the surface of PAMAM.2?A method for rapid detection of Salmonella by LF-NMR based on biotinylated nanoparticles was established.The entire capture operation was carried out in an enzyme-labeled plate.The antigen was immobilized in a sandwich of double antibodies,and then a signal source was added.The antigen captured by the probe is finally eluted,the eluate is collected,and the T₂ signal value is measured.Through LF-NMR detection,the results can be obtained within 2 minutes,and the entire operation process is about 3 hours.The LF-NMR detection system based on biotin-modified nanoparticles constructed in this study has a detection limit of 10⁵ CFU/mL in PBS and milk spiked samples.Compared with other methods,this method has good specificity and convenient operation.3?Rapid detection of Salmonella by LF-NMR based on PAMAM-mediated functionalized nanoparticle probes.Use the synthesized probe to construct the detection system,use the double antibody sandwich method to immobilize the target bacteria in the ELISA plate,add SA-PAMAM and functionally modified superparamagnetic nanoparticles in sequence,and then use the eluent to supersed the signal substance.The magnetic nanoparticles are eluted,and the target bacteria are detected by measuring the T₂ signal with a low-field nuclear magnetic resonance instrument.By optimizing the detection system,the best parameters are obtained:the amount of coating antibody is 0.5?g/mL,the blocking agent is 2%BSA,the amount of biotinylated antibody is 0.5?g/mL,the amount of SA-PAMAM is 0.75?g/mL,the amounts of magnetic beads were 10?g,the incubation time was 30 min.Under optimal conditions,the low-field NMR biosensor can quickly detect Salmonella in milk within 3 hours,with a detection limit of 10⁴ CFU/mL.In addition,the LF-NMR biosensor shows good specificity and anti-interference performance.Therefore,the innovative detection platform in this paper can detect foodborne pathogens sensitively,quickly and efficiently.