Research On Rapid Detection Method For Salmonella Aptamer Based On Photothermal And Catalytic Properties Of Nanomaterials

Salmonella typhimurium is a foodborne pathogen that poses a serious risk to both human health and food safety,so currently,traditional culture methods for the detection of Salmonella typhimurium have many advantages,but there are still deficiencies such as cumbersome operation,time consuming and laborious,high cost and unsuitable for field real time detection.It is important to establish a portable,rapid and sensitive detection method for the diagnosis and treatment of foodborne pathogens.Although rapid detection technology has the advantages of convenience,high efficiency and low cost,it is easily affected by subjective factors and its sensitivity needs to be improved.Therefore,a current research focus is still on developing a rapid,low cost,simple,and highly sensitive method of detecting Salmonella typhimurium.In this study,the excellent properties of nanomaterials,such as simulating natural enzyme catalysis and photothermal effect,were used to effectively improve the detection sensitivity.At the same time,a quick and accurate biosensor for Salmonella typhimurium was built using aptamers,which have a small molecular weight,excellent chemical stability,ease of chemical modification,and low cost.After the analysis of the risks and current state of Salmonella typhimurium detection,two rapid detection methods were established:the detection method of the Salmonella typhimurium aptamer based on Au nanoparticles（AuNPs）photothermal effect,and the catalytic detection method of Prussian blue colloidal gold（PB@Au）nanomaterials.The main research contents of this thesis are as follows:In the first part,the detection method of Salmonella typhimurium based on the photothermal effect of AuNPs was established.Streptavidin and biotinylated aptamer chain were coated to nitrocellulose membrane（Nitrocellulose membrane,NCM）and closed the NCM to obtain the aptamer strip.The closed colloidal gold-aptamer（AuNPs-Apt）probe was incubated with Salmonella Typhimurium to the strip test area to observe the color and calculate the temperature difference（ΔT）to establish a standard curve with the quantity of Salmonella Typhimurium to realize quantitative photothermal detection.The results of the thesis optimization of the conditions for developing the rapid detection technique are as follows:the concentration of sodium chloride bound by the colloidal gold and the aptamer was 1 mol L-1,the pH was 8.0,and the aptamer concentration was 1.2μmol·L^-1,the strip blocked BSA concentration was 2%,Streptavidin biotinylated aptamer molar ratio of 1:6,the blocking solution concentration of the AuNPs-Apt probe was 0.5%,the concentrations of Mg²⁺,Ca²⁺and K⁺in the probe binding buffer with Salmonella typhimurium were 1 mmol·mL^-1,0.2 mmol·mL^-1 and 1 mmol·mL^-1,respectively,the laser irradiation time was 35 s.Under the best experimental conditions,the detection limit of the strip was determined to be 96 CFU·mL^-1 according to the linear regression equation Y=0.3396X-0.1940,R²=0.9761,the recovery rate of the detection method was between 80.1%and 117%,and it had excellent specificity,repeatability,and stability.In the second part,the Salmonella detection method based on the catalytic effect of PB@Au nanomaterials was established.Catalytic colorimetric assay and photothermal quantification of Salmonella typhimurium using composite peroxidase activity and photothermal effect.First,the aptamer was initially immobilized on the surface of the PB@Au complex after the synthesis conditions were optimized.Moreover,the biotinylated aptamer was bound to streptavidin and enclosed at the base of the enzyme labeling wells.Based on the double aptamer sandwich principle,the PB@Au-Apt probe was immobilized in the mapping wells by incubation with Salmonella typhimurium.After adding the spectrophotometric method was established by measuring the absorbance value at 460 nm.TheΔT of the enzyme well was calculated by laser irradiation,and a standard curve was established with the number of Salmonella typhimurium to realize quantitative photothermal detection.In this paper,the establishment conditions of the rapid detection method were optimized,and the results were as follows:the amount of chloric acid prepared by PB@Au was 320μL,the catalytic optimum pH was 6.5,the time period was 25min,H₂O₂ volume of 3μL,the DAB dosage was 2.5 mg,the measured Miian constant（Km）is3.09×10^-3mol·L^-1,laser irradiation power and time were 2.5 W and 120 s,the concentrations of the enzyme strip blocked BSA and coated streptavidin biotinylated aptamer were 1%and 200μmol·L^-1,respectively,PB@Au-coupled to streptavidin-biotinylated aptamer molar ratios and pH were 1:2 and 6.5,respectively.Under the optimal experimental conditions,according to the linear regression equation Y=0.04361X+0.01340,R²=0.9572,the catalytic colorimetric detection limit was 47 CFU·mL^-1;According to the linear regression equation Y=0.5000X-0.05333,R²=0.9794,the photothermal detection limit was 23 CFU·mL^-1,the detection method specificity,repeatability and stability were good,and the recovery rate in the actual food sample were 80.3%to 115%.Two rapid detection methods for Salmonella typhimurium have been developed based on the catalytic and photothermal conversion capabilities of nanomaterials.These methods were used in food samples for quick,sensitive,and accurate detection of Salmonella typhimurium.These new detection methods were being investigated to show that them had a wide range of potential applications in the detection and control of Salmonella typhimurium.