Rapid Detection Method Study Of Staphylococcus Aureus In Food Based On Nanoenzyme Catalysis

Foodborne diseases caused by Staphylococcus aureus have attracted high attention and attention due to their wide pollution range and bad influence.It needs to be able to detect it accurately and quickly.At present,several commonly used detection methods for S.aureus,such as traditional culture method,enzyme-linked immunoassay experiment,DNA analysis,and other methods,have various deficiencies in the basic rapid field detection scenarios due to their own technical limitations and other reasons.It is necessary to develop a rapid,sensitive and accurate detection method for S.aureus to meet the needs of rapid field detection at the basic level.This project aims to establish and optimize a rapid semiquantitative detection method for S.aureus in polycolorimetric foods.Combined with yolk antibody technology,immunomagnetic separation technology,nano enzyme technology and precious metal color technology,establish S.aureus rapid semi-quantitative visual detection system and implementation for rapid and accurate detection of S.aureus in food,for the scene to provide new detection method,also for other pathogenic bacteria detection technology development to provide new ideas.The main contents were as below:（1）Preparation and evaluation of chicken yolk antibody（Ig Y）specifically recognizing S.aureus.The chicken yolk antibody was precipitated by PEG-6000 and purified.After determination,the heavy and light chains of the prepared yolk antibody were located near 70 k Da and 25 k Da,respectively,and the highest protein content of the yolk antibody was 31.18 mg/m L,and the highest titer was 1:512000,which showed good specificity for S.aureus.S.aureus-specific yolk antibodies were successfully prepared.（2）Fe₃O₄Preparation and evaluation of magnetic nanoparticles,Ag-Mn O₂nanocomplexes,and Fe₃O₄-Ag-Mn O₂nanocomposites.Transmission electron microscopy（TEM）analysis showed that the synthesized Fe₃O₄magnetic nanoparticles had the same size,with an average of 195 nm.Zeta potential analysis shows that the Zeta potential of Fe₃O₄nanoparticles and Ag-Mn O₂nanocomplexes is-57.95 m V and64.06 m V,respectively,which can synthesize Fe₃O₄-Ag-Mn O₂nanocomposites due to electrostatic interactions.The synthetic Fe₃O₄-Ag-Mn O₂nanocomposite has an average size of 213 nm and fluffy ball,showing Fe,Ag,Mn elements by elemental mapping and elemental analysis.The hysteresis loop analysis showed that the saturated magnetization of Fe₃O₄-Ag-Mn O₂nanocomposite is 56.91 emu·g^-1,which is almost 0,and it is superparamagnetic.Triple enzyme activity:peroxidase-like（POD）activity,oxidase-like activity,catalase-like（CAT）activity.（3）Construct the immunomagnetic nanoprobe（IMB）.The Fe₃O₄-Ag-Mn O₂nanocomposite was coupled to S.aureus-specific Ig Y and analyzed by thermogravimetric analysis（TGA）and TEM.The antibody loss coupled on the composite surface was 2.83%.Results showed successful preparation of IMB.（4）Gold nanorods（Au NRs）were prepared and characterized by the seed growth method.The synthesized Au NRs have uniform shape size,an average length of 57.21nm and aspect ratio of 3.36,providing a good basis for etching morphology.（5）Fe₃O₄-Ag-Mn O₂@Ig Y gold nanorod multiple color color system construction.After optimization,the dosage of IMB was 0.5 mg/m L,the p H of the reaction system was 7.0,and the color development time of TMB was 5 min,and the dosage was 100μg/m L.The detection limit of the detection system is 3.7 CFU/m L,the regression equation of the standard curve is Y=-20.465lg C+140.86,R²=0.983,the linear range is10 CFU/m L-10⁶CFU/m L.All detection can be completed within 1 h,using four common pathogens:Listeria monocytogenes monocytogenes,Salmonella typhimurium,Escherichia coli O157:H7,and Vibrio parahaemolyticus.Milk samples and pork samples were selected for real sample simulation and recovery analysis,the detection limit was 5.8 CFU/m L and 9.2 CFU/m L respectively,visual color detection limit was 10CFU/m L,recovery rate of 106%,RSD<20%,good practicability and high accuracy.The experimental results show that a Fe₃O₄-Ag-Mn O₂nanocomposite with triple enzyme activity was synthesized,and a rapid semi-quantitative visual detection method of S.aureus was designed,and the evaluation results have good potential for the rapid detection of S.aureus in food samples.It provides a more extensive detection idea and basis for the rapid detection of S.aureus and the rapid detection of other foodborne pathogenic bacteria.