The Preparation Process Of PDA Immunosensor And Application In Salmonella Detection In Milk

Food poisoning caused by pathogenic bacteria contaminating food poses a serious threat to human health and also causes huge economic losses to the global food industry.The cases of food poisoning caused by Salmonella often top the list.To solve this problem,it is necessary to establish an efficient and sensitive method for pathogenic bacteria detection.The currently available detection techniques,including traditional detection methods and polymerase chain reaction（PCR）,all have their own limitations.To solve this problem,it is necessary to establish an efficient and sensitive method for Salmonella detection.As a new rapid detection technology,electrochemical immunosensor has received a lot of attention from the industry in recent years due to its advantages of easy operation,rapid efficiency and low detection cost.In this paper,using Salmonella as a model bacterium,an electrochemical immunosensor based on polydopamine/CoFe-MOFs@Nafion nanocomposite was developed for the detection of Salmonella in milk.The research contents are as follows:（1）Construction,characterization and optimization of BSA/Ab/PDA/CoFe-MOF@Nafion/GE immunosensor:The nanomaterials of CoFe-MOFs compounded with Nafion are uniformly coated on the electrode surface by drop coating method;formation of polydopamine nanoparticles by cyclic voltammetry on the CoFe-MOF@Nafion/GE surface and immobilization of antibodies by covalent binding of reactive groups on the surface of polydopamine;closing the excess active site on the surface of polydopamine with bovine serum protein,the BSA/Ab/PDA/CoFe-MOF@Nafion/GE immunosensor was obtained.Modifying the electrode with CoFe-MOFs can increase the effective electroactive area of the electrode,providing more active sites for antibody immobilization and improving the sensitivity of the sensor.Based on the application of the Randles-Sevcik equation,the effective electroactive area of the gold electrode（GE）,CoFe-MOFs@Nafion/GE and PDA/CoFe-MOFs@Nafion/GE were 0.1313 cm²,0.1597 cm² and 0.1395 cm² respectively,indicating that CoFe-MOFs significantly increased the active site of antibody immobilization.When the electrodes are modified with single CoFe-MOFs,the material is easy detachment,therefore,in this paper,Nafion,which has good film-forming properties,is compounded with CoFe-MOFs so that the CoFe-MOFs are firmly bonded to the electrode surface.On this basis,dopamine is polymerised by means of an electro-polymerisation process,which allows parameters to be set to control the amount of dopamine polymerised on the surface of the electrode,allowing the sensor to achieve optimum detection performance.The sensor construction process was characterised by scanning electron microscopy（SEM）,differential pulse voltammetry（DPV）and AC impedance（EIS）to optimise the detection performance of the BSA/Ab/PDA/CoFe-MOF@Nafion/GE immunosensor through optimisation experiments.（2）Study of the mechanism of dopamine polymerisation:The immobilization of antibodies by polydopamine depends on the functional group of the dopamine polymerization product,so the mechanism of the electropolymerization of dopamine has been investigated.The electropolymerisation curve of dopamine was found to have two pairs of distinct redox peaks.By varying the CV sweep rate and fitting the redox current values to the sweep rate,on the basis of previous research results,deducing the possible polymerisation process of dopamine on the surface of gold electrodes.The results show that the electropolymerization of dopamine on the surface of gold electrodes is a reversible redox reaction.The first pair of redox products are dopamine and dopaminequinone,and the second pair of redox products are 5,6-dihydroxyindoline and Dopaminechrom,where the oxidation reaction occurring in dopamine is controlled by diffusion and the oxidation occurring in 5,6-dihydroxyindoline has adsorption diffusion co-control.（3）Detection performance analysis of BSA/Ab/PDA/CoFe-MOF@Nafion/GE immunosensors:using[Fe（CN）₆]^3-/4-as a redox probe,evaluation the immunosensor performance under optimal experimental parameters by recording changes in current values before and after the capture of Salmonella by DPV.The results showed that the sensor exhibited good linearity in the range of 1.38×10²～1.38×10⁸ CFU m L^-1,the detection limit is 10² CFU m L^-1 and it has good resistance to interference,reproducibility and stability.