Study On Electrochemical Immunosensor For Detection Of Listeria Monocytogenes In Milk

Food safety is related to our daily life and health closely. In recent years, there are a growing number of food safety incidents, in which the most important is foodborne illness caused by pathogen. While Listeria monocytogenes is widely distributed, virulent and high mortality, it plays an important role in the pathogen. Therefore, the development of rapid and sensitive detection of Listeria monocytogenes is of great significance in the field of food safety.At present, there are some methods to detect Listeria monocytogenes, such as traditional culture method, enzyme-linked immunosorbent assay and polymerase chain reaction, and so on. Although these methods played important roles in detecting Listeria monocytogenes, they are time-consuming and complicated to operate. So they can not meet the demand of food safety supervision and inspection. The high specificity antibody-based immune-sensing technology will be the main way to solve rapid and sensitive detection of Listeria monocytogenes.Recently, more and more people pay attentions to the electrochemical immunosensor who is fast response, simplicity, sensitivity and low cost and so on. The research will aim to develop a rapid and sensitive electrochemical immunosensor to meet the need for real-time detection of Listeria monocytogenes. Specific research contents are as follows:1. Preparation and labeling of Listeria monocytogenes polyclonal antibodyUsing short-range immunological methods, anti-Listeria monocytogenes serum was gotten after eight times immunization of rabbits. The antiserum was purified by the method of octanoic acid-saturated ammonium sulfate precipitation. Then, SDS-PAGE was used for identification of the effect of antibody purifications. The concentration of the purified antibody was20mg/mL which was measured by the BCA Protein Assay Kit. The results of indirect ELISA showed that the titer of purified antibody were over1:96,000. Then, using a modified sodium iodide method to prepare HRP-labeled Listeria monocytogenes polyclonal antibody, the conjugate of HRP-pAb was identified by direct ELISA, the labeling efficiency was0.8, which was measured by UV-visible spectrophotometric. 2. Prcparation of anti-Listeria monocytogenes monoclonal antibodyBALB/c mice were immunized with subtractive immunization, the mixture of Listeria innocua and Listeria welshimeri was used to immunize mice in the tolerization procedure, then Listeria monocytogenes cells as immunogen in the immunization procedure. Cell fusion was selected a mouse with higher titers. After2-3times subcloned,15hybridoma cell strains that can secret antibody stably were screened, and then5strong positive cell lines named C41G11H7, B41B9D5F10, B42D2G4G4, C41B8G7H10, C42B5D12H10were selected to prepare ascites. B41B9D5F10was purified by the method of octanoic acid-saturated ammonium sulfate precipitation. The results of indirect ELIS A showed that the titer of purified antibody were over1:106. The concentration of the purified antibody was1.0mg/mL which was measured by the BCA Protein Assay Kit. Its affinity constant was2.47x1010L/mol, and the immunoglobulin subclass was IgG1.3. Construction of the electrochemical immunosensor and application for the detection of Listeria monocytogenesThe self-assembled monolayer on a gold electrode was formed by3-mercaptopropionic acid. Mouse monoclonal antibody of LM was immobilized on the SAM through a stable acyl amino ester intermediate generated by the co-addition of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide. Then the sandwich-based electrochemical immunosensor with Chronoamperometry was developed. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed to characterize the stepwise assembly of the immunosensor in a solution containing2.5mmol/L Fe(CN)63-/Fe(CN)64"(1:1).Under optimized conditions, the immunosensor has application to detect LM in PBS, the results showed the response current was correlated to the amount of LM linear ranging from102to106CFU/mL (R2=0.9883), the detection limit was102CFU/mL and the detection time only need200s. Compared with traditional culture counting method, it was found that the relative standard deviation of two methods was low than8%. The sensor was shown to have good specificity when using Staphylococcus aureus, Shigella, Salmonella and E. coli O157:H7as controls. There was no need for sample pretreatment when applied to detect LM in milk, and the detection range was103-106CFU/mL. What’s more, sample turbidity was not required. It is suitable for the rapid detection of the actual sample.Anti-LM monoclonal antibidy with high specificity and anti-LM polyclonal antibody were prepared in this research. And, electrochemical immunosensor that was constructed immunoassay with electrochemical technologies used for rapid and sensitive detection of LM. This method is not only simple, fast and low cost, but also easy operation. There are prospects for practical application in terms of food security, public health and environmental monitoring.