Rapid detection of Listeria monocytogenes in milk by self-assembled electrochemical immunosensor

Listeria monocytogenes (LM) is a gram-positive food-borne pathogen, which can cause serious disease. Rapid screening plays an important role in the prevention and identification of LM to cause food poisoning. In this study, we describe a novel electrochemical immunosensor by self-assembled monolayers (SAM)-modified gold (Au) electrodes for the detection of LM. Mouse monoclonal antibody of LM was immobilized on the SAM through a stable acyl amino ester intermediate generated by the co-addition of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then, a direct sandwich format was developed, in which a polyclonal antibody for LM was conjugated to horseradish peroxidase (HRP) as the enzyme label. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed to characterize the stepwise assembly of the immunosensor. In the presence of H2O2, HRP catalyzes the oxidation of thionine, whose back electrochemical reduction was detected on an Au electrode at −0.3 V for 200 s with chronoamperometry measurements versus an Ag/AgCl reference electrode. The reaction conditions were optimized, such as antibody concentration, immune reaction time, activation time, and blocking time. Under optimized conditions, when the concentration of LM is ranging from 102 to 106 CFU/ml, the response current which is obtained from the labeled HRP to thionine–H2O2 system and the amount of LM are linearly related (R2 = 0.9883). There was no need for sample pretreatment when applied to detect LM in milk. Compared with plate count assay, it was found that the relative error of two methods was lower than 8%. The sensor is a straightforward and reliable method for analysis of LM with a simple operation, sensitivity at a low cost.