Ultrasensitive peptide-based multiplexed electrochemical biosensor for the simultaneous detection of Listeria monocytogenes and Staphylococcus aureus.

A novel electrochemical biosensor is reported for simultaneous detection of two of the most common food-borne pathogens: Listeria monocytogenes and Staphylococcus aureus. The biosensor is composed of an array of gold nanoparticles-modified screen-printed carbon electrodes on which magnetic nanoparticles coupled to specific peptides were immobilized via streptavidin-biotin interaction. Taking advantage of the proteolytic activities of the protease enzymes produced from the two bacteria on the specific peptides, the detection was achieved in 1 min. The detection was realized by measuring the percentage increase of the square wave voltammetric peak current at 0.1 V versus a Ag/AgCl reference electrode in ferro/ferricyanide redox couple after incubation with the bacteria protease. The integration of the specificity of the bacterial enzymes towards their peptide substrates with the sensitivity of the electrochemical detection on the sensor array allows the rapid, sensitive and selective quantification of the two bacteria. Outstanding sensitivities were achieved using this biosensor array platform with limit of detection of 9 CFU mL for Listeria monocytogenes and 3 CFU mL for Staphylococcus aureus. The multiplexing capability and selectivity of the array voltammetric biosensor were demonstrated by analysing samples of Staphylococcus aureus, Listeria monocytogenes or E. coli and also containing a mixture of two or three bacteria. Using this biosensor, the two bacteria were successfully quantified simultaneously in one step without the need for DNA extraction or amplification techniques. This platform offers promise for rapid, simple and cost-effective simultaneous detection of various bacteria. Graphical abstract.