Voltammetric sandwich immunoassay for Cronobacter sakazakii using a screen-printed carbon electrode modified with horseradish peroxidase, reduced graphene oxide, thionine and gold nanoparticles.

The authors describe a sandwich-type of electrochemical immunoassay for rapid determination of the foodborne pathogen Cronobacter sakazakii (C. sakazakii). Polyclonal antibody against C. sakazakii (anti-C. sakazakii) and horseradish peroxidase were immobilized on a nanocomposite consisting of reduced graphene oxide, thionine and gold nanoparticles (AuNPs) that was placed on a screen-printed carbon electrode (SPCE). Thionine acts as an electron mediator which also shortens the electron transfer pathway from the conjugated HRP to the electrode surface and amplifies the electrochemical signal. The AuNPs, in turn, improve the electron transfer rate and increase the surface area for capturing antibody. The morphologies of the electrodes were characterized by means of field emission scanning electron microscopy. The electrochemical performance of the immunoassay was evaluated by cyclic voltammetry and differential pulse voltammetry. Under optimal experimental conditions, the electrochemical immunoassay, best operated at a woking potential of -0.18 V (vs. Ag/AgCl) and scan rate of 20 mV/s has a linear response that covers the 8.8 × 104 to 8.8 × 108 CFU·mL-1 C. sakazakii concentration range, with a 1.0 × 104 CFU·mL-1 detection limit (at an S/N ratio of 3). The assay was applied to the detemination of C. sakazakii in spiked infant milk powder and gave recoveries ranging from 92.0 to 105.7%. Graphical abstract A sandwich-type electrochemical immunosensor was designed for C. sakazakii based on the use of rGO. TH, HRP, antibody and AuNPs were anchored on rGO. The nanocomposites were used as traces tag and H2O2 as enzyme substrates. AuNPs were modified on SPCE by electrodeposition.