Esterase-2 Mutant-based Nanostructured Amperometric Biosensors for the Selective Determination of Paraoxon (Neurotoxin)

Organophosphate pesticides (OPs) are causing non-selective inhibition in enzymatic bioreceptors, thus the enzymatic-inhibition-based traditional assays are not suitable for their specific detection in food and environmental samples. Accordingly, a selective nanostructured electrochemical biosensing system was designed using six mutants of the esterase-2 (EST2 protein) enzymes from A. acidocaldarius to be exploited as targeting bio-receptors for the specific detection of OPs. Each of the EST2 mutant enzymes was immobilized on disposable screen-printed electrodes modified with Aluminum oxide (AlO)/Copper (Cu) nanocomposite. Consequently, chronoamperometric assay was fully optimized, and cross-reactivity study was carried out using paraoxon, malathion and chlorpyrifos. The comparative cross-reactivity study was performed on the six mutant proteins in terms of inhibitory percentage over a wide range of pesticide concentrations. Eventually, a wide dynamic inhibition range was achieved while the limit of detection for the paraoxon toxicity was 0.01 nM and the limit of quantification was 0.05 nM. Finally, paraoxon was selectively determined using the newly developed EST-based biosensor in different spiked food samples.