Experimental and Computational Studies of a Laccase Immobilized ZnONPs/GO-Based Electrochemical Enzymatic Biosensor for the Detection of Sucralose in Food Samples

Monitoring of high intensity artificial sweeteners in the food industry for environmental and human health has become relevant in recent years. This work exploits the electrocatalytic properties of the laccase enzyme for sensing of a high intensity sweetener sucralose experimentally and supported by computational modeling. The fabrication of the laccase biosensor was achieved using laccase immobilized onto zinc oxide nanoparticles (ZnONPs) capped withp-amino thiophenol (ATP) and covalently attached to graphene oxide (GO) modified glassy carbon electrode (Lac/ZnONPs-ATP-GO/GCE). The developed biosensor exhibited an 8-fold enhancement of differential pulse voltammetry signals compared with the bare GCE at pH 5.0 in a 0.1 M phosphate buffer. The amplification of signals was due to a firm binding of laccase onto the surface of GO through high isoelectric point ZnONPs, exhibiting an enzymatic catalytic activity towards the oxidation of sucralose (SUC) at + 0.25 V (vs. Ag/AgCl). Under the optimized experimental conditions, the anodic peak current linearly increased with the sucralose concentrations ranging from 0.025-0.1 mM (R-2 = 0.9984) and 0.25-1.0 mM (R-2 = 0.9979) with a detection limit (S/N = 3) of 0.32 mu M. Furthermore, the proposed strategy was confirmed by assessing the interactions between the sucralose and the laccase using computational tools. First, the density functional theory (DFT) calculations of SUC revealed a HOMO-LUMO energy gap of - 0.2555 eV, suggesting a great tendency to act as an electron donor. Furthermore, adsorption and sucralose-laccase docking studies were carried out to better understand the redox mechanisms. These results revealed that SUC forms hydrogen bonds with ILE 230 and GLN 228 and other amino acids of the hydrophobic channel of the binding sites, thereby facilitating the redox reaction for the detection of sucralose.