An interface embedded gold nanoparticles in sol-gel thin film was constructed by one-step electrochemical deposition. Acetylcholinesterase (AChE) was physically absorbed onto to the interface to form a thin enzymatic layer. The proposed thin enzymatic layer, having kinetics similar to that of the enzyme in solution, provides an ideal sensing platform to electrochemically evaluate the chemical mechanism of enzyme inhibition. Lineweaver-Burk plot and surface plasmon resonance confirmed that the inhibition of AChE by malathion followed an irreversible mechanism and was a mixed type of competitive and noncompetitive. On the contrary, the degrees of inhibition by Pb2+ and Fe3+ were independent of the incubation time and the AChE concentrations, showing the reversibility of the inhibition. Furthermore, UV-vis absorption spectra indicated that the AChE mediated the hydrolysis of acetylthiocholine to yield a reducing agent thiocholine that reduced Fe3+ to Fe2+ and Fe2+ presented an effect of activation. To meet the demand of the biosensor design, we further investigated the relationship between inhibition percentage and both incubation time and inhibitor concentration. The enzyme's sensitivity to solvent effects and reactivation of the biosensor were also evaluated. It is anticipated that a rapid evaluation of the chemical mechanism of AChE inhibition could paves the way to rationally design biosensors and new compounds, as candidates for the treatment of Alzheimer's disease and pesticides.An interface embedded gold nanoparticles in sol-gel thin film was constructed by one-step electrochemical deposition. Acetylcholinesterase (AChE) was physically absorbed onto to the interface to form a thin enzymatic layer. The proposed thin enzymatic layer, having kinetics similar to that of the enzyme in solution, provides an ideal sensing platform to electrochemically evaluate the chemical mechanism of enzyme inhibition. Lineweaver-Burk plot and surface plasmon resonance confirmed that the inhibition of AChE by malathion followed an irreversible mechanism and was a mixed type of competitive and noncompetitive. On the contrary, the degrees of inhibition by Pb2+ and Fe3+ were independent of the incubation time and the AChE concentrations, showing the reversibility of the inhibition. Furthermore, UV-vis absorption spectra indicated that the AChE mediated the hydrolysis of acetylthiocholine to yield a reducing agent thiocholine that reduced Fe3+ to Fe2+ and Fe2+ presented an effect of activation. To meet the demand of the biosensor design, we further investigated the relationship between inhibition percentage and both incubation time and inhibitor concentration. The enzyme's sensitivity to solvent effects and reactivation of the biosensor were also evaluated. It is anticipated that a rapid evaluation of the chemical mechanism of AChE inhibition could paves the way to rationally design biosensors and new compounds, as candidates for the treatment of Alzheimer's disease and pesticides
