A novel cholinesterase assay for the evaluation of neurotoxin poisoning based on the electron-transfer promotion effect of thiocholine on an Au electrode

A simple and cost-effective cholinesterase assay is presented. Thiocholine (TCh), which is generated from substrates by the action of cholinesterase, promotes the electron transfer between [Fe(CN)6]3–/4– and Au electrodes. The electric current increases with the TCh concentration; therefore, the current decreases when cholinesterase is inhibited by neurotoxic chemicals, such as organophosphorus and carbamate compounds. First, we monitored the responses of acetylcholinesterase to cholinesterase inhibitors. The substrate acetylthiocholine was digested with acetylcholinesterase in Tris−HCl buffer, then the reaction mixture was combined with acetonitrile. A polished Au electrode was immersed in the mixed solution for 30 s to immobilize the enzymatic hydrolysis product, TCh, on the electrode surface. The electrode was then subjected to differential pulse voltammetry in 0.5 mM [Fe(CN)6]3– in the presence of KCl as a supporting electrolyte, with a concentration as low as 1 mM. The optimum conditions for the enzymatic treatment were for 10 min at 30 °C with 1.25 mU/mL acetylcholinesterase and 100 μM acetylthiocholine as final concentrations. The inhibition of acetylcholinesterase activities from DDVP (organophosphorus) and methomyl (carbamates) could be monitored. This system was applied to human serum samples containing butyrylcholinesterase. The inhibition of butyrylcholinesterase by DDVP and methomyl were successfully monitored even in human serum. Finally, real human blood donated by 10 volunteers were subjected to a cholinesterase assay using this system. The results from the present method showed good correlation with those obtained from the conventional method using 5,5′-dithiobis-(2-nitrobenzoicacid). The present method was considered valid as a novel ChE assay for human blood.