Development of amperometric horseradish peroxidase based biosensors for clozapine and for the screening of thiol compounds

Two amperometric biosensors with immobilized horseradish peroxidase (HRP) were developed for the investigation of the clozapine drug oxidation and for thiols screening based on biosensor signal inhibition. The HRP was retained either in magnetized nanoporous silica microparticles (MMPs) or in a carbon paste (CP). The latter served for the carbon paste electrode while the MMPs were attracted in close proximity of a magnetized carbon electrode. The potential use of these configurations for drug oxidation and inhibition studies was illustrated by the enzymatic oxidation of clozapine (CLZ) in the presence of hydrogen peroxide. The biosensor signal corresponded to the electro-reduction of CLZ oxidation products namely a nitrenium ion (CLZox) generated by the enzyme HRP. Several thiols reactive towards CLZox were investigated and the biosensor signal inhibition (IC50) was comparatively determined. A protective effect of the nanoporous silica and carbon paste matrices towards HRP inactivation was inferred by comparing the biosensor inhibition results with those obtained with the free enzyme in solution.