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Abstract: 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. [Copyright &y& Elsevier]

Abstract: Magnetised silica-based microparticles (MMPs) (5μm) with a high density of nanopores were used for horseradish peroxidase (HRP) immobilization and amperometric biosensor development in batch conditions. The resulting biosensor was applied to study the peroxidation of clozapine (CLZ) which is a dibenzoazepine drug often used in the treatment of neurological disorders. The amperometric response corresponded to the electroreduction of CLZ-oxidized products namely a nitrenium cation and quinoneimine derivative of CLZ. Despite a relatively low amount of immobilized HRP (0.3μmol/g), clozapine quantification in the micromolar concentration range was achieved by the use of a magnetized solid paraffin carbon paste electrode for microparticles attraction. Diffusion of substrate and products of the enzyme reaction within the nanopores were identified as limiting factors in the biosensor response. This amperometric biosensor configuration has number of interesting advantages such as ease and reproducible microparticle layer renewing, low enzyme consumption, controlled surface immobilization, protective enzyme microenvironment etc. [Copyright &y& Elsevier]