Amperometric biosensor based on horseradish peroxidase-immobilised magnetic microparticles

Magnetised silica-based microparticles (MMPs) (5 μm) 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 (0.3 μmol/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.