Horseradish peroxidase and toluidine blue covalently immobilized leak-free sol-gel composite biosensor for hydrogen peroxide

Abstract

The enzyme horseradish peroxidase and the water-soluble mediator toluidine blue were covalently immobilized to 3-aminopropyl trimethoxy silane precursor through glutaraldehyde crosslinker. A rigid ceramic composite electrode was fabricated from this modified silane along with graphite powder, which resulted in an amperometric biosensor for H₂O₂. The electrochemical behaviour of the modified biosensor was monitored using cyclic voltammetry in the potential range of 0.2V to -0.4V vs SCE. The biosensor exhibited a stable voltammogram with cathodic peak at -0.234V and anodic peak at -0.172V, with a formal potential of -0.203V. Various factors influencing the performance of the biosensor such as buffer solution, pH, temperature and potential were examined for optimizing the working conditions. The modified biosensor exhibited a good catalytic behaviour for the reduction of H₂O₂ at a lower potential of -0.25V without any barrier from possible interferents. The analytical working range was found to be 0.429μM to 0.455mM of H₂O₂ with a detection limit of 0.171μM. The fabricated biosensor is robust for long-term usage in addition to the high sensitivity, rapid response and having an advantage of surface renewability by simple mechanical polishing.

Keywords: Ceramic composite biosensor; Covalent immobilization; Cyclic voltammetry; Horseradish peroxidase; Hydrogen peroxide.