Amperometric and impedimetric H₂O₂ biosensor based on horseradish peroxidase covalently immobilized at ruthenium oxide nanoparticles modified electrode

In this work, Ruthenium oxide nanoparticles (RuNPs) were electrochemically deposited on a glassy carbon electrode (GCE) surface using RuCl₃. nH₂O as a precursor. The model redox enzyme, horseradish peroxidase (HRP) was covalently immobilized at the RuNPs modified electrode surface using Chitosan-glutaraldehyde (Chi-GAD) cross-linking. The surface morphology of RuNPs, HRP and HRP/Chi-GAD/RuNPs films were investigated using scanning electron microscopy (SEM) and atomic force microscopy (AFM) studies. From the SEM and AFM results the average size of the RuNPs was calculated as 60-160 nm. UV-visible absorption spectra of HRP and HRP/Chi-GAD/RuNPs films displayed a soret band at 406 nm and thus the immobilized HRP retains its native structure at Chi-GAD/RuNPs film. Electrochemical impedance spectroscopy (EIS) results confirmed the rapid electron transfer process occurring at the composite film surface. The electron transfer resistance (R_et) values observed at the composite film surface varied linearly between 5.92 x10^-5 M-6.15 x 10^-4 M H₂O₂ additions, which validates the efficient impedimetric H₂O₂ quantification. The composite film also showed excellent amperometric i-t response towards H₂O₂ in the linear concentration range between 5.09 x 10^-3 M-1.5 x 10^-2 M. The developed HRP/Chi-GAD/RuNPs biosensor was highly selective towards H2O2 and acceptable H₂O₂ recovery was achieved in the contact lens cleaning solution.