Coadsorption of Horseradish Peroxidase with Thionine on TiO₂ Nanotubes for Biosensing

In this study, we investigate the coadsorption of protein with thionine on TiO₂ nanotubes for biosensor design. The TiO₂ nanotube arrays fabricated by anodic oxidation of titanium substrate possess large surface areas and good uniformity and conformability and are ready for enzyme immobilization. Electrochemical and spectroscopic measurements show that the TiO₂ nanotube arrays provide excellent matrixes for the coadsorption of horseradish peroxidase (HRP) and thionine and that the adsorbed HRP on these TiO₂ nanotube arrays effectively retains its bioactivity. The immobilized thionine can be electrochemically reduced but cannot be reoxidized in the electrode potential range between −0.7 and 0.0 V. The addition of H₂O₂ leads to the biocatalytic oxidation of the reduced thionine in the presence of HRP, resulting in developing a novel H₂O₂ sensor with good stability and reproducibility. The fabricated TiO₂ nanotubes offer a stage for further study of immobilization and electrochemistry of proteins. The proposed method opens a way to develop biosensors using nanostructured materials with low electrical conductivity.