Immobilization of Horseradish Peroxidase Enzyme on Nanoporous Titanium Dioxide Electrodes and Its Structural and Electrochemical Characterizations

Hierarchically ordered, honeycomb-like nanoporous TiO2 electrodes are prepared by a simple electrochemical anodization process using ammonium fluoride dissolved in ethylene glycol as an electrolytic medium. Formation of hexagonally arranged nanopores along with the tubular structure and anatase crystalline phase of TiO2 is confirmed by field emission scanning electron microscope (FESEM) and X-ray diffraction (XRD) studies. Further, these nanoporous TiO2 electrodes are employed as a substrate for enzyme (horseradish peroxidase, HRP) immobilization in an attempt to enhance the electron transport across the semiconductor electrode-electrolyte interface. Two different strategies, namely, physical entrapment and covalent linking, are used for anchoring the enzyme. Various parameters such as conductivity, stability, enzyme loading, enzymatic activity, sensitivity, linear range, etc., are investigated by using electrochemical techniques. Structural and morphological analyses of enzyme-modified electrodes are carried out using spectroscopic (UV -aEuro parts per thousand vis) and microscopic (AFM) methods. In the case of physical entrapment, a simple drop casting method of HRP solution on the nanoporous TiO2 electrodes is used in contrast to chemical linking method where a monolayer of 3-aminopropyltrimethoxy silane (APTMS) is formed initially on TiO2 followed by HRP immobilization using an amide coupling reaction. Interestingly, both of these methods result in anchoring of HRP enzyme, but the amount of enzyme loading and the stability are found to be higher in the covalent linking method. Cyclic voltammetric studies reveal the formation of a well-defined reversible peak for HRP enzyme. Dependence of peak current with the scan rate suggests that HRP enzyme is immobilized and stable and that the overall electron transfer process is predominantly controlled by a diffusion process. Enzymatic activity of HRP is investigated by monitoring the reduction process of hydrogen peroxide by incremental addition using cyclic voltammetry and amperometry techniques, from which several kinetic parameters are determined.