A Sensing Platform for Direct Electron Transfer Study of Horseradish Peroxidase

A novel sensing platform for the study of enzymatic kinetics using gold nanoparticles dispersed in a film of fourth generation PAMAM-OS-trimethoxysilyl dendrimer self assembled on indium tin oxide (ITO) electrodes is proposed. An overlayer of chitosan formed on the gold nanoparticles modified ITO electrode is used for the immobilization of an enzyme namely, horseradish peroxidase. In addition to a single layer of gold nanoparticles, multilayer assembly method is also studied. The strategy used for the modification resulted in a new third generation amperometric biosensor for hydrogen peroxide (H2O2). Different steps corresponding to the modification strategy employed in this work are characterized using microscopic (SEM & AFM) and electrochemical techniques. The resulting biosensor exhibited excellent sensing toward H2O2 in neutral phosphate buffer. Influence of gold nanoparticles in increasing the loading of active enzyme molecules and the amplification of catalytic activity are observed. The lowest detection limit is determined to be 0.165 mM with a sensitivity of 0.3151 mu A/mM (for 3-layers) and 0.07614 mu A/mM (for a single layer) as determined from the cyclic voltammetric data. The apparent Michaelis - Menten rate constant is calculated to be 0.119 mM. The enzyme turn-over is estimated to be 4.873 s(-1). This biosensor provides a wide response range toward H2O2 detection with an excellent reproducibility. (C) 2013 The Electrochemical Society. All rights reserved.