A Sensing Platform for Direct Electron Transfer Study of Horseradish Peroxidase

Abstract

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 μA/mM (for 3-layers) and 0.07614 μA/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.

One-Step Preparation of Direct Electrochemistry HRP Biosensor via Electrodeposition

“…Taking hydrogen peroxide as the example, the sensitivity is higher than that of the reported references of AuNPs-based HRP electrode. 11,18 The apparent Michaelis-Menten constant (K m app ) for H 2 O 2 , indicative of the enzyme-substrate kinetics, was calculated based on the Lineweaver-Burk equation: 31 1…”

“…[6][7][8][9][10][11][12] Among these, AuNPs have gained immense attention in the third generation HRP biosensor because of their unique properties such as high electroconductivity, good biocompatibility and high surface-to-volume ratio. AuNPs alone 12,13 or AuNPs-based nanocomposites such as combining AuNPs with titanate nanotubes, 14 graphene, 15,16 Ag nanobelts, 17 dendrimers, 18 electroconductive polymers 19,20 have been designed for the HRP biosensor interfaces.…”

A Hydrogen Peroxide Biosensor Based on the Immobilization of the Highly Stable Royal Palm Tree Peroxidase (Roystonea regia) with Chitosan and Glutaraldehyde on Screen-printed Graphene Electrodes

“…Compound II then is reduced back to RPTP native form by accepting one electron from the electrode as is shown in Eq. 3 [18]. These results suggest that the electrocatalytic reduction of H 2 O 2 can be achieved by RPTP immobilized on the surface of a CS-GA SPGE.…”

“…At this lower work potential the number of interferences in real samples could be minimized (see Fig.6). As is presented in previous reports [10,[16][17][18], the electrocatalytic cycle of peroxidases in the reduction of H 2 O 2 at peroxidase modified electrodes can be expressed as follows:…”

Bio-Electrocatalytic Reduction of Hydrogen Peroxide by Peroxidase from Guinea Grass (Panicum Maximum) Immobilized on Graphene and Graphene Oxide Screen-Printed Electrodes

“…Traditionally, the analytical methods for detecting H2O2 usually employ equipment that measures permissible levels of peroxides. Among these methods are high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and gas chromatography-mass spectrometry (CG-MS) [10][11][12]. Although these methods are effective due to their highly sensitive and accurate detection, they suffer from disadvantages such as complex clean-up process, multiple derivative operation steps, being time-consuming and expensive.…”