Indirect Amperometric Determination of Selected Heavy Metals Based on Horseradish Peroxidase Modified Electrodes

Due to the high toxicity of heavy metals, it is crucial to detect ultra low levels of the metals, especially in drinking water. The common techniques include spectrometric techniques such as inductively coupled plasmaatomic emission spectroscopy, ICP-AES (Bettinelli et al. 2000; Rahmi et al. 2007; Tuzen et al. 2008) as well as anodic stripping voltammetry (Brainina et al. 2004). Even though ICP techniques have low detection limits (ranges from parts per billion, ppb to parts per trillion, ppt (Berezhetskyy et al. 2008), however, they are unsuitable for in-situ analysis, they are expensive, sophisticated and require skilled operators. For these reasons, the development of alternative techniques such as electrochemical biosensor techniques, offer alternative methods because they are sensitive, low cost and simple to operate (Wang et al. 2009b). Recent developments have shown the use of electrochemical biosensors as indirect methods for detection of Cd2+, Cu2+, Cr3+, Zn2+, Ni2+ and Pb2+ using urease biosensor (Ilangovan et al. 2006; Tsai et al. 2003); Cd2+, Co2+, Zn2+, Ni2+ and Pb2+ using alkaline phosphatase (Berezhetskyy et al. 2008); Cd2+, Cu2+, Zn2+ and Pb2+ by glucose oxidase (Ghica and Brett 2008); Hg2+ using glucose oxidase invertase and mutarose (Mohammadi et al. 2005); Cu2+, Cd2+, Mn2+ and Fe3+ using acetylcholinesterase (Stoytcheva 2002); and Cu2+, Cd2+, Zn2+ and Pb2+ by nitrate reductase (Wang et al. 2009b). Horseradish peroxidase (HRP) biosensor has so far only been reported for detection of mercury (Han et al. 2001). This study sought to extend its application for detection of other metals such as lead, cadmium and copper. We have chosen cadmium due to its similarities with mercury with regards to toxicity as both metals, belong to the same group. In addition, we have also chosen copper and lead because of their common occurrence in environmental matrices (Pb from leaded petrol and Cu from wiring activities). Furthermore, copper is reported to show interaction with biological systems (Cecconi et al. 2002; Uriu-Adams and Keen 2005) and therefore interesting to see how it interacts with HRP enzyme. The main aim of this present work is to investigate the inhibition of HRP enzyme by Cd, Pb and Cu, a phenomenon that can be employed for their indirect determination. Kinetic studies were done to determine the nature of enzyme inhibition (whether it is reversible or irreversible and if reversible whether it is competitive or noncompetitive). The apparent