Current Analytical Chemistry, 2011, 7, 00-00 1 1573-4110/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd. Enhancement of DNA Immobilization and Hybridization on Gold Electrode Modified using ZnO Nanoparticles/Chitosan Film Shafiquzzaman Siddiquee* 1 , Nor Azah Yusof* 2,3 , Abu Bakar Salleh 3,4 , Soon Guan Tan 4 and Fatimah Abu Bakar 3,5 1 Biotechnology Research Institute, Universiti Malaysia Sabah, JLN UMS, 88400 Kota Kinabalu, Sabah, Malaysia 2 Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 3 Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4 Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 5 Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Abstract: Electrochemical DNA biosensor was fabricated using the ZnO nanoparticles/chitosan (CHIT) nanocomposite membrane on modified gold electrode (AuE) as the working electrode. The ZnO/CHIT was used as a modified-AuE for the immobilization of the single-stranded DNA probe. This particular DNA biosensor provided some advantages such as the biocompatibility of the ZnO nanoparticles, good film forming ability of CHIT, and the high conductivity of AuE. Methylene blue was used as the electrochemical indicator for monitoring the hybridization reaction following the hybridization of the target DNA sequence. Differential pulse voltammetry was used for recording the electrochemical response of MB. The specific target DNA sequence could be detected in the concentration range of 1.0 x 10 -14 to 1.82 x 10 -4 mol L -1 , with the detection limit at 1.0 x 10 -15 mol L -1 . This novel approach of constructing an electrochemical biosensor allowed the hybridization of synthetic target DNA. In addition, it also facilitated hybridization with template— DNA taken from real samples. The results proved that the ZnO/CHIT/AuE electrode has the potential for the sensitive detection of specific sequence related to a Trichoderma harzianum gene. Keywords: DNA electrochemical biosensor, Hybridization, ZnO nanoparticles, T. harzianum. 1. INTRODUCTION DNA hybridization is one of the most important techniques in molecular biology. It has been applied for the detection and analysis of a specific DNA sequence. The analysis of nucleotide sequences is an important in various fields, such as clinical diagnosis, forensic analysis, drug research, and food safety monitoring [1]. A variety of approaches including fluorescence, electrochemical, piezoelectric, and acoustic techniques have been developed for detecting DNA hybridization [2]. Among these techniques, electrochemical transduction offers a number of advantages, such as good sensitivity, accurate specificity, simplicity, reliability and a low-cost for converting nucleic acid hybridization events into useful analytical signals [3-5]. Thus, in recent years, electrochemical DNA biosensors have attracted considerable attention. They have been developed and used for DNA sequence analysis with immobilization of single-stranded DNA (ssDNA) probes on different electrodes surfaces. *Address correspondence to these authors at the Biotechnology Research Institute, Universiti Malaysia Sabah, Jln UMS, 88400 Kota Kinabalu, Sabah, Malaysia; Tel: +6088-320000 ext 8467; Fax: +6088-320993; E-mail: shafiqpab@ums.edu.my; Department of Chemistry, Universiti Putra Malaysia, 43400 UPMSerdang, Selangor D.E, Malaysia; Tel: +603-8943 5380; Fax: +603-8943 5380; E- mail: azah@science.upm.edu.my A comprehensive research has been performed in the field of analytical electrochemistry during the last decade. Electrochemical DNA biosensors are useful analytical tools for sequence-specific DNA diagnosis and detection owing to their inherent advantages of sensitivity and rapidity of response [6]. Nanoparticles play an important role both in DNA immobilization on the gold electrode surfaces and as suitable labels to improve the detection level of hybridization events. The electrode surface-immobilization system is a key feature in the development of a DNA biosensor for achieving a stable and highly intense ssDNA monolayer. This research is undertaken to design proficient electrochemical biosensors in order to provide better analytical characteristics such as sensitivity, selectivity, reliability, simplicity of fabrication, and low cost usage. At present, it is well recognized that the performance of biosensors is dependent on the immobilization of biomolecules. One of the most novel approaches in this context is the use of nanomaterials for the development of biosensing devices. The properties of nanomaterials are solely responsible for increasing the potential prospects of these devices [7]. Thus, this study is aimed at the use of ZnO nanoparticles for the construction of electrochemical biosensors with enhanced analytical performance. Recently, the development of electrochemical DNA biosensor devices based on the principle of nanotechnology