Biosensors and Bioelectronics 24 (2009) 1796–1800 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Short communication Direct electrochemical genosensing for multiple point mutation detection of Mycobacterium tuberculosis during the development of rifampin resistance Pinar Kara a,∗∗ , Cengiz Cavusoglu b , Seda Cavdar a , Mehmet Ozsoz a,∗ a Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, 35100 Bornova, Izmir, Turkey b Department of Microbiology and Clinical Microbiology, Faculty of Medicine, Ege University, 35100 Bornova, Izmir, Turkey article info Article history: Received 14 April 2008 Received in revised form 16 July 2008 Accepted 11 August 2008 Available online 27 August 2008 Keywords: Biosensor DNA Rifampin Tuberculosis Electrochemistry abstract We present a robust and simple method for the direct detection of multiple point mutations in the Mycobacterium tuberculosis rpoB gene during the development of rifampin (RIF) resistance using an electrochemical genosensor. The device contained five different capture probes which are designed to hybridize with several sequence segments within the bacterial rpoB gene hotspot region. Point mutations were detected by monitoring the guanine oxidation with differential pulse voltammetry after hybridiza- tion between PCR amplicons and inosine modified capture probes at graphite surface. Changes in the peak voltage corresponding to guanine oxidation provide an electrochemical signal for hybridization that can be used to determine the presence of point mutations conferring rifampin resistance. The analyti- cal parameters (sensitivity, selectivity and reproducibility) were evaluated. High selective discrimination against point mutation of bacteria at hot-spot region was observed. Several mutations were detected at several parts of the amplicon from 21 positive samples. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Mycobacterium tuberculosis infection remains one of the most significant causes of death worldwide and leads to millions of deaths annually. A rise in the incidence of drug-resistant strains of M. tuberculosis has accompanied the increased incidence of tuber- culosis (Telenti et al., 1993; Mani et al., 2001). In the Aegean region 8.2% of M. tuberculosis strains isolates were found to be resistant to rifampin (RIF). Many DNA sequencing studies have demonstrated that more than 95% of RIF-resistant bacteria strains possess muta- tions within the 81-bp hotspot region between the 507th and 533rd codons of the rpoB gene (Cavusoglu et al., 2002, 2006). Recent advances in biosensors based on nucleic acid hybridiza- tion recognition have led to the development of genosensor technology for DNA sequence analysis (Millan and Mikkelsen, 1993; Palecek, 1996; Mikkelsen, 1996; Wang et al., 1997, 1998; Erdem et al., 2000). Specifically, electrochemical hybridization biosensors demonstrate great promise for pathogen identification, mutation detection, and genomic sequencing (Thorp, 1998; Sawata et al., 1999; Mascini et al., 2001; Pividori and Allegret, 2003; Kara et al., 2004; Wong et al., 2005). ∗ Corresponding author. Tel.: +90 232 388 40 00 3992; fax: +90 232 388 52 58. ∗∗ Co-corresponding author. Tel.: +90 232 388 40 00 1353; fax: +90 232 388 52 58. E-mail addresses: pinar.kara@ege.edu.tr (P. Kara), mehmet.ozsoz@ege.edu.tr (M. Ozsoz). Herein, we have demonstrated a genosensor for the direct electrochemical detection of multiple point mutations in the M. tuberculosis genome during the development of RIF resistance. This analytical method relied on graphite electrode arrays, modified with five different guanine-free aminohexyl-tethered oligonu- cleotide probes. Samples of genetic material for genotyping the bacteria of interest were obtained from cell culture and subse- quent PCR amplification of the corresponding rpoB gene. These amplicons were captured via hybridization to five different capture probes, which recognized several different parts of the amplicon, at five different electrode interfaces. Differential pulse voltammetry (DPV) was employed to detect hybridization and to discriminate between point mutations via guanine oxidation signals. The results of this study demonstrate great promise for practical applications in the development of clinical assay techniques and the design of oligonucleotide chips. 2. Experimental 2.1. Chemicals and Apparatus 2.1.1. Chemicals For surface preparation, covalent attachment buffer containing 5 mmol/L EDC and 8 mmol/L NHS was used. Binding buffer contain- ing 0.5 M acetic acid, 20 mM NaCl (pH 4.8), and capture probes was used during immobilization of oligonucleotides on electrode sur- 0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.08.033