Full Paper An Electrochemical DNA Biosensor for the Detection of the Apa I Polymorphism in the Vitamin D Receptor Gene Using Meldolas Blue as a Hybridization Indicator Nilay Aladag, a Dilsat Ozkan-Ariksoysal, a * Duygu Gezen-Ak, b Selma Yilmazer, b Mehmet Ozsoz a * a Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, Bornova, 35100, Izmir, Turkey b Istanbul University, Cerrahpasa Faculty of Medicine, Department of Medical Biology, Istanbul, Turkey *e-mail: dilsat.ariksoysal@ege.edu.tr; mehmet.ozsoz@ege.edu.tr Received: August 13, 2009 Accepted: November 9, 2009 Abstract Electrochemical detection of nucleic acid base mismatches related to Apa I single nucleotide polymorphism (SNP) in the vitamin D receptor gene was performed successfully using 7-dimethyl-amino-1,2-benzophenoxazinium salt (Meldolas blue, MDB) with 10.9 pmol/100 mL of detection limit. MDB reduction signals obtained from probe, mismatch(probe-SNP containing target) and hybrid(probe-target) modified pencil graphite electrode(PGE) increased respectively. The sensor was able to clearly distinguish perfect match from mismatch DNA in a 30 min. detection time. Several factors affecting on the hybridization and indicator response are studied to maximize sensitivity and selectivity. The advantages of the biosensor are discussed in comparison with previous electrochemical assays for DNA hybridization. Keywords: Electrochemical DNA biosensor, Meldolas blue; Intercalator, Vitamin D receptor, Apa I polymorphism, Biosensors, Polymorphism DOI: 10.1002/elan.200900405 1. Introduction Single nucleotide polymorphisms (SNPs) in duplex DNA of the human chromosomes can lead to mutations causing many human diseases. SNPs can be identified in several different ways, each of which has specific advantages and limitations [1, 2]. Any mutation detection technique, such as restriction enzyme digestion, denaturing gradient gel electrophoresis (DGGE), denaturing high-performance liquid chromatography (DHPLC), primer extension, and the oligonucleotide ligation assay (OLA) can be used to identify sequence changes [3]. The most widely used technique involves using PCR to amplify the DNA containing the SNP of interest, then testing that amplified DNA. The technique can also be used to identify the SNP. More commonly, a specific assay is developed involving a restriction enzyme. If the SNP changes a restriction site, the two alleles can be distin- guished by whether the enzyme cuts or not, which can be detected through gel electrophoresis. Instead of amplifying the DNA and testing for an SNP, the method may be designed to amplify the DNA only if it contains a specific allele. This is called allele-specific polymerase chain reac- tion (AS-PCR). All of these detection systems have some limitations, such as being labor-intensive, time-consuming, and expensive. These aspects make them unsuitable for automation and rapid medical analysis, particularly for point-of-care tasks [1, 2]. In recent years, there has been significant interest in improving DNA electrochemical biosensors to allow the rapid and inexpensive diagnosis of genetic diseases and other biological analysis using methods superior to more conventional ones due to their ease of miniaturization, simplicity, speed, and low cost. The active form of vitamin D, 1,25-dihydroxyvitamin D 3 (1,25 [OH] 2 D 3 ), is a steroid hormone and a nuclear transcription regulator that acts via a nuclear hormone receptor [4]. The vitamin D endocrine system helps to modulate the immune response, bone metabolism, and regulation of cell proliferation and differentiation [5, 6]. Thus, mutations in this endocrine system give rise to several diseases, including diabetes, cancer, osteoarthritis, tuber- culosis, and cardiovascular disease [7, 8]. A convenient method for detecting polymorphisms is restriction fragment length polymorphism (RFLP) [4, 9, 10]. If one allele contains a recognition site for a restriction enzyme (e.g., Apa I) but the other does not, digestion of the two alleles will give rise to fragments of different length. So far, three adjacent restriction fragment length polymorphisms in the vitamin D receptor (VDR) gene have been studied the most often: Bsm I, Apa I, and Taq I [9]. The Apa I polymorphism in the VDR gene is known to be involved in neurodegener- ative disorders such as Alzheimers disease, and it may affect the parathyroid response [4, 10, 11]. Two main approaches, which can be widely referred to as labeled methods and label-free methods, have been applied to the electrochemical transduction of DNA hybridization Full Paper 590  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2010, 22, No. 5, 590 – 598