ORIGINAL PAPER A new peptide nucleotide acid biosensor for electrochemical detection of single nucleotide polymorphism in duplex DNA via triplex structure formation Ezat Hamidi-Asl • Jahan Bakhsh Raoof • Reza Ojani • Seyed Mahdi Golabi • Mohammad Saeid Hejazi Received: 9 December 2012 / Accepted: 17 March 2013 Ó Iranian Chemical Society 2013 Abstract In this paper, we report a new PNA biosensor for electrochemical detection of point mutation or single nucleotide polymorphism (SNP) in p53 gene corresponding oligonucleotide based on PNA/ds-DNA triplex formation following hybridization of PNA probe with double-stran- ded DNA (ds-DNA) sample without denaturing the ds-DNA into single-stranded DNA (ss-DNA). As p53 gene is mutated in many human tumors, this research is useful for cancer therapy and genomic study. In this approach, methylene blue (MB) is used for electrochemical signal generation and the interaction between MB and oligonu- cleotides is studied by differential pulse voltammety (DPV). Probe-modified electrode is prepared by self- assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of Au electrode. A significant increase in the reduction signal of MB following hybrid- ization of the probe with the complementary double- stranded oligonucleotide (ds-oligonucleotide) confirms the function of the biosensor. The selectivity of the PNA sensor is investigated by non-complementary ds-oligonu- cleotides and the results support the ability of the sensor to detect single-base mismatch directly on ds-oligonucleotide. The influence of probe and ds-DNA concentrations on the effective discrimination against complementary sequence and point mutation is studied and the concentration of 10 -6 M is selected as appropriate concentration. Diag- nostic performance of the biosensor is described and the detection limit is found to be 4.15 9 10 -12 M. Keywords Peptide nucleic acid Á Triplex structure Á p53 oligonucleotide Á Biosensor Á Point mutation Introduction A biosensor is a device incorporating a molecular recog- nition element associated with a physicochemical trans- ducer. Transducing microsystem may be optical, electrochemical, thermometric, piezoelectric or magnetic. Materials that can be used as recognition elements consist of proteins, tissues, cells, enzymes, nucleic acids, etc. [1–4]. Sequence-specific detection of DNA provides the basis for detecting a wide variety of infectious and inherent diseases. There are various platforms for DNA probe immobilization and detection including fluorescence [5], total internal reflection fluorescence [6], surface plasmon field-enhanced fluorescence spectroscopy [7], quartz crys- tal micro-balance [8] and electrochemical methods. Elec- trochemical hybridization biosensors for the detection of DNA sequences reduce the assay time and simplify med- ical analysis [9–13]. The electrochemical investigations for DNA hybridization are classified into two groups. The first one, direct method is related to the intrinsic E. Hamidi-Asl Á J. B. Raoof (&) Á R. Ojani Eletroanalytical Chemistry Research Laboratory, Department of Analytical Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran e-mail: j.raoof@umz.ac.ir S. M. Golabi Eletroanalytical Chemistry Research Laboratory, Department of Analytical Chemistry, Faculty of Chemistry, Tabriz University, Tabriz, Iran M. S. Hejazi Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran M. S. Hejazi Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 123 J IRAN CHEM SOC DOI 10.1007/s13738-013-0254-0