The development of gold disk electrodes modified with single-stranded PNA (peptide nucleic acid) for the determination of Hg2+ is described. PNA is the most useful and popular example of nucleic acid analogues. Due to the presence of glycine units linked via peptide bond instead of sugar-phosphate backbone, its interaction with molecules like proteins or metal ions is possible. The performance of anionic (AQMS) and cationic (MB and RuHex) redox markers for the generation of current signal is compared. It is shown that the use of anionic marker results in a significantly better analytical parameters, as compared to measurements with cationic markers. In the proposed system, the AQMS current difference increases proportionally within the Hg2+ concentration range 0.05 to 10 mu mol.L-1, while for higher concentrations, the saturation of PNA layer with Hg2+ is observed. The optimized sensor distinguishes itself with high selectivity toward Hg2+ and lower detection limit of 3.33 nmol.L-1. The interactions between mercury ion and PNA strands were studied using electrochemical impedance spectroscopy (EIS) and spectrophotometry (UV-VIS). (C) 2016 The Electrochemical Society. All rights reserved.