DNA-based biosensors for Hg 2 þ determination by polythymine–methylene blue modified electrodes Cristina Tortolini a,b , Paolo Bollella b , Marta Letizia Antonelli b , Riccarda Antiochia a , Franco Mazzei a , Gabriele Favero a,n a Department of Chemistry and Drug Technologies, Sapienza University of Rome, Italy b Department of Chemistry, Sapienza University of Rome, Italy article info Article history: Received 6 June 2014 Received in revised form 12 September 2014 Accepted 15 September 2014 Available online 17 September 2014 Keywords: DNA-based biosensor Mercury Polythymine abstract In this work we have developed a new electrochemical DNA-based biosensor for the selective determination of the Hg 2 þ ion by the use of different electrodes modified with polythymine, bearing methylene blue, as redox probe, in 3′ position. The determination of Hg 2 þ can be employed with an excellent degree of selectivity by the use of DNA biosensors through the formation of the complex Thymine–Hg–Thymine (T–Hg–T): in fact, Hg 2 þ tends to bind two thymines, generating a T–Hg–T complex with a formation constant higher than that one of the coupling Adenine–Thymine, which can be employed for a selective, fast and cost-effective Hg 2 þ detection. The presence of the Hg 2 þ in solution leads to the formation of T–Hg–T complex thus causing the “hairpin-like” folding of oligonucleotide, leading to an improved electronic exchange of methylene blue with the electrode surface due to the reduced distance and thus to an increase of the faradic current which is detected by means of square wave voltammetry (SWV). To test the feasibility of this kind of biosensor to be applied to the analysis of Hg 2 þ we have developed several biosensors configuration by modifying the electrochemical sensor transducer: (a) Au electrode; (b) Au screen-printed electrode (SPE). The proposed system, allows the determination of Hg 2 þ in the range 0.2–100 nM (0.05–20 ppb), with a sensitivity 0.327 mA/nM, LOD 0.1 nM (0.02 ppb), LOQ 0.2 nM (0.05 ppb) and RSD r4.3% when Au electrode is used as electrochemical transducer; on the other hand, in the case of Au SPE the linear range is 0.2–50 nM (0.05–10 ppb), with a sensitivity 0.285 mA/nM, while LOD and LOQ are the same as previously and RSD is r3.8%. This enabled the detection of mercury in real samples (waters and fishes) with good accuracy (recoveries 92–101% on waters and 92–107% on fishes, respectively) and reprodu- cibility (RSD r9.6% for measurements on waters and r8.8% on fishes, respectively). & 2014 Elsevier B.V. All rights reserved. 1. Introduction Heavy metal ions contamination may cause risks on human health and the environment. The main threats to human health from heavy metals are associated with exposure to lead, cadmium, mercury and arsenic. These metals have been extensively studied and also their effects on human beings (Sardar et al., 2013). Development of new metal ion-sensing strategies for detecting and quantifying metal ions in many different fields (Agasti et al., 2010; Aragay et al., 2011) continues to be the focus of numerous researches. Mercury together with other heavy metals is one of the most worrisome environmental contaminant: mainly because of its metabolic inertia it remains permanently in the body procuring metabolic and structural damage to the cells for its ability to interact with proteins. In particular, mercury contamination is widespread and arises from a variety of natural sources, such as oceanic and volcanic emissions (Renzoni et al., 1998) as well as anthropogenic sources (Pirrone et al., 2010; Stracher et al., 2010) such as gold mining and the combustion of solid waste and fuels. The transformations and cycles of mercury within the environ- ment have been thoroughly studied (Hammerschmidt and Fitzgerald, 2001). Once introduced into the marine environment, bacteria convert inorganic mercury into methylmercury, which enters the food chain and accumulates in higher organisms, such as large edible fish (Harris et al., 2003). Methylmercury is neuro- toxic and has been implicated as a cause of mercury-pollution- related diseases (Grandjean et al., 1998). Several methods have been developed for the determination of mercury in environmental samples, but the problems related to Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics http://dx.doi.org/10.1016/j.bios.2014.09.031 0956-5663/& 2014 Elsevier B.V. All rights reserved. n Corresponding author. Biosensors and Bioelectronics 67 (2015) 524–531