An electrochemical DNA biosensor for trace amounts of mercury ion quantification

The development of electrochemical sensors for the recognition of heavy metal ions is very important due to their fundamental role in biological, environmental and chemical processes (Prodi et al. 2000). Therefore, many sensor types have been used for environmental analysis, industrial quality control and clinical diagnostics (Andreescu & Sadik 2004). Among various types of sensors, electrochemical receptor layers, such as enzymes, nucleic acids, and antibodies, came to a special prominence. Recently, the interactions of heavy metal ions, in particular mercury, with nucleic acids have been studied, due to the possible toxicity and cancerogenicity of these ions (Anastassopoulou 2003). Furthermore, the mercury ion is a highly toxic environmental pollutant and has posed a serious threat to human health (Bolger & Schwetz 2002). Mercury can also affect many different areas of the brain and their associated functions, resulting in symptoms such as tremors, vision problems, deafness and loss of muscle coordination, sensation and memory (Stern 2005). In addition to the brain, inorganic mercury can damage the heart, kidney, stomach, and intestines (Zheng et al. 2003; Mutter et al. 2005; Wojcik et al. 2006). Due to their serious harm, the binding of metal ions to nucleic acids is used for the construction of biosensors for the detection of heavy metal ions (Liu et al. 2009). Recently, it has been reported that there is specific and strong coordination between Hg²⁺and the two DNA thymine bases (T) to form a mediated base pair (T–Hg²⁺–T) (Ono & Togashi 2004). Classical methods for mercury detection, such as atomic absorption spectroscopy (Li et al. 2006), colorimetric (Lin et al. 2010) and fluorescence (Chiang et al. 2008) are widely used. However, electrochemical methods have received particular attention due to their high sensitivity and selectivity, such as differential pulse stripping analysis (Yantasee et al. 2003; Wang et al. 2007), electrochemical impedance spectroscopy (EIS) (Lin et al. 2011) and square wave voltammetry (Jiang et al. 2015). In this study, we describe an electrochemical biosensor with high sensitivity and selectivity for Hg²⁺ detection based on DNA oligonucleotides. This probe report based on a DNA-AuNPs-glutathione/cysteine/Au modified electrode to capture mercury (II) ions. First, the interaction between thymine and mercury ions was evaluated by the EIS. Then, the electrochemical reduction of the surface provides a readout signal for the quantitative detection of Hg²⁺. We also demonstrate that the sensitivity of this Hg²⁺ sensor could be significantly improved with the DNA oligonucleotides immobilized by using gold nanoparticles (AuNPs).

Cysteine (95%), glutaraldehyde (Glu, 25%), glutathione (GSH reduced form), hexacyanoferrate (II/III), gold colloid solution (20 nm), N-hydroxysuccinimide (NHS), phosphate buffered saline (PBS), trishydroxymethylaminomethane and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma Aldrich (St. Louis, USA). CuCl₂.2H₂O, 3CdSO₄.8H₂O, Pb(NO₃) and HgCl₂ were purchased from Fluka-Chemika (Buchs, Switzerland). The DNA oligonucleotide was purchased from Bioneer Oligo Synthesis Report (Bioneer, BPS Bioscience, Biotools – Tunisia). The sequence is 5′-NH₂-(CH₂)₆-ATTTGTTCATGCCT-3′. All electrolytic solutions were prepared using ultra-pure water.

The electrochemical measurements were performed using an Autolab (PGSTAT 302 N, Eco Chemie). The measurements were made using a conventional three electrode electrochemical system consisting of a gold electrode, a platinum wire auxiliary electrode and Ag/AgCl/KCl reference electrode. The geometrical area of the working electrode was 0.031 cm². Impedance measurements were performed in the frequency range from 0.1 to 100,000 Hz. All electrochemical measurements were performed in a Faraday cage at room temperature (25 °C) to avoid any stray light or electrical perturbation from external sources.

To obtain a clean, activated and reproducible electrochemical surface, the gold electrode was mechanically polished with alumina slurry followed by rinsing with distilled water and sonication in acetone for 2 min. After mechanical cleaning, the gold electrode was treated using a chemical process by immersion in a piranha solution (H₂SO₄/H₂O₂, 1:3 v/v) for 1 min and rinsed with ultrapure water. Afterwards, the clean gold electrode was immersed into 5 mM cysteine in 0.1 M PBS solution at pH 7.4 for 2 h. Then, the modified electrode was rinsed with ultrapure water to remove non-covalent attached cysteine molecules.

The value of 50 pM is below the maximum contaminant level for inorganic mercury in drinking water set by US EPA (0.002 mgL^–1, ca. 10 nmol L^–1) (US Environmental Protection Agency 2009).

The analytical performance of the developed electrochemical DNA modified electrode was applied for the determination of the mercury ion in wastewater samples. The tested water samples were obtained from the Tunisian public industrial company ONAS, which is interested in the management of the sanitation sector. The wastewater samples were analyzed with voltammetric and impedance metric DNA sensors, and were compared to the results obtained using the spectrophotometric method. The results presented in Table 2 indicate the applicability of the voltammetric and impedancemetric methods to quantify mercury ions in wastewater samples. This result proves the usefulness of DNA/AuNPs-glutathione/cysteine modified electrodes for the detection of mercury in aqueous samples. Furthermore, in the present system the sensor probe can be regenerated in 100 mM of ascorbic acid solution for 1 hour to reduce Hg²⁺ into Hg⁺, and might exhibit a weak coordination with thymine as described by Liu et al. (2009). The mercury sensor showed good reproducibility and efficacious regeneration (relative standard deviation below 5.0%).

In this work an electrochemical DNA biosensor has been successfully fabricated based on thymine-Hg²⁺-thymine complexation for trace mercury ion quantification. Incidentally, the developed biosensor, based on an AuNPs-glutathione/cysteine-DNA matrix, was simple, sensitive and rapid. The Hg²⁺ ions were detected and identified at trace level quantities with a detection limit of 50 pM. A linear relationship of the biosensor response to the analyte concentration was obtained in the dynamic range from 50 pM to 0.1 μM. The obtained results revealed that this sensor exhibited a high sensitivity for mercury among other tested metal ions. The applicability of the DNA sensor for the determination of low concentration levels of Hg(II) ions in wastewater samples was successfully tested with a high reproducibility.