Furthermore for the dsDNA GE the presence of the intermediat
Furthermore, for the dsDNA/GE, the presence of the intermediate form of 8-oxoguanine (8-oxoG) at E = +0.25 V is a reliable indicator that the oxidation of Pimozide is taking place (Fig. 2B) from the second scan onwards. In addition, the guanine oxidation peak current increases as the oxidative DNA damage increases for dsDNA/GE.
3.4. Optimization of the concentration of 7ESTAC01 and detection of DNA damage
The electro-oxidation processes involved in purine DNA bases are similar to those affecting enzymatic oxidation. For this reason, the electro-oxidation of the DNA immobilized on the GE was applied to detect DNA damage through the interaction with the oxidizing com-pound (7ESTAC01). The oxidative DNA damage was induced by electro-oxidation in the presence of the given 7ESTAC01. A reduction potential was applied to 7ESTAC01 to obtain 7ESTAC01 radicals to
Scheme 1. Schematic representation the fabrication the Hairpin-DNA modified Gold electrode (GE). A) Schematic of the assay procedure of the SL-DNA probe and dsDNA biosensor at GE B) SL-DNA probe and dsDNA structures. SL-DNA probe, 5´-C6-S-S-TC GCG ACA TAC AAT AGA TCG CG-MeB-3′. The number of Guanines (G). The number of adenines (A). 6-mercapto-1-hexanol (MCH). Complementary DNA (cDNA). Methylene-blue (MeB).
Fig. 2. Electrochemical behaviour of SL-DNA/GE and dsDNA/GE under electro-oxidation by DPV. (A) Comparison of DPV signals between SL-DNA probe (curve a) and dsDNA (curve b) and blank response of GE (GE/blank). Histogram graph represents the guanine peak current for SL-DNA/GE (a) and dsDNA/GE (b) modified electrode (A, inner graph). (B) DPV response for dsDNA/GE from first to the fifth scan. The electro-oxidation by DPV was carried out in acetate buffer at pH 4.2 for potentials range from 0.0 to + 1.6 V; amplitude 0.05 V; sample width 0.0167 s. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Fig. 3. DPV peak currents responses under electro-oxidation on SL-DNA/GE in the presence of 7ESTAC01 to the detection of DNA damage. (A) Guanine peak current in the presence of 7ESTAC01 at concentrations of (a) 10, (b) 100, (c) 400 µM for 1, 2 and 24 h of interaction. (B) Guanine (blue histogram on the left) and Adenine peak current (red histogram, on the right) in the presence of 100 µM 7ESTAC01 (b) for 1 and 2 h of interaction. Histograms represent the guanine and adenine peak current for SL-DNA/GE extrapolated from the DPV signal. Results were expressed as the average of three independent experiments. Error bars represent standard deviations. (*) All the intercalation measurements were done with 7ESTAC01 in solution. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
damage DNA. According to Abreu et al. (2002) and Dogan-Topal et al. (2014) certain anticancer drugs require the formation of short-lived radicals to interact and damage DNA.
The direct determination of the oxidation of electroactive DNA bases in the presence of 7ESTAC01 were carried out by DPV from 0 to + 1.6 V. The oxidation peak current differences between SL-DNA/GE and their corresponding SL-DNA/GE-7ESTAC01 system were further investigated in acetate buffer (pH 4.2). The reproducibility of the SL-DNA/GE was investigated in presence and absence of different con-centrations of 7ESTAC01 (Fig. 3A and B). As seen in Fig. 3A, 7ESTAC01 interactions with the SL-DNA/GE were examined at different times, 1, 2 and 24 h.
In general, the guanine oxidation peak current increased notoriously as the interaction time increased, which indicated the formation of SL-DNA/GE-7ESTAC01, increasing the electron transfer ability of the SL-DNA/GE. Another valuable data collected from Fig. 3A showed that the guanine oxidation peak current increased as the 7ESTAC01 concentra-tions increased. It is important to note that acridine, which binds to DNA by intercalation, might either donate electrons to or accept electrons from, the double helix, thus actively participating in electron transfer reactions (Kovacic and Wakelin, 2001; Baguley et al., 2003; Nepali et al., 2014). These results support literature data of acridine and its im-portance on the electron transfer (Noh et al., 2015); showing that oxi-dation of 7ESTAC01 is facilitating the electron transfer on the SL-DNA/ GE biosensor. The adenine oxidation was evaluated with the minimum required concentration of 7ESTAC01 (Fig. 3B). The electrochemical oxidation of adenine followed a multiple step, six electron, six protons oxidation (Wei et al., 2011), which implies a more demanding oxidation process compared to guanine. As seen in Fig. 3B, the adenine oxidation was registered in the presence of higher concentrations of 7ESTAC01, equal to 100 µM 7ESTAC01 (Fig. 3B). Taking into account the interaction time of 1 and 2 h under the same level of 100 µM 7ESTAC01, adenine peak currents registered at 5.45 µA and 8.34 µA, respectively.