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  • Nutlin-3 br Gold electrodes GEs were

    2020-07-27


    Gold electrodes (GEs) were purchased from CH Instruments (Austin, USA). Alumina slurry (1.0, 0.3 and 0.05 µm) was obtained from Buehler (Lake Bluff, USA). SL-DNA Probe, modified with a methylene-blue (MeB) redox marker (SL-DNA-MeB, 5´ -C6-S-S-TC GCG ACA TAC AAT AGA TCG CG-MeB-3′), and complementary DNA (cDNA, 5´-CGA TCT ATT GTA TGT TAA CG −3′) were obtained from Biosearch Technologies, Inc. (Petaluma, USA) and used as received.
    The synthesis of 7ESTAC01 was synthesized at the Laboratory of Synthesis and Drug Delivery (LSVM) at the State University of Paraiba, Brazil. In short, as represented in Fig. 1A, the Nutlin-3 was obtained by reacting acridine-9-carboxaldehyde with 2-amino-thiophene moiety previously obtained via the classic Gewald reaction (Gewald, 1965; Gewald et al., 1966). Subsequent purification steps and recrystallization in ethanol yielded 7ESTAC01 as a red powder, with a melting point of 155–158 °C. Comparison of physical-chemical characteristics and spectral data confirms the achievement of the hybrid.
    2.3. Preparation and immobilization of SL-DNA/GE and dsDNA/GE
    GEs were used as substrate for SL-DNA probe and dsDNA im-mobilization. GEs were cleaned in a piranha solution (1:3 ratio of H2O2: H2SO4) and then polished on a micro cloth with 1.0 µm, followed by 0.3 and 0.05 µm alumina slurry. The GEs were then sonicated in water and ethanol to remove any residual alumina particles trapped at the surface
    of the electrode. The GE was activated in 0.5 M H2SO4 via CV from + 0.1 to + 1.6 V at a scan rate of 0.5 V s−1. The SL-DNA probe was immobilized on the electrode surface via a gold-thiol bond. The disulfide bonds of the SL-DNA probe were reduced with 1 mM TCEP by shaking the solution at room temperature for 1 h. The solution was then diluted with IB to yield 0.1 µM and 1.0 µM of the SL-DNA probe. 15 µL of this solution was drop casted onto the electrode and incubated at room temperature for 30 min. The electrodes were rinsed with IB and dried with nitrogen. To minimize nonspecific ad-sorption on the electrode surface, 15 µL of 2 mM MCH in IB was dropcasted on the electrode and incubated for 30 min. Then, the elec-trodes were rinsed with IB and dried with nitrogen. For dsDNA/GE preparation, SL-DNA modified GE was hybridized by dropcasting 15 µL of 50 nM cDNA in HB for 1.5 h at room temperature, which formed dsDNA at the surface of GE. Following hybridization, the GEs were rinsed using HB and then dried with nitrogen.
    2.4. Optimization of 7ESTAC01 concentration and experimental timing
    The protocol for analysis for the proposed biosensors consists of the following steps: (i) measurement of 7ESTAC01 signal in acetate buffer pH = 4.2; (ii) measurement of guanine and adenine signal obtained from SL-DNA/GE and dsDNA/GE modified electrode before the inter-action with 7ESTAC01; (iii) measurements of guanine and adenine signal after interaction between 7ESTAC01 and DNA/GE (7ESTAC01-DNA complex). The optimization of 7ESTAC01 concentration was performed at various final concentrations of 10 µM, 100 µM, and 400 µM. Intercalation time of the compound with the SL-DNA/GE and dsDNA/GE was varied from 1 h, 2 h and 24 h.
    2.5. Electrochemical measurements
    Electrochemical measurements were performed with a CHI660D Electrochemical workstation (CH Instruments, USA) at room tempera-ture. A typical 3-electrode system was used where the GE served as the working electrode, a platinum wire was used as the counter electrode (CE), and Ag/AgCl (3 M KCl) was used as a reference electrode (RE). The electrochemical characterization of 7ESTAC01 was investigated using CV in 0.2 M Phosphate buffer (pH=7.2) and 20% Dimethylformamide (DMF) in nitrogen saturated solutions Nutlin-3 (the solu-bility of the hybrid compound in DMF gave better solubility for the analyses in protic media). CVs of 10 mM 7ESTAC01 were recorded from − 1.2 to + 1.0 V vs. Ag/AgCl at scan rates of 0.1 V s−1.
    CV was performed to analyze the electrochemical behaviour of SL-DNA probe and dsDNA immobilized on the GE and were recorded from 0.1 to 800 V s−1 scan rates. The Optimization of SL-DNA/GE and dsDNA/GE focused on the analysis of the signal of suppression (% SS) before and after the hybridization with the cDNA. The % SS was cal-culated using the equation (Eq. (1)) (Lai et al., 2013) as follows: % SS = ( I
    where I, is the current obtained upon hybridization with the cDNA and I0 is the current obtained of the immobilized SL-DNA. We analyzed % SS of 0.1 µM and 1.0 µM SL-DNA probe concentration with a fixed 50 nM cDNA to obtain the dsDNA/GE biosensor.
    The electro-oxidation of 7ESTAC01 and detection of DNA damage