Estudo eletroquímico e eletroanalítico do biomarcador de estresse oxidativo 3-nitrotirosina em diferentes substratos eletroquímicos e de sua interação com o DNA

Abstract

An electrochemical study of 3-nitro-tyrosine (3-NO2-Tyr), in aqueous medium, was performed on different electrochemical substrates, glassy carbon electrode (GCE), platinum and gold electrode, using voltammetric techniques, such as cyclic voltammetry (CV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS), to investigate its oxidation mechanism, as well as its reactivity / toxicity to the double-strands DNA molecule (dsDNA). para-Tyrosine electro-oxidation in aqueous media was also investigated and compared with 3-NO2-Tyr. Potentialities of 3-NO2-Tyr oxidation properties for electroanalytical applications were also studied. The electrochemical results, in general, showed that 3-NO2-Tyr undergoes electro-oxidation in aqueous medium in a single irreversible pH-dependent step, losing one electron and one proton in the phenolic group and forming non-electroactive products, that are adsorbed strongly on all used electrodes. In addition, the phenolic group present in the 3-NO2-Tyr structure was found to be more difficult to oxidize (~ 170 mV) when compared to its electro-oxidation in the para-tyrosine molecule, possibly due to the influence of the nitro group. an electron withdrawing group. The mass transport of 3-NO2-Tyr to the electrode surface was investigated at platinum electrode and it was established that it is predominantly diffusion controlled. Electroanalytical methods for detection and quantification of 3-NO2-Tyr were proposed at GCE, gold and platinum electrode, the detection limits (LOD) and quantification (LOQ) of these methods were calculated and compared. At GCE the LOD was 0.17 μmol L-1, in the platinum electrode was 6,81 μmol L-1, while in the gold electrode was 7,33 μmol L-1, demonstrating a better sensitivity for detection and quantification of 3-NO2-Tyr. at GCE. Finally, the DPV and gel electrophoresis results clearly demonstrated a 3-NO2-Tyr interaction with dsDNA molecule in physiological medium (pH = 7.0), since the DPV results over incubation time of 3-NO2-Tyr with DNA, detected a significant decrease or the complete disappearance of the anodic peak associated with 3-NO2-Tyr oxidation, at E = 0.70 V, while electrophoresis results detected a very significant decrease in the ethidium bromide fluorescence. Thus, it was established the interaction of 3-NO2-Tyr possibly via its intercalation in the DNA double helix and with formation of a stable 3-NO2-Tyr-dsDNA complex, hindering the oxidation of 3-NO2-Tyr on the GCE, as well as the intercalation of the fluorescent biomarker, ethidium bromide, on the dsDNA.