Electrochemical devices have received particular attention due to their rapid detection and great sensitivity for the evaluation of DNA-hazard compounds interaction mechanisms. Several types of bioanalytical method use nucleic acids probes to detect DNA damage. This article reviews current directions and strategies in the development and applications of electrochemical DNA sensors for the detection of DNA damage.

O objectivo deste trabalho consistiu no desenvolvimento de um biossensor electroquímico com DNA para monitorização directa dos processos de hibridização e para detecção dos danos oxidativos causados ao DNA por diferentes compostos. Deste modo é necessária uma boa compreensão do comportamento redox dos ácidos nucleicos e dos seus produtos de degradação. A base purínica adenina e o seu produto de oxidação, a 2,8-dihidroxiadenina (2,8-DHA), foram investigadas por métodos voltamétricos. Os resultados obtidos são de grande interesse para a compreensão dos mecanismos redox destas substâncias e para a explicação das propriedades mutagénicas da 2,8-DHA. A oxidação de polinucleotídeos de sequências conhecidas, de DNA de hélice simples e dupla foi investigada em solução. Os polinucleotídeos de bases purínicas são facilmente oxidados enquanto que a oxidação dos polinucleotídeos de bases pirimidínicas ocorre apenas em condições especiais. A imobilização de oligonucleotídeos (ODNs) de sequências conhecidas na superfície do eléctrodo de carbono vítreo (GCE) foi seguida da hibridização com a sequência complementar ou por experiências de controle com ODNs não-complementares. Os dados obtidos mostraram que a adsorção não-específica influencia os resultados electroquímicos. Para reduzir a contribuição dos ODNs não-específicamente adsorvidos foi efectuada a modificação da superfície do GCE com ODNs oxidados. A interacção in situ do flavonóide quercitina com o DNA foi estudada com o objectivo de compreender melhor a sua acção in vivo sobre o DNA. A oxidação da quercitina causa danos oxidativos nos resíduos de guanina do DNA levando à formação de 8-oxoG. Foi proposto um mecanismo para a interacção entre a quercitina e o DNA. Foi investigado o comportamento redox do glivec, tendo sido dada particular atenção à forte adsorção na superfície do GCE do seu produto de oxidação. Foi desenvolvido um método para detecção de glivec em fluidos biológicos. A interacção do glivec com DNA foi investigada utilizando vários tipos de biossensores com DNA. Foi proposto um mecanismo para explicar os danos oxidativos que ocorrem nos resíduos de adenina e levam à formação de 2,8-DHA.

The adsorption and the redox behaviour of thrombin-binding aptamer (TBA) and extended TBA (eTBA) were studied using atomic force microscopy and voltammetry at highly oriented pyrolytic graphite and glassy carbon. The different adsorption patterns and degree of surface coverage were correlated with the sequence base composition, presence/absence of K(+), and voltammetric behaviour of TBA and eTBA. In the presence of K(+), only a few single-stranded sequences present adsorption, while the majority of the molecules forms stable and rigid quadruplexes with no adsorption. Both TBA and eTBA are oxidized and the only anodic peak corresponds to guanine oxidation. Upon addition of K(+) ions, TBA and eTBA fold into a quadruplex, causing the decrease of guanine oxidation peak and occurrence of a new peak at a higher potential due to the oxidation of G-quartets. The higher oxidation potential of G-quartets is due to the greater difficulty of electron transfer from the inside of the quadruplex to the electrode surface than electron transfer from the more flexible single strands.

The electrochemical behavior of the antileukemia drug glivec was investigated at a glassy carbon electrode (GCE). The oxidation is a complex, pH-dependent, irreversible electrode process involving the transfer of 2 electrons and 2 protons and the formation of an electroactive product, Pglivec, which strongly adsorbs on the GCE surface and undergoes reversible oxidation. The adsorption of Pglivec at the GCE surface yields a compact monolayer that inhibits further oxidation of glivec. The electrochemical reduction is a simple pH dependent irreversible process involving the transfer of 2 electrons and 2 protons and occurs with the formation of a nonelectroactive product. The diffusion coefficient of glivec was calculated to be DO=7.35×10-6 cm2 s-1 in pH 4.5 0.1 M acetate buffer.

Glivec is a relatively small molecule with activity against the protein tyrosine kinase, a protein expressed by all patients with chronic myelogenous leukemia (CML). Based on its activity on CML, glivec is undergoing extensive evaluation of its activity against other types of tumor. As a result, there is an ongoing need for clarification of glivec pharmacokinetics so that issues such as drug-biomolecular complex interactions can be understood.

Glivec is a newly developed drug that belongs to the class of 2-phenylaminopyrimidine. It is a potent inhibitor of ABL-kinase, the main clinical manifestation of chronic myelogenous leukemia (CML). Based on its activity on CML, glivec is undergoing extensive evaluation for its activity against other tumor types. Detection and quantitation of glivec in biological fluids or cells is thus very important. The antileukemia drug glivec undergoes oxidation at glassy carbon electrodes and involves the formation of an oxidation product, Pglivec. The adsorption of Pglivec at the GCE surface yields a compact monolayer allowing an electrochemical study of this compound adsorbed at the GCE surface. The reversible redox reaction of the adsorbed Pglivec is pH dependent and occurs with the transfer of 2 electrons and 2 protons. The surface standard potential and the rate constant of the heterogeneous electrochemical reaction were calculated using cyclic voltammetry to be Eθ=+180 mV and k=15.5 s-1, respectively. The total surface concentration of adsorbed Pglivec is 2.5×10-12 mol cm-2. The analytical determination of glivec was carried out by differential pulse voltammetric measurement of the anodic peak current corresponding to either the oxidation peak of glivec or the oxidation peak of Pglivec adsorbed on the GCE surface. The limits of detection of glivec and adsorbed Pglivec based on three times the noise level are 3.3×10-8 M and 2.9×10-10 M, respectively.

The structure–activity relationship of the complex between thrombin and thrombin binding aptamers (TBA) was evaluated by differential pulse voltammetry at a glassy carbon electrode and atomic force microscopy at a highly oriented pyrolytic graphite electrode. The effects on the interaction with thrombin of TBA primary and secondary structures as well as of its folding properties in the presence of alkaline metals were investigated. The complex between thrombin and single stranded aptamers involved the coiling of the single stranded molecules around thrombin structure leading to the formation of a robust TBA–thrombin complex that maintains the symmetry and conformation of the thrombin molecule. Monitoring both thrombin and TBA oxidation peaks, showed that the thrombin oxidation peaks occur at more positive potentials after TBA–thrombin complex formation. In the presence of K+ ions, the aptamers fold into quadruplex structures that facilitate the interaction with thrombin molecules. The TBA–thrombin complex adsorbs at the surface with the aptamer quadruplex always in preferential contact with the surface, and the thrombin molecules on top of the aptamer quadruplex structure, thus being less accessible to the electrode surface leading to the occurrence of thrombin oxidation peaks at less positive potentials.

Graphite–castor oil polyurethane composite electrodes with different graphite weight percentages, 30–70% graphite–polyurethane w w−1, were morphologically studied by atomic force microscopy (AFM) and voltammetry. AFM images and r.m.s. roughness measurements demonstrated that the polyurethane roughness decreased with increasing the graphite content, composites of 50% and 60% graphite–polyurethane w w−1 showing the smother electrode surface. The electrochemical characterisation was performed in solutions of K4Fe(CN)6 by cyclic voltammetry and impedance spectroscopy. For compositions of 60% and 70% graphite–polyurethane w w−1, the cyclic voltammetry results showed the K4Fe(CN)6 system reversibility. The charge transfer resistance, determined from the EIS spectra, decreased significantly with increasing the graphite/polyurethane ratio, and the capacitance increased for higher graphite percentages. AFM and voltammetric results enable to conclude that 60% graphite–polyurethane w w−1 was the optimal composition for the preparation of the graphite–polyurethane composite electrodes.

Glassy carbon electrodes (GCE) were modified with poly(glutamic acid) acid films prepared using three different procedures: glutamic acid monomer electropolymerization (MONO), evaporation of poly(glutamic acid) (PAG) and evaporation of a mixture of poly(glutamic acid)/glutaraldehyde (PAG/GLU). All three films showed good adherence to the electrode surface. The performance of the modified GCE was investigated by cyclic voltammetry and differential pulse voltammetry, and the films were characterized by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). The three poly(glutamic acid) modified GCEs were tested using the electrochemical oxidation of ascorbic acid and a decrease of the overpotential and the improvement of the oxidation peak current was observed. The PAG modified electrode surfaces gave the best results. AFM morphological images showed a polymeric network film formed by well-defined nanofibres that may undergo extensive swelling in solution, allowing an easier electron transfer and higher oxidation peaks.

The adsorption and the redox processes of two first-generation salicylaldiamine dendritic ligands and their copper, cobalt and nickel metallo-functionalized complexes have been studied at two types of carbon electrode surface. Glassy carbon (GC) was used in an electrochemistry study and highly oriented pyrolitic graphite (HOPG) in ex situ atomic force microscopy (AFM) imaging. All salicylaldimine ligands and their metallo-functionalized complexes adsorb on the surface of the HOPG electrode, resulting in the formation of nanoclusters and films, which vary between 0.9 and 6 nm in size, depending on the metallo-functionalized salicylaldimine dendrimer chemical composition and solution concentration. Differential pulse voltammetry of the surface-confined films has shown that the anodic reactions observed correspond to the oxidation of the hydroxyl groups present in the ligand structure of all compounds. However, by following the changes in peak currents, potentials and width at half height it has been shown that destabilization of the ligand internal structure occurred in the metallo-functionalized complexes depending on the metal involved. The electrochemical behaviour of the surface-confined films observed in buffer solution was related to the morphology, obtained by AFM, of the immobilised first-generation salicylaldiamine dendritic ligands and corresponding salicylaldimine metallo-functionalized complexes.