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We herein explore a novel route for oxidative stress in DNA by using electrochemistry as mimicking tool. Essentially, the electrochemical oxidation of guanine and adenine nucleosides and oligo-homo-nucleotides at pyrolytic graphite electrodes in neutral and alkaline aqueous solutions was studied. Under these experimental conditions all these compounds give rise to an adsorbed product not previously described, which was electrochemically and kinetically characterized. The virtually identical kinetic and electrochemical features exhibited by the oxidized compounds generated from all precursors strongly suggest a common species arising from both adenine and guanine derivatives. Supported by DFT calculations, we propose two convergent pathways for the electrochemical oxidation of adenosine and guanosine. Those calculations indicate that the common oxidized base product forms stable H-bond base pairs with both thymine and cytosine. Keywords: Cyclic voltammetry, Density functional calculations, Electrochemistry, Guanosine, Substitution mutation

The generation of a new electrocatalytic system for NADH after oxidizing flavin adenine dinucleotide (FAD) is shown. The oxidation is performed in alkaline medium until +1.4 V (Ag/AgCl) at graphite electrodes. The catalytic activity is ascribed to the electrooxidized moiety of FAD and not to quinone surface groups. A comparison between this catalyst and that attributed to poly(FAD) (Karyakin, A. A.; Ivanova Y. N.; Revunova, K. V.; Karyakina, E. E. Anal. Chem. 2004, 76, 2004-2009.) is presented. It is concluded that the surface quinone groups generated during the strong anodization of the electrode in acidic medium at 2-2.5 V and not the poly(FAD) are responsible for the catalytic activity described in the above mentioned work.

A new and simple electrochemical method capable to detect single-base mutations in DNA has been developed. It relies on the electrocatalytic oxidation of NADH by the intercalator Brilliant Cresyl Blue (BCB). Whereas negligible catalytic responses were obtained with graphite electrodes modified by BCB-single-stranded DNA, high catalytic activity is obtained at electrodes modified by the complexes between double-stranded DNA and BCB. The proposed explanation for these results is that charge transport from the intercalated BCB through the DNA base stack is a necessary event for the electrocatalytic activity as disruption by a single-base mismatch leads to a complete suppression of the electrocatalytic voltammetric response. These finds open new possibilities for the detection of DNA hybridization and mutation.

The knowledge of the redox chemistry of nucleic acids (NA) is of paramount importance in cancer and aging research. Charge migration through DNA is also involved in biologically relevant functions such as DNA damage and repair. In the first part of this article the main aspects of the electrochemistry of nucleic acids at solid electrodes are revised, including redox processes, photoelectroactivity and electrical conductivity. In the second part, an overview of its applications is presented. Methods for electrochemical detection of NA, NA-based biosensors for detection of nonnucleic acid molecules, studies on the nature and dynamics of interactions and structural conformations of NA, are some applications that take advantage of NA electrochemistry at solid electrodes.

A review of current strategies aimed at detecting nucleic acids (NA) using NA-modified solid electrodes reveals the versatility and potential of electrochemical detection in this field. What emerged at the beginning of 90s as a very promising detection system in DNA technology is now resulting in the first commercial devices. Many aspects of the experimental design, for example surface immobilisation and detection schemes, are outlined and evaluated. Although most approaches use hybridisation as the recognition reaction, those not based on hybridisation are also included. As is finally shown, great advances have been achieved, although further developments are required if electrochemical devices are to be suitable for routine measurement.

A new electrocatalytic scheme for the detection of DNA is described. This scheme relies on the different interactions of single- and double-stranded oligonucleotides with the organic dyes methylene blue, thionin, azure A, azure B, toluidine blue O, brilliant cresyl blue and neutral red, which in addition can act as NADH catalysts. This catalytic activity can be used for amplifying differentiation between duplex and single-stranded DNA. Pyrolytic graphite electrodes modified with adsorbed dsDNA–organic dye complexes gave higher electrocatalytic NADH oxidation currents than those measured with electrodes modified with adsorbed ssDNA–organic dye. This difference is maximum for brilliant cresyl blue. These results suggest that the proposed scheme may be useful for the detection of DNA hybridisation and damage. Keywords: DNA hybridisation, Catalytic NADH oxidation, Phenoxazines, Phenothiazines, Phenazines

The electrochemical oxidation of 5′-polyadenylic acid (poly-A) on graphite electrodes has been studied by cyclic voltammetry. The oxidation of poly-A in neutral and alkaline solutions gives rise to redox-active products strongly adsorbed on the electrode surface, which exhibit catalytic activity toward NADH oxidation. The amount and properties of the catalyst are remarkably influenced by the pH of the oxidation medium. Depending on the conditions of catalyst formation, it is possible to reduce the overpotential for NADH oxidation by 300 mV. Amperometric detection of NADH using electrodes modified from different media were evaluated and compared. Electrodes modified from 0.1 M NaOH solutions containing the polynucleotide allow carrying out the amperometric detection of NADH at 50 mV (vs. Ag/AgCl), at pH 9, within a wide concentration range, 2.5×10−8−1×10−4 M, and with a detection limit of 1.1×10−8 M. These electrodes also exhibit very good stability and reproducibility.

An electrochemical genosensor for the detection of nucleic acid sequences specific of Legionella pneumophila is reported. An immobilized thiolated hairpin probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the signaling probe, and streptavidin-alkaline phosphatase as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 2 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The sensor allows discrimination between L. pneumophila and L. longbeachae with high sensitivity under identical assay conditions (no changes in stringency). A limit of detection of 340 pM L. pneumophila DNA, and a linear relationship between the analytical signal and the logarithm of the target concentration to 2 muM were obtained. Experimental results show the superior sensitivity and selectivity of the hairpin-based assay when compared with analogous sandwich-type assays using linear capture probes.

A new label-free electrocatalytic method for the detection of DNA, is presented, in which DNA is the catalyst. The method takes advantage of the catalytic properties of the electrooxidised adenines within DNA toward the oxidation of NADH. This catalytic event results in an enhancement in the oxidation current of the electrooxidised adenines within DNA. Further improvement in this analytical signal is achieved in the presence of Ca 2+ ions. Parameters affecting the electrocatalytic current, such as pH or concentration of Ca 2+ ions have been investigated and optimised. Finally, the analytical features of the developed method are obtained. This method constitutes a more sensitive and reproducible alternative to other methods that use the oxidation current of the electrooxidised adenines without coupling to any catalytic event. A limit of detection of 33 fmol deoxyadenylic acid icosanucleotide (dA) 20 , is obtained without labels.

A new electrochemical method to determine underivatized oligonucleotides is developed. The electro-oxidation of the adenine moieties of adsorbed oligonucleotides at elevated potentials on pyrolytic graphite electrodes (PGE) in neutral or alkaline media gives rise to electroactive products strongly adsorbed on the electrode surface. The extent of the redox processes of these products, with formal potential close to 0 V (vs Ag /AgCl) at pH 10, correlates well with the amount of parent oligonucleotide. Various electrochemical techniques have been compared and applied to the detection of specific DNA sequences and synthetic homopolynucleotides. Detection limits of 2 and 10 ng for (dA)20 and a 21-mer sequence of HIV-1, respectively, have been achieved using sample volumes of 10 microL. Moreover, the adsorbed oxidized oligonucleotide shows electrocatalytic activity toward the oxidation of NADH. The capability of the new method to detect DNA hybridization is discussed.