Ana Maria Oliveira-Brett

Electrochemical oxidation mechanism of guanine and adenine using a glassy carbon microelectrode

The electrochemical oxidation mechanism of guanine and adenine was investigated using a glassy carbon microelectrode and cyclic and differential pulse voltammetry. It is pH-dependent and the electron transfer process occurs in consecutive steps with the formation of strongly adsorbed dimers on the electrode surface for both compounds.

Electrochemical detection of in situ adriamycin oxidative damage to DNA

Adriamycin intercalation and in situ interaction with double helix DNA was investigated using a voltammetric DNA-biosensor. Oxidation and reduction of adriamycin molecules intercalated in double helix DNA were investigated in order to understand the in vivo mechanism of action with this anti-neoplasic drug. The results showed that the interaction of adriamycin with DNA is potential-dependent causing contact between DNA guanine and adenine bases and the electrode surface such that their oxidation is easily detected. A mechanism for adriamycin reduction and oxidation in situ when intercalated in double helix DNA immobilised onto the glassy carbon electrode surface is presented and the formation of the mutagenic 8-oxoguanine explained.

Ultrasound extracted flavonoids from four varieties of Portuguese red grape skins determined by reverse-phase high-performance liquid chromatography with electrochemical detection

Several flavonoids present in red grape skins from four varieties of Portuguese grapes were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) with electrochemical detection (ECD). Extraction of flavonoids from red grape skins was performed by ultrasonication, and hydrochloric acid in methanol was used as extraction solvent. The developed RP-HPLC method used combined isocratic and gradient elution with amperometric detection with a glassy carbon-working electrode. Good peak resolution was obtained following direct injection of a sample of red grape extract in a pH 2.20 mobile phase. Eleven different flavonoids: cyanidin-3-O-glucoside (kuromanin), delphinidin-3-O-glucoside (myrtillin), petunidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside (oenin), (+)-catechin, rutin, fisetin, myricetin, morin and quercetin, can be separated in a single run by direct injection of sample solution. The limit of detection obtained for these compounds by ECD was 20-90 pg/L, 1000 times lower when compared with photodiode array (PDA) limit of detection of 12-55 ng/L. RP-HPLC-ECD was characterized by an excellent sensitivity and selectivity, and appropriate for the simultaneous determination of these electroactive phenolic compounds present in red grape skins.

Boron doped diamond and glassy carbon electrodes comparative study of the oxidation behaviour of cysteine and methionine.

The electrochemical oxidation behaviour at boron doped diamond and glassy carbon electrodes of the sulphur-containing amino acids cysteine and methionine, using cyclic and differential pulse voltammetry over a wide pH range, was compared. The oxidation reactions of these amino acids are irreversible, diffusion-controlled pH dependent processes, and occur in a complex cascade mechanism. The amino acid cysteine undergoes similar three consecutive oxidation reactions at both electrodes. The first step involves the oxidation of the sulfhydryl group with radical formation, that undergoes nucleophilic attack by water to give an intermediate species that is oxidized in the second step to cysteic acid. The oxidation of the sulfhydryl group leads to a disulfide bridge between two similar cysteine moieties forming cysteine. The subsequent oxidation of cystine occurs at a higher potential, due to the strong disulfide bridge covalent bond. The electro-oxidation of methionine at a glassy carbon electrode occurs in two steps, corresponding to the formation of sulfoxide and sulfone, involving the adsorption and protonation/deprotonation of the thiol group, followed by electrochemical oxidation. Methionine undergoes a one-step oxidation reaction at boron doped diamond electrodes due to the negligible adsorption, and the oxidation also leads to the formation of methionine sulfone.

Electrochemical sensing of DNA–adriamycin interactions

Adriamycin, a cancerostatic anthracycline antibiotic, causes considerable death of tumour cells, together with the induction of breaks in DNA single and double strands. The interaction of this compound with DNA was investigated using an electrochemical DNA-biosensor. Adriamycin intercalation in DNA disrupts the double helix and the detection of guanine and 8-oxoguanine could mimic one possible mechanism for the in vivo adriamycin drug action.

In situ electrochemical and AFM study of thalidomide-DNA interaction

The interaction of thalidomide (TD) with double-stranded DNA (dsDNA) was studied using atomic force microscopy (AFM) at highly oriented pyrolytic graphite (HOPG), differential pulse voltammetry (DPV) at glassy carbon electrodes (GCE), UV-Vis and electrophoresis. After incubation of dsDNA with different concentrations of TD, the AFM images show the formation of thin and incomplete TD-DNA network films with a number of embedded molecular aggregates and regions of uncovered HOPG. Both the TD-dsDNA aggregates and network thickness directly depended on the TD concentration and incubation time. The voltammetric data also showed that the modifications caused by TD to the DNA double helical structure are time-dependent. In agreement with AFM, DPV, UV-Vis and electrophoresis results, a model is proposed for the TD-DNA interaction, considering that TD intercalates into the dsDNA, causing defects in the dsDNA secondary structure and DNA double helix unwinding. Moreover, both AFM and DPV show that condensation is caused to DNA by TD and occurs until 24 h of incubation, as well as DNA oxidative damage, detected electrochemically by the appearance of the 8-oxoGua and/or 2,8 oxoAde oxidation peak.

Peptide methionine sulfoxide reductase A (Msra): Direct electrochemical oxidation on carbon electrodes

The direct electrochemical behaviour of peptide methionine sulfoxide reductase A (MsrA) adsorbed on glassy carbon and boron doped diamond electrodes surface, was studied over a wide pH range by cyclic and differential pulse voltammetry. MsrA oxidation mechanism occurs in three consecutive, pH dependent steps, corresponding to the oxidation of tyrosine, tryptophan and histidine amino acid residues. At the glassy carbon electrode, the first step corresponds to the oxidation of tyrosine and tryptophan residues and occurs for the same potential. The advantage of boron doped diamond electrode was to enable the separation of tyrosine and tryptophan oxidation peaks. On the second step occurs the histidine oxidation, and on the third, at higher potentials, the second tryptophan oxidation. MsrA adsorbs on the hydrophobic carbon electrode surface preferentially through the three hydrophobic domains, C1, C2 and C3, which contain the tyrosine, tryptophan and histidine residues, and tryptophan exists only in these regions, and undergo electrochemical oxidation.

In situ DNA oxidative damage by electrochemically generated hydroxyl free radicals on a boron-doped diamond electrode.

In situ DNA oxidative damage by electrochemically generated hydroxyl free radicals has been directly demonstrated on a boron-doped diamond electrode. The DNA-electrochemical biosensor incorporates immobilized double-stranded DNA (dsDNA) as molecular recognition element on the electrode surface, and measures in situ specific binding processes with dsDNA, as it is a complementary tool for the study of bimolecular interaction mechanisms of compounds binding to DNA and enabling the screening and evaluation of the effect caused to DNA by radicals and health hazardous compounds. Oxidants, particularly reactive oxygen species (ROS), play an important role in dsDNA oxidative damage which is strongly related to mutagenesis, carcinogenesis, autoimmune inflammatory, and neurodegenerative diseases. The hydroxyl radical is considered the main contributing ROS to endogenous oxidation of cellular dsDNA causing double-stranded and single-stranded breaks, free bases, and 8-oxoguanine occurrence. The dsDNA-electrochemical biosensor was used to study the interaction between dsDNA immobilized on a boron-doped diamond electrode surface and in situ electrochemically generate hydroxyl radicals. Non-denaturing agarose gel-electrophoresis of the dsDNA films on the electrode surface after interaction with the electrochemically generated hydroxyl radicals clearly showed the occurrence of in situ dsDNA oxidative damage. The importance of the dsDNA-electrochemical biosensor in the evaluation of the dsDNA-hydroxyl radical interactions is clearly demonstrated.

Anodic voltammetry and AFM imaging of picomoles of adriamycin adsorbed onto carbon surfaces

Adriamycin adsorbs strongly and irreversibly onto surfaces and this enabled electrochemical detection of in situ adriamycin oxidative damage to DNA. The adsorption of adriamycin onto glassy carbon and highly oriented pyrolytic graphite (HOPG) electrodes was studied by voltammetry and mode atomic force microscopy (MAC). At a glassy carbon electrode (GCE), the adsorbate has similar voltammetric behaviour to adriamycin in solution, which enabled the cyclic, differential pulse and square wave voltammetric study of the electron transfer reaction. The total surface concentration of adriamycin adsorbed onto GCE, from a 50 nM adriamycin solution during 3 min, was calculated to be 2.57� /10 � 12 mol cm � 2 . The oxidation of adsorbed adriamycin is pHdependent and corresponds to a two electron/two proton mechanism, and the detection limit for adriamycin adsorbed onto the GCE was 3.33� /10 � 10 M. In situ AFM images show quick and spontaneous adsorption of the adriamycin onto a HOPG surface. Adriamycin forms a stable monolayer when adsorbed from different concentrations of adriamycin solutions and for short adsorption times. The strong and irreversible chemisorption of adriamycin onto carbon electrodes enables detection limits of the order of picomolar, which is much lower than the detection limits attainable by voltammetric methods for most organic compounds. # 2002 Elsevier Science B.V. All rights reserved.

DNA interaction with palladium chelates of biogenic polyamines using atomic force microscopy and voltammetric characterization.

The interaction of double-stranded DNA with two polynuclear Pd(II) chelates with the biogenic polyamines spermidine (Spd) and spermine (Spm), Pd(II)-Spd and Pd(II)-Spm, as well as with the free ligands Spd and Spm, was studied using atomic force microscopy (AFM) at a highly oriented pyrolytic graphite (HOPG) surface, voltammetry at a glassy carbon (GC) electrode, and gel electrophoresis. The AFM and voltammetric results showed that the interaction of Spd and Spm with DNA occurred even for a low concentration of polyamines and caused no oxidative damage to DNA. The Pd(II)-Spd and Pd(II)-Spm complexes were found to induce greater morphological changes in the dsDNA conformation, when compared with their ligands. The interaction was specific, inducing distortion and local denaturation of the B-DNA structure with release of some guanine bases. The DNA strands partially opened give rise to palladium intra- and interstrand cross-links, leading to the formation of DNA adducts and aggregates, particularly in the case of the Pd(II)-Spd complex.