O objectivo deste trabalho foi o estudo dos processos de adsorção da guanina, da desoxiguanosina, de vários tipos de ácidos nucleicos e do fármaco adriamicina, na superfície de eléctrodos de ouro e grafite pirolítica altamente orientada (HOPG) por microscopia de túnel de varrimento (STM) e microscopia de força atómica (AFM) com vista à caracterização morfológica da superfície de eléctrodos modificados com DNA, os biossensores electroquímicos com DNA. A adsorção espontânea na superfície de HOPG, a partir de soluções saturadas de guanina, determinou a formação de múltiplas camadas moleculares estáveis e resistentes e a adsorção com potencial aplicado permitiu a condensação da guanina, conjuntamente com dímeros, trímeros e outros produtos de oxidação da guanina, formando filmes espessos, heterogéneos, menos compactos e resistentes. A desoxiguanosina adsorveu espontaneamente na superfície de ouro, por intermédio de processos de nucleação e crescimento, sendo observados por STM núcleos de desoxiguanosina, alinhados em filas. As características da adsorção da desoxiguanosina foram estudadas em função do potencial aplicado ao eléctrodo de ouro e de HOPG. As imagens de AFM de DNA hélice simples, de DNA hélice dupla, do ácido poliadenílico e de oligonucleotídeos sintéticos, imobilizados na superfície de HOPG, mostraram a tendência das moléculas para uma auto–organização espontânea na superfície. Os ácidos nucleicos condensaram-se em redes bidimensionais densas, extensas e cobrindo uniformemente toda a superfície. A interacção com a superfície hidrofóbica de HOPG induziu sobreposições de moléculas e interacções intramoleculares e intermoleculares. A aplicação dum potencial positivo ao eléctrodo de HOPG durante a adsorção aumentou a robustez e a estabilidade mecânica dos filmes de DNA, devido a múltiplas interacções electrostáticas estabelecidas entre o esqueleto de açúcar–fosfato do DNA carregado negativamente e a superfície carregada positivamente. O controlo do pH da solução de DNA foi determinante para a cobertura da superfície. A adsorção espontânea de moléculas de adriamicina na superfície de HOPG e a formação de monocamadas estáveis para baixas concentrações de adriamicina e tempos curtos de adsorção foi investigada, e é determinante na detecção da interacção DNA-adriamicina utilizando um biossensor electroquímico com DNA.

DNA adsorption on electrode surfaces is of fundamental interest for the development of DNA-based biosensors. The free adsorption of 10-mer synthetic oligodeoxynucleotides (ODNs) onto highly oriented pyrolytic graphite (HOPG) surfaces was studied using Magnetic AC mode atomic force microscopy (MAC Mode AFM). The mechanism of interaction of nucleic acids with carbon electrode surfaces was elucidated, using 10-mer synthetic homo- and hetero-ODNs sequences of known base sequences, because they allow clear interpretation of the experimental data. AFM images in air revealed different adsorption patterns and degree of HOPG surface coverage for the ODNs, and correlation with the individual structure and base sequence of each ODN molecule will be presented. The results demonstrated that the hydrophobic interactions with the HOPG hydrophobic surface explain the main adsorption mechanism, although other effects such as electrostatic and Van der Waals interactions may contribute to the free adsorption process. The ODNs interacted differently with the HOPG surface, according to the ODN sequence hydrophobic characteristics, being directly depending on the molecular mass, the hydrophobic character of the individual bases and on the secondary structure of the molecule. The importance of the type of base existent at the ODN chain extremities on the adsorption process was investigated and different adsorption patterns were obtained with ODN sequences composed by the same group of bases aligned in a different order.

In order to better understand the adsorption mechanism of nucleic acids at electrode surface, MAC Mode atomic force microscopy (MAC Mode AFM) was used to investigate the adsorption of a 10-base synthetic oligodeoxynucleotide (ODN) with the sequence 5'-CTTTTTCTTT-3' containing only pyrimidinic bases, onto a highly oriented pyrolytic graphite (HOPG) electrode. Free adsorption and adsorption at applied potentials of +0.30 and +0.65 V versus AgQRE, were carried out. AFM images in air demonstrated that the molecules adsorb spontaneously on the electrode surface. The ODNs have the tendency to self-assemble from solution onto the solid support in a tight and well-spread two-dimensional lattice covering the entire surface, showing the existence of different molecular conformations and exposing parts of the HOPG surface. The degree of surface coverage and the adsorption pattern were directly dependent on ODN concentration and immobilization procedure. During free adsorption, the hydrophobic interactions of the ODNs with the HOPG represent the main adsorption mechanism. When a positive potential is applied to the HOPG, electrostatic interactions between the positively charged electrode surface and the negatively charged sugar-phosphate backbones of ODNs are predominant.

Electrode surface characteristics represent an important aspect on the construction of sensitive DNA electrochemical biosensors for rapid detection of DNA interaction and damage. Two different immobilization procedures of double-stranded DNA (dsDNA) at the surface of a HOPG electrode were evaluated by MAC mode AFM performed in air. A thin dsDNA adsorbed film forming a network structure with holes exposing the electrode surface and a thick dsDNA film completely covering the electrode surface, presenting a much rougher structure, were investigated. The DNA surface characteristics and structure are discussed with respect to the degree of surface coverage.

Guanine rich nucleic acids can self-assemble into four-stranded guanine (G)-quadruplex structures that have been identified in eukaryotic telomeres, as well as in non-telomeric genomic regions, such as gene promoters, recombination sites and DNA tandem repeats, finding wide applications in areas ranging from medical chemistry to nanotechnology and biosensor technology. In addition to classical methodologies for studying G-quadruplex structures, such as circular dichroism, nuclear magnetic resonance or crystallography, the electrochemical methods present very high sensitivity and selectivity and have been used for the rapid detection of the conformational changes from single-strand to G-quadruplex. This review is focused on the recent advances of G-quadruplexes electrochemistry and the design of strategies for the fabrication of G-quadruplex-based biosensors with electrochemical detection, in particular G-quadruplex aptasensors and hemin/G-quadruplex peroxidase-mimicking DNAzymes biosensors.

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.

Single-stranded and double-stranded DNA electrochemical biosensors prepared by adsorption during 3 min on HOPG, with or without an applied potential, at pH 5.3 and 7.0, were characterised by MAC mode AFM. During adsorption DNA condenses on the substrate forming complex network films with pores exposing the HOPG surface. The thin films formed in pH 5.3 acetate buffer always presented a better coverage of the HOPG surface with DNA molecules than films formed in pH 7.0 phosphate buffer. The application of a positive potential of 300 mV during adsorption enhanced the robustness and stability of the DNA films with the formation of bigger network holes and a more condensed and compact self-assembled DNA lattice. The knowledge of the morphology of adsorbed DNA on electrode surfaces explains non-specific adsorption on the electrode surface and can be used to improve and develop DNA-electrochemical biosensors.

Single-molecule AFM imaging of single-stranded and double-stranded DNA molecules self-assembled from solution onto a HOPG electrode surface is reported. The interaction of DNA with the hydrophobic surface induced DNA aggregation, overlapping, intra- and intermolecular interactions. Controlling the electrode potential and using the phase images as a control method, to confirm the correct topographical characterization, offers the possibility to enlarge the capability of AFM imaging of DNA immobilized onto conducting substrates, such as HOPG. The application of a potential of +300 mV (versus AgQRE) to the HOPG enhanced the robustness and stability of the adsorbed DNA molecules, increasing the electrostatic interaction between the positively charged electrode surface and the negatively charged DNA sugar-phosphate backbone.

Magnetic AC mode atomic force microscopy (MAC Mode AFM) was used to characterize the process of adsorption of DNA on a highly oriented pyrolytic graphite (HOPG) electrode surface using different concentrations of DNA and adsorption procedures. AFM of DNA immobilized on the HOPG showed that both single-stranded DNA and double-stranded DNA molecules have the tendency to spontaneously self-assemble from solution onto the solid support and the process was very fast. DNA condensed on the substrate in a tight and well-spread two-dimensional lattice covering the entire surface uniformly. The interaction of DNA with the hydrophobic HOPG surface induced DNA superposition, overlapping, and intra- and intermolecular interactions. The application of a positive potential of 300 mV (vs Ag wire) to the HOPG electrode during adsorption was studied. The applied potential considerably enhanced the robustness and stability to mechanical stress of the DNA films, through multiple electrostatic interactions between the negatively charged hydrophilic sugar−phosphate backbone and the positively charged carbon surface. The characteristics of the DNA films and the apparent height of the network wires were dependent on the DNA concentration and the immobilization procedure. The DNA lattices were held together on the substrate surface only by noncovalent interactions such as hydrogen bonding, base stacking, electrostatic, van der Waals, and hydrophobic interactions.

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 pH-dependent 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.