Microscopia de varrimento de sonda de DNA e de outras moléculas biológicas absorvidas na superfície de eléctrodos

Tese de doutoramento em Bioquímica (Técnicas Bioquímicas) apresentada à Fac. de Ciências e Tecnologia de CoimbraO 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

Electrochemistry of Flavonoids: A Comprehensive Review

Flavonoids represent a large group of aromatic amino acids that are extensively disseminated in plants. More than six thousand different flavonoids have been isolated and identified. They are important components of the human diet, presenting a broad spectrum of health benefits, including antibacterial, antiviral, antimicrobial, antineoplastic, anti-mutagenic, anti-inflammatory, anti-allergic, immunomodulatory, vasodilatory and cardioprotective properties. They are now considered indispensable compounds in the healthcare, food, pharmaceutical, cosmetic and biotechnology industries. All flavonoids are electroactive, and a relationship between their electron-transfer properties and radical-scavenging activity has been highlighted. This review seeks to provide a comprehensive overview concerning the electron-transfer reactions in flavonoids, from the point of view of their in-vitro antioxidant mode of action. Flavonoid redox behavior is related to the oxidation of the phenolic hydroxy groups present in their structures. The fundamental principles concerning the redox behavior of flavonoids will be described, and the phenol moiety oxidation pathways and the effect of substituents and experimental conditions on flavonoid electrochemical behavior will be discussed. The final sections will focus on the electroanalysis of flavonoids in natural products and their identification in highly complex matrixes, such as fruits, vegetables, beverages, food supplements, pharmaceutical compounds and human body fluids, relevant for food quality control, nutrition, and healthcare research

Atomic Force Microscopy of DNA Immobilized onto a Highly Oriented Pyrolytic Graphite Electrode Surface

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

DNA imaged on a HOPG electrode surface by AFM with controlled potential

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.http://www.sciencedirect.com/science/article/B6W72-4D5X9NS-1/1/d1528546598a5fb98abb8331a760556

8-oxoguanine and 8-oxodeoxyguanosine Biomarkers of Oxidative DNA Damage: A Review on HPLC-ECD Determination

Reactive oxygen species (ROS) are continuously produced in living cells due to metabolic and biochemical reactions and due to exposure to physical, chemical and biological agents. Excessive ROS cause oxidative stress and lead to oxidative DNA damage. Within ROS-mediated DNA lesions, 8-oxoguanine (8-oxoG) and its nucleotide 8-oxo-2'-deoxyguanosine (8-oxodG)-the guanine and deoxyguanosine oxidation products, respectively, are regarded as the most significant biomarkers for oxidative DNA damage. The quantification of 8-oxoG and 8-oxodG in urine, blood, tissue and saliva is essential, being employed to determine the overall effects of oxidative stress and to assess the risk, diagnose, and evaluate the treatment of autoimmune, inflammatory, neurodegenerative and cardiovascular diseases, diabetes, cancer and other age-related diseases. High-performance liquid chromatography with electrochemical detection (HPLC-ECD) is largely employed for 8-oxoG and 8-oxodG determination in biological samples due to its high selectivity and sensitivity, down to the femtomolar range. This review seeks to provide an exhaustive analysis of the most recent reports on the HPLC-ECD determination of 8-oxoG and 8-oxodG in cellular DNA and body fluids, which is relevant for health research

Advances in Electrochemical Biosensor Technologies for the Detection of Nucleic Acid Breast Cancer Biomarkers

Breast cancer is the second leading cause of cancer deaths in women worldwide; therefore, there is an increased need for the discovery, development, optimization, and quantification of diagnostic biomarkers that can improve the disease diagnosis, prognosis, and therapeutic outcome. Circulating cell-free nucleic acids biomarkers such as microRNAs (miRNAs) and breast cancer susceptibility gene 1 (BRCA1) allow the characterization of the genetic features and screening breast cancer patients. Electrochemical biosensors offer excellent platforms for the detection of breast cancer biomarkers due to their high sensitivity and selectivity, low cost, use of small analyte volumes, and easy miniaturization. In this context, this article provides an exhaustive review concerning the electrochemical methods of characterization and quantification of different miRNAs and BRCA1 breast cancer biomarkers using electrochemical DNA biosensors based on the detection of hybridization events between a DNA or peptide nucleic acid probe and the target nucleic acid sequence. The fabrication approaches, the biosensors architectures, the signal amplification strategies, the detection techniques, and the key performance parameters, such as the linearity range and the limit of detection, were discussed

Atomic force microscopy characterization of synthetic pyrimidinic oligodeoxynucleotides adsorbed onto an HOPG electrode under applied potential

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.http://www.sciencedirect.com/science/article/B6TG0-4JVSVMG-2/1/0c861dc43bb4382dd9c195a0ebd408b

Adsorption of synthetic homo- and hetero-oligodeoxynucleotides onto highly oriented pyrolytic graphite: Atomic force microscopy characterization

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.http://www.sciencedirect.com/science/article/B6TFB-4J6WR05-2/1/64ef57ec21856f05e10206d1d7b1033

DNA Electrochemical Biosensors for In Situ Probing of Pharmaceutical Drug Oxidative DNA Damage

Deoxyribonucleic acid (DNA) electrochemical biosensors are devices that incorporate immobilized DNA as a molecular recognition element on the electrode surface, and enable probing in situ the oxidative DNA damage. A wide range of DNA electrochemical biosensor analytical and biotechnological applications in pharmacology are foreseen, due to their ability to determine in situ and in real-time the DNA interaction mechanisms with pharmaceutical drugs, as well as with their degradation products, redox reaction products, and metabolites, and due to their capacity to achieve quantitative electroanalytical evaluation of the drugs, with high sensitivity, short time of analysis, and low cost. This review presents the design and applications of label-free DNA electrochemical biosensors that use DNA direct electrochemical oxidation to detect oxidative DNA damage. The DNA electrochemical biosensor development, from the viewpoint of electrochemical and atomic force microscopy (AFM) characterization, and the bottom-up immobilization of DNA nanostructures at the electrode surface, are described. Applications of DNA electrochemical biosensors that enable the label-free detection of DNA interactions with pharmaceutical compounds, such as acridine derivatives, alkaloids, alkylating agents, alkylphosphocholines, antibiotics, antimetabolites, kinase inhibitors, immunomodulatory agents, metal complexes, nucleoside analogs, and phenolic compounds, which can be used in drug analysis and drug discovery, and may lead to future screening systems, are reviewed