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

Graphite–castor oil polyurethane composite electrode surfaces – AFM morphological and electrochemical characterisation

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

Atomic Force Microscopy and Voltammetric Investigation of Quadruplex Formation between a Triazole-Acridine Conjugate and Guanine-Containing Repeat DNA Sequences

The interactions of the Tetrahymena telomeric repeat sequence d(TG4T) and the polyguanylic acid (poly(G)) sequence with the quadruplex-targeting triazole-linked acridine ligand GL15 were investigated using atomic force microscopy (AFM) at a highly oriented pyrolytic graphite and voltammetry at a glassy carbon electrode. GL15 interacted with both sequences, in a time dependent manner, and G-quadruplex formation was detected. AFM showed the adsorption of quadruplexes as small d(TG4T) and poly(G) spherical aggregates and large quadruplex-based poly(G) assemblies, and voltammetry showed the decrease and disappearance of GL15 and guanine oxidation peak currents and appearance of the G-quadruplex oxidation peak. The GL15 molecule strongly stabilized and accelerated G-quadruplex formation in both Na+ and K+ ion-containing solution, although only K+ promoted the formation of perfectly aligned tetra-molecular G-quadruplexes. The small-molecule complex with the d(TG4T) quadruplex is discrete and approximately globular, whereas the G-quadruplex complex with poly(G) is formed at a number of points along the length of the polynucleotide, analogous to beads on a string.Financial support from Fundação para a Ciência e Tecnologia (FCT), Grant SFRH/BPD/92726/2013 (A.-M. Chiorcea-Paquim), Project Grant (A.D.R. Pontinha), projects PTDC/SAU-BMA/118531/2010, PTDC/QEQ-MED/0586/2012, PEst-C/EME /UI0285/2013 and CENTRO-07-0224-FEDER-002001 (MT4MOBI) (co-financed by the European Community Fund FEDER), FEDER funds through the program COMPETE - Programa Operacional Factores de Competitividade is gratefully acknowledged. Work in the S.N. laboratory was supported by Programme Grant No. C129/A4489, from Cancer Research UK, and by the FP6 framework grant “Molecular Cancer Medicine” from the EU. S.S. was a Maplethorpe Fellow of The University of London.Peer reviewe

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

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

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

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

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

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

Redox behaviour of G-quadruplexes

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