DNA imaged on a HOPG electrode surface by AFM with controlled potential
Introduction
DNA is an important biomacromolecule with remarkable chemical and biophysical properties [1], [2], [3]. The adsorption of single-stranded and double-stranded DNA at the solid electrodes surface plays a vital role in a variety of biotechnological, medical and nanoscience applications and enables the chemical and structural modification of the sensor surface [4], being very important for understanding many physiological processes. Different structures and conformations that DNA molecules can adopt at the electrode surface lead to different interactions with other molecules, such as modifications of the accessibility of different drugs to the DNA grooves and modifications in DNA hybridization efficiency.
Atomic force microscopy (AFM) has proved to be a powerful tool for obtaining high-resolution images of DNA in air and in solution. Images of DNA conformations, unusual structures and DNA–protein complexes have been obtained almost exclusively on mica or silicon [5], [6], [7], [8], but rarely on conducting materials. Effectively, the DNA molecules do not bind strongly enough to conducting substrates and the AFM tip tends to sweep away the adsorbed macromolecules. AFM imaging onto conducting substrates has been limited to gold substrates [9]. However, the oxidation of the gold electrodes occurs at potentials of approximately +0.8 V, [10], [11] and the gold surface becomes covered with gold oxides. Electrochemical oxidation of nucleic acids on carbon electrodes showed that, with the exception of guanine base, which has an oxidation peak at approximately +0.8 V, depending on the experimental conditions and electrodes used, all the other nucleic acid bases and nucleosides are oxidized at higher electrode potentials [12], [13]. Consequently, the gold surface is not a good choice for electrochemical studies of DNA due to its limited potential range. A major challenge in the area of direct visualization of DNA molecules is to extend the capability of AFM imaging to other conducting substrates required in electrochemical applications.
Carbon electrodes such as glassy carbon, carbon fibres, graphite or carbon black have a wide useful potential range, particularly in the positive direction, which enables the detection of damage caused to DNA by following the oxidation peaks of the purine bases [12], [13], [14].
Highly oriented pyrolytic graphite (HOPG) has been used to study single DNA molecules by STM. However, the geometry of the grain boundary, the step edge texture and surface defects of the freshly cleaved HOPG surface produce STM images that mimic both single-stranded and double-stranded DNA molecules, interfering with the ability to distinguish between the true biological features and the features related with the clean HOPG steps [15], [16], [17]. The problems encountered in STM imaging of the molecules adsorbed onto HOPG steps also limited the use of HOPG in AFM studies of single-DNA molecules. AFM has been has been limited to studying the formation of DNA networked films assembled from high and very high DNA solution concentrations [18], [19], [20], which enabled the formation of thick and stable DNA lattices, completely covering the HOPG boundary defects. Nevertheless, the nature of the single DNA–surface interaction and the morphology adopted by isolated DNA molecules when small solution concentrations are used is still not yet well understood.
Magnetic AC mode AFM (MAC Mode AFM) permits the visualization of the molecules weakly bound to the substrate material and it can be very helpful in the investigation of single-molecules loosely attached to the conducting surface of electrochemical transducers. AFM is not as sensitive as STM to unusual electronic structures on the surface, the AFM images being less affected by artefacts than STM [5].
In this context, this paper explores the possibility of using MAC Mode AFM to image single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) from calf-thymus immobilized by free adsorption and by applying a potential of +300 mV vs. AgQRE to the HOPG electrode surface immersed in solutions of low DNA concentration. Calf-thymus DNA is a large molecule that is easily commercially available and has already been used for biosensor construction [13], [14].
Section snippets
Materials
Calf-thymus double-stranded DNA (sodium salt, type I) and single-stranded DNA were purchased from Sigma-Aldrich Química, Spain and were used without further purification. The electrolyte used was pH 7.0 0.1 M phosphate buffer solution and was prepared using analytical grade reagents and purified water from a Millipore Milli-Q system (conductivity <0.1 μS cm−1). Solutions of different concentrations were obtained by direct dilution of the appropriate volume in phosphate buffer.
Highly oriented
Free adsorption of ssDNA and dsDNA on HOPG
DNA is a highly charged, hydrophilic molecule, whereas HOPG has a hydrophobic surface. These characteristics reduce the spontaneous interaction of DNA with the HOPG surface. Despite the fact that MAC mode AFM is a gentle technique, with a view to minimizing as much as possible any damage to the biological films by the AFM tip, it represents one of the most significant problems when imaging single DNA molecules. In order to improve the stability of the molecules on the surface, the DNA samples
Conclusions
The results suggest that the adsorption of ssDNA and dsDNA at the HOPG surface can be controlled by the applied potential and the electrochemically assisted adsorption provides better attachment of the molecules at the HOPG surface compared with free adsorption. Parts of the molecules interact together by hydrogen bonding during equilibration on the substrate, and hydrophobic interactions and van der Waals forces may also contribute to adsorption on the HOPG electrode. For both ssDNA and dsDNA,
Acknowledgements
Financial support from Fundação para a Ciência e Tecnologia (FCT), Post-Doc Grant SFRH /BPD/14425/2003 (A.-M. C.P.), POCTI (co-financed by the European Community Fund FEDER), ICEMS (Research Unit 103) and European Projects QLK3-2000-01311 and HPRN-CT-2002-00186 are gratefully acknowledged.
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