Electronic properties and assambly of DNA-based molecules on gold surfaces: A study by electrochemistry, single-molecule in situ STM, and SERS

This thesis presents a multifaceted study of deoxyribonucleic acid (DNA) in the form of strands and individual components, attached/adsorbed on single-crystal Au(111) and Au(110) gold surfaces, and on citrate-reduced gold nanoparticles. Strategically designed DNA moieties were addressed directly in aqueous chemical and biological media, under electrochemicalcontrol using electrochemistry and in situscanning tunnelling microscopy/spectroscopy orlaser radiation (via surface-enhanced Raman spectroscopy).

The aim was to provide new insight into surface assembly mechanisms, molecular interactions, and interfacial charge transfer kinetics of DNA-based molecules at the level of the single molecule. Pure and new synthetic oligonucleotides (ONs) were functionalizedwith terminating alkanethiols and attached to the gold surfaces via their terminal thiol groups, with subsequent formation of (full or dilute) self-assembled monolayers(SAMs), grafted to the substrate through the strong gold-sulphur bond.

The voltammetric behaviour of such DNA-based systems was analysed in the presence of “smart” redox molecules, the intercalating aromatic anthraquinone monosulfonate (AQMS) and a covalently attached terpyridine (terpy) redox unit. The spontaneous insertion of AQMS into the DNA double helix, and the chelating geometry of the triply dentate terpy coordinated to several transition metals offered a route to study electronic properties and charge transfer kinetics of the ONs confined at the electrochemical gold/electrolyte interface.

Mercaptohexanol treatment embedding the thiolated ONs in a matrix of smaller thiol-based molecules was exploited to form dilute, more open ON structures and thus favourable conditions for intercalation at the gold/electrolyte interface. The voltammetric analysis of such ON systems proved the efficiency of the hybridization approach adopted, and disclosed an AQMS-induced voltammetric signal specific to the double helical structure intercalated by AQMS.

This supports the charge transfer through the π-stack of such double helices, which is, however, not completely understood at the moment. The fragile behaviour evidenced by such AQMS-intercalated systems inspired further studies of the double helical structures in the presence of polycationic compounds, such as hexamine cobalt(III) chloride, magnesium chloride, and spermidine trihydrochloride.

The larger multivalent cations are expected to give a more rigid ds-ON structure that might assist efficient charge transfer through DNA, by the delicate π-stacking of bases which is extremely sensitive to subtle structural variations. Voltammetry and in situSTM offered a powerful tool to address the structural changes induced by polycation binding and disclosed an unexpected, highly base specific voltammetric peak in the presence of spermidine ions.

A capacitive origin was attributed to this peak, and a novel route to detection of hybridization and base pair mismatches proposed on the basis of the high sensitivity to base pair mismatches showed by such ON-based monolayers in buffered solutions. The terpy unit was shown to be a suitable template for in situ preparation of several transition metal complexes (Os, Fe, and Ru).These could be mapped to single-molecule resolution by in situ STM and STS.

Investigations on the terpy-marked ONs were inspired by these studies and addressing metal coordination of ONs with the terpyridine ligand in the highly flexible structure ofthe new synthetic unlocked nucleic acid (UNA). Composite voltammetric behaviour for each of the metal-functionalized ONbased monolayers was observed and supported by in situscanning tunnelling microscopy/spectroscopy (STM/STS).

The well-defined local environment of single-crystal Au(111)-electrode surfaces enabled achieving optimal voltammetric resolution, to gain further insight into electronic and kinetic properties of such systems. More interestingly, strong in situSTM contrasts induced by metal coordination of terpy-functionalized ONs as a novel way to explore the ET mechanism in ON strands were disclosed.

Unmodified moieties (i.e. DNA constituents and pure ONs) on the open Au(110)-electrode surface and on the citrate-reduced NP surface were finally addressed using electrochemical technique (cyclic voltammetry and in situSTM) and SERS, respectively. Such studies proved adsorption of DNA bases (adenine, cytosine, guanine, and thymine) on the gold substrate, disclosing distinct adsorption patterns for each of them, with new insight into nucleobase assembly on a freshly cleaned Au(110) surface (not nearly as widely employed as Au(111) surfaces).

In particular, SERS offered a valuable and rapid way ofcharacterising interactions between the DNA-based molecules and the NP surface, with no need for complex sample preparation.