A thermodynamic study of ferrocene modified hairpin oligonucleotides upon duplex formation: applications to the electrochemical detection of DNA

Abstract

New electrochemical probes are being developed for nucleic acid detection. A ferrocenyl phosphoramidite synthon (Fc) is synthesized and then incorporated into stem-loop structured oligodeoxyribonucleotides (ODNs) by automated solid-phase DNA synthesis. The thermodynamic study shows slight destabilization of the structures obtained due to the presence of ferrocene moieties (ΔG°₂₉₈ = −1.28 to −1.82 kcal mol⁻¹ for hairpin folding and ΔG°₂₉₈ = −14.66 to −17.16 kcal mol⁻¹ for duplex) compared to the unmodified sequence (ΔG°₂₉₈ = −2.04 kcal mol⁻¹ for hairpin folding and ΔG°₂₉₈ = −18.97 kcal mol⁻¹ for duplex). When spotting these ferrocenyl stem-loop structured ODNs on a gold electrode microarray, a self-structuring of the probes occurs on the electrode surface. The hairpin opening, generated by the hybridization with the complementary nucleic acid target, induces variations in both current intensity and potential of the ferrocenyl electrochemical signal by cyclic voltammetry (CV). The effects of the number of ferrocenes in the hairpin sequences on thermodynamic stability and the electrochemical response of the probes upon DNA target hybridization are studied. These ferrocene modified stem-loop ODNs allow complementary target detection by cyclic voltammetry.