A label-free electrochemical DNA biosensor based on a Zr(IV)-coordinated DNA duplex immobilised on a carbon nanofibre|chitosan layer

摘要

A label-free electrochemical biosensor for detecting DNA hybridisation was developed by monitoring the change in the voltammetric activity of ferrocenecarboxylic acid at the biosensor–solution interface. The biosensor was constructed by initially immobilising on a glassy carbon electrode an anchoring layer consisting of chitosan, carboxyl group functionalised carbon nanofibres and glutaraldehye. Chitosan acted as an adhering agent and carbon nanofibres were strategically used to provide a large surface area with binding points for DNA immobilisation, while glutaraldehye was a linker for DNA probes on the electrode surface. Based on a two-factorial design, cyclic voltammetry of [Fe(CN)₆]3−/4− was performed to optimise the composition of the anchoring layer. Next, a 17-base pair DNA probe was attached to the anchoring layer, followed by its complementary target. Zr(IV) ion, known to exhibit affinity for oxygen-containing electroactive markers, for example, ferrocenecarboxylic acid, was then coordinated in the DNA duplex. In this way, ferrocenecarboxylic acid was attracted towards the biosensor for oxidation. A change in the voltammetric oxidation current of ferrocenecarboxylic acid pre- and post-hybridisation was used to provide an indication of hybridisation. A linear dynamic range between 0.5 and 40 nM and a detection limit of 88 pM of DNA target were then achieved. In addition, the biosensor exhibited good selectivity, repeatability and stability for the determination of DNA sequences.