Electrochemcial characterisation and hybridisation efficiency of co-assembled monolayers of PEGylated ssDNA and mercaptohexanol on planar gold electrodes.

The realisation of efficient genosensors relies crucially on the surface chemistry strategy selected for the immobilisation of DNA probes. We report on the performance of surfaces prepared via the direct co-immobilisation of a 20-nucleotide (nt) thiolated single stranded DNA probe (ssDNA) in the presence of the short alkanethiol mercaptohexanol (MCH) at varying ratio. Electrochemical impedance spectroscopy was used for the physical characterisation of the formed monolayers. Functional characterisation of the surfaces was achieved by means of differential pulse voltammetry and calibration curves in the range 6.25-100 nM of the complementary sequence 104 nt in length were obtained for each surface studied, using the guanine specific redox marker methylene blue (MB) as hybridisation indicator. The best sensor performance was obtained for a ratio of 1:100, as previously suggested by electrochemical impedance spectroscopy. No hybridisation could be measured at 1:1 ratio and significantly lower hybridisation were recorded for surfaces prepared at 1:10 and 1:1000 ratio. For comparison, sensors prepared via initial thiolated ssDNA immobilisation followed by the backfilling of the surface with MCH, as widely reported, were also assessed. Our results suggested that the performances of such sensors were sub-optimum and comparable to that obtained at 1:10 co-immobilisation. We concluded that the co-immobilisation approach offers several advantages, with highly reliable surfaces being prepared in a single step.