Methylene blue as an electrochemical discriminator of single- and double-stranded oligonucleotides immobilised on gold substrates

Self-assembly of thiol-terminated oligonucleotides on gold substrates provi des a convenient and versatile route to DNA-functionalised surfaces. Here w e show that the square-wave voltammetric peak position of methylene blue co mplexed to thiol-terminated single-stranded oligonucleotides immobilised on gold electrodes differs from that of methylene blue complexed to thiol-ter minated double-stranded oligonucleotides immobilised on gold electrodes, Th e peak potential of methylene blue at the single-stranded oligonucleotide a rray was consistently found to occur at potentials ca. 10-15 mV more positi ve than that at double-stranded oligonucleotide arrays, the precise differe nce being dependent on the direction of the voltammetry. This voltammetric behaviour mirrors that found for methylene blue bound to freely diffusing s ingle- and double-stranded calf thymus DNA and suggests that the immobilise d oligonucleotides retain the methylene blue binding properties of their fr eely diffusing counterparts. Thus methylene blue provides a simple electroc hemical indicator for the status of oligonucleotide-functionalised gold sur faces.