Electrochemical-based DNA detection using methylene blue Methylene blue (MB), a redox-active molecule, is known to interact with DNA and the difference in its affinity towards ssDNA and dsDNA has been commonly exploited in the development of electrochemical biosensors . The molecule can (i) bind directly to guanine bases , (ii) is electrostatically attracted to the negatively charged phosphate backbone of DNA , or (iii) intercalate between dsDNA . The mechanisms at which it will interact with DNA can be influenced by the experimental parameters used. In this work, we focused on enhancing MB intercalation between dsDNA and detecting DNA hybridisation based on an increased SWV signal compared to ssDNA. One parameter that was shown to affect MB to dsDNA binding efficiency is the ionic strength of the solution . Kara, Kerman have suggested that the concentration of Cl -ions can influence the efficiency of MB intercalation between dsDNA. In their work, the MB intercalation between dsDNA was preferred over the other type of MB interactions at 10 mM NaCl. In this work, DNA detection was undertaken using 50 µM MB dissolved in 0.02 M HEPES buffer with 10 mM KCl and an accumulation step to achieve the highest efficiency of intercalation. The electrochemical detection of DNA in this work was undertaken by accumulating MB at a DNA modified electrode followed by its reduction to leukomethylene blue recorded with SWV. An OCP commonly used for the accumulation of methylene blue in DNA sensor development was applied to an accumulator electrode as described above. Figure . (A) and Figure . (B) show square wave voltammograms before and after exposure to 50 nM of complementary DNA, with and without applying OCP to the accumulator IDE, respectively. In both cases, the accumulation was done by incubating the chip for 10 minutes in 50 µM MB dissolved in 0.02 M HEPES and 0.01 M KCl (pH 7). The peak increase for dsDNA (50 nM) over ssDNA, without applying an OCP, was 2.28 nA equivalent to ~50% increase in the measured signal. When an OCP was applied to an accumulator IDE, it was observed that the ssDNA peak current had a similar magnitude compared to the static accumulation. By contrast, a significant increase in peak current on the binding of complementary dsDNA (50 nM) was observed with a Δ peak height current (between ssDNA and dsDNA) increased to 5.05 nA, over a 100% increase. This suggests that applying an OCP (8 mV, see Figure .) increases the efficiency of MB intercalation between dsDNA without affecting its interaction with ssDNA. In Figure (D) we have previously shown that electrodeposition of AuNPs as a first layer significantly increased the conductivity of the modified sensor IDE. We further evaluated how AuNPs affected DNA detection using MB. Figure (C) summarises peak current height before (orange) and after (green) hybridisation with complementary strand when the electrode was not modified and modified (400 ppm) with the first layer of AuNPs. It was found that the difference between ssDNA and dsDNA was hard to distinguish without the layer of AuNPs. This is in agreement with Figure where the conductivity of the electrode decreased after Cht-Au modification. However, in the presence of the underlying AuNPs layer, a significant increase in MB measured current was observed between ssDNA and dsDNA, again confirming the need for this layer.