Finally, the effect of ssDNA concentration immobilised at AuNPs/Cht-Au modified sensors IDE on the MB reduction signal was assessed. Figure presents the peak height increase observed after dsDNA (50 nM) binding with different concentrations of immobilised ssDNA (i) 0.25 µM, (ii) 0.5 µM, (iii) 1 µM and (iv) 1.5 µM, respectively. The best response was achieved when 0.5 µM probe concentration was used. This suggests that with 0.25 µM there was not enough probe ssDNA attached to the surface, while at higher concentrations 1 µM and 1.5 µM the probe density was too high to achieve optimal hybridisation due to steric hindrance. Figure . (D) presents square wave voltammograms after incubation of a sensor with 50 nM of (i) target DNA, (ii) non-complementary strand and (iii) a DNA strand containing 3 base mismatches. An increase in current magnitude was not observed for the noncomplementary or mismatched strands. However, a significant increase was observed for the complementary strand, confirming the specificity of the developed biosensor. To find the limit of detection and establish a calibration curve, the AuNPs/Cht-Au/ssDNA modified IDEs were incubated for 20 minutes with a series of DNA concentrations diluted with 0.1 M PBS buffer. The peak height was found to increase linearly between 100 aM and 1 µM of target DNA. Figure . (A) shows the calibration curve where the difference between dsDNA and ssDNA peak height current was plotted against the logarithm of DNA concentration. All data points represent an average value of four measurements (from different sensors) and standard deviation for each point. The calibration line showed excellent linearity, with a correlation coefficient of R 2 =0.995. The lowest measured concertation, and thus the limit of detection, was observed to be 100 aM. Figure . (B) presents square wave voltammograms for each concentration. The baseline was normalised to allow better visualisation To evaluate the specificity of the developed sensor in complex samples, chromosomal DNA extracted from different strains of E. coli, viz, stx1 positive (O103 and O157) and stx1 negative (O91 and 12900) and non-E. coli (Listeria monocytogenes and Bacillus cereus) was tested. After the extraction, the DNA was diluted 5 times with the PBS buffer, heated to 95°C for 5 mins and immediately cooled on ice for 60 s. The samples were then incubated on a sensor for 30 minutes. Figure . (C) presents combined square wave voltammograms from different sensors following incubation with the chromosomal DNA. It can be seen that only DNA extracted from E. coli containing the stx1 gene (O103 and O157) resulted in an increase in the signal, further demonstrating the selectivity of the sensor to complex samples. The signal from the O103 strain was higher than O157, suggesting that it either contained more stx1 gene or it was more readily available to hybridise with the sensor.