3. Results and discussion 3.1. Characterisation of modified electrodes Figure . (A) presents a picture of the silicon chip. In this work, an external Ag/AgCl reference electrode was used during the deposition of AuNPs and Cht-Au nanocomposite, while the on-chip pseudo-reference electrode was used for DNA detection. Figure . (B) shows a silicon chip in a chip holder. Chitosan was electrodeposited at sensor IDE in presence of Au ions, similar to Du, Luo . To find the optimal potential for cathodic deposition of chitosan, cyclic voltammetry (CV) in Cht-Au solution (0.04 % chitosan, 50 ppm Au) at pH ~5 was undertaken. The potential was cycled between 0 V and -2 V, see Figure . (C), and it was found that the cathodic current increased significantly around -1.4 V suggesting the commencement of hydrogen evolution and therefore an increase in pH around the electrode. The observed current remained stable (plateaued) until -1.8 V whereon it started increasing rapidly at more cathodic applied voltages. Consequently, based on CV data, the potential range of interest for Cht-Au deposition was found to be between -1.4 and -1.8 V. To confirm this, multiple electrodepositions were undertaken in the potential of -1.2 V, and -2 V and visualised under optical microscopy. Figure . shows that a defined polymer layer was electrodeposited at sensor IDEs for potentials of -1.4, -1.5 and -1.6, which supports the results based on CV. At -1.2 V the pH change was not sufficient for chitosan deposition at the electrode while at greater than -1.8 V the hydrogen evolution was so rapid that the polymer deposited around the electrode instead of specifically at an array. Following these experiments, a potential of -1.5 V was selected as optimal for chitosan deposition. CV in 5 mM Fe 4-/Fe 3-in 0.1 M KCl as supporting electrolyte (scan rate 100 mV/s) were undertaken to characterise the different gold surfaces of a pristine IDE and post modified IDEs. In addition, the electrodeposition of a gold nanoparticle layer onto gold electrodes was explored to increase surface roughness prior to Cht-Au deposition. Figure . (D) presents CV for (i) unmodified gold, (ii) Cht-Au, (iii) AuNPs, and (iv) AuNPs/Cht-Au. For pristine gold electrodes, a well-defined voltammogram exhibited typical characteristics for the Fe 4-/Fe 3-redox couple with a peak oxidation current of ~25 nA and low hysteresis was observed. Following electrodeposition of a Cht-Au complex at the unmodified electrode, a significant decrease in the magnitude of the redox currents for the Fe - 4 /Fe 3-redox couple was observed (~3 nA). This suggests a Cht-Au layer deposited effectively onto the electrode surface and that this layer had a lower conductivity and thus partially insulated the surface of the electrode; due to the lack of conduction pathways from the underlying electrode to the modified layer. We attribute the lack of these pathways to the smoothness of the evaporated gold of the electrode. An increase in Fe -4 /Fe 3-current magnitudes, compared to the pristine gold electrodes (~50 nA), were observed for IDE electrodes first modified with an Au-NP layer, which may be attributed to an increase in surface area. A Cht-Au layer was then deposited onto these modified electrodes and characterised by CV and electrochemical impedance spectroscopy (EIS). From the CV data, it was observed that (unlike the unmodified electrodes) the addition of the Cht-Au layer, resulted in a slight increase in the Fe - 4 /Fe 3-current magnitudes and also decreased the separation between the oxidation and reduction peaks, suggesting a successful deposition of this layer. The CV results are supported by the EIS data presented in Figure (E) where a decrease in impedance was observed from 12.2 MΩ at unmodified IDE to 5 MΩ following modification of the underlying AuNPs layer, due to the increased surface area and 5.5 MΩ following deposition of a subsequent Cht-Au layer. The effect of varying the Cht concentrations within the Cht-Au complex was also investigated at IDEs pre-modified with AuNPs, see Figure (F). Cht-Au complexes were deposited at -1.5 V for 15 seconds with the CHI concentrations varied between 0.01% and 0.05%. It was found that the conductivity of the electrode increased with the increasing chitosan concentration reaching a maximum at 0.04% of chitosan and decreasing thereafter. This suggests that, with increasing chitosan concentration in a solution, the Cht enables the formation of more gold nanoparticles at an electrode, which results in an increase in electrode conductivity. However, at higher Cht concentrations, the Cht: Au ratio becomes unfavourable, thereby decreasing the conductivity of the electrode. Figure .