*3.1. Cyclic Voltammetry (CV)*

One of the most powerful and popular voltammetry techniques used to investigate the reduction and oxidation processes of molecular species is cyclic voltammetry (CV). CV is an analysis to study electron transfer-initiated chemical reactions, which includes catalysis. Generally, a typical graph with a "duck" shaped curve will be obtained from CV analysis which is called a voltammogram or a cyclic voltammogram, as shown in Figure 7.

**Figure 7.** Examples of "duck" shaped cyclic voltammograms [49]. Reproduced with permission from [N. Elgrishi], [A Practical Beginner's Guide to Cyclic Voltammetry]; published by [ACS Publications], [2017].

From the graph, it can be seen that the *x*-axis represents the applied potential (*E*) that is imposed on the system; meanwhile, the *y*-axis is the resulting current (*i*) passed, which is the response during the measurement. Some direct information can be obtained from the CV graph, such as that at the potential axis (*x*-axis), it contains an arrow which indicates the direction of the scanned potential used to record the data. Besides that, it also indicates the beginning and sweep direction of the first segment (or "forward scan"). Sometimes, a crucial parameter also can be found in the graph which is scan rate (*υ*). It indicates that the potential was varied linearly at the speed (scan rate) during the experiment: for example, *υ* = 100 mV/s [49].

Figure 8 shows the "duck"-shaped voltammogram of a reversible reduction of 1 mM Fc+ solution to Fc, at a scan rate of 100 mV/s. As the potential is scanned negatively (cathodically) from point A to point D, (Fc+) is reduced to Fc and it is steadily depleted near the electrode. Simultaneously, a peak cathodic current (*i*p,c) can be observed at point C [50]. It is dictated by the delivery of additional Fc+ via diffusion from the bulk solution. The volume of solution at the surface of the electrode containing the reduced Fc, called the diffusion layer, continues to grow throughout the scan. This will then slow down the mass transport of Fc<sup>+</sup> to the electrode. Thus, upon scanning of more negative potentials, the diffusion rate of Fc<sup>+</sup> from the bulk solution to the electrode surface becomes slower, resulting in a decrease in the current as the scan continues (C→D). When it reaches the switching potential, D, the scan direction is reversed, and the potential is scanned in the positive (anodic) direction. While the concentration of Fc+ at the electrode surface is depleted, the concentration of Fc at the electrode surface is increased, satisfying the Nernst equation. The Nernst equation (Equation (4)) can be used in order to predict how

a system will respond to a change of concentration of species in solution or a change in the electrode potential. The Fc presented at the electrode surface is oxidized back to Fc+ as the applied potential becomes more positive. At points B and E, the concentrations of Fc<sup>+</sup> and Fc at the electrode surface are equal, following the Nernst equation, *E* = *E*1/2. This corresponds to the halfway potential between the two observed peaks (C and F) and provides a straightforward way to estimate the *E*0 for a reversible electron transfer, as noted above. The two peaks are separated due to the diffusion of the analyte to and from the electrode.

$$E = E^0 + \frac{RT}{nF} \ln{\frac{(Ox)}{(Red)}} = E^0 + 2.3026 \frac{RT}{nF} \log{10} \frac{(Ox)}{(Red)} \tag{4}$$

**Figure 8.** Concentration profiles (mM) for Fc+ (blue) and Fc (green) as a function of the distance from the electrode (D, from the electrode surface to the bulk solution, e.g., 0.5 mm) at various points during the voltammogram analysis [49]. (Current flow from A to G.) Reproduced with permission from [N. Elgrishi], [A Practical Beginner's Guide to Cyclic Voltammetry]; published by [ACS Publications], [2017].

Generally, in an electrochemical sensor, cyclic voltammetry is used to study the effect of a conducting polymer's modification towards its current intensity peak. Previously, Kwak et al. reported on the modification of PPy-base with carbon doped polydimethylsiloxane (PPy/ CPDMS). Their results showed current peaks during the reduction and oxidation exhibited at a voltage nearby 1.5 V and −1 V, respectively [51]. As the scan rate increases, the currents peak magnitude tends to increase due to the higher scan rate facilitating a thin diffusion layer between the electrolyte and the PPy surface [49]. However, as the scan rate was increased, the voltage at the corresponding current peaks were not identical during the redox reaction. This implied some degree of chemical irreversibility possibly caused by insufficient electron transfer because of the fast scan rate, or the decomposition of the PPy surface [52,53].

Zaabal et.al modified a glassy carbon electrode with polypyrrole (PPy/GCE) to be used as a promising electrode for electrochemical sensing of adefovir (ADV). They reported a weak anodic peak current obtained at 1.559 V for the unmodified electrode. By modifying the GCE with PPy, the anodic peak was shifted to a more negative potential which was 1.484 V accompanied by an enhancement in the peak height of ADV. The higher anodic response of ADV at the PPy/GCE electrode showed that this modified electrode was more sensitive than GCE alone. The enhanced signals and shift of potential peak towards the negative direction indicated that the modified electrode improves electrochemical reactivity of ADV oxidation as compared to bare GCE. This was probably mainly due to the large effective surface area and subtle electronic conductivity of PPy film, which was beneficial to promoting the electron transfer reaction [54].

Besides having a shift in potential axes, the changes in current peak also give important information; for example, in research done by Chen et al. [55] where they prepared a novel polypyrrole/glassy carbon electrode (PPy/GCE) core-zeolitic imidazolate framework-8 (ZIF-8) shell structure composite for quercetin (QR) determination. They found that the current peak of the QR sensor composed of ZIF-8/PPy/GCE was higher than the bare PPy/GCE electrode. It was due to a larger electrocatalytic surface obtained from ZIF-8 and high charge collectability of the host PPy [54]. A similar trend was observed by Hu at el. [56], where they prepared a novel electrochemical sensor based on ion imprinted polypyrrole and reduced graphene oxide (PPy/rGO) composite for trace level determination of cadmium ion (Cd(II)) in water. They found that with the addition of rGO into PPy/GCE, it increased the rate of electron transfer on the electrode surface and amplified the signal response [56].

Furthermore, Yu et al. developed a new electrochemical sensor based on titanium dioxide (TiO2) and a PPy molecularly imprinted polymer (MIP) nanocomposite for the highly selective detection of p-nonylphenol in food samples. On just the bare GCE, a well-defined reversible redox peak could be observed. When the GCE was modified with PPy and TiO2, the current intensity peak was obviously enhanced. It suggested that the modification could result in a larger electrochemical surface area, due to the cavities found in the PPy matrix which could accelerate electron transfer of (Fe(CN)6) <sup>3</sup>−/4−. After incubation with p-nonylphenol, the MIP absorbed p-nonylphenol molecules and blocked the cavities in the PPy matrix. Thus, the redox peak current intensity decreased as a result of the limitations of electron transfer. In contrast, the electrode modified with PPy and nanoimprinted TiO2 exhibited a lower current intensity peak compared to PPy with TiO2 MIP [57].

However, this was found to be different from the findings Ma et al., where they developed an electrochemical biosensor based on sodium alginate-polypyrrole/Au nanoparticles (SA-PPy/AuNPs) nanocomposite for the detection of miRNAs [58]. They reported that the current peak decreased after the modification of bare GCE. The redox peak current of Fe(CN)6 <sup>4</sup>−/3<sup>−</sup> slightly decreased due to the poor conductivity of SA and modified hair pin (H1). This could slow down the electron transfer on the surface of the electrode. The GCE/SA-PPy/AuNPs/H1 modified with miRNA-21 and modified hair pin (H2) formed a large number of double helix DNA structures on the surface of the electrode due to the occurrence of the CHA reaction, with a reduction in the redox peak current of Fe(CN)6 <sup>4</sup>−/3−. Finally, a slight decrease in redox peak current was observed after the copper ion (Cu(II)) complex was inserted onto the double helix DNA structure. This could be attributed to the dissolution of the Cu(II) complex in the mixture of dimethyl sulfoxide (DMSO) and water (H2O) (volume ratio 7:3). However, the observed poor solubility may have triggered a blockage of the electron transfer between the surface of the electrode and the electrolyte [58].

Besides conducting polymer modifications, the current peak of CV can also be affected by the concentration of a sample [30,59–61]. Zhang et al. designed and constructed an electrochemical ammonia sensor based on Ni foam-supported silver/polypyrrole and platinum nanoparticles electrode (Pt-Ag/PPy-NiF). They studied the effect of ammonia concentration on the oxidation current peak of the PPy/Pt/Ag/NiF electrode and found that its current peak increased when the ammonia concentration increases. This happened because of the strength of the synergistic effect between Ni foam and Pt nanoparticles [59]. Suvina et al. developed a polypyrrole-reduced graphene oxide hydrogel composite electrode for the detection of metal ions. They investigated the effect of metal ion concentration on the PPy-rGO hydrogel electrode and observed that the formation of a multilayer metal ion complex accumulated as a pre-deposited monolayer helped increase the peak current [61]. Meanwhile, Devi et al. prepared a mixture of PPy with synthesized zinc oxide nanoparticles (ZnO-NPs) which were then electropolymerized onto a platinum (Pt) electrode to form a ZnO-NPs–polypyrrole (PPy) composite film. Then, xanthine oxidase (XOD) was immobilized onto the nanocomposite film through physiosorption to study the effect

of XOD concentration on the ZnO-NPs/PPy/Pt electrode. They reported that the increases in oxidation current was due to the increased concentration of hydrogen peroxide (H2O2) produced during enzymatic reaction [30]. However, it is in contrast to Alagappan et al.'s study, which prepared an electrochemical cholesterol biosensor based on the cholesterol oxidase (ChOx) enzyme immobilized on a gold nanoparticle—functionalized -multiwalled carbon nanotube (MWCNT)—polypyrrole (PPy) nanocomposite modified electrode. They reported that the anodic and cathodic peak currents decreased with an increase in cholesterol concentration. This happened because of an absence of a redox mediator in the system which reduced the electron hopping from the analyte to the enzyme modified electrode [60].

Besides that, the potential difference between the anodic and the cathodic peaks also can be extracted from a cyclic voltammogram. Lo et al. prepared a PPy/CNT/NH2- ITO composite by electropolymerization onto polypyrrole-aminophenyl-modified flexible indium tin oxide (PPy/NH2/ITO) electrodes coated with multi-walled carbon nanotubes (CNTs), in the presence of ethylene glycol-bis(2-aminoethylether)-tetraacetic acid (EGTA) as a chelator. They reported that the potential difference of PPy films deposited onto bare ITO was 430 mV [62]. This was high compared to bare ITO which only exhibited 165 mV [63]. This difference can be linked to the absence of any adhesion between the PPy layer and the bare ITO surface. Meanwhile, in the case of PPy/NH2-ITO, the presence of NH2 on ITO contributed to an increase in electronic transfers leading to a lower ΔE (181 mV). For PPy/CNT/NH2-ITO, ΔE = 321 mV. The CVs were consistent with those obtained by impedance measurement.

Pineda et al. [64] investigated the effect of polymerization time on the potential and current peak on polypyrrole (PPy) films with a micro tubular structure decorated with gold nanoparticles. The result showed that the anodic current peak from the voltammogram of the PPy film exhibited a cauliflower-like structure, occurring at 0.05 V, and there was found a small cathodic peak at −0.8 V that was led by a small hump at −0.5 V. Furthermore, these anodic and cathodic current peaks were well-defined at ca. 0.28 V and −0.45 V, respectively, as the electropolymerisation time was increased and the tubular structure was formed. This shows that the tubular structure exhibited a better separation between the faradaic and capacitive contributions in a polymeric deposited film [64].

Another aspect that can be studied through cyclic voltammogram is electrocatalytic behavizr [65,66]. Xing et al. studied the electrocatalytic behavior of polypyrrole/platinum (PPy/Pt) nanocomposites toward hydrogen peroxide (H2O2) reduction. They found that in the absence of H2O2, no reduction peak was observed with bare glassy carbon electrode (GCE), PPy/GCE, and PPy/Pt/GCE. Upon the addition of H2O2, no obvious current from the reduction of H2O2 was observed at bare GCE other than a minor increase in the background current. While only a weak reduction peak for H2O2 at about −0.28 V was observed on the PPy/GCE electrode, in contrast, on the PPy/Pt/GCE electrode, there was a remarkable reduction peak of H2O2 obtained of around −0.2V. This was even higher than the bare Pt electrode in terms of reduction peak current value, indicating that the PPy/Pt/GCE might provide a better electrocatalytic effect than the bare Pt electrode [65].

The electrocatalytic oxidation of an adenine and guanine mixture at bare and modified PPy/graphene/GCE electrodes were studied by Gao et al. [66]. They reported that there was no oxidation signal observed in the CV curves of the PPy/graphene/GCE electrode due to a blocking effect but there was a high current peak observed with the modified PPy/graphene/GCE electrode. The report also concluded that the overoxidized polypyrrole/graphene/glassy carbon electrode (PPyox/graphene/GCE) electrode had the highest current peak of adenine and guanine oxidation which indicated the highest electrocatalytic activity [66].
