**3. Results**

#### *3.1. Electrode Modification and Characterizations*

The PPy-NT/AuNPs-modified electrodes were characterized by TEM and SEM, as shown in Figure 2. The nanotube morphology is clearly present and fully covered the mesh substrate (Figure 2A,B). The AuNPs can be seen in Figure 2C and in more detail in Figure 2D, using backscattered electron images (Figure 2D); the gold presence was also corroborated by EDS spectrum (Figure A1). The TEM images show individual AuNPs (Figure 2E) with very few nanometers spread along the PPy-NT's surface. Using TEM, it was also possible to verify the filling of the mesh structure with the polymer nanotubes (Figure 2F).

**Figure 2.** Representative SEM images from the steel mesh coverage: (**A**,**B**) closer approximation of a wire mesh, (**C**) the wire-mesh image of secondary electrons of the hybrid PPy/AuNPs, and (**D**) the SEM with backscattered electrons. (**E**,**F**) TEM representative images from a single nanotube and a small gap in between the steel mash, respectively.

The electrochemical characterization of modified electrodes relies on two fundamental techniques, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). These two must be studied in consonance to obtain valuable information about the electrode kinetics, adsorption and fouling effects, electron transfer, mass transport effects, steady state conditions, and so on. For EIS studies, it is important to adopt an equivalent circuit model to better understand and quantify different processes at the electrode surface; to date, the Randles modified circuit is very common in the study of conductive-polymer-modified electrodes [31,32]. For the biosensor proposed herein, the main information obtained by the EIS technique is associated with the biomolecule interaction, such as antigen–antibody, a so-called affinity interaction caused by the changes at the interface of the electrochemical active material, in terms of both charge transfer and double-layer effects [1,5,31].

Electrochemical experiments of CV and EIS were performed to characterize and compare the proprieties between PPy-NTs- and PPy-NTs/AuNPs-modified electrodes. Figure 3A shows the CVs for each modified electrode, and it is possible to observe an increment in the current in the presence of AuNPs. It is important to note that no additional redox processes are observed; there is solely an increment of the capacitive current, indicating an increase of the electroactive surface provoked by the exposure of a large area of the AuNPs. Figure 3B shows the Nyquist plots of the modified electrodes; they show a traditional semicircle response that is characteristic of conducting polymers. Clearly there is a drastic decrease in the semicircle radius in the presence of AuNPs; in general lines, this behavior indicates an increase in the electroactivity of the interface, thus corroborating the presence of a metallic structure on a polymeric matrix. The equivalent circuit used to fit the electrochemical parameters is found in Figure 3C; they can be summarized as follows: the QDL parameter is related to the energy of the double layer at the interface electrode/electrolyte, the RCT is the resistance of the charge transfer at the electrode surface, RS is the resistance of the solution, and QLF deals with the number of interacted ions inserted within the polymeric matrix.

**Figure 3.** (**A**) CV for the electrodes modified with just PPy (black) and PPy/AuNPs (red). The Nyquist plot is shown in (**B**) from electrodes modified with PPy (black) and with PPy/AuNPs (red). The equivalent circuit used to model the EIS results is also inserted as (**C**).

The experimental results obtained in Figure 3B were modeled according to the equivalent circuit shown in Figure 3C; the results are shown in Table 1. As discussed previously, there is a significant improvement in the charge transfer in the polymer/electrode interface with the AuNPs, as indicated by the lower value of RCT. It is important to add that the presence of a metallic particle itself contributes to the increment of conductivity of the PPy-NTs, and this also facilitates any electron transfer at the surface. Due to the high superficial area of AuNPs, the QDL value shows an increment of almost 2.5 times, in agreement with the increase that the capacitive current showed in CV. At a low frequency, the QLF value had no significant variations, indicating that the intercalation of charges in the polymeric matrix is not affected by the presence of AuNPs; this seems reasonable, as the amount of polymer was kept the same, at the same cutoff charge. Regarding the morphology, after the AuNPs' deposition, it was possible to observe a decrease in the nDL and nLF parameters, which represent the escape from ideality of a traditional parallel capacitor, which represents *n* = 1; thus, the further away it is from the unity, the rougher the surface is present at the electrode surface [33,34].


**Table 1.** Parameters' values obtained by EIS to PPy e PPy/AuNPs, obtained from fitting of EIS results, R2 > 0.99. The equivalent circuit was modeled by the NOVA software.
