*3.2. Functionalized Steel Mesh Electrode (PPy/AuNPs/MPA) for Biosensing Applications* Avidin-HRP/Biotin Complex: A Model System

The steps of the biosensor construction were characterized electrochemically by CV and EIS, as shown in Appendix A Figures A1 and A2, where the blocking of the surface can be easily identified. The availability for the attachment of biomolecules was performed by the Avidin-HRP protein to detect Biotin, as a well-known system, possessing a very strong interaction. Avidin is a basic tetrameric glycoprotein composed of four identical subunits, and each of these subunits can bind to Biotin with high stability and affinity, being one of nature's strongest non-covalent interactions (dissociation constant = 10−<sup>15</sup> mol L<sup>−</sup>1). Thus, this interaction can be used to verify the effectiveness of the modified electrode, as shown elsewhere [35,36].

In Figure 4A, it is shown how the concentration of Biotin affects the voltammetric response of the electrode. The voltammogram just after the blocking of glycine is shown for the sake of comparison, as no Biotin is added. Clearly the CVs present a diminishment of the current response, indicating the adsorption of Biotin at the electrode surface, where some active sites are no longer available. This effect is also observed in the Nyquist plots (Figure 4B), with the change of the RCT parameter, as observed in other contributions [28,37]. As the concentration of the insulating Biotin increases, more electroactive sites are being hindering, so there is the increment of the resistance of any potential redox reaction; since this behavior is related to the amount of analyte, a proper analytical curve can be drawn, as shown. The EIS results of Figure 4B were modeled, as mentioned before, and the results are shown in Table 2. Besides the variation of the RCT, the QDL parameter also changes, indicating that the double layer is also affected by the presence of Biotin, corroborating the strong adsorption at the electrode's surface. The other parameters have shown no drastic changes, and this outcome is in consonance with no redox reactions promoted by the PPy-NT electrodes.


**Table 2.** Parameters' values obtained by EIS to PPy e PPy/AuNPs after fitting, R2 > 0.98.

These results obtained with the avidin/biotin biological system indicate the interesting behavior of PPy-NTs/AuNPs-modified electrodes for the construction of biosensors based on electrochemical response, as is later discussed.

**Figure 4.** Cyclic voltammetry (**A**) and Nyquist plot (**B**) of the EIS measurement to Biotin detection (100 up to 900 fmol L<sup>−</sup>1) indicated by colors in both CV and EIS.

*3.3. Biosensor for Folate Detection from the Disposable Electrode Modified by PPy/AuNPs/MPA* 3.3.1. Biofunctionalization Step: Recombinant Human Folate Binding Protein (FBP, Abcam) as Recognition Element

After the interesting results presented by the PPy-NTs/AuNPs electrodes for the Avidin/Biotin biomolecules, the same platform was used for the construction of FBP-Ab/FBP biosensor. In the same perspective observed in Figure 4, the CV and EIS responses in the presence of FBP-Ab are shown in Figure 5, and a similar behavior was found, indicating that the same effects of strong interaction and adsorption are occurring.

To test the stability of the recognition process, several measurements of EIS were performed for the same antibody concentration, as shown in Figure 5C and Tables A1 and A2. After immersion in FBP-Ab, five measurements in a row were performed, applying analysis of variance (ANOVA) with 95% confidence. The RCT parameter showed no significant difference, maintaining the confidence in the analytical response; this point is related to the strong interaction between the biosensor and analyte, with no desorption of the FBP-Ab from the electrode's surface [38].

We also tested and proved that the glycine blocking step is crucial. It is already known that the adsorption of biomolecules in conductive polymers can cause non-specific interactions on the electrode's surface, interfering with the signal [39]. We performed a test shown in Appendix A Figure A3, where we verified that, without a blocking step, it is possible to have nonspecific antibody adsorption on the polymer matrix, which directly interferes with the signal.

3.3.2. Detection Step: Determination of Femtomolar Concentrations of Folic Acid

Finally, the FBP/Folic Acid biosensor was assembled on the PPy-NT/AuNPs platform, all electrochemical experiments were the same ones descried earlier for the detection of the analyte. Folic Acid has a great affinity for FBP, and the impedimetric response is found

in Figure 6, in the concentration range from 0.02 up to 113.3 nmol L−1, in triplicate. The analytical curve was inserted; the limit of detection (LOD) was calculated as 0.030 nmol L<sup>−</sup>1, and the limit of quantification (LOQ) was 0.090 nmol L−1, indicating that the proposed biosensor herein can detect and quantify the range of concentration of clinical interest, which is around 11 up to 34 nmol L−<sup>1</sup> [24,25]. As this biomarker can be found as a group of molecules, many different configurations of biosensors based on folate can be found in the literature, and the simple comparison between analytical parameters is not always easy to study. Nonetheless, in Table 3, different information is presented to better analyze the recent development in this issue.

**Figure 5.** Cyclic voltammetry (**A**) and Nyquist plot (**B**) to FBP-Ab detection (0.001 up to 6.70 pmol L<sup>−</sup>1); (**C**) the EIS response in stability test to 0.001 pmol L−<sup>1</sup> of FBP-Ab. The gray measurement was performed in the blank step, while the others correspond to the same antibody concentration.

**Figure 6.** Folic Acid detection (0.02 up to 113.3 nmol L<sup>−</sup>1) using the PPy/AuNPs-modified electrode.

**Table 3.** Comparison between experimental conditions and LOD values between different biosensors for FA detection.

