*2.2. Characterization and Electrochemical Measurements*

For the electrochemical experiments, Metrohm DropSens μStat-i 400s potentiostat was employed. The EIS and CV were performed in PBS buffer 0.1 mol L−<sup>1</sup> at pH 7.4; as the reference electrode, we used Ag/AgCl/KClsat, and platinum wire served as a counter electrode. The working electrode was 316 steel mesh–400 mesh, previously cleaned by immersion in ethanol and ultrapure water. The spectroscopic and microscopic characterizations were performed in UFPR Electronic Microscopy Center (CME-UFPR), with Tescan Vega3 LMU equipment and Transmission Electron Microscopy (MET) with JEOL JEM 1200EX-II equipment with 0.5 nm resolution. All experiments were performed in triplicate to assure homogeneity and reliability of the results.

#### *2.3. Electrode Preparation and Electrochemical Synthesis of PPy/AuNPs*

The electrochemical synthesis of PPy nanotubes was performed in aqueous solution containing 100 mmol L−<sup>1</sup> of pyrrole monomer, methyl orange (MO) 5 mmol L−1, and 8 mmol L−<sup>1</sup> KNO3; the pH 3 was adjusted by dropping HNO3 solution. The electrochemical synthesis was performed over the steel mesh by potentiostatic method, applying 0.8 V over time, controlling the amount of polymer over the mesh with charge control of 0.5 C cm−<sup>2</sup> [26].

The AuNPs deposition into PPy was performed in a solution of 1.0 mmol L−<sup>1</sup> HAuCl4, 0.17 mol L−<sup>1</sup> K2HPO4, 0.036 mol L−<sup>1</sup> Na2SO3, and 0.48 mmol L−<sup>1</sup> EDTA. The chemicals were added in this sequence to avoid the darkening of the solution, due to gold precipitation. The electrodeposition was performed by chronoamperometry, applying −1.1 V vs. Ag/AgCl/Cl-sat, with charge control of 300 mC cm−<sup>2</sup> [27,28].

#### *2.4. Biosensor Construction and Characterization*

For biosensor construction, the formation of a favorable environment for the biomolecule immobilization is necessary. Gold has a strong interaction with sulfur, so organic molecules with thiol groups can be easily anchored onto the AuNPs surface by stable covalent bonds [29]. This affinity and stability are explored in SAMs formation, producing an organized and compatible electrode surface for the immobilization of biomolecules.

The methodology for biosensor construction was the same for all the biological systems studied. The modified electrode (PPy/AuNPs) was immersed into MPA 1 mmol L−<sup>1</sup> aqueous solution for five hours to SAM formation and then was washed in ultrapure water for 15 min. Thus was followed by activation with 100 and 150 mmol L−<sup>1</sup> EDC:NHS aqueous solution for 20 min. Then it was washed in ultrapure water for 1 min. After activation, the biorecognition element was immobilized by immerging the electrode in a solution of the respective biomolecule for 45 min, followed by a cleansing step in PBS for 15 min. For the complex Avidin/Biotin, both were tested as a bioreceptor in the concentration of 25 μg mL<sup>−</sup>1. Moreover, in the other two tests evaluated for the folate biomarker, the same bioreceptor was explored: FBP 8 nmol L−1. The next step was blocking unspecific active sites with glycine 100 mmol L−<sup>1</sup> by submerging the electrode into the glycine solution for 15 min. In Figure 1, the basic steps of the SAM formation are shown.

**Figure 1.** The SAM formation is due to the covalent interaction between gold and sulfur, which makes possible biomolecule immobilization through the carboxylic groups.

The detection of the biomolecule analyte followed the same methodology, where the electrode was immersed in a solution containing the analyte at a known concentration, followed by a washing step in PBS for 5 min before CV and EIS measurements [28,30]. The impedimetric results were modeled by using the proper equivalent circuit and values obtained from NOVA software.
