**1. Introduction**

The development of electrochemical biosensors has been extensively explored so far. With these devices, the selective detection of low concentrations of different analytes, such as contaminants and biomolecules, is performed in a rapid and straightforward way; in addition, other features are highly desirable, such as low-cost, easy operation, portability, and no need of further analytical steps, as these are key parameters to obtaining an advantageous alternative to the traditional monitoring methods, which are often expensive and also not accessible to the entire population [1,2].

The construction of a high-performance electrochemical biosensor relies on a previous study on the material interface and transduction. Different assemblies of materials and architectures are possible in terms of nanomaterials, metals, and biomolecules to enhance both detection and quantification [3,4]. Special care must be taken on the biomolecule immobilization on the electrode surface, as this experimental step, consisting of the bioreceptor attachment needing to be stable, preserves its conformation and maintains a good orientation to interact with the analyte and provides a reliable signal of recognition [5].

Many different methodologies have been described along the past years [6–8], and in this context, the use of conducting polymers (CPs) and nanoparticles as hybrid synergist materials presents several advantages, not only for biosensors but for any electrochemicalbased technology [9–11]. Among CPs, polypyrrole (PPy) plays an important role in electrode modification, as it can be further chemically prepared to attach biomolecules [7,12].

**Citation:** Deller, A.E.; Soares, A.L.; Volpe, J.; Ruthes, J.G.A.; Souto, D.E.P.; Vidotti, M. Development of Folate-Group Impedimetric Biosensor Based on Polypyrrole Nanotubes Decorated with Gold Nanoparticles. *Biosensors* **2022**, *12*, 970. https://doi.org/10.3390/ bios12110970

Received: 8 October 2022 Accepted: 31 October 2022 Published: 4 November 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

For biosensing, gold nanoparticles (AuNPs) are widely employed, as they present some interesting advantage based on biocompatibility, chemical affinity with sulfur ending molecules, besides the intrinsic metallic conductivity, which represents a rapid and reliable electrochemical transduction signal [13–15]. This last point is a key feature for the development of impedimetric biosensors which presents a remarkable sensitivity of detection; thus, it is possible to obtain trustable results in different stages, even early periods, of any disease [16,17]. Besides that, the impedimetric sensor proposed herein depends greatly on the better accuracy on the measure of the electric resistance of the transducer, so the higher the conductivity, the better will be the analytical parameters.

The folate group molecules have been found to possess different biological functions, such as cellular regulation, DNA synthesis, reparation, and methylation. It is important to adequately maintain the folate levels, as cardiovascular diseases, anemia, embryonic disorders, and various types of cancer are highly related to those levels [18–20]. Mammals do not synthetize folate, so its ingestion as vitamin B9 controls the adequate concentration in organisms [21]. The absorption of folate is performed by three different mediators: the reduced folate carrier (RFC); the proton-coupled folate transporter (PCFT); and the folatebinding proteins (FBPs), e.g., the folate receptor (FR-α) [22,23]. The interaction between FA and FR-α has a high specificity (KD = 10−<sup>9</sup> mol L−1), so this strong interaction can be explored for the biosensor transduction mechanism. Recent studies indicate that the normal levels of FA in the human serum are around 11.3–34.0 nmol L−1, emphasizing the need for a highly sensitive biosensor [24,25]. In this study, we developed a hybrid nanomaterial formed by polypyrrole nanotubes and gold nanoparticles, electrochemically synthesized in a rapid and straightforward methodology. This modified electrode was employed as a platform to build up the well-known self-assembly monolayer (SAM) based on thiol chemical bonds and the attachment of biomolecules for further detection and quantification, using electrochemical impedance spectroscopy. All steps were properly characterized as well.

#### **2. Materials and Methods**

#### *2.1. Reagents and Solutions*

All solutions were prepared with ultrapure water (ElgaLab water 18 MΩ cm−1). Pyrrole (PI, Aldrich, San Luis, MO, USA) was distilled before use. Methyl orange (MO, Aldrich), nitric acid (HNO3, Synth), gold chloride trihydrate (III) (HAuCl4.3H2O, Aldrich), ethylenediaminetetraacetic acid (EDTA, Aldrich), and potassium chloride (KCl, Aldrich) were used as received, without any further purification step. Mercaptopropionic acid (MPA, Aldrich), N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC, Aldrich), Nhydroxysuccinimide (NHS, Aldrich), and amino acetic acid (Glycine, Aldrich) were kept in a refrigerator at 5 ◦C. The biological samples, Avidin/Biotin couplings, avidin conjugated with horseradish peroxidase (Avidin-HRP, Abcam, Cambridge, UK), anti-avidin antibody (Biotin, Abcam), recombinant human folate binding protein (FBP, Abcam), and anti-folate binding protein antibody (FBP-Ab, Abcam), were kept in a refrigerator at 5 ◦C.
