Available via license: CC BY 4.0
Content may be subject to copyright.
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
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
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/).
biosensors
Article
Development of Folate-Group Impedimetric Biosensor Based
on Polypyrrole Nanotubes Decorated with Gold Nanoparticles
Andrei E. Deller 1, Ana L. Soares 1, Jaqueline Volpe 2, Jean G. A. Ruthes 1, Dênio E. P. Souto 2
and Marcio Vidotti 1,*
1Grupo de Pesquisa em Macromoléculas e Interfaces, Universidade Federal do Paraná(UFPR),
Curitiba 81531-980, PR, Brazil
2Laboratório de Espectrometria, Sensores e Biossensores, Universidade Federal do Paraná(UFPR),
Curitiba 81531-980, PR, Brazil
*Correspondence: mvidotti@ufpr.br
Abstract:
In this study, polypyrrole nanotubes (PPy-NT) and gold nanoparticles (AuNPs) were
electrochemically synthesized to form a hybrid material and used as an electroactive layer for the
attachment of proteins for the construction of a high-performance biosensor. Besides the enhancement
of intrinsic conductivity of the PPy-NT, the AuNPs act as an anchor group for the formation of
self-assembly monolayers (SAMs) from the gold–sulfur covalent interaction between gold and
Mercaptopropionic acid (MPA). This material was used to evaluate the viability and performance of
the platform developed for biosensing, and three different biological approaches were tested: first,
the Avidin-HRP/Biotin couple and characterizations were made by using cyclic voltammetry (CV)
and electrochemical impedance spectroscopy (EIS), wherein we detected Biotin in a linear range of
100–900 fmol L
−1
. The studies continued with folate group biomolecules, using the folate receptor
α
(FR-
α
) as a bioreceptor. Tests with anti-FR antibody detection were performed, and the results
obtained indicate a linear range of detection from 0.001 to 6.70 pmol L
−1
. The same FR-
α
receptor
was used for Folic Acid detection, and the results showed a limit of detection of 0.030 nmol L
−1
and
a limit of quantification of 90 pmol L
−1
. The results indicate that the proposed biosensor is sensitive
and capable of operating in a range of clinical interests.
Keywords: modified electrode; impedimetric biosensor; folate
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 advan-
tageous 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 biore-
ceptor 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
Biosensors 2022,12, 970. https://doi.org/10.3390/bios12110970 https://www.mdpi.com/journal/biosensors
Biosensors 2022,12, 970 2 of 13
materials presents several advantages, not only for biosensors but for any electrochemical-
based technology [
9
–
11
]. Among CPs, polypyrrole (PPy) plays an important role in elec-
trode modification, as it can be further chemically prepared to attach biomolecules [
7
,
12
].
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 devel-
opment 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 folate-
binding proteins (FBPs), e.g., the folate receptor (FR-
α
) [
22
,
23
]. The interaction between
FA and FR-
α
has a high specificity (KD = 10
−9
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 M
Ω
cm
−1
).
Pyrrole (PI, Aldrich, San Luis, MO, USA) was distilled before use. Methyl orange (MO,
Aldrich), nitric acid (HNO
3
, Synth), gold chloride trihydrate (III) (HAuCl
4
.3H
2
O, 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), N-
hydroxysuccinimide (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.
2.2. Characterization and Electrochemical Measurements
For the electrochemical experiments, Metrohm DropSens
µ
Stat-i 400s potentiostat
was employed. The EIS and CV were performed in PBS buffer 0.1 mol L
−1
at pH 7.4; as
the reference electrode, we used Ag/AgCl/KCl
sat
, 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 characteriza-
tions were performed in UFPR Electronic Microscopy Center (CME-UFPR), with Tescan
Vega3 LMU equipment and Transmission Electron Microscopy (MET) with JEOL JEM
Biosensors 2022,12, 970 3 of 13
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
−1
of pyrrole monomer, methyl orange (MO) 5 mmol L
−1
, and
8 mmol L
−1
KNO
3
; the pH 3 was adjusted by dropping HNO
3
solution. The electro-
chemical 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−2[26].
The AuNPs deposition into PPy was performed in a solution of 1.0 mmol L
−1
HAuCl
4
,
0.17 mol L
−1
K
2
HPO
4
, 0.036 mol L
−1
Na
2
SO
3
, and 0.48 mmol L
−1
EDTA. The chemicals
were added in this sequence to avoid the darkening of the solution, due to gold precipita-
tion. The electrodeposition was performed by chronoamperometry, applying
−
1.1 V vs.
Ag/AgCl/Cl-sat, with charge control of 300 mC cm−2[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
−1
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
−1
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
µ
g mL
−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
−1
by submerging the electrode into the glycine solution for
15 min. In Figure 1, the basic steps of the SAM formation are shown.
Biosensors 2022, 12, x FOR PEER REVIEW 3 of 14
II equipment with 0.5 nm resolution. All experiments were performed in triplicate to as-
sure 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−1 of pyrrole monomer, methyl orange (MO) 5 mmol L−1, and 8
mmol L−1 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−2 [26].
The AuNPs deposition into PPy was performed in a solution of 1.0 mmol L−1 HAuCl4,
0.17 mol L−1 K2HPO4, 0.036 mol L−1 Na2SO3, and 0.48 mmol L−1 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−2 [27,28].
2.4. Biosensor Construction and Characterization
For biosensor construction, the formation of a favorable environment for the biomol-
ecule 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 co-
valent 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 sys-
tems studied. The modified electrode (PPy/AuNPs) was immersed into MPA 1 mmol L−1
aqueous solution for five hours to SAM formation and then was washed in ultrapure wa-
ter for 15 min. Thus was followed by activation with 100 and 150 mmol L−1 EDC:NHS
aqueous solution for 20 min. Then it was washed in ultrapure water for 1 min. After acti-
vation, the biorecognition element was immobilized by immerging the electrode in a so-
lution 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 µ g mL−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−1 by submerging the electrode into the glycine solu-
tion 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].
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.
Biosensors 2022,12, 970 4 of 13
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).
Biosensors 2022, 12, x FOR PEER REVIEW 4 of 14
The impedimetric results were modeled by using the proper equivalent circuit and values
obtained from NOVA software.
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 fundamen-
tal 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 sur-
face; 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 infor-
mation 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].
20 μm20 μm
(A) (B)
(C) (D)
10 µm
20 µm
200 µm
(E) (F)
100 μm
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 addi-
Biosensors 2022,12, 970 5 of 13
tional 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 Q
DL
parameter is related to the energy of the double layer at the interface
electrode/electrolyte, the R
CT
is the resistance of the charge transfer at the electrode surface,
R
S
is the resistance of the solution, and Q
LF
deals with the number of interacted ions
inserted within the polymeric matrix.
Biosensors 2022, 12, x FOR PEER REVIEW 5 of 14
Electrochemical experiments of CV and EIS were performed to characterize and com-
pare 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 tradi-
tional 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 elec-
trode/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 equiv-
alent 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 su-
perficial area of AuNPs, the QDL value shows an increment of almost 2.5 times, in agree-
ment 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 morphol-
ogy, 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,
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 equiva-
lent 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 R
CT
. It is important to add that the pres-
ence 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 Q
DL
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 Q
LF
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 n
DL
and n
LF
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].
Biosensors 2022,12, 970 6 of 13
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.
RS/kΩQDL 10−5F sn−1nDL RCT/kΩQLF 10−3F sn−1nLF
PPy-NTs 0.04 1.89 0.82 0.25 3.60 0.83
PPy-NTs/AuNPs 0.03 4.27 0.76 0.05 6.70 0.77
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 AFigures 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
−15
mol L
−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 R
CT
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 R
CT
, the Q
DL
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.
Glycine Biotin Concentration (fmol L−1)
100 300 500 700 900
RS/kΩ0.05 0.03 0.06 0.12 0.04 0.06
QDL/10 −5F sn−12.36 2.96 2.63 3.06 2.37 3.38
nDL 0.87 0.84 0.85 0.80 0.86 0.81
RCT/kΩ0.16 0.48 1.11 1.31 2.11 2.56
QLF/10−3F sn−14.6 4.6 5.35 4.37 5.17 4.83
nLF 0.80 0.81 0.90 0.84 0.91 0.86
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.
Biosensors 2022,12, 970 7 of 13
Biosensors 2022, 12, x FOR PEER REVIEW 7 of 14
Figure 4. Cyclic voltammetry (A) and Nyquist plot (B) of the EIS measurement to Biotin detection
(100 up to 900 fmol L−1) indicated by colors in both CV and EIS.
Table 2. Parameters’ values obtained by EIS to PPy e PPy/AuNPs after fitting, R2 > 0.98.
Glycine
Biotin Concentration (fmol L−1)
100
300
500
700
900
RS/kΩ
0.05
0.03
0.06
0.12
0.04
0.06
QDL/10 −5F sn−1
2.36
2.96
2.63
3.06
2.37
3.38
nDL
0.87
0.84
0.85
0.80
0.86
0.81
RCT/kΩ
0.16
0.48
1.11
1.31
2.11
2.56
QLF/10−3 F sn−1
4.6
4.6
5.35
4.37
5.17
4.83
nLF
0.80
0.81
0.90
0.84
0.91
0.86
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 Av-
idin/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.
Figure 4.
Cyclic voltammetry (
A
) and Nyquist plot (
B
) of the EIS measurement to Biotin detection
(100 up to 900 fmol L−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 per-
formed 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 R
CT
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 AFigure 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
Biosensors 2022,12, 970 8 of 13
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−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
−1
[
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.
Biosensors 2022, 12, x FOR PEER REVIEW 8 of 14
Figure 5. Cyclic voltammetry (A) and Nyquist plot (B) to FBP-Ab detection (0.001 up to 6.70 pmol
L−1); (C) the EIS response in stability test to 0.001 pmol L−1 of FBP-Ab. The gray measurement was
performed in the blank step, while the others correspond to the same antibody concentration.
To test the stability of the recognition process, several measurements of EIS were per-
formed 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 sig-
nificant difference, maintaining the confidence in the analytical response; this point is re-
lated 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
Figure 5.
Cyclic voltammetry (
A
) and Nyquist plot (
B
) to FBP-Ab detection (0.001 up to
6.70 pmol L−1)
;
(
C
) the EIS response in stability test to 0.001 pmol L
−1
of FBP-Ab. The gray measurement was
performed in the blank step, while the others correspond to the same antibody concentration.
Biosensors 2022,12, 970 9 of 13
Biosensors 2022, 12, x FOR PEER REVIEW 9 of 14
Finally, the FBP/Folic Acid biosensor was assembled on the PPy-NT/AuNPs plat-
form, 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−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−1 [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 6. Folic Acid detection (0.02 up to 113.3 nmol L−1) using the PPy/AuNPs-modified electrode.
Table 3. Comparison between experimental conditions and LOD values between different biosen-
sors for FA detection.
Material
Detection
Method
Concentration
Range (nmol L−1)
LOD
(nmol L−1)
Reference
Steel mesh covered by
PPy/AuNPs
EIS
0.02–113.3
0.030
This work
Gold/PPy/POM
Cyclic voltam-
metry
0.01–1
0.0075
[40]
Gold electrode modified
with SAM
Square wave
voltammetry
0.008–1
0.004
[41]
Hydroxyapatite NPs/GCE
Differential
pulse voltam-
metry
0.1–350
0.075
[42]
Platinum
NPs/MWCNT/GCE
Linear voltam-
metry
0.2–100
0.05
[43]
MoS2/rGO/GCE
Differential
pulse voltam-
metry
0.1–100
0.01
[44]
Boron doped diamond elec-
trode
Stripping volt-
ammetry
0.23–45
0.08
[45]
PPy-modified sol–gel car-
bon ceramic
Differential
pulse voltam-
metry
7–55
1.8
[46]
Chromatographic column
HPLC/UV–Vis
0.3–100
44.14
[42]
Figure 6.
Folic Acid detection (0.02 up to 113.3 nmol L
−1
) using the PPy/AuNPs-modified electrode.
Table 3.
Comparison between experimental conditions and LOD values between different biosensors
for FA detection.
Material Detection Method Concentration Range (nmol L−1)LOD
(nmol L−1)Reference
Steel mesh covered by
PPy/AuNPs EIS 0.02–113.3 0.030 This work
Gold/PPy/POM Cyclic voltammetry 0.01–1 0.0075 [40]
Gold electrode modified
with SAM
Square wave voltammetry
0.008–1 0.004 [41]
Hydroxyapatite NPs/GCE Differential pulse
voltammetry 0.1–350 0.075 [42]
Platinum NPs/MWCNT/GCE Linear voltammetry 0.2–100 0.05 [43]
MoS2/rGO/GCE Differential pulse
voltammetry 0.1–100 0.01 [44]
Boron doped diamond electrode Stripping voltammetry 0.23–45 0.08 [45]
PPy-modified sol–gel
carbon ceramic
Differential pulse
voltammetry 7–55 1.8 [46]
Chromatographic column HPLC/UV–Vis 0.3–100 44.14 [42]
SPCE/GO Amperometry 100–1.6 ×10620 [43]
SPCE/SWCNT
Square wave voltammetry
70–500 ×103800 [46]
4. Conclusions
The electrode modification with PPy-NTs/AuNPs has shown to be rapid, straightfor-
ward, and reliable for the construction of biosensors. This hybrid material was used as a
platform for SAM layers, followed by the anchoring of different biomolecules, indicating a
potential application in different types of biosensors and recognition elements. All char-
acterization experiments corroborated the influence of the nanometric architecture on the
electrochemical response for the detection and quantification of different analytes, with
the R
CT
parameter showing the most sensible response for the biological recognition of the
biological markers. The nanostructures also are responsible for the possibility of detection
in the range of femtomolar to picomolar, corroborating the great sensitivity achieved by
the combination of the nanostructures, specific adsorption, and impedance technique.
Author Contributions:
Conceptualization, A.E.D., J.V. and A.L.S.; methodology, A.E.D. and J.V.;
validation, A.E.D. and A.L.S.; investigation, A.E.D., J.V., D.E.P.S. and M.V.; data curation, A.E.D.,
J.V. and J.G.A.R.; writing—original draft preparation, A.E.D.; J.V. and D.E.P.S.; writing—review and
editing, A.E.D. and M.V.; supervision, D.E.P.S. and M.V.; project administration, D.E.P.S. and M.V.;
funding acquisition, D.E.P.S. and M.V. All authors have read and agreed to the published version of
the manuscript.
Funding:
This research was funded by CAPES (Finance Code 001), FAPESP (grant no. 2014/50867-3),
and CNPq (grant no. 465389/2014-7).
Biosensors 2022,12, 970 10 of 13
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments:
The authors would like to thank Centro de Microscopia Eletrônica da UFPR for
microscopic analysis.
Conflicts of Interest:
The funders had no role in the design of the study; in the collection, analyses,
or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Appendix A
Biosensors 2022, 12, x FOR PEER REVIEW 10 of 14
SPCE/GO
Amperometry
100–1.6 × 106
20
[43]
SPCE/SWCNT
Square wave
voltammetry
70–500 × 103
800
[46]
4. Conclusions
The electrode modification with PPy-NTs/AuNPs has shown to be rapid, straightfor-
ward, and reliable for the construction of biosensors. This hybrid material was used as a
platform for SAM layers, followed by the anchoring of different biomolecules, indicating
a potential application in different types of biosensors and recognition elements. All char-
acterization experiments corroborated the influence of the nanometric architecture on the
electrochemical response for the detection and quantification of different analytes, with
the RCT parameter showing the most sensible response for the biological recognition of the
biological markers. The nanostructures also are responsible for the possibility of detection
in the range of femtomolar to picomolar, corroborating the great sensitivity achieved by
the combination of the nanostructures, specific adsorption, and impedance technique.
Author Contributions: Conceptualization, A.E.D., J.V, and A.L.S.; methodology, A.E.D. and J.V.;
validation, A.E.D. and A.L.S.; investigation, A.E.D., J.V., D.E.P.S., and M.V.; data curation, A.E.D.,
J.V., and J.G.A.R.; writing—original draft preparation, A.E.D.; J.V., and D.E.P.S.; writing—review
and editing, A.E.D. and M.V.; supervision, D.E.P.S. and M.V.; project administration, D.E.P.S. and
M.V.; funding acquisition, D.E.P.S. and M.V. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by CAPES (Finance Code 001), FAPESP (grant no. 2014/50867-
3), and CNPq (grant no. 465389/2014-7).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: The authors would like to thank Centro de Microscopia Eletrônica da UFPR for
microscopic analysis.
Conflicts of Interest: The funders had no role in the design of the study; in the collection, analyses,
or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Appendix A
Figure A1. EDS spectra were obtained for the characterization of the electrode modified with PPy
(A) and the electrode modified with PPy/AuNPs (B).
Figure A1.
EDS spectra were obtained for the characterization of the electrode modified with PPy (
A
)
and the electrode modified with PPy/AuNPs (B).
Biosensors 2022, 12, x FOR PEER REVIEW 11 of 14
Figure A2. Characterization of the PPy/AuNPs-based biosensor construction steps for the Avidin-
HRP/Biotin couple. Cyclic voltammograms (A,B) show the characterization of different steps in the
construction of the platform-based biosensor, and the Nyquist diagram (C) is for the same charac-
terization.
Table A1. Parameters’ values obtained by EIS to PPy e PPy/AuNPs, in electrode modification
steps.
Parameter
MPA
Biotin
Glycine
Avidin-HRP
RS/kΩ
0.05
0.07
0.05
0.14
QDL/10−5F sn−1
1.87
1.88
1.61
2.43
nDL
0.90
0.90
0.91
0.85
RCT/kΩ
0.56
1.62
3.95
4.67
QLF/10−3 F sn−1
5.80
6.70
8.06
8.99
nLF
0.81
0.87
0.96
0.97
Table A2. Parameters’ values obtained by EIS to PPy e PPy/AuNPs for stabilization tests using
0.001 pmol L-1.
Glycine
EIS Measurements to FBP-Ab to 0.001 pmol L−1
RCT
(Ohm)
206.3
283.3
288.2
291.2
312.7
309.3
312.3
315.8
318.2
Figure A2.
Characterization of the PPy/AuNPs-based biosensor construction steps for the Avidin-
HRP/Biotin couple. Cyclic voltammograms (
A
,
B
) show the characterization of different steps in
the construction of the platform-based biosensor, and the Nyquist diagram (
C
) is for the same
characterization.
Biosensors 2022,12, 970 11 of 13
Table A1.
Parameters’ values obtained by EIS to PPy e PPy/AuNPs, in electrode modification steps.
Parameter MPA Biotin Glycine Avidin-HRP
RS/kΩ0.05 0.07 0.05 0.14
QDL/10−5F sn−11.87 1.88 1.61 2.43
nDL 0.90 0.90 0.91 0.85
RCT/kΩ0.56 1.62 3.95 4.67
QLF/10−3F sn−15.80 6.70 8.06 8.99
nLF 0.81 0.87 0.96 0.97
Table A2.
Parameters’ values obtained by EIS to PPy e PPy/AuNPs for stabilization tests using
0.001 pmol L−1.
Glycine EIS Measurements to FBP-Ab to 0.001 pmol L−1
RCT (Ohm) 206.3 283.3 288.2 291.2 312.7 309.3 312.3 315.8 318.2
Biosensors 2022, 12, x FOR PEER REVIEW 12 of 14
Figure A3. Adsorption test of different antibody concentrations (0.001, 0.67, 3.30, and 6.60 pmol L−1),
using a modified electrode only with PPy. Cyclic voltammograms (A), Nyquist diagram (B).
References
1. Islam, M.N.; Channon, R.B. Electrochemical sensors. In Bioengineering Innovative Solutions for Cancer; Ladame, S., Chang, J.Y.H.,
Eds.; Elsevier: Amsterdam, The Netherlands, 2020; pp. 47–71, ISBN 978-0-12-813886-1.
2. Abdul Ghani, M.A.; Nordin, A.N.; Zulhairee, M.; Che Mohamad Nor, A.; Shihabuddin Ahmad Noorden, M.; Muhamad Atan,
M.K.F.; Ab Rahim, R.; Mohd Zain, Z. Portable Electrochemical Biosensors Based on Microcontrollers for Detection of Viruses:
A Review. Biosensors 2022, 12, 666. https://doi.org/10.3390/bios12080666.
3. Adarakatti, P.S.; Kempahanumakkagari, S.K. Modified electrodes for sensing. In Electrochemistry; The Royal Society of Chemis-
try: London, UK, 2019; pp. 58–95.
4. Baig, N.; Rana, A.; Kawde, A.-N. Modified Electrodes for Selective Voltammetric Detection of Biomolecules. Electroanalysis 2018,
30, 2551–2574. https://doi.org/10.1002/elan.201800468.
5. Sandhyarani, N. Surface modification methods for electrochemical biosensors. In Electrochemical Biosensors; Ensafi, A.A., Ed.;
Elsevier: Amsterdam, The Netherlands, 2019; pp. 45–75, ISBN 978-0-12-816491-4.
6. Nemiwal, M.; Zhang, T.C.; Kumar, D. Enzyme Immobilized Nanomaterials as Electrochemical Biosensors for Detection of Bio-
molecules. Enzym. Microb Technol. 2022, 156, 110006. https://doi.org/10.1016/j.enzmictec.2022.110006.
7. Suni, I.I. Substrate Materials for Biomolecular Immobilization within Electrochemical Biosensors. Biosensors 2021, 11, 239.
https://doi.org/10.3390/bios11070239.
8. Holzinger, M.; Buzzetti, P.H.M.; Cosnier, S. Polymers and Nano-Objects, a Rational Combination for Developing Health Mon-
itoring Biosensors. Sens. Actuators B Chem. 2021, 348, 130700. https://doi.org/10.1016/j.snb.2021.130700.
9. Wang, Y.; Liu, A.; Han, Y.; Li, T. Sensors Based on Conductive Polymers and Their Composites: A Review. Polym. Int. 2020, 69,
7–17. https://doi.org/10.1002/pi.5907.
10. Kaur, G.; Kaur, A.; Kaur, H. Review on Nanomaterials/Conducting Polymer Based Nanocomposites for the Development of
Biosensors and Electrochemical Sensors. Polym.-Plast. Technol. Mater. 2020, 1–18. https://doi.org/10.1080/25740881.2020.1844233.
11. Kim, J.; Park, M. Recent Progress in Electrochemical Immunosensors. Biosensors 2021, 11, 360.
https://doi.org/10.3390/bios11100360.
12. Qing, Y.; Yang, Y.; Ouyang, P.; Fang, C.; Fang, H.; Liao, Y.; Li, H.; Wang, Z.; Du, J. Gold Nanoparticle-Based Enzyme-Assisted
Cyclic Amplification for the Highly-Sensitive Detection of MiRNA-21. Biosensors 2022, 12, 724.
https://doi.org/10.3390/bios12090724.
13. Singh, S.; Gill, A.A.S.; Nlooto, M.; Karpoormath, R. Prostate Cancer Biomarkers Detection Using Nanoparticles Based Electro-
chemical Biosensors. Biosens. Bioelectron. 2019, 137, 213–221. https://doi.org/10.1016/j.bios.2019.03.065.
14. Montoro-Leal, P.; Frías, I.A.M.; Vereda Alonso, E.; Errachid, A.; Jaffrezic-Renault, N. A Molecularly Imprinted Polypyr-
role/GO@Fe3O4 Nanocomposite Modified Impedimetric Sensor for the Routine Monitoring of Lysozyme. Biosensors 2022, 12,
727. https://doi.org/10.3390/bios12090727.
15. O’Connor, C.; Wallace-Povirk, A.; Ning, C.; Frühauf, J.; Tong, N.; Gangjee, A.; Matherly, L.H.; Hou, Z. Folate Transporter Dy-
namics and Therapy with Classic and Tumor-Targeted Antifolates. Sci. Rep. 2021, 11, 6389. https://doi.org/10.1038/s41598-021-
85818-x.
Figure A3.
Adsorption test of different antibody concentrations (0.001, 0.67, 3.30, and 6.60 pmol L
−1
),
using a modified electrode only with PPy. Cyclic voltammograms (A), Nyquist diagram (B).
References
1.
Islam, M.N.; Channon, R.B. Electrochemical sensors. In Bioengineering Innovative Solutions for Cancer; Ladame, S., Chang, J.Y.H.,
Eds.; Elsevier: Amsterdam, The Netherlands, 2020; pp. 47–71, ISBN 978-0-12-813886-1.
2.
Abdul Ghani, M.A.; Nordin, A.N.; Zulhairee, M.; Che Mohamad Nor, A.; Shihabuddin Ahmad Noorden, M.; Muhamad Atan,
M.K.F.; Ab Rahim, R.; Mohd Zain, Z. Portable Electrochemical Biosensors Based on Microcontrollers for Detection of Viruses:
A Review. Biosensors 2022,12, 666. [CrossRef] [PubMed]
3.
Adarakatti, P.S.; Kempahanumakkagari, S.K. Modified electrodes for sensing. In Electrochemistry; The Royal Society of Chemistry:
London, UK, 2019; pp. 58–95.
Biosensors 2022,12, 970 12 of 13
4.
Baig, N.; Rana, A.; Kawde, A.-N. Modified Electrodes for Selective Voltammetric Detection of Biomolecules. Electroanalysis
2018
,
30, 2551–2574. [CrossRef]
5.
Sandhyarani, N. Surface modification methods for electrochemical biosensors. In Electrochemical Biosensors; Ensafi, A.A., Ed.;
Elsevier: Amsterdam, The Netherlands, 2019; pp. 45–75, ISBN 978-0-12-816491-4.
6.
Nemiwal, M.; Zhang, T.C.; Kumar, D. Enzyme Immobilized Nanomaterials as Electrochemical Biosensors for Detection of
Biomolecules. Enzym. Microb Technol. 2022,156, 110006. [CrossRef] [PubMed]
7.
Suni, I.I. Substrate Materials for Biomolecular Immobilization within Electrochemical Biosensors. Biosensors
2021
,11, 239.
[CrossRef]
8.
Holzinger, M.; Buzzetti, P.H.M.; Cosnier, S. Polymers and Nano-Objects, a Rational Combination for Developing Health
Monitoring Biosensors. Sens. Actuators B Chem. 2021,348, 130700. [CrossRef]
9.
Wang, Y.; Liu, A.; Han, Y.; Li, T. Sensors Based on Conductive Polymers and Their Composites: A Review. Polym. Int.
2020
,
69, 7–17. [CrossRef]
10.
Kaur, G.; Kaur, A.; Kaur, H. Review on Nanomaterials/Conducting Polymer Based Nanocomposites for the Development of
Biosensors and Electrochemical Sensors. Polym.-Plast. Technol. Mater. 2020, 1–18. [CrossRef]
11. Kim, J.; Park, M. Recent Progress in Electrochemical Immunosensors. Biosensors 2021,11, 360. [CrossRef] [PubMed]
12.
Qing, Y.; Yang, Y.; Ouyang, P.; Fang, C.; Fang, H.; Liao, Y.; Li, H.; Wang, Z.; Du, J. Gold Nanoparticle-Based Enzyme-Assisted
Cyclic Amplification for the Highly-Sensitive Detection of MiRNA-21. Biosensors 2022,12, 724. [CrossRef]
13.
Singh, S.; Gill, A.A.S.; Nlooto, M.; Karpoormath, R. Prostate Cancer Biomarkers Detection Using Nanoparticles Based Electro-
chemical Biosensors. Biosens. Bioelectron. 2019,137, 213–221. [CrossRef]
14.
Montoro-Leal, P.; Frías, I.A.M.; Vereda Alonso, E.; Errachid, A.; Jaffrezic-Renault, N. A Molecularly Imprinted Polypyr-
role/GO@Fe3O4 Nanocomposite Modified Impedimetric Sensor for the Routine Monitoring of Lysozyme. Biosensors
2022
,
12, 727. [CrossRef] [PubMed]
15.
O’Connor, C.; Wallace-Povirk, A.; Ning, C.; Frühauf, J.; Tong, N.; Gangjee, A.; Matherly, L.H.; Hou, Z. Folate Transporter
Dynamics and Therapy with Classic and Tumor-Targeted Antifolates. Sci. Rep. 2021,11, 6389. [CrossRef] [PubMed]
16.
Boss, S.D.; Ametamey, S.M. Development of Folate Receptor
−
Targeted PET Radiopharmaceuticals for Tumor Imaging—A
Bench-to-Bedside Journey. Cancers 2020,12, 1508. [CrossRef] [PubMed]
17.
Bobrowski-Khoury, N.; Ramaekers, V.T.; Sequeira, J.M.; Quadros, E.V. Folate Receptor Alpha Autoantibodies in Autism Spectrum
Disorders: Diagnosis, Treatment and Prevention. J. Pers. Med. 2021,11, 710. [CrossRef] [PubMed]
18. Miller, J.W. Folic acid. In Encyclopedia of Human Nutrition; Elsevier: Amsterdam, The Netherlands, 2013; pp. 262–269.
19. Bailey, L.B.; Caudill, M.A. Folate. In Present Knowledge in Nutrition; Wiley: Oxford, UK, 2012; pp. 321–342.
20.
Scaranti, M.; Cojocaru, E.; Banerjee, S.; Banerji, U. Exploiting the Folate Receptor
α
in Oncology. Nat. Rev. Clin. Oncol.
2020
,
17, 349–359. [CrossRef]
21. Müller, C.; Schibli, R. Prospects in Folate Receptor-Targeted Radionuclide Therapy. Front. Oncol. 2013,3, 249. [CrossRef]
22.
Batra, B.; Narwal, V.; Kalra, V.; Sharma, M.; Rana, J. Folic Acid Biosensors: A Review. Process Biochem.
2020
,92, 343–354. [CrossRef]
23.
Hryniewicz, B.M.; Lima, R.V.; Wolfart, F.; Vidotti, M. Influence of the PH on the Electrochemical Synthesis of Polypyrrole
Nanotubes and the Supercapacitive Performance Evaluation. Electrochim. Acta 2019,293, 447–457. [CrossRef]
24.
Colombo, R.N.P.; Petri, D.F.S.; Córdoba De Torresi, S.I.; Gonçales, V.R. Porous Polymeric Templates on ITO Prepared by Breath
Figure Method for Gold Electrodeposition. Electrochim. Acta 2015,158, 187–195. [CrossRef]
25.
Soares, A.L.; Hryniewicz, B.M.; Deller, A.E.; Volpe, J.; Marchesi, L.F.; Souto, D.E.P.; Vidotti, M. Electrodes Based on PEDOT
Nanotubes Decorated with Gold Nanoparticles for Biosensing and Energy Storage. ACS Appl. Nano. Mater.
2021
,4, 9945–9956.
[CrossRef]
26.
Singh, M.; Kaur, N.; Comini, E. The Role of Self-Assembled Monolayers in Electronic Devices. J. Mater. Chem. C Mater.
2020
,
8, 3938–3955. [CrossRef]
27.
Souto, D.E.P.; Silva, J.V.; Martins, H.R.; Reis, A.B.; Luz, R.C.S.; Kubota, L.T.; Damos, F.S. Development of a Label-Free Immunosen-
sor Based on Surface Plasmon Resonance Technique for the Detection of Anti-Leishmania Infantum Antibodies in Canine Serum.
Biosens. Bioelectron. 2013,46, 22–29. [CrossRef] [PubMed]
28.
Bertok, T.; Lorencova, L.; Chocholova, E.; Jane, E.; Vikartovska, A.; Kasak, P.; Tkac, J. Electrochemical Impedance Spectroscopy
Based Biosensors: Mechanistic Principles, Analytical Examples and Challenges towards Commercialization for Assays of Protein
Cancer Biomarkers. ChemElectroChem 2019,6, 989–1003. [CrossRef]
29.
Soares, A.L.; Zamora, M.L.; Marchesi, L.F.; Vidotti, M. Adsorption of Catechol onto PEDOT Films Doped with Gold Nanoparticles:
Electrochemical and Spectroscopic Studies. Electrochim. Acta 2019,322, 134773. [CrossRef]
30.
Gonçalves, R.; Pereira, E.; Marchesi, L. The Overoxidation of Poly(3-Hexylthiophene) (P3HT) Thin Film: CV and EIS Measure-
ments. Int. J. Electrochem. Sci. 2017,12, 1983–1991. [CrossRef]
31.
Wolfart, F.; Hryniewicz, B.M.; Góes, M.S.; Corrêa, C.M.; Torresi, R.; Minadeo, M.A.O.S.; Córdoba de Torresi, S.I.; Oliveira, R.D.;
Marchesi, L.F.; Vidotti, M. Conducting Polymers Revisited: Applications in Energy, Electrochromism and Molecular Recognition.
J. Solid State Electrochem. 2017,21, 2489–2515. [CrossRef]
32.
Samanta, D.; Sarkar, A. Immobilization of Bio-Macromolecules on Self-Assembled Monolayers: Methods and Sensor Applications.
Chem. Soc. Rev. 2011,40, 2567–2592. [CrossRef]
33.
Bănică, F. Affinity-based recognition. In Chemical Sensors and Biosensors; John Wiley & Sons: Hoboken, NJ, USA, 2012; pp. 101–117.
Biosensors 2022,12, 970 13 of 13
34.
Bănică, F. Electrical-impedance-based sensors. In Chemical Sensors and Biosensors; John Wiley & Sons: Hoboken, NJ, USA, 2012;
pp. 367–403.
35.
Battistel, A.; Fan, M.; Stojadinovi´c, J.; la Mantia, F. Analysis and Mitigation of the Artefacts in Electrochemical Impedance
Spectroscopy Due to Three-Electrode Geometry. Electrochim. Acta 2014,135, 133–138. [CrossRef]
36.
Teles, F.R.R.; Fonseca, L.P. Applications of Polymers for Biomolecule Immobilization in Electrochemical Biosensors. Mater. Sci.
Eng. C 2008,28, 1530–1543. [CrossRef]
37.
Babakhanian, A.; Kaki, S.; Ahmadi, M.; Ehzari, H.; Pashabadi, A. Development of
α
-Polyoxometalate–Polypyrrole–Au Nanoparti-
cles Modified Sensor Applied for Detection of Folic Acid. Biosens. Bioelectron. 2014,60, 185–190. [CrossRef]
38.
Wan, Q.; Yang, N. The Direct Electrochemistry of Folic Acid at a 2-Mercaptobenzothiazole Self-Assembled Gold Electrode. J.
Electroanal. Chem. 2002,527, 131–136. [CrossRef]
39.
Kanchana, P.; Sekar, C. Development of Electrochemical Folic Acid Sensor Based on Hydroxyapatite Nanoparticles. Spectrochim.
Acta A Mol. Biomol. Spectrosc. 2015,137, 58–65. [CrossRef] [PubMed]
40.
Kun, Z.; Ling, Z.; Yi, H.; Ying, C.; Dongmei, T.; Shuliang, Z.; Yuyang, Z. Electrochemical Behavior of Folic Acid in Neutral Solution
on the Modified Glassy Carbon Electrode: Platinum Nanoparticles Doped Multi-Walled Carbon Nanotubes with Nafion as
Adhesive. J. Electroanal. Chem. 2012,677–680, 105–112. [CrossRef]
41.
Chekin, F.; Teodorescu, F.; Coffinier, Y.; Pan, G.-H.; Barras, A.; Boukherroub, R.; Szunerits, S. MoS2/Reduced Graphene Oxide as
Active Hybrid Material for the Electrochemical Detection of Folic Acid in Human Serum. Biosens. Bioelectron.
2016
,85, 807–813.
[CrossRef]
42.
Yardim, Y.; ¸Sentürk, Z. Electrochemical Behavior of Folic Acid at A Boron-Doped Diamond Electrode: Its Adsorptive Stripping
Voltammetric Determination in Tablets. Turk J. Pharm. Sci. 2012,11, 87–100.
43.
Majidi, M.R.; Dastangoo, H.; Hasannejad, M.; Malakouti, J. Voltammetric Determination of Folic Acid with a Overoxidized
Polypyrrole Film Modified Sol-Gel Carbon Ceramic Electrode. Int. J. Polym. Anal. Charact. 2011,16, 486–495. [CrossRef]
44.
Jastrebova, J.; Witthöft, C.; Grahn, A.; Svensson, U.; Jägerstad, M. HPLC Determination of Folates in Raw and Processed Beetroots.
Food Chem. 2003,80, 579–588. [CrossRef]
45.
Mani, V. Highly Sensitive Determination of Folic Acid Using Graphene Oxide Nanoribbon Film Modified Screen Printed Carbon
Electrode. Int. J. Electrochem. Sci. 2017,12, 475–484. [CrossRef]
46.
Karimi-Maleh, H.; Hatami, M.; Moradi, R.; Khalilzadeh, M.A.; Amiri, S.; Sadeghifar, H. Synergic Effect of Pt-Co Nanoparticles
and a Dopamine Derivative in a Nanostructured Electrochemical Sensor for Simultaneous Determination of N-Acetylcysteine,
Paracetamol and Folic Acid. Microchim. Acta 2016,183, 2957–2964. [CrossRef]
