materials

Article
A Nanocomposite Based on Reduced Graphene and
Gold Nanoparticles for Highly Sensitive
Electrochemical Detection of Pseudomonas aeruginosa
through Its Virulence Factors
Islem Gandouzi 1,2,† , Mihaela Tertis 1,† , Andreea Cernat 1,† , Dalila Saidane-Mosbahi 2 ,
Aranka Ilea 3 and Cecilia Cristea 1, *
1 Analytical Chemistry Department, Faculty of Pharmacy, Iuliu Haţieganu University of Medicine and
Pharmacy, 4 Louis Pasteur St., 400349 Cluj-Napoca, Romania; islemgandouzi@yahoo.fr (I.G.);
mihaela.tertis@umfcluj.ro (M.T.); ilioaia.andreea@umfcluj.ro (A.C.)
2 Laboratory of Analysis, Treatment and Valorization of the Pollutants of the Environment and Products,
Faculty of Pharmacy, University of Monastir, Avicenne St., 5000 Monastir, Tunisia;
Dalila.Saidane@fphm.rnu.tn
3 Department of Oral Rehabilitation, Oral Health and Dental Office Management, Faculty of Dentistry,
Iuliu Haţieganu University of Medicine and Pharmacy, 400012 Cluj-Napoca, Romania;
arankailea@yahoo.com
* Correspondence: ccristea@umfcluj.ro
† Authors with equal contributions.




Received: 24 March 2019; Accepted: 9 April 2019; Published: 11 April 2019


Abstract: Pyoverdine is a fluorescent siderophore produced by Pseudomonas aeruginosa that can

be considered as a detectable marker in nosocomial infections. The presence of pyoverdine
in water can be directly linked to the presence of the P. aeruginosa, thus being a nontoxic and
low-cost marker for the detection of biological contamination. A novel platform was developed
and applied for the electrochemical selective and sensitive detection of pyoverdine, based on a
graphene/graphite-modified screen-printed electrode (SPE) that was electrochemically reduced and
decorated with gold nanoparticles (NPs). The optimized sensor presenting higher sensitivity towards
pyoverdine was successfully applied for its detection in real samples (serum, saliva, and tap water),
in the presence of various interfering species. The excellent analytical performances underline the
premises for an early diagnosis kit of bacterial infections based on electrochemical sensors.

Keywords: Pseudomonas aeruginosa; pyoverdine; graphene; electrochemical sensor; virulence; factors

1. Introduction
Nosocomial infections, known as hospital-acquired/associated infections, are caused by
various bacteria species, the most frequent being Streptococcus species (spp.), Acinetobacter spp.,
Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Legionella, Proteus mirablis,
Klebsiella pneumonia, Escherichia coli and Serratia marcescens. Out of these, P. aeruginosa, S. aureus,
and E. coli are the most common bacteria related to the highest rate of disease and complications. The
repercussions are prolonged hospital stays and a major risk of serious health issues leading to death,
both from medical and economic point of view [1].
A mandatory check-point in the development of rapid tests for identifying bacteria is the
identification of virulence factors, which are the key to survival, replication, and development
of a disease [2,3]. Being essential for bacterial growth and replication, iron became the target for
highly efficient iron-acquisition systems known as siderophores [4]. These iron chelators are clinical

Materials 2019, 12, 1180; doi:10.3390/ma12071180 www.mdpi.com/journal/materials

Materials 2019, 12, 1180 2 of 13

biomarkers for specific bacteria, commercially available at low costs and with valuable perspectives
for early diagnosis and therapy modulation.
The most common bacteria siderophores are pyoverdine (PyoV) and pyochelin for P. aeruginosa,
aerobactin and enterobactin for E. coli, and staphyloferrin A and staphyloferrin B for S. aureus [4]. In the
past few decades, the search for new and improved tools for the detection of virulence factors has led to
the development of biochemical methods, genetic screens, and transcriptomic and genomic approaches,
especially for the most common bacteria [5,6]. Mass spectrometries, nanoparticle (NP)-assisted
microextraction approaches for bacterial profiling, have been employed to track down and identify
these iron-scavenging molecules [7,8]. Moreover, proteomic methods were applied for full protein
mapping as an important tool for the detection of biomedical biomarkers. The major drawbacks
of these methods are represented by restrictive laboratory conditions, the need for highly trained
personnel, and high analysis costs. The sampling and transport procedures could also be prone to a
high degree of contamination, which could generate false positive/negative conclusions. A suitable
alternative may be represented by the elaboration of electrochemical sensors for the detection and
quantification in situ of the analytes. This approach associates the high selectivity, sensitivity, and
rapidity related to electrochemical methods with low production costs and stability. Also, the possibility
of miniaturization and decentralized analysis enables the sensors to be integrated in point-of-care
(POC) devices [9,10]. The generation of 3D nanoarchitectures for the immobilization of biomolecules
represents the first major goal in the fabrication of a biosensor. The platforms based on conductive
polymers are widely employed due to their biocompatibility, increased electron transfer rates, and
augmented active surface areas [11–13]. The supplementary tailoring with carbon-based nanomaterials
or metallic nanoparticles (NPs) enhances their features, with outstanding results in the analytical
performances of the (bio)sensors. The explanation is that the association between nanomaterials
can create a summed effect towards the detection of the analyte, combining both the increase of the
active surface area with new catalytic properties and increased selectivity [14,15]. In our previous
studies, two platforms for the detection of PyoV, based on Au chemically modified graphene and
polypyrrole-functionalized carboxylic groups with AuNPs were developed, both with close results
for the detection of the target analyte [16,17]. In order to achieve miniaturization and integration in
portable sensing devices, the protocol was readapted to overcome the observed difficulties, specifically
the instability of the Au-modified graphene layers and the low conductivity of the polymeric film
due to its passivation. Thus, the protocol was adapted to the electrochemical deposition of AuNPs
on the graphene/graphite working electrode instead of using chemically Au-modified graphene.
The upgrade in the development protocol was closely linked to the increase of the stability of the
platform as well as its sensitivity. Moreover, the polymeric film was eliminated, simplifying and
reducing the time of the elaboration. The outcome is represented by a tailored hybrid surface with
the same selectivity, but an improved sensitivity and limit of detection (LOD) compared to the other
reported approaches.

2. Materials and Methods

2.1. Reagents
The reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), Fluka Chemie GmbH
(Buchs, Switzerland), Merck Chemicals (Darmstadt, Germany), Alfa Aesar (Karlsruhe, Germany),
and used without any further treatments. PyoV from Pseudomonas fluorescens (P8374), pyocyanin
(PyoC) (P0046), sodium chloride (746398), potassium ferrocyanide (K4 [Fe(CN)6 ]) (P3289), potassium
ferricyanide (K3 [Fe(CN)6 ]) (31253), acetylsalicylic acid (ASA) (PHR1003), L-ascorbic acid (AA) (A92902),
β-nicotinamide adenine dinucleotide phosphate-reduced tetrasodium salt (NADH) (10107735001),
glucose (G) (G8270), and dopamine (DA) (H8502) were purchased from Sigma-Aldrich, while uric
acid (UA) (51449), disodium hydrogen phosphate (71650), and sodium dihydrogen phosphate (71504)
were purchased from Fluka Chemie GmbH. Sulphuric acid (100732), nitric acid (101799), and ethanol
Materials 2019, 12, 1180 3 of 13

(K48380027 645) were purchased from Merck Chemicals, and hexachloroauric acid (12325) from
Alfa Aesar.
The stock solutions were prepared using double-distilled water and ultrapure water (Milli-Q,
Millipore; 18 MΩ·cm−1 ). The preparation of samples was performed depending on the desired
concentrations, as follows: PyoV dilutions of 0.5, 0.75, 1, 5, 10, 25, 50, 75, and 100 µM were
prepared in 20 mM saline phosphate buffer (PBS) pH 7.4 from a stock solution of PyoV–Fe complex
from P. fluorescens of 860 µM prepared in ultrapure water (MilliQ), and stored at −20 ◦ C. The
concentrations of possible interfering compounds were established close to real values in order to
evaluate experimental conditions similar to those of real matrices, as follows: G 7000 µM (pathological
concentration), while AA, ASA, UA, NADH, and DA solutions were of 25 µM. In all cases, the
concentration of PyoV was kept constant at 25 µM. The effect of PyoC, another P. aeruginosa metabolite,
was also assessed in the presence of 25 µM PyoV at a concentration of 10 µM in 20 mM PBS pH 7.4.
The real sample analyses were performed on Pierce normal human serum purchased from Thermo
Scientific and tap water. The stimulated saliva was collected in plastic sterilized containers (Salivette
device, Thermo Ficher Scientific, Waltham, MA, USA) from a healthy volunteer. Consent from the
participant was obtained prior to the collection of samples, and all local guidelines (No. 94/08.03.2017)
regarding the work with human saliva were respected. The biological samples were prepared following
a simple pretreatment protocol that involved a 1:100 dilution with 20 mM PBS pH 7.4 and a filtration
process through a 0.2-µm pore diameter filter (Phenex), with storage at −20 ◦ C. Tap water samples
were diluted 1:1 with 20 mM PBS pH 7.4 and stored at 4 ◦ C. Prior to experiments, the samples were
spiked with 25 µM PyoV and tested without accumulation time.

2.2. Sensor Elaboration and Electrochemical Characterization

The sensors were developed on graphene/graphite-based screen-printed electrodes (SPE)
(DropSens, Asturias, Spain) with an integrated three-electrode cell based on a graphene/graphite
working electrode with a 4-mm diameter, a silver reference, and a carbon counter electrode. The
electrochemical experiments were recorded using an Autolab PGSTAT100 potentiostat (Metrohm, Eco
Chemie Netherlands, Utrecht, Netherlands) with a module for electrochemical impedance spectroscopy
(EIS) using Nova 1.10.4 software.
The elaboration consisted in a simple two-step protocol that required less than 10 min
preparation time:

(i) The electrochemical reduction of graphene was performed by sweeping the potential 10 times
from +0.100 to −1.500 vs. Ag/V (pseudo Ag/AgCl) with a scan rate of 0.100 V·s−1 in a 30-µL
drop of 20 mM PBS pH 7.4;
(ii) The electrochemical decoration of reduced graphene with AuNPs was done by cycling 10 times
the potential from −0.2 to +1.2 vs. Ag/V (pseudo Ag/AgCl) with a scan rate of 0.100 V·s−1 in a
50-µL drop of 2 mM HAuCl4 prepared in 0.5 M H2 SO4 .

The electrochemical characterization of the sensor was tested step by step, after each modification
level, in 10 mM [Fe(CN)6 ]3−/4− redox shuttle in 20 mM PBS pH 7.4 solution by cyclic voltammetry (CV)
and EIS. The CV experiments were recorded by sweeping the potential from −0.5 to +0.8 vs. Ag/V
(pseudo Ag/AgCl), at a scan rate of 0.1 V·s−1 . The EIS analyses were carried on a frequency window
of 10 mHz to 100 KHz at open circuit potential (OCP). The electrochemical tests were performed in a
50-µL drop of redox mediator. The sensitivity of the sensor towards PyoV was assessed by differential
pulse voltammetry (DPV). The oxidation potential of PyoV was recorded by sweeping the potential
from −0.3 to +0.8 vs. Ag/V (pseudo Ag/AgCl) with a scan rate of 0.02 V·s−1 in a 25-µL drop of stock
solution. When evaluating the interference effect of PyoC, the potential range was cathodically shifted
from −0.5 to +0.8 vs. Ag/V (pseudo Ag/AgCl), while keeping the other parameters constant.
Materials 2019, 12, 1180 4 of 13

2.3. Morphological Characterization of the Sensor

The2019,
Materials topography of the
12, x FOR PEER sensor was evaluated by scanning electron microscopy (SEM). The 4SEM
REVIEW of 13
images for the surface characterization were registered on SU8230 SEM (Hitachi, Tokyo, Japan) at
an accelerating
accelerating voltage
voltage of of
30 30
kV,kV,
1010mA
mAextraction
extractioncurrent,
current,and
andaaworking
workingdistance
distance of
of 11
11 mm from
multiple points on the surface.
surface.

3. Results
3. Results

3.1. Analytical Principle

3.1. Analytical Principle of
of the
the Electrochemical
Electrochemical Sensor
Sensor for
for PyoV
PyoV Detection
Detection
The
The principle of the electrochemical PyoV sensor based on
principle of the electrochemical PyoV sensor based on reduced
reduced graphene
graphene and
and AuNPs
AuNPs was
was
presented
presented in
in Scheme
Scheme 1. 1.

Scheme 1. Schematic representation of the PyoV sensor elaboration protocol ((1) the electrochemical
Scheme 1.ofSchematic
reduction representation
the graphene of theby
by CV, followed PyoV sensor
(2) the elaboration deposition
electrochemical protocol ((1)
of the
the electrochemical
AuNPs by CV),
reduction
and testingofprotocol
the graphene
by DPVbyinCV, followed of
the presence byPyoV
(2) the
as electrochemical deposition of the AuNPs by
the target analyte.
CV), and testing protocol by DPV in the presence of PyoV as the target analyte.
Firstly, the graphene/graphite-based electrode was electrochemically reduced in the presence
of 20Firstly,
mM PBS thepH 7.4 by cyclic voltammetry
graphene/graphite-based (CV) (Figure
electrode 1A). As can be observed
was electrochemically reduced ininthe
Figure 1A, the
presence of
signal
20 mMdecreases
PBS pH proportionally with the number
7.4 by cyclic voltammetry (CV) of cycles.1A). As can be observed in Figure 1A, the
(Figure
signalAfter washing
decreases the electrode with
proportionally with the
ultrapure
number water, the AuNPs were electrochemically generated in
of cycles.
the presence of HAuCl
After washing by CV (Figure
the4electrode 1B). The electrochemical
with ultrapure water, the AuNPs deposition of AuNPs was confirmed
were electrochemically generated
by
in the
the increase
presenceof ofboth anodic
HAuCl andCV
4 by cathodic
(Figure peaks
1B).corresponding to the oxidation/reduction
The electrochemical deposition of AuNPs of Auwas
on
the graphene/graphite surface, as can be seen in Figure 1B.
confirmed by the increase of both anodic and cathodic peaks corresponding to the
After the complete
oxidation/reduction modification
of Au of the graphene/graphite
on the graphene/graphite working
surface, as can surface,
be seen an oxidation
in Figure 1B. peak
was observed
After the in the presence
complete of PyoV of
modification at about +0.325 vs. Ag/V (pseudo
the graphene/graphite workingAg/AgCl).
surface, an oxidation peak
The oxidation
was observed in thesignal registered
presence by at
of PyoV using
aboutan+0.325
optimized DPV(pseudo
vs Ag/V procedure was proportional with the
Ag/AgCl).
concentration of PyoV,
The oxidation the analyte
signal being
registered bydetected
using anwith a higherDPV
optimized sensitivity than was
procedure similar electrochemical
proportional with
sensors [16,17].
the concentration of PyoV, the analyte being detected with a higher sensitivity than similar
electrochemical sensors [16,17].
Materials 2019, 12, 1180 5 of 13
Materials 2019, 12, x FOR PEER REVIEW 5 of 13

Figure 1. (A) Electrochemical reduction of graphene (CV in 20 mM PBS pH 7.4); 10 cycles from +0.1 to
Figure 1. (A) Electrochemical reduction of graphene (CV in 20 mM PBS pH 7.4); 10 cycles from +0.1
−1.5 vs. Ag/V; 0.1 V·s−1 ); (B) electrochemical deposition of gold on reduced graphene (CV in 2 mM
to −1.5 vs. Ag/V; 0.1 V·s−1); (B) electrochemical deposition of gold on reduced −graphene (CV in 2 mM
HClO4 solution in 0.5 M H2 SO4 ; 10 cycles from −0.2 to +1.2 vs. Ag/V; 0.1 V·s 1 ).
HClO4 solution in 0.5 M H2SO4; 10 cycles from −0.2 to +1.2 vs. Ag/V; 0.1 V·s−1).
3.2. Electrochemical Characterization of the PyoV Sensor Using CV, DPV, and EIS
3.2. Electrochemical Characterization of the PyoV Sensor Using CV, DPV, and EIS
Electrochemical techniques such as CV, DPV, and EIS were used for the electrochemical
Electrochemical
characterization after techniques
each modificationsuch asstep CV,during
DPV, sensor
and EIS were usedBoth
elaboration. for CV
the and
electrochemical
EIS studies
characterization after each
were performed in 10 mM [Fe(CN)6 ] modification 3 −step
/4 − during
(1:1) in 20 mM PBS pH 7.4. CV was used to trackwere
sensor elaboration. Both CV and EIS studies the
performed in 10 mM [Fe(CN) 6]3−/4− (1:1) in 20 mM PBS pH 7.4. CV was used to track the changes of
changes of electrochemical behavior at different stages of sensor elaboration, and is presented in
electrochemical
Figure 2A. A reversiblebehavior at different
redox stages ofinsensor
peak appeared elaboration,
all tested situationsanddueistopresented in Figure
the equivalent amount2A. of
A
reversible 4redox
− peak appeared
3 − in all tested situations due to the equivalent
[Fe(CN)6 ] and [Fe(CN)6 ] ions in the solution. As presented in Figure 2A (black curve (a)), the amount of [Fe(CN) 6]4−

and
CV for[Fe(CN)
the bare6]3− ions in the solution. As presented in Figure 2A (black curve (a)), the CV for the bare
graphene/graphite electrode registered the anodic peak at +0.168 vs. Ag/V (pseudo
graphene/graphite
Ag/AgCl), while the electrode
cathodic registered
peak appearedthe anodicat −peak
0.036atvs.
+0.168
Ag/V vs.(pseudo
Ag/V (pseudo Ag/AgCl),
Ag/AgCl), while
resulting in
the cathodic peak appeared at −0.036 vs. Ag/V (pseudo Ag/AgCl), resulting
peak-to-peak separation of 0.204 V. After the electrochemical reduction of the graphene layer (Figure 2A in peak-to-peak
separation
(red of 0.204
curve (b)), V. After
a slight the electrochemical
increase in oxidation/reduction reduction of the
peak graphene
currents, layer
paired (Figure
with 2A (red
an anodic curve
shift was
(b)), a slight increase in oxidation/reduction peak currents, paired with an
observed, maintaining the same peak separation of 0.200 V. This may be attributed to the removal of anodic shift was observed,
maintaining
any impuritythe thatsame
could peak
block separation
the accessofof0.200 V. This
the redox maytobe
probe theattributed to theFigure
surface. From removal 2A of any
(green
impurity
curve (c)),that
it was could block the
observed thataccess of the redox probe
oxidation/reduction to currents
peak the surface.
wereFrom Figure 2A
consistently (green
larger curve
after the
Materials
(c)), it 2019,
was 12, x FOR PEER
observed REVIEW
that oxidation/reduction peak currents were consistently larger 6 ofthe
after 13
electrochemical decoration of the reduced graphene entities with AuNPs, a registered increase in peak
electrochemical
current of 55%. decoration of the reduced graphene entities with AuNPs, a registered increase in
peak current of 55%.
The EIS plots represented in Figure 2B (red curve (b)) showed that the reduction of graphene
did not influence the parameters in Nyquist representation of impedance compared with the bare
graphene/graphite SPE (black curve (a)). Thus, similar values were obtained for the Rct using the
software facilities in both cases (23.87 Ω compared with 24.32 Ω). After the deposition of AuNPs, an
important decrease of Rct to 11.09 Ω was registered (green curve (c)) due to the large electron transfer
speed and better electrical conductivity of AuNPs compared with that of the bare electrode.
Furthermore, the Nyquist plot changed, confirming the modification of the electrochemical
transformation mechanism at the surface of the working electrode after the completed protocol.

Figure 2. (A) CVs and (B) EIS spectra recorded for graphite SPE modified with graphene (black (a));
graphite
Figure 2. SPE modified
(A) CVs with
and (B) EISgraphene reducedfor
spectra recorded (red (b)); and
graphite graphite
SPE SPEwith
modified modified with(black
graphene graphene
(a));
reduced and 3−/4− in 20 mM PBS pH 7.4.
graphite SPE AuNPs (green
modified with(c)) in 10 mM
graphene [Fe(CN)
reduced 6 ] (b)); and
(red graphite SPE modified with graphene
reduced and AuNPs (green (c)) in 10 mM [Fe(CN)6]3−/4− in 20 mM PBS pH 7.4.

3.3. Amplification Performance of the Electrochemical PyoV Sensor

In order to verify the amplification performance of the electrochemical PyoV sensor, DPV tests
were recorded step by step throughout the experimental protocol with and without the addition of
the analyte (Figure 3). There was a remarkable current response of 14.67 µA at +0.325 vs. Ag/V
(pseudo Ag/AgCl) on the optimized sensor in the presence of 100 µM PyoV (Figure 3, blue curve (d)),
Materials 2019, 12, 1180 6 of 13

The EIS plots represented in Figure 2B (red curve (b)) showed that the reduction of graphene
did not influence the parameters in Nyquist representation of impedance compared with the bare
graphene/graphite SPE (black curve (a)). Thus, similar values were obtained for the Rct using
the software facilities in both cases (23.87 Ω compared with 24.32 Ω). After the deposition of
AuNPs, an important decrease of Rct to 11.09 Ω was registered (green curve (c)) due to the large
Figure 2. (A) CVs and (B) EIS spectra recorded for graphite SPE modified with graphene (black (a));
electron transfer speed and better electrical conductivity of AuNPs compared with that of the bare
graphite SPE modified with graphene reduced (red (b)); and graphite SPE modified with graphene
electrode. Furthermore, the Nyquist plot changed, confirming the modification of the electrochemical
reduced and AuNPs (green (c)) in 10 mM [Fe(CN)6]3−/4− in 20 mM PBS pH 7.4.
transformation mechanism at the surface of the working electrode after the completed protocol.
3.3. Amplification Performance of the Electrochemical PyoV Sensor
3.3. Amplification Performance of the Electrochemical PyoV Sensor
In order to verify the amplification performance of the electrochemical PyoV sensor, DPV tests
In order to verify the amplification performance of the electrochemical PyoV sensor, DPV tests
were recorded step by step throughout the experimental protocol with and without the addition of
were recorded step by step throughout the experimental protocol with and without the addition of
the analyte (Figure 3). There was a remarkable current response of 14.67 µA at +0.325 vs. Ag/V
the analyte (Figure 3). There was a remarkable current response of 14.67 µA at +0.325 vs. Ag/V
(pseudo Ag/AgCl) on the optimized sensor in the presence of 100 µM PyoV (Figure 3, blue curve (d)),
(pseudo Ag/AgCl) on the optimized sensor in the presence of 100 µM PyoV (Figure 3, blue curve (d)),
compared with a mild current response of only 5.04 µA at +0.350 vs. Ag/V (pseudo Ag/AgCl) on the
compared with a mild current response of only 5.04 µA at +0.350 vs. Ag/V (pseudo Ag/AgCl)
bare graphene/graphite-based SPE (Figure 3, red curve (b)), and of 8.16 µA at +0.340 vs. Ag/V (pseudo
on the bare graphene/graphite-based SPE (Figure 3, red curve (b)), and of 8.16 µA at +0.340 vs.
Ag/AgCl) on the graphene/graphite-based SPE after the electrochemical reduction of graphene
Ag/V (pseudo Ag/AgCl) on the graphene/graphite-based SPE after the electrochemical reduction of
(Figure 3, green curve (c)). The control DPV (black curve (a)) registered in the absence of PyoV did
graphene (Figure 3, green curve (c)). The control DPV (black curve (a)) registered in the absence of
not show any oxidation signal in the optimized potential range. Thus, the reduced graphene and
PyoV did not show any oxidation signal in the optimized potential range. Thus, the reduced graphene
AuNPs could co-mediate the electrochemical oxidation of PyoV amplifying the signal in DPV.
and AuNPs could co-mediate the electrochemical oxidation of PyoV amplifying the signal in DPV.

Figure 3. DPVs performed in PBS in the absence of PyoV for reduced graphene/graphite SPE modified
with AuNPs (black (a)), and performed in 100 µM PyoV in PBS for bare graphene/graphite SPE
Figure 3. DPVs performed in PBS in the absence of PyoV for reduced graphene/graphite SPE modified
(red (b)); reduced graphene/graphite SPE (green (c)), and reduced graphene/graphite SPE with AuNPs
with AuNPs (black (a)), and performed in 100 µM PyoV in PBS for bare graphene/graphite SPE (red
(blue (d)) (20 mM PBS pH 7.4; from −0.3 to +0.8 V vs. Ag/AgCl; 0.02 V·s−1 ).
(b)); reduced graphene/graphite SPE (green (c)), and reduced graphene/graphite SPE with AuNPs
(blueCharacterization
3.4. SEM (d)) (20 mM PBS of
pHPyoV
7.4; from −0.3 to +0.8 V vs. Ag/AgCl; 0.02 V·s−1).
Sensor
Figure
3.4. SEM 4 shows theofSEM
Characterization PyoVimages
Sensor of the stepwise modification process of the PyoV sensor.
Figure 4A,B reveals the SEM images of the bare SPE based on the graphene/graphite working surface
before and after the decoration with AuNPs. On the other hand, SEM images were obtained on
reduced graphene/graphite before and after the electrochemical deposition of AuNPs were presented,
as shown in Figure 4C,D. It was observed that the surface of the electrode was homogenous, covered
with metallic NPs after the electrochemical reduction of graphene, which confirms the importance of
this step in the experimental protocol. As can be also seen, the coverage with AuNPs was higher after
the conditioning step, furthermore confirming the importance of the conditioning protocol regarding
the density in catalytic metallic NPs.
shown in Figure 4C,D. It was observed that the surface of the electrode was homogenous, covered
with metallic NPs after the electrochemical reduction of graphene, which confirms the importance of
this step in the experimental protocol. As can be also seen, the coverage with AuNPs was higher after
the conditioning step, furthermore confirming the importance of the conditioning protocol7 of
Materials 2019, 12, 1180
regarding
13
the density in catalytic metallic NPs.

Figure 4. (A) SEM graphs for graphene/graphite SPE, (B) graphene/graphite SPE after AuNPs
Figure electrochemical generation,
4. (A) SEM graphs (C) graphene/graphite
for graphene/graphite SPE(B)
SPE, after electrochemical reduction,
graphene/graphite SPE afterandAuNPs
(D) graphene/graphite SPE after electrochemical reduction and AuNP electrochemical generation.
electrochemical generation, (C) graphene/graphite SPE after electrochemical reduction, and (D)
graphene/graphite SPE afterofelectrochemical
3.5. Analytical Performance the PyoV Sensor reduction and AuNP electrochemical generation.
Under optimized experimental conditions, the PyoV electrochemical sensor was used to quantify
3.5. Analytical
differentPerformance of the
concentrations PyoV
of the Sensor
analyte using an optimized DPV procedure. The variation of the
DPV current
Under response
optimized with the concentration
experimental conditions,of PyoV
theisPyoV
presented in Figure 5A. Nosensor
electrochemical oxidation signal
was used to
was observed without the addition of PyoV in the tested solution (black curve), while noticeable
quantify different concentrations of the analyte using an optimized DPV procedure. The variation of
oxidation signals appeared with the addition of PyoV starting from 0.5 µM. Moreover, the current
the DPV current response with the concentration of PyoV is presented in Figure 5A. No oxidation
response increased linearly with the increase of PyoV concentration from 0.5 to 100 µM (Figure 5B). The
signal was observed
regression equationwithout the
was fitted as Iaddition of PyoV(µM)–0.357
(µA) = 0.139[PyoV] in the tested solution
(R2 = 0.993; RSD =(black curve), while
5.36% (Relative
noticeable oxidation
standard signals
deviation (RSD)appeared with as
was calculated theaverage
addition of PyoV
of RSD starting
for each from 0.5
concentration µM.
in the Moreover, the
calibration
current response increased linearly with the increase of PyoV concentration from 0.5 to 100 µM
curve)). Three tests were performed for each concentration from the calibration curve on different
(Figuresensors,
5B). Theand the data are
regression presented
equation wasas fitted
the average values
as I (µA) of results. The
= 0.139[PyoV] sensitivity of
(µM)–0.357 (Rthe
2 = sensor,
0.993; RSD =
expressed as the slope of the calibration graph, was calculated from the plot as 0.14 µA·µM−1 by linear
5.36% (Relative standard deviation (RSD) was calculated as average of RSD for each concentration in
regression. In addition, the limit of detection (LOD) was estimated at 66.90 nM based on S/N = 3.
the calibration curve)). Three tests were performed for each concentration from the calibration curve
on different sensors, and the data are presented as the average values of results. The sensitivity of the
sensor, expressed as the slope of the calibration graph, was calculated from the plot as 0.14 µA µM−1
by linear regression. In addition, the limit of detection (LOD) was estimated at 66.90 nM based on
S/N = 3.
Materials2019,
Materials 2019,12,
12,1180
x FOR PEER REVIEW 88of
of13
13

Figure 5. (A) DPVs in 20 mM PBS pH 7.4 without PyoV (black line) and for different concentrations
Figure
of PyoV:5.0.5
(A)(green
DPVs line);
in 20 mM
0.75 PBS
(red pH 7.41without
line); PyoV
(dark blue (black
line); line) and
5 (light bluefor different
line); concentrations
10 (purple line); 25
of PyoV:line);
(yellow 0.5 (green line);line);
50 (olive 0.75 (red line);line);
75 (blue 1 (dark blue
and line);
100 5 (light
(dark blueline)
purple line);µM
10 (purple
in 20 mM line);
PBS25 pH
(yellow
7.4
line);
on 50 (olive line); 75
graphene/graphite (blue
after line); and
reduction and100 (darkdecoration
AuNPs purple line) SPE.µM (B)inCalibration
20 mM PBS plotpHof 7.4
PyoV on
graphene/graphite
concentration after (error
vs. current reduction and AuNPs
bars represent decoration
the standard SPE. for
deviation (B) three
Calibration
tests). plot of PyoV
concentration vs current (error bars represent the standard deviation for three tests).
3.6. Specificity, Intra- and Inter-Assay Precision, Reusability and Stability of the PyoV Sensor
3.6. Specificity,
Specificity Intra- and Inter-Assay
tests were Precision,
performed for Reusability
the optimized and under
sensor Stability
theofsame
the PyoV Sensor and different
conditions,
common interfering
Specificity testssubstances were used
were performed (e.g.,optimized
for the G, AA, ASA, DA,under
sensor NADH, theandsame UA). When using
conditions, and
electrochemical methods on real samples (serum, urine, saliva), it
different common interfering substances were used (e.g., G, AA, ASA, DA, NADH, and UA). When is usually necessary to dilute
with
usingthe electrolyte to enhance
electrochemical methodsthe conductivity.
on real Depending
samples (serum, urine,onsaliva),
the dilution level (1:100)
it is usually necessary thetochosen
dilute
concentration of PyoV was found to be around 25 µM. Furthermore, the physiological
with the electrolyte to enhance the conductivity. Depending on the dilution level (1:100) the chosen concentration
range of UA is 2201–5472
concentration of PyoV was [18], and
µMfound after
to be a 1:100
around 25dilution level the employed
µM. Furthermore, value could
the physiological be included
concentration
in the 22–54.72
range of UA isµM range. AA
2201–5472 µMhas [18],a physiological
and after a 1:100 concentration range
dilution level theofemployed
26–84.6 µM, valueclose to the
could be
established concentration value of 25 µM [19]. The concentration of G was
included in the 22–54.72 µM range. AA has a physiological concentration range of 26–84.6 µM, closeselected to be slightly higher
that the physiological one situated between 4400 and 6100 µM (79.2–110 ·mL −1 ) [20]. Pyocianin
to the established concentration value of 25 µM [19]. The concentration of Gmg was selected to be slightly
(PyoC),
higher thethatother virulence factor
the physiological one P. aeruginosa,
forsituated betweenwas 4400
used andas the6100
negative control target
µM (79.2–110 mg·mL to test the
−1) [20].

selectivity of the sensor for PyoV detection. As can be seen from Figure 6A,
Pyocianin (PyoC), the other virulence factor for P. aeruginosa, was used as the negative control target the current responses
obtained
to test the with the elaborated
selectivity of the sensor for 25 PyoV PyoV in the
µM detection. Aspresence of 7000
can be seen from G, 25 µM
µMFigure 6A, AA, 25 µM
the current
ASA,
responses obtained with the elaborated sensor for 25 µM PyoV in the presence of 7000 µM G, 25with
25 µM NADH, 25 µM UA, and 25 µM DA presented reduced (or low) influences compared µM
the
AA,DPV25 µM signal
ASA, registered
25 µM NADH,for PyoV 25 alone.
µM UA, Thus,
and the correspondent
25 µM DA presented calculated
reducedrecoveries between
(or low) influences
96.51%
compared and with
115.46%.
the DPVIn thesignal
case ofregistered
the PyoC for standard
PyoV solution prepared
alone. Thus, for the interference
the correspondent tests,
calculated
an
recoveries between 96.51% and 115.46%. In the case of the PyoC standard solution prepared for(c)).
electrochemical signal was observed for PyoV also, as can be seen in Figure 6B (green curve the
This may have
interference been
tests, andue to the fact that
electrochemical both was
signal compounds
observedare formetabolites
PyoV also, for P. aeruginosa,
as can be seen inand could
Figure 6B
have
(greenbeen simultaneously
curve (c)). This may present
have beenduring duethetosynthesis and both
the fact that separation of PyoC
compounds areused for this study.
metabolites for P.
Thus, whenand
aeruginosa, 25 µMcouldPyoVhaveandbeen10simultaneously
µM PyoC werepresent both induring
the solution, the intensity
the synthesis of the oxidation
and separation of PyoC
peak
usedforforthe
thisPyoV
study.atThus,
aboutwhen0.3 V/Ag/AgCl
25 µM PyoVincreased,
and 10 µMcompared
PyoC were with
both theinstandard
the solution,test the
of the same
intensity
concentration (Figure 6B, blue (d), and Figure 6B, red (b), respectively),
of the oxidation peak for the PyoV at about 0.3 V/Ag/AgCl increased, compared with the standardprobably due to traces of PyoV
from the
test of the PyoC
samesolution.
concentration (Figure 6B, blue (d), and Figure 6B, red (b), respectively), probably due
The of
to traces intra-
PyoVand from inter-assay precision of the sensor were investigated by testing the same
the PyoC solution.
concentration of PyoV (25 µM) performing five different tests on the same sensor and five different
sensors, respectively. The DPV curves obtained during these tests were evaluated and the RSD was
calculated. The average of the current intensity for the oxidation peak of PyoV was 3.269 µA, and the
RSD of the intra-assay and inter-assay precisions of the sensor were 4.16% and 3.97%.
Materials 2019,
Materials 2019, 12,
12, 1180
x FOR PEER REVIEW 99 of
of 13
13

Figure 6. (A) The selectivity of the sensor for PyoV over other analogs G, AA, ASA, NADP, DA, UA,
and PyoC. (B) DPVs registered in 20 mM PBS pH 7.4 (black (a)), 25 µM PyoV (red (b)), 10 µM PyoC
(green (c)), and 25 µM PyoV+10 µM PyoC (blue (d)). The oxidation peaks for PyoV and PyoC are
highlighted in (B) with green and blue circles, respectively.

The intra-
Figure 6. (A) and inter-assay
The selectivity precision
of the sensor forofPyoV
the over
sensor were
other investigated
analogs G, AA, ASA,byNADP,
testing
DA,the
UA,same
Figure 6. (A) The selectivity of the sensor for PyoV over other analogs G, AA, ASA, NADP, DA, UA,
concentration
and PyoC.of (B)PyoV
DPVs(25 µM) performing
registered in 20 mM PBSfivepHdifferent tests
7.4 (black (a)),on
25 the
µM same sensor
PyoV (red (b)),and fivePyoC
10 µM different
and PyoC. (B) DPVs registered in 20 mM PBS pH 7.4 (black (a)), 25 µM PyoV (red (b)), 10 µM PyoC
sensors, respectively.
(green (c)), and 25 The DPV curves
µM PyoV+10 obtained
µM PyoC (blueduring these
(d)). The tests were
oxidation peaksevaluated
for PyoV andand PyoC
the RSD
are was
(green (c)), and 25 µM PyoV+10 µM PyoC (blue (d)). The oxidation peaks for PyoV and PyoC are
highlighted in (B) with green and blue circles, respectively.
calculated. The average of the current intensity for the oxidation peak of PyoV was 3.269 µA, and the
highlighted in (B) with green and blue circles, respectively.
RSD of the intra-assay and inter-assay precisions of the sensor were 4.16% and 3.97%.
The possibility to reuse the same sensor for more tests was also evaluated. Thus, different tests
The possibility to reuse the same sensoroffor more testswere
was also evaluated.by Thus, different tests
wereThe intra- and
performed usinginter-assay precision
the same sensor in 25 µMthe sensor
of PyoV solution,investigated
after a washing steptesting the Itsame
in PBS. was
were performed
concentration of using
PyoV the
(25 same
µM) sensor in 25five
performing µM different
of PyoV solution,
tests on after
the a washing
same sensor step
and in PBS.
five It was
different
observed that the current intensity slightly decreased after each test, the signal recovery being 97.25%
observed that the current
sensors, Theintensity slightly decreased after each test,were
the signal recovery being 97.25%
after the respectively.
second test, 91.94% DPV
aftercurves obtained
the fourth during
test, and these
76.10% tests
after the ninthevaluated and7).
test (Figure theWeRSD
assumewas
after the second
calculated. test, 91.94% after the fourth test, and 76.10% after the ninth test (Figure 7). We assume
that each sensor could be reused with good analytical parameters for two successive tests, suitable the
The average of the current intensity for the oxidation peak of PyoV was 3.269 µA, and for
that each
RSD of thesensor couldand
intra-assay be reused withprecisions
inter-assay good analytical
of the parameters
sensor werefor twoand
4.16% successive
3.97%. tests, suitable
a disposable detection device.
for a The
disposable detection
possibility to reuse device.
the same sensor for more tests was also evaluated. Thus, different tests
were performed using the same sensor in 25 µM of PyoV solution, after a washing step in PBS. It was
observed that the current intensity slightly decreased after each test, the signal recovery being 97.25%
after the second test, 91.94% after the fourth test, and 76.10% after the ninth test (Figure 7). We assume
that each sensor could be reused with good analytical parameters for two successive tests, suitable
for a disposable detection device.

Figure 7. The reusability tests for the PyoV sensor.

Figure 7. The reusability tests for the PyoV sensor.
To check the operational stability of this sensor, the electrochemical performance was tested
againstTo the
check the operational
storage stability
time. Different PyoVofsensors
this sensor, the electrochemical
were elaborated using the performance was tested
optimized protocol and
against the storage time. Different PyoV sensors were elaborated using
◦ the optimized
were stored in a dry and clean environment at room temperature (20 C) if not in use. Three of these protocol and
were stored in a dry and clean environment at room temperature (20 °C) if not
sensors were tested immediately after elaboration in 25 µM PyoV, while others were tested from timein use. Three of these
sensors
to were
time for 30 tested immediately
days. It was observedafter elaboration
that the signalin 25 µM PyoV,
obtained 25 while others were tested from after
time
Figure 7. The reusability tests for thefor
PyoV µM PyoV
sensor. increased by 8.75%
3todays,
time for 30 days.
14.99% afterIt7was observed
days, 10.44%thatafterthe
14signal
days, obtained
while only forby
25 4.15%
µM PyoV afterincreased
30 days. by 8.75%
These after
results
3 days,
suggest 14.99%
To that
check after
thethe 7 days,
operational
elaborated 10.44% after
stability
sensor 14
of this
exhibited days,
good while
sensor, only
the for
stability by 4.15%
electrochemical after 30 days.
performance
the DPV determination These results
was tested
of PyoV.
suggest the
against thatstorage
the elaborated sensor exhibited
time. Different PyoV sensorsgoodwere
stability for the DPV
elaborated usingdetermination
the optimizedofprotocol
PyoV. and
3.7. Recovery
were stored inTests of the
a dry andPyoV
cleanSensor in Real Samples
environment at room temperature (20 °C) if not in use. Three of these
sensors were tested immediately after elaboration
The sensor response towards 25 µM of PyoV spiked in 25 µM PyoV, while
in different others
complex were tested
matrices such from time
as human
to time for 30 days. It was observed that the signal obtained for 25 µM PyoV increased
serum, saliva, and tap water was also tested. The spiked samples were prepared after the dilution of by 8.75% after
3thedays,
real 14.99%
samplesafter
with7electrolytes,
days, 10.44%and after 14 days,
injected withwhile only analyte
the target by 4.15% after 30
in order to days.
achieveThese results
the desired
suggest that the elaborated sensor exhibited good stability for the DPV determination of PyoV.
Materials 2019, 12, 1180 10 of 13

concentrations. The results were expressed as recoveries, and it can be noticed that the values obtained
for 25 µM PyoV ranged from 96.75% in saliva, to 98.83% in serum, and 100.55% in tap water (data
not presented). The RSD of three different measurements of the same sample varied from 1.9% to
4.1%, respectively

4. Discussion
Until now bacterial infections are usually diagnosed by using microbiological techniques (cell
culturing in controlled experimental conditions) in which case detection is based on cell growth and
development. Clinical diagnosis is not possible if the concentration of bacteria in collected real samples
does not exceed 105 cells/mL [21]. In laboratories, the use of a simple method and an optimized
platform for direct bacterial detection is not yet possible. A useful alternative for P. aeruginosa direct
detection could be the use of PyoV, a virulence factor, as a marker with electrochemical activity.
A procedure based on the electrochemical oxidation of PyoV could allow the rapid and sensitive
detection of P. aeruginosa. After two simple electrochemical conditioning steps, a commercially available
SPE was successfully applied for the sensitive detection of PyoV even in the presence of several
analytes known as possible interfering compounds in real samples. In this work, the ability to detect
and quantify PyoV, as bacterial marker, in complex samples was demonstrated, offering a promising
tool for early diagnosis of bacterial infections.
Electrochemical reduction of graphene was introduced as a conditioning step in the experimental
protocol, in order to eliminate any small differences between electrodes. This step also allowed
the reduction of the impurities that could possibly be found on the working electrode. Usually,
this procedure is used for graphene oxide-modified electrodes in order to avoid the decrease in
conductivity and carrier mobility observed when compared with graphene-modified electrodes [22,23].
The electrochemically reduced graphene has a larger number of electroactive sites, having a similar
structure with that of graphene [24–26]. Moreover, the SEM images confirmed the generation of a more
homogenous film after the electrochemical reduction of the graphene suitable for the detection of the
target analyte.
The increase in peak current of 55% after the deposition of AuNPs could be assigned to the
enhancement of the active surface by metallic NPs that facilitated the redox probe electrochemical
transformation. Another explanation could be the good conductivity of AuNPs on the surface of the
electrode, which were favorable for the electron transfer. The peak-to-peak separation became 0.227 V.
The electroactive surface of the electrode was calculated after each modification step according to the
Randles–Sevcik equation (Equation (1)) [27]:

Ip = 2.69·105 ·A·D1/2 ·n3/2 ·v1/2 ·C (1)

where Ip represents the value of the current peak intensity A; A is the effective area of the electrode
surface (cm2 ); D is the diffusion coefficient of [Fe(CN)6 ]3−/4− (6.70 × 10−6 cm2 ·s−1 ); n is the number
of electrons involved in the redox reaction (1); v is the scan rate of the potential (V·s−1 ); and C is the
bulk concentration of the redox probe (mol·cm−3 ).
By calculation, the effective surface area of the bare electrode was 0.150 cm2 , while after decoration
with AuNPs it became 0.234 cm2 , showing that this step generated an important increase in the surface
area of the working electrode.
Moreover, EIS was used as an effective and rapid technique to measure the electrical properties
of the electrode before and after functionalization with different materials. The typical Nyquist
representation includes a semicircle corresponding to the electron transfer limited process, the diameter
displaying the electron transfer resistance values (Rct ), and a linear zone that corresponds to the
diffusion limited process.
When evaluating the analytical performance of the sensor, the low value of LOD could be
attributed to the following factors: (i) reduced graphene with a high electron-transfer capability
Materials 2019, 12, 1180 11 of 13

and large specific surface area, and (ii) excellent electrical conductivity of AuNPs determining the
improvement of the electron transfer rate.
The detection of PyoV in the presence of G, AA, ASA, NADH, UA, and DA was not influenced,
while in the presence of PyoC it could not be detected with high accuracy. Considering that the
electrochemical oxidation of PyoC occurred at negative potential (−0.300 vs. Ag/V (pseudo Ag/AgCl))
it is unlikely that the presence of this compound would influence the signal of PyoV that appeared at a
positive potential value (+0.325 vs. Ag/V (pseudo Ag/AgCl)). Indeed, from the DPV curve recorded
in the presence of both compounds in the same sample (Figure 6B, blue curve (d)) it was observed
that the signal for PyoV summed up the initial signal observed in the PyoC solution and the signal
corresponding to the PyoV added concentration (25 µM). On the contrary, the electrochemical signal of
PyoC was dramatically influenced by the presence of PyoV, proving that the two compounds cannot
be determined simultaneously with this sensor. Hence, the sensor had a high specific affinity for PyoV
detection, but cannot be used for the simultaneous detection of both P. aeruginosa virulence factors.

5. Conclusions
The ability of the DPV detection to sense PyoV in a micromolar range in complex matrices such
as tap water, human serum, and saliva was demonstrated, being a practical validation for the sensor.
Those satisfying results demonstrate that the sensor has a great potential for practical applications in
the testing and quantification of PyoV in biological samples.
The good sensitivity in detection was achieved through defining the optimal potential range for
the PyoV voltammetric quantification with constant background contribution. The sensor was able
to detect PyoV in a nanomolar range, proving the presence of P. aeruginosa in the analyzed samples.
An important issue is the fact that the sensor can be reused a minimum of two times after cleaning with
water, significantly reducing time and cost, qualifying it for fast diagnosis of P. aeruginosa, and making
it suitable for point-of-care devices, as no sample pretreatment is needed. This novel electrochemical
sensor successfully integrated the advantages of the high conductivity and large specific surface area
of graphene, and the excellent electrochemical activity of AuNPs, with a high sensitivity, selectivity,
wide linear range, and fast analysis time for PyoV.

Author Contributions: Conceptualization, M.T., A.C., and C.C.; methodology, A.C. and C.C.; validation, M.T.,
A.C., and C.C.; formal analysis, I.G.; investigation, I.G. and M.T.; resources, C.C. and A.I.; writing—original draft
preparation, I.G., A.C., and M.T.; writing—review and editing, C.C., D.S.-M., and A.I.; project administration, C.C.
and A.I.; funding acquisition, C.C.
Funding: This research was funded by the Romanian National Authority for Scientific Research and Innovation,
CNCS/CCCDI-UEFISCDI, grant number PN-III-P1-1.2-PCCDI2017-0221/59PCCDI/2018, within PNCDI III, and
also by the COFUND-ERA-HDHL ERANET, European and International Cooperation-Subprogram 3.2-Horizon
2020, PNCDI III Program-Biomarkers for Nutrition and Health–“Innovative technological approaches for
validation of salivary AGEs as novel biomarkers in evaluation of risk factors in diet-related diseases” (SALIVAGES),
No. 25/1.09.2017.
Acknowledgments: The authors acknowledge Maria Suciu (National Institute for Research and Development of
Isotopic and Molecular Technologies, Cluj-Napoca, Romania) for SEM experiments.
Conflicts of Interest: The authors declare no conflict of interest.

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