sensors

Article
Label-Free Aptasensor for Lysozyme Detection Using
Electrochemical Impedance Spectroscopy
Dionisia Ortiz-Aguayo ID
and Manel del Valle * ID

Sensors and Biosensors Group, Department of Chemistry, Universitat Autònoma de Barcelona, Bellaterra 08193,
Spain; Dionisia.ortiz@uab.cat
* Correspondence: manel.delvalle@uab.es; Tel.: +34-935-811-017; Fax: +34-935-812-477

Received: 20 December 2017; Accepted: 23 January 2018; Published: 26 January 2018

Abstract: This research develops a label-free aptamer biosensor (aptasensor) based on graphite-epoxy
composite electrodes (GECs) for the detection of lysozyme protein using Electrochemical Impedance
Spectroscopy (EIS) technique. The chosen immobilization technique was based on covalent bonding
using carbodiimide chemistry; for this purpose, carboxylic moieties were first generated on the
graphite by electrochemical grafting. The detection was performed using [Fe(CN)6 ]3− /[Fe(CN)6 ]4−
as redox probe. After recording the frequency response, values were fitted to its electric model using
the principle of equivalent circuits. The aptasensor showed a linear response up to 5 µM for lysozyme
and a limit of detection of 1.67 µM. The sensitivity of the established method was 0.090 µM−1 in
relative charge transfer resistance values. The interference response by main proteins, such as bovine
serum albumin and cytochrome c, has been also characterized. To finally verify the performance of
the developed aptasensor, it was applied to wine analysis.

Keywords: aptamer; biosensor; electrochemical impedance spectroscopy; electrochemical grafting

1. Introduction
Nowadays, there is an undoubted need of monitoring and controlling different parameters
in fields such as food industry, forensic, environmental monitoring, drug development or clinical
diagnoses. Conventional analytical methods may provide high sensitivity and selectivity but they can
present problems such as time-delays, high cost and have requirements of trained personal. Thus, it is
very important to find reliable analytical devices capable of performing accurate and fast analyses. One
proposal to overcome these problems is the development of biosensors [1]. Biosensors can be classified,
depending on the technique employed for the transduction, as optical, calorimetric, piezoelectric
and electrochemical or, by the type of biorecognition element, as enzyme, nucleic acid, aptamer, cells
and biomimetic systems. In this work, the developed biosensors are aptasensors and the type of
transduction for biosensing is electrochemical. Aptasensors are biosensors that use aptamer [2] as
the biorecognition element. Aptamers (from the Latin aptus–fit, and Greek meros–part) are artificial
single strands of DNA or RNA with a length in the range of 10–100 nucleotides, which are selected
in vitro and have the ability, through specific folding conformations, to bind proteins, ions, whole
cells, drugs and low molecular weight ligands, ranging from picomolar (pM) to nanomolar (nM) level.
They recognize their target with high affinity and specificity [3], often matching or even exceeding
those of antibodies [4]. Their affinity constants reach the order of 3 × 107 M−1 , which makes them
promising recognition agents. Due to all these properties, aptamers can be used in a wide range of
applications, such as therapeutics and diagnostics [5], molecular switches, drug development, affinity
chromatography and biosensors [6,7]. The in vitro process [8] to obtain a certain specific aptamer
is called SELEX (Systematic Evolution of Ligands by Exponential enrichments). Therefore, this study
describes the construction of an electrochemical aptamer-based sensor (aptasensor) for lysozyme (Lys)

Sensors 2018, 18, 354; doi:10.3390/s18020354 www.mdpi.com/journal/sensors

Sensors 2018, 18, 354 2 of 11

detection using Electrochemical Impedance Spectroscopy (EIS) technique. EIS [9] has been used in
many fields of electrochemistry, electrode kinetics, double-layer studies, batteries, corrosion, solid-state
electrochemistry and bioelectrochemistry [10]. In addition, it can be considered as a characterization
technique, which provides electric information in the frequency domain. With this technique, a process
that occurring in an electrochemical cell can be modelled using a combination of electrical circuits
that give the same AC current response provided by the electrochemical system [11]. By the use of
equivalent circuits, the experimental spectra can be fitted with the theoretical curve corresponding to
the selected circuit model, thus obtaining the values of electrical parameters (resistance, capacitance,
etc.) which are directly correlated to specific electrochemical phenomena occurring in the system
under study [10]. Its wide ability for the characterization of electrode-electrolyte interfaces and its high
sensitivity for probing the interfacial properties of a modified electrode surface have made EIS a good
alternative for biosensing during the last years. Besides, an important feature presented by the EIS
technique is that it does not require any labelled species for the transduction. Thus, it can be used for
designing label-free protocols avoiding more expensive and time-consuming assays.
The analyte studied in the present work was lysozyme (Lys). Lysozyme is a mucopolysaccharide
alkaline enzyme that is capable of destroying bacterial cellular membranes by catalyzing the breakage
of the β-1,4 bond found in peptidoglycan residues of cell walls of Gram-positive bacteria [12].
This protein has many points of interest, because it is considered as a good model to study enzyme
catalysis, protein structure and interactions or amyloid-fibrillation formation. In addition, Lys from hen
egg has become also a model protein for the pharmaceutical industry when it comes to the development
of new drug delivery systems or the design of innovative treatment strategies. Furthermore, Lys
is involved in different fields such as wine-making and food industry including cheese and beer
production. In the case of wine industry, this protein replaces sulfites and it is added at doses of
250–500 mg·L−1 to inhibit malolactic fermentation and to stabilize the wine afterwards. Residual levels
of 0.06–327 mg·L−1 were found in lysozyme-treated wines [13]. According with the International
Organization of Vine and Wine (OIV) the maximum permissible dose of Lys in wine-making is
500 mg·L−1 (~35 µM) [14], thus it is considered as regulated substance. Despite the above, Lys has
many drawbacks associated to allergic reactions in susceptible individuals and elevated human Lys
level in serum and urine may cause kidney problems [15] and leukemia [16]. At last, there are different
analytical methods [17] for its detection such as chromatography and ELISA among others, but they are
expensive and need complex procedures. For this reason, there is a need to develop rapid, cheap and
effective devices for Lys detection. All of these requirements are supplied by label-free aptasensors [18].
As mentioned previously, we have designed in this work a disposable and label-free aptasensor
for the detection of Lys protein. To achieve this feature, different optimizations were performed,
a calibration curve of the sensor and its behavior were also assessed in a complex matrix such as wine.
The specificity and the regeneration of the sensor were also evaluated. The transduction principle
used is based on the change of electron-transfer resistance in the presence of a redox probe, which
can be measured by EIS. The aptamer was immobilized by covalent binding using carbodiimide
chemistry [19,20]. The aptasensor showed a high sensitivity, good specificity and they have a low
production cost and rapid response.

2. Materials and Methods

2.1. Chemicals and Reagents

Potassium ferricyanide K3 [Fe(CN)6 ], potassium ferrocyanide K4 [Fe(CN)6 ], sodium chloride (NaCl),
potassium chloride (KCl), disodium phosphate (Na2 HPO4 ) were obtained from Merck (Darmstadt,
Germany). N-hydroxysuccinimide (NHS), N-(3-dimethyl aminopropyl)-N0 -ethylcabodiimide
hydrochloride (EDC), poly (ethylene glycol) 1000 (PEG) were obtained from Fluka (Buchs,
Switzerland). Monopotassium phosphate (KH2 PO4 ), sodium nitrite (NaNO2 ), MES monohydrate,
4-aminobenzoic acid (ABA) and magnesium chloride (MgCl2 ) were obtained from Sigma Aldrich
Sensors 2018, 18, 354 3 of 11

(St. Louis, MO, USA). Graphite-epoxy composite electrodes (GEC) were prepared using 50 µm particle
size graphite powder (Merck, Darmstadt, Germany), Resineco epoxy resin and its corresponding
hardener. Biomers (Ulm, Germany) was the source of the aptamer used in this study. It has a
modification in 50 extreme with aminolink C6. The sequence is 50 -ATC TAC GAA TTC ATC AGG
GCT AAA GAG TGC AGA GTT ACT TAG-30 for proper immobilization using an amide linkage
as described in the literature [18]. Lysozyme (Lys), bovine serum albumin (BSA) and cytochrome c
from bovine heart (Cyt c) were purchased from Sigma Aldrich (St. Louis, MO, USA). Commercial
wine samples used are from Don Simon. All solutions were made up using sterilized Milli-Q water
(Millipore, Billerica, MA, USA). The buffer solutions employed were PBS (187 mM NaCl, 2.7 mM KCl.
8.1 mM Na2 HPO4 ·H2 O and 1.76 mM KH2 PO4 , pH 7.0), BB (1 mM MgCl2 , 2.7 mM KCl, 140 mM NaCl,
0.1 mM Na2 HPO4 and 1.8 mM KH2 PO4 , pH 7.0) and MES (100 mM 2-(N-morpholino)ethanesulfonic
acid and 0.09% NaCl, pH 7.0). Aptamer stock solutions were first diluted with deionized water,
separated in fractions of 10 µM and stored at a temperature of −20 ◦ C. When required, a single
fraction was defrosted and used.

2.2. Equipment
Alternating current (AC) impedance measurements were performed with an Autolab PGStat
20 (Metrohm Autolab B.V, Utrecht, The Netherlands). FRA software was used for the acquisition
of the data and the control of the experiments. Finally, ZView (Scribner Associates Incorporated,
Southern Pines, NC, USA) software was used for data processing. Cyclic Voltammetry measurements
were performed with an Autolab PGStat 20 (Metrohm Autolab B.V, Utrecht, The Netherlands). GPES
software was used for the acquisition of the data. A three-electrode cell was used to perform the
impedance and cyclic voltammetry measurements: a platinum-ring auxiliary electrode (Crison 4.75,
Barcelona, Spain), an Ag/AgCl reference electrode and GEC as working electrode. Furthermore, other
equipment such as Eppendorf Thermomixer C to control temperature incubations were used for the
experiment, pH-meter GLP22 (Crison 5224, Barcelona, Spain), Vortex shaker MS3 basic (IKA, Staufen,
Germany) and Sterilizer CertoClav-Tisch-Autoclav 12L GS (Schaffhausen, Switzerland).

2.3. Preparation of Working Electrodes

The construction of the electrodes was carried out using PVC tube body (6 mm i.d.) and a small
copper disk was soldered at the end of an electrical connector. The next step of the procedure is to
prepare the graphite-epoxy composite mixture. This mixture was prepared by hand mixing of the
epoxy resin, the hardener and the graphite powder [21,22]. The resulting paste was homogenized by
mixing for 1 h and then it was cured at 40 ◦ C during 2 d. Before each use, the electrode surface was
moistened with MilliQ water and then thoroughly smoothed with abrasive sandpaper and finally with
alumina paper (polishing strips 301044-001, Thermo Scientific Orion, Waltham, MA, USA) in order to
obtain a reproducible electrochemical surface.

2.4. Experimental Procedure

The experimental procedure for biosensing consists of immobilization of the aptamer onto the
transducer surface using carbodiimide chemistry via electrochemical grafting, followed by the labeless
recognition of the Lys protein by its aptamer via incubation at room temperature. The steps of the
working electrode construction and biosensing are described in more detail below (Figures 1 and 2).
Sensors 2018, 18, 354 4 of 11
Sensors 2018, 18, x FOR PEER REVIEW 4 of 11
Sensors 2018, 18, x FOR PEER REVIEW 4 of 11

Figure
Figure 1.
1. Construction
Figure 1. Construction of
Construction working
of working
of electrodes
working electrodes based
electrodes based on
based on graphite-epoxy
on graphite-epoxy composite.
graphite-epoxy composite.
composite.

Figure 2.
Figure 2. Steps
2. Steps for
Steps for the
for the experimental
the experimental protocol
experimental protocol for
protocol for the
for the label-free
the label-free aptasensor
label-free aptasensor for
aptasensor for Lys
for Lys detection
Lys detection based
detection based on
based on
on
Figure
covalent
covalent bond
bond immobilization
immobilization technique
technique via
via electrochemical
electrochemical grafting.
grafting.
covalent bond immobilization technique via electrochemical grafting.
2.4.1.
2.4.1. Aptamer
Aptamer Preconditioning
Preconditioning
2.4.1. Aptamer Preconditioning
Before
Before aptamer
aptamer immobilization,
immobilization, it it is
is very
very important
important to
to make
make aa pretreatment
pretreatment ofof the
the aptamer.
aptamer. This
This
Before
aptamer aptamer immobilization,
preconditioning is carried it
out is
in very
orderimportant
to promoteto make
its a
loosepretreatment
conformation. of the
Thus,aptamer.
the This
aptamer
aptamer preconditioning is carried out in order to promote its loose conformation. Thus, the aptamer
aptamer
solutions preconditioning is carriedfor
out in order to promote its loose conformation. Thus,cold
the aptamer
solutions were
were heated
heated at
at 80–90
80–90 ◦°C
°C for 33 min.
min. Then,
Then, the
the solution
solution was
was dipped
dipped in
in aa bath
bath of
of cold water
water to
to
solutions
fix the were
loose heated at 80–90
conformation. C for 3 min. Then, the solution was dipped in a bath of cold water to
fix the loose conformation.
fix the loose conformation.
2.4.2. Aptasensor
2.4.2. Aptasensor Immobilization
Aptasensor Immobilization
Immobilization onto onto the
onto the Electrode
the Electrode Surface
Electrode Surface
Surface
2.4.2.
After the
After the preconditioning
the preconditioning
preconditioning of of the
of the aptamer,
the aptamer,
aptamer, the the aptamer
the aptamer
aptamer is is immobilized
is immobilized
immobilized throughthrough
through aaa covalent
covalent
covalent bondbond
bond
After
between
between the
the aptamer
aptamer and
and the
the electrode
electrode surface.
surface. This
This is
is done
done by
by electrochemical
electrochemical grafting
grafting [23],
[23], being
being
between the aptamer and the electrode surface. This is done by electrochemical grafting [23], being
its goal
its goal to
goal to obtain
to obtain stable
obtain stable carboxyl-derivatized
stable carboxyl-derivatized conductive
carboxyl-derivatized conductive surfaces
conductive surfaces
surfaces forfor the
for the immobilization
the immobilization
immobilization of of aminated
of aminated
aminated
its
biomolecules.
biomolecules. To To achieve this, 4-aminobenzoic acid (ABA) and sodium nitrite are used to form
form aaa
biomolecules. To achieve
achieve this,
this, 4-aminobenzoic
4-aminobenzoic acid acid (ABA)
(ABA) andand sodium
sodium nitrite
nitrite are
are used
used to to form
diazonium
diazoniumsalt, salt, which
salt,which
which is immobilized
is immobilized with electrochemical
with electrochemical control
control on the electrode
on thesurface.
electrode surface.
surface.
diazonium is immobilized with electrochemical control on the electrode Afterwards,
Afterwards,
Afterwards, the amino
the amino terminated
terminated aptamer is immobilized
aptamer isthrough
immobilized through
through formation of an amide bond
the amino terminated aptamer is immobilized formation of anformation
amide bond of helped
an amide withbond
the
helped
helped with
with the
the carbodiimide
carbodiimide chemistry.
chemistry. In
In detail,
detail, the
the GEC
GEC was
was first
first subjected
subjected to
to electrochemical
electrochemical
carbodiimide chemistry. In detail, the GEC was first subjected to electrochemical pretreatment by
pretreatment
pretreatment by
by 10 cyclic
10scans potential
cyclicbetween
potential1.0scans
scans between 1.0 and
and −1.5 V at scan rate of 0.2 V/s in
in 0.5 M
MH H22SO
SO44
10 cyclic potential andbetween
−1.5 V 1.0 at scan −1.5
rate V ofat0.2
scan
V/sratein of0.50.2
MV/s H2 SO 0.5
4 and 0.1 M
and
and 0.1
0.1 M
M KCl
KCltoin order
order to
inactivate activate
tothe
activate the surface.
theThen,
surface. Then, thethe diazotation reaction took place
place adding 40 µL
KCl in order surface. theThen,
diazotation diazotation
reaction reaction
took place tookadding adding
40 µL of401µL M
of
of 1
1 M
M NaNO
NaNO 2 and 20 mL of 2 mM ABA prepared in 0.5 M HCl. The mixture was left to react for 5 min
2 and 20 mL of 2 mM ABA prepared in 0.5 M HCl. The mixture was left to react for 5 min
NaNO2 and 20 mL of 2 mM ABA prepared in 0.5 M HCl. The mixture was left to react for 5 min at low
at
at low
low temperature
temperature [24]. After
[24].that,
After that, 10 mL of
of the
the mixture and
andof10
100.5mL of 0.5 M
M HCl were
were deposited
temperature [24]. After 10that,
mL of 10the
mLmixture mixture
and 10 mL mLM ofHCl 0.5were HCl
deposited deposited
onto the
onto
onto the
the electrode
electrode surface
surface and
and the
the electrochemical
electrochemical modification
modification was
was performed
performed by
by 10
10 linear
linear sweep
sweep
electrode surface and the electrochemical modification was performed by 10 linear sweep voltammetry
voltammetry
voltammetry scans
scans from
from 0.6
0.6 to
to −0.8
−0.8 V.
V. After
After this
this modification,
modification, the
the electrode
electrode was
was rinsed
rinsed three
three times
times
scans from 0.6 to −0.8 V. After this modification, the electrode was rinsed three times with distilled
with distilled
with distilled water. After
water.a After that, a solution
that,using
a solution using EDC reagents (100 mM EDC and 25 mM NHS in 100 mM
water. After that, solution EDCusing EDC(100
reagents reagents
mM(100EDCmM andEDC 25 mMand 25 mMinNHS
NHS 100 in mM 100MES
mM
MES
MES buffer)
buffer) is
is prepared
prepared in
in order
order to
to activate
activate the
the carboxylic
carboxylic groups.
groups. Then,
Then, 130
130 µL
µL of
of EDC
EDC reagents
reagents are
are
buffer) is prepared in order to activate the carboxylic groups. Then, 130 µL of EDC reagents are added
added
added in
in an
an Eppendorf
Eppendorf jointly
jointly with
with 30
30 µL
µL of
of aptamer
aptamer 1
1 µM.
µM. The
The incubation
incubation is
is performed
performed for
for 12
12 h.
h. The
The
in an Eppendorf jointly with 30 µL of aptamer 1 µM. The incubation is performed for 12 h. The next
next day,
next the the
the electrodes
day,electrodes
electrodes were washed twice with BB
BB buffer during 5 min to to remove unbound Apt.
day, were were
washed washed
twice twice
with BBwith buffer buffer
during during
5 min5tomin remove remove
unboundunbound
Apt. Apt.
Sensors 2018, 18, 354 5 of 11

2.4.3. Blocking Step

After that, a blocking step is performed using polyethylene glycol as blocking agent; the assay
to detect Lys consists of a simple incubation step with the sample and non-specific adsorption of
unwanted species needs to be avoided. For the latter, the electrode surface is blocked by submerging
in 160 µL of 50 mM polyethylene glycol (PEG) solution for 20 min at 25 ◦ C. Next, two 10 min washing
steps using BB solution were followed.

2.4.4. Label Free Detection of Lysozyme

The last step of the procedure consists of the recognition between the aptamer and the protein.
For the detection of the target protein, the Apt modified electrodes were incubated for 1 h with the
desired concentration of Lys and finally the aptasensor was washed twice for 10 min with BB solution.

2.4.5. Regeneration of Aptasensor

To carry out the regeneration of the aptasensor the following protocol was used. The first step is
to dissociate the AptLys-Lys complex. To achieve this objective, the used aptasensor is maintained in
stirring conditions in saline media (NaCl) and increasing the temperature until 42 ◦ C.

2.5. Impedimetric Measurements and Data Processing

All the experiments were performed in PBS buffer containing 0.01 M K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ]
(1:1) mixture as redox marker. The applied potential was +0.17 V (vs. Ag/AgCl reference electrode)
and the range of frequency of 50 KHz–0.05 Hz, an AC amplitude of 10 mV and a sampling rate of
10 points per decade above 66 Hz and 5 points per decade at the lower range. The most common
graphical representation of impedimetric data is the “Nyquist Diagram”, in which the imaginary part
of the impedance (−Zi ), is plotted versus the real part Zr . The equivalent Randles circuit [25] proposed
to fit the experimental data is shown in Figure 3 where R1 is the resistance of the solution, R2 is the
charge transfer resistance associated to the redox reaction, that established between the solution and
the electrode surface, and C is the capacitance. In our case, a constant phase element (CPE) is used
instead of a capacitor. CPE is required to optimize the fit to the experimental data, and this is due to
the nonideal nature of the electrode surface. In this treatment, the diffusion component was discarded,
as it is non-relevant for the purpose of the study. In our case, we are interested in two parameters:
the charge transfer resistance (R2 ) and the chi-square (χ2 ). Both are obtained by ZView software.
The first one evaluates the modification of the electrode surface, while the chi-square evaluates the
goodness of fitting. As in all cases, the calculated values of chi-square for each experiment is <0.2,
much lower than the tabulated value for 60 degrees of freedom (67.505 at 95% confidence level), that
verifies that the adjustments were done correctly. With the aim of comparing the results obtained from
the different electrodes used, a normalization is performed. This normalization is called ∆ratio [6,22],
whose advantage is the improvement of the reproducibility of the measurements. It was calculated
as follows:
∆ratio = ∆s /∆p (1)

∆s = Rct(Apt-Prot) − Rct(electrode-bare) (2)

∆p = Rct(Apt) − Rct(electrode-bare) (3)

In this case, Rct(Apt-Prot) is the electron transfer resistance measured after incubating with the
protein desired (Lys), Rct(Apt) is the electron transfer resistance after immobilization and PEG blocking
and Rct(electrode-bare) is the electron transfer resistance of the bare electrode and buffer. In future
experiments the measurements were expressed as ∆ratio .
Sensors 2018, 18, x FOR PEER REVIEW 6 of 11

Sensors 2018, 18, 354 6 of 11

Sensors 2018, 18, x FOR PEER REVIEW 6 of 11

Figure 3. Randles equivalent circuit proposed to best fit.

In this case, Rct(Apt-Prot) is the electron transfer resistance measured after incubating with the
protein desired (Lys), Rct(Apt) is the electron transfer resistance after immobilization and PEG blocking
and Rct(electrode-bare) is the electron transfer resistance of the bare electrode and buffer. In future
Figure 3. Randles
Randles equivalent
equivalent circuit proposed to best fit.
experiments the measurements were expressed as Δratio.
In thisand
3. Results case, Rct(Apt-Prot) is the electron transfer resistance measured after incubating with the
Discussions
3. Results and Discussions
protein desired (Lys), Rct(Apt) is the electron transfer resistance after immobilization and PEG blocking
3.1. Incubation
and Time
Rct(electrode-bare) is of
theAptamer
electron transfer resistance of the bare electrode and buffer. In future
3.1. Incubation Time of Aptamer
experiments
This section was developedwere
the measurements expressed
in order as Δratio
to optimize . immobilization time of aptamer employed.
the
This section was developed in order to optimize the immobilization time of aptamer employed.
For this, four different incubation times such as 1.0 h, 2.0 h, 5 h and 12 h were evaluated.
For3. this,
Results
fourand Discussions
different incubation times such as 1.0 h, 2.0 h, 5 h and 12 h were evaluated. The
The concentrations of the aptamer and the blocking agent used were 1 µM and 40 mM, respectively.
concentrations of the aptamer and the blocking agent used were 1 µM and 40 mM, respectively. As
As it shows Figure 4, the minimum incubation time of the aptamer that can be used is 2 h, due to
3.1. Incubation
it shows Figure 4,Time the of Aptamer incubation time of the aptamer that can be used is 2 h, due to the
minimum
the ∆ratio begins to increase. Less than 2 h is not a good immobilization time of the aptamer onto
Δratio begins to increase. Less than in 2 horder
is notto aoptimize
good immobilization time time
of theofaptamer onto the
This section
the surface of thewas developed
electrode, as the ∆ ratio values are closethe immobilization
to 1. For that reason aptamer
and employed.
for practical use,
surface of the electrode, as the Δ ratio values are close to 1. For that reason and for practical use, the best
the best option considered was 12 h. In this time, high ∆ratio values were obtained and also with
For this, four different incubation times such as 1.0 h, 2.0 h, 5 h and 12 h were evaluated. The
option consideredofwas
concentrations 12 h. In and
the aptamer this the
time, high Δagent
blocking ratio values were obtained and also with proper
used were 1 µM and 40 mM, respectively. As
proper reproducibility.
reproducibility.
it shows Figure 4, the minimum incubation time of the aptamer that can be used is 2 h, due to the
Δratio begins to increase. Less than 2 h is not a good immobilization time of the aptamer onto the
surface of the electrode, as the Δratio values are close to 1. For that reason and for practical use, the best
option considered was 12 h. In this time, high Δratio values were obtained and also with proper
reproducibility.

Figure 4. 4.
Figure Optimization of the
Optimization incubation
of the timetime
incubation of of
aptamer using
aptamer electrochemical
using grafting
electrochemical as as
grafting
immobilization technique.
immobilization technique.

3.2.3.2.
Optimization
Optimization of Aptamer andand
of Aptamer PEG PEGBlocking Agent
Blocking AgentConcentrations
Concentrations
First, the
Figure concentration of the aptamer and the
First, the concentration of the aptamer and theofblocking
4. Optimization of the incubation blocking
time agentagent
aptamer mustmust
using be optimized
electrochemical
be optimizedbygrafting
building asits
by building
interaction curves.curves.
immobilization
its interaction Totechnique.
perform these experiments
To perform 0.05, 0.5, 0.05,
these experiments 1 and0.5,
2.5 µM
1 andconcentrations of aptamer of
2.5 µM concentrations
were tested. The concentration of the Lys and PEG employed were
aptamer were tested. The concentration of the Lys and PEG employed were 2.5 µM and 40 mM, 2.5 µM and 40 mM, respectively.
In 3.2.
FigureOptimization
respectively.
of Aptamer and PEG
5A, theIninteraction
Figure 5A,curve
Blocking
of AptLys
the interaction
Agent Concentrations
immobilized
curve of AptLys ontoimmobilized
the electrodeonto surface
the is represented.
electrode surface
In is
this plot,
First, itthe
represented. can be this
In observed
concentration
plot, ofitthat
canthe
the values
aptamer of the
and
be observed Δratio
that increase
blocking
the until
agent
values ∆aratio
of mustfixed
be value until
optimized
increase is reached.
by fixedThis
a buildingvalueits
phenomenon
interaction is
is reached. curves.
This produced
phenomenonbecause
To perform it corresponds
isthese experiments
produced becauseto0.05,
ait Langmuir
0.5, 1 andisotherm.
corresponds 2.5
to µM From aisotherm.
concentrations
a Langmuir concentration
of aptamer
From a
above
were 1 µM
concentrationtheThe
tested. saturation
above of the
1 µM the aptamer
concentration onofthe
of the Lys
saturation theelectrode
and PEG
aptamer surface
employed is produced,
were 2.5 µM
on the electrode therefore
and
surface therespectively.
is40produced,
mM, optimum
therefore
concentration
In
theFigure
optimum established
5A, concentration to perform
the interactionestablished the
curve of AptLys calibration curve
immobilized
to perform of Lys
onto the
the calibration was 1
electrode
curve µM. In
of Lyssurface order
was 1 µM. to
In avoid
is represented.
order to
In thisnonspecific
avoid plot, it canadsorption
be observedthethat the valuesofofblocking
concentration Δratio increase until to
agent needs a fixed value is as
be optimized reached.
well. InThis
this
phenomenon is produced
case, the blocking because
agent selected was itPEG
corresponds to a Langmuir
and the concentrations isotherm.
tested were 20,From a 50
30, 40, concentration
and 60 mM.
above 1 µM the saturation of the aptamer on the electrode surface is produced, therefore the optimum
concentration established to perform the calibration curve of Lys was 1 µM. In order to avoid
Sensors 2018, 18, x FOR PEER REVIEW 7 of 11
Sensors 2018, 18, 354 7 of 11
nonspecific adsorption the concentration of blocking agent needs to be optimized as well. In this case,
the blocking agent selected was PEG and the concentrations tested were 20, 30, 40, 50 and 60 mM. As
Asshown
it it shown in Figure
in Figure 5B,5B,
thethe behaviorisissimilar
behavior similartotothe
theprevious
previouscase.
case. The
The values of ∆
values of Δratio increase until
ratio increase until
the concentration tested is 60 mM; this fact shows that PEG followed an adsorption isotherm.
the concentration tested is 60 mM; this fact shows that PEG followed an adsorption isotherm. Hence, Hence,
the optimum
the optimum concentration
concentration of
of PEG
PEG was
was 50 50 mM.
mM.

Figure 5. (A)
Figure 5. (A) Optimization
Optimization of
ofthe
theconcentration
concentrationofofAptLys;
AptLys,(B)(B) Optimization
Optimization of the
of the concentration
concentration of
of the
the blocking agent, PEG. Uncertainly values corresponding to replicated experiments
blocking agent, PEG. Uncertainly values corresponding to replicated experiments (n = 5). (n = 5).

3.3. Analytical Performance of the Aptasensor for Lysozyme Detection

3.3. Analytical Performance of the Aptasensor for Lysozyme Detection
The analytical performance of the aptasensor was evaluated with different concentrations of Lys.
The analytical performance of the aptasensor was evaluated with different concentrations of Lys.
For this, the concentration of Lys employed were 0.25, 0.5, 1.5, 2.5 and 5 µM. The concentrations of
For this, the concentration of Lys employed were 0.25, 0.5, 1.5, 2.5 and 5 µM. The concentrations of
aptamer and PEG employed were 1 µM and 50 mM, respectively. The aptamer strand recognizes the
aptamer and PEG employed were 1 µM and 50 mM, respectively. The aptamer strand recognizes the
protein through a three-dimensional folding following host-guest principles. The results obtained in
protein through a three-dimensional folding following host-guest principles. The results obtained in
this experiment are shown in Figure 6A. In this representation, it can be seen that the charge-transfer
this experiment are shown in Figure 6A. In this representation, it can be seen that the charge-transfer
resistance Rct increased in each step of the procedure. This is due to the effect on the kinetics of the
resistance Rct increased in each step of the procedure. This is due to the effect on the kinetics of the
electron transfer reaction by the redox probe, which is delayed at the interface of the electrode, mainly
electron transfer reaction by the redox probe, which is delayed at the interface of the electrode, mainly
caused by steric hindrance and electrostatic repulsion presented by the complex aptamer-protein
caused by steric hindrance and electrostatic repulsion presented by the complex aptamer-protein
formed [26]. This is an additional advantage of using EIS as the transduction principle: each step in
formed [26]. This is an additional advantage of using EIS as the transduction principle: each step in the
the biosensing protocol can be assessed, thus confirming the procedure is correctly performed. On
biosensing protocol can be assessed, thus confirming the procedure is correctly performed. On the other
the other hand, when the concentration of Lys increases the Rct also increases. To evaluate the
hand, when the concentration of Lys increases the Rct also increases. To evaluate the detection limit
detection limit (LOD), sensitivity and the lineal response range, a calibration curve was built. In this
(LOD), sensitivity and the lineal response range, a calibration curve was built. In this calibration curve
calibration curve (Figure 6B) the analytical signal (Δratio) is plotted versus the concentration of Lys. In
(Figure 6B) the analytical signal (∆ratio ) is plotted versus the concentration of Lys. In a first instance,
a first instance, it is deduced that there is a correct recognition of the protein by the aptamer. This fact
it is deduced that there is a correct recognition of the protein by the aptamer. This fact is observed
is observed when the concentration of Lys increases because, R2, the interfacial electron transfer
when the concentration of Lys increases because, R2 , the interfacial electron transfer resistance between
resistance between the electrode surface and solution increases as well. The linear relationship (R =
the electrode surface and solution increases as well. The linear relationship (R = 0.999) between the
0.999) between the Δratio and the concentration of Lys is according to the equation: Δratio = 0.090 [Lys]
∆ratio and the concentration of Lys is according to the equation: ∆ratio = 0.090 [Lys] + 1.182. All the
+ 1.182. All the measurements were performed with n = 5 replicates. The detection limit, 1.67 µM, was
measurements were performed with n = 5 replicates. The detection limit, 1.67 µM, was estimated as
estimated as 3.3 times the standard deviation (n = 5) of the lowest stock concentration (0.25 µM) and
3.3 times the standard deviation (n = 5) of the lowest stock concentration (0.25 µM) and the sensitivity
the sensitivity (0.090 µM−1) is the slope of the calibration curve, all these in label-free conditions. In
(0.090 µM−1 ) is the slope of the calibration curve, all these in label-free conditions. In addition, it was
addition, it was obtained a calibration curve with a linear range from 1.67 µM to 5 µM of Lys.
obtained a calibration curve with a linear range from 1.67 µM to 5 µM of Lys. However, Figure 6B
However, Figure 6B shows perceptible signal below the LOD, because it was calculated using the
shows perceptible signal below the LOD, because it was calculated using the standard deviation of the
standard deviation of the lowest stock concentration, what increase noticeable the LOD.
lowest stock concentration, what increase noticeable the LOD.
Sensors 2018, 18, x FOR PEER REVIEW 8 of 11
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Sensors 2018,18,
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x FOR PEER REVIEW 88of
of11
11

Figure 6. (A) Nyquist Plot for a biosensing protocol with increasing Lys concentrations: 0.5, 2.5, 4, 7
Figure6.6.(A)
Figure (A)Nyquist
NyquistPlotPlotfor
foraabiosensing
biosensingprotocol
protocolwith
withincreasing
increasingLys Lysconcentrations:
concentrations:0.5,
0.5,2.5,
2.5,4,4,77
and 9 and
µM, 9(B)
µM,
Calibration
(B)
curvecurve
Calibration
vs. Lys
vs.
concentrations:
Lys
0, 0.25,
concentrations: 0,
0.5,0.5,
0.25,
1.5,1.5,
2.5 2.5
andand
5 µM.
5
Uncertainty
µM. Uncertainty
and 9 µM; (B) Calibration curve vs. Lys concentrations: 0, 0.25, 0.5, 1.5, 2.5 and 5 µM. Uncertainty
valuesvalues
corresponding to replicated
corresponding experiments (n = (n
5). All the measurements were performed inin
PBS
values corresponding totoreplicated
replicated experiments
experiments (n ==5).
5).All
Allthe
the measurements
measurements were
were performed
performed PBS
in PBS
solution in the presence of the redox probe.
solution in the presence of the redox probe.
solution in the presence of the redox probe.

3.4. Regeneration of the of

3.4. Regeneration Aptasensor
the Aptasensor
3.4. Regeneration of the Aptasensor
It wasItItpossible
was
to regenerate
waspossible
possible toregenerate
to regeneratethe the
aptasensor
theaptasensor
aptasensor by by
dissociating
bydissociating
dissociating thetheAptLys-Lys
the AptLys-Lys
AptLys-Lys
complex.
complex.This
complex. Thiswas
This was
was
achieved by
achieved stirring
by the
stirring aptasensor
the aptasensor in saline
in media
saline (NaCl)
media and
(NaCl) increasing
and increasing the
achieved by stirring the aptasensor in saline media (NaCl) and increasing the temperature [27]. At this thetemperature
temperature [27]. At
[27]. At
this point,
point, thecomplex
this point,
the complex
the complex is dissociated
is dissociated
is dissociated and the andresistance
the the
and resistance decreased
resistance
decreased todecreased to to
the baseline thethebaseline
value baseline value
valueofofthe
of the correspondent the
correspondent
correspondent AptLys.AptLys.
In the In the
study,study,
five five sensing
sensing cycles
cycles werewere evaluated
evaluated
AptLys. In the study, five sensing cycles were evaluated with a blank measure in between each one. withwith a a blank
blank measure
measure in in
betweenbetween
As iteach
shows each
one. one.
in As As
it shows
Figure it shows
7, thein in Figure
Figure 7, could
aptasensor 7, the aptasensor
the aptasensor
regeneratecould could regenerate
twiceregenerate
at most. This twice
twice
fact at at most.
is most.
observed This
Thiswithfactisis
factthe
observed
observed
first and withfirst
withsecond
the the firstsecond
and and second
regeneration. regeneration.
regeneration.
From the From From
the the
third regeneration, third
third
the regeneration,
regeneration,
detection of Lys the isthe detection
detection
lower, ofofLys
probably Lys isis
due
lower,
lower,to probablyprobably due
due to from
the loss of aptamer to
the lossthe loss of
of aptamer
the electrode aptamerfrom
surface. from
Forthe the electrode
this electrode
reason, thissurface.surface.
regeneration For
For isthis this reason,
not reason,
carried out this
thisin
regeneration
regeneration
the is notisassays,
performed not carried
carried out in
which out
the
were inperformed
the performed
done on each assays,
assays,
cycle which
which
with were
brand were
new done
done onon each
immobilizationeach cycle
cycleofwithwith
the brand
brand
aptamer
new
on immobilization
new immobilization
each theofaptamer
cycle. Toofrecover the aptamer
electrode onuse onfor
each each cycle.
cycle.
future To To recover
recover
experiments, electrode
electrode
the useuse
followed for for
option future
future was experiments,
experiments,
to perform a
the followed
surface polishing
the followed option
option was was
andtonew to perform
immobilization.
perform a surface polishing and new
a surface polishing and new immobilization. immobilization.

Figure 7. Δratio values for five consecutive cycles of regeneration. The concentration of Lys employed
is7.2ΔµM. The deviation is calculated for n = 5 of
replicates.
FigureFigure ratio ∆ratio values
7. values for five
forconsecutive cycles
five consecutive cyclesregeneration. TheThe
of regeneration. concentration ofof
concentration Lys
Lysemployed
employed
is 2 µM.
is 2The
µM.deviation is calculated
The deviation for nfor
is calculated = 5n replicates.
= 5 replicates.
Sensors 2018, 18, x FOR PEER REVIEW 9 of 11

Sensors 2018, 18, 354 9 of 11

3.5. Specificity of the Aptasensor
An3.5.efficient
Specificityelectrochemical
of the Aptasensor aptasensor has to display good specificity. The influences of
interferences can be estimated from the value of the delta ratio of assays in their presence. In order to
An efficient electrochemical aptasensor has to display good specificity. The influences of
verify that the aptasensor developed is specific for the detection of Lys different experiments were
interferences can be estimated from the value of the delta ratio of assays in their presence. In order
performed. The proteins analyzed were Lys, Cyt c and BSA, essentially because they were common
to verify that the aptasensor developed is specific for the detection of Lys different experiments were
proteins and available
performed. in the laboratory.
The proteins analyzed were TheLys,
procedure
Cyt c andwasBSA, carried in thebecause
essentially same manner
they were as common
above but
changing the protein
proteins in theinlast
and available thestep of the The
laboratory. protocol. Thewas
procedure concentration
carried in theofsame
the protein
manner as used
above in these
but
selectivity
changing the protein in the last step of the protocol. The concentration of the protein used in these in
assays was 0.5 µM. The concentrations of aptamer and PEG used were those optimized
the previous sections.
selectivity assaysThe wasresults
0.5 µM.areTheshown in Figure
concentrations of 8. As it isand
aptamer observed
PEG used there,
wereCyt c isoptimized
those not detectedin
by the aptasensor. Alternatively, in the case of BSA, the interfering effect is more significant. It is
the previous sections. The results are shown in Figure 8. As it is observed there, Cyt c is not detected
importantby the aptasensor.that
to highlight Alternatively, in thegiven
the interference case ofbyBSA, the interfering
proteins depends effect
of theisblocking
more significant.
agent used,It isas
important to highlight that the interference given by proteins depends of the blocking
it is originated on the non-specific adsorption of the former. In our protocol, the employed blocking agent used, as it
agent wasis originated on the non-specific
PEG, normally consideredadsorption
a weakof blocking
the former.agent.
In our protocol, the employed
In the literature, blocking
many agent
researchers
was PEG, normally considered a weak blocking agent. In the literature, many researchers reported the
reported the use of stronger blocking agents such as casein or BSA. One possible solution to improve
use of stronger blocking agents such as casein or BSA. One possible solution to improve the assay and
the assay and overcome the BSA interference would be to use a stronger blocking agent such as the
overcome the BSA interference would be to use a stronger blocking agent such as the same BSA or
same BSA or casein,
casein, whichoccupy
which would wouldthe occupy the adsorption
adsorption points. Thispoints. Thiswould
alternative alternative wouldonly
be necessary be necessary
if a very
only if complex
a very complex sample
sample matrix matrix
were used.were used. To
To conclude, conclude,
a negative a negative
control was alsocontrol waswith
evaluated alsothe
evaluated
aim of
with the aim of verifying that the protocol operates properly. This negative control
verifying that the protocol operates properly. This negative control was made with deionized water was made with
deionizedandwater and it demonstrated
it demonstrated that the
that the procedure procedure
provided provided
consistent consistent results.
results.

Figure 8. Specificity of AptLys to Lys, BSA, Cyt c at 0.5 µM. Errors bars are obtained based on five
Figure 8. Specificity of AptLys to Lys, BSA, Cyt c at 0.5 µM. Errors bars are obtained based on five
independent measurements.
independent measurements.

3.6. Application of the Aptasensors in Spiked Wine Samples

3.6. Application of the Aptasensors in Spiked Wine Samples
To check the feasibility of the developed aptasensor, it was employed to evaluate Lys in real
Tosamples
check thesuchfeasibility
as wine. of
Forthe developed
this, aptasensor,
wine aliquots it was
were spiked withemployed
Lys 1 µMtousing
evaluate Lys in real
the developed
samples such as wine.
aptasensors. For this,
The obtained wine aliquots
recovery were spiked
rate is illustrated with
in Table Lys
1. The 1 µM using
developed the developed
aptasensors exhibit
aptasensors. The obtained
good recovery of 77%recovery
indicatingrate
the is illustrated
suitability for in
LysTable 1. The
detection developed
in wine aptasensors
samples. The relativeexhibit
low
good recovery of 77% indicating the suitability for Lys detection in wine samples. The relative
recovery was obtained probably due to some matrix interference. In spite of, the aptasensors still meet low
thewas
recovery criteria and merits
obtained of biosensors
probably due toinsome
termsmatrix
of recovery.
interference. In spite of, the aptasensors still
meet the criteria and merits of biosensors in terms of recovery.

Table 1. Recoveries of Lys in wine samples of aptasensors in real matrix such as wine.

Aptasensor Spike Lys (µM) Δratio Found Lys (µM) Recovery (%)
0 1.19 0 -
Apt Lys
1 1.25 0.77 77
Sensors 2018, 18, 354 10 of 11

Table 1. Recoveries of Lys in wine samples of aptasensors in real matrix such as wine.

Aptasensor Spike Lys (µM) ∆ratio Found Lys (µM) Recovery (%)
0 1.19 0 -
Apt Lys
1 1.25 0.77 77

4. Conclusions
This research reports a simple label-free aptasensor for the detection of lysozyme. The
electrochemical grafting using carbodiimide chemistry has been a good choice for the immobilization
due to its ease of realization, and for the possibility of electrical addressing. With the use of EIS
technique we can evaluate the recognition event, and check the rest of the processes modifying the
electron transfer kinetics of the redox probe at the electrode interface. In addition, the described
aptasensor showed a reasonable range of response (from 1.67 µM to 5 µM) with a lower detection limit
(LOD) of 1.67 µM. The calculated LOD is lower than the maximum amount allowed to be added in wine
as prescribed by the International Organization of Vine and Wine (OIV), allowing the determination in
this field. The interference produced by BSA showed distorted signal that probably may be solved
using stronger blocking agent. Unfortunately, thermal wet regeneration of the biosensor was not
effective for more than twice, impeding its repeated use. Finally, the aptasensor developed in this
research could be applied for the detection of lysozyme in a complex matrix such as wine, providing
satisfactory recovery yields of about 77%, showing the great potential of the proposed methodology
for detecting Lys in other food matrices.

Acknowledgments: Financial support for this work was provided by Spanish Ministry of Science and Innovation
through projects CTQ2013-41577-P and CTQ2016-80170-P and by program ICREA Academia from Generalitat
de Catalunya. Dionisia Ortiz-Aguayo thanks the support of Universitat Autònoma de Barcelona for the
PIF fellowship.
Author Contributions: D.O.-A. performed the experiments, the data processing, fittings and wrote the paper.
M.d.V. planned, get the founding and supervised the experiments.
Conflicts of Interest: The authors declare no conflict of interest.

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