Sensors and Actuators B 238 (2017) 651659

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Development of a new electrochemical imprinted sensor based on

poly-pyrrole, solgel and multiwall carbon nanotubes for
determination of tramadol
Behjat Deiminiat, Gholam Hossein Rounaghi , Mohammad Hossein Arbab-Zavar
Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

a r t i c l e i n f o a b s t r a c t

Article history: A novel electrochemical imprinted sensor was constructed for sensitive and selective determination of
Received 10 May 2016 tramadol (TRA) by combination of a functionalized multiwall carbon nanotubes (f-MWCNTs) layer and a
Received in revised form 29 June 2016 thin molecularly imprinted lm. The imprinted sensor was fabricated on the surface of a glassy carbon
Accepted 21 July 2016
electrode (GCE) through layer by layer modication of f-MWCNTs and a thin lm of molecularly imprinted
Available online 22 July 2016
polymer (MIP) composed of polypyrrole (PPy), solgel and tramadol by electrochemical deposition. Mul-
tiwall carbon nanotubes (MWCNTs) were introduced for the enhancement of electronic transmission and
Keywords:
sensitivity. Electrochemical methods including cyclic voltammetry (CV) and electrochemical impedance
Electrochemical imprinted sensor
Tramadol
spectroscopy (EIS) were used to characterize the processes of electropolymerization, template removal
Functionalized multiwall carbon nanotubes and rebinding in the presence of [Fe(CN)6 ]3 /[Fe(CN)6 ]4 which was used as an electrochemical active
Molecularly imprinted polymer probe. The effect of several parameters inuencing the performance of the imprinted sensor, were inves-
Solgel tigated and optimized. Under the optimized experimental conditions, the calibration curve of the MIP
Polypyrrole electrode had two linear concentration ranges from 0.2 to 2.0 nM and 2.020.0 nM, with a limit of detec-
tion (LOD) of 0.03 nM. The effects of interfering substances on the determination of tramadol were also
investigated and it was found that the proposed sensor is highly selective to tramadol. Also, the repro-
ducibility, repeatability, stability and the response time of the imprinted sensor were all found to be
satisfactory. Finally, the constructed electrode was successfully employed for determination of tramadol
in real samples.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Tramadol, (1RS, 2RS)-2-[(dimethylamine)-methyl]-1-(3-
methoxy-phenyl)-cyclohexanol hydrochloride) which is a
synthetic and centrally acting analgesic with a low afnity
for -opioid receptors is used for the treatment of moderate to
severe pain and it appears to have monoaminergic activity that
inhibits noradrenaline and serotonin reuptake [1]. Furthermore,
tramadol is used as an anti-addictive by addicts [2,3] and a severe
dependence to tramadol in addicts has been reported due to its
effect on opioid receptors in central nervous system [4,5]. There
is also a risk of death and life-threatening side effects such as
nausea, vomiting, central nervous system depression, tachycardia,
seizure and cardiopulmonary arrest in people who have frequently
received tramadol in overdose especially in young adults with the
Corresponding author.
E-mail addresses: ghrounaghi@yahoo.com, ronaghi@um.ac.ir (G.H. Rounaghi).
history of drug abuse and mental disorders. These serious side
effects may occur because tramadol can be accumulated in the
body achieving critical levels of toxicity [6]. Therefore, develop-
ment of a rapid, simple, sensitive and selective analytical method
for determination of tramadol is necessary from the industrial,
environmental and health viewpoints.
Various analytical methods have been reported for the deter-
mination of TRA including HPLC [7,8], LCMS [9], LCMSMS
[10,11], GCMS [12,13], capillary electrophoresis with electro-
chemiluminescence detection [14], thin layer chromatography
with densitometric detection [15], ow injection analysis sys-
tem with square wave voltammetric (SWV) and amperometric
detection methods [16], spectrophotometry [17,18] and electro-
chemical methods [19,20]. Despite these techniques have high
sensitivity and accuracy, they are usually expensive, time consum-
ing and highly skilled techniques that require trained operators
and complicated procedures. Electrochemical methods, especially
electrochemical molecularly imprinted polymer (MIP) based sen-
sors, are preferred for pharmaceutical determination due to their

http://dx.doi.org/10.1016/j.snb.2016.07.110
0925-4005/ 2016 Elsevier B.V. All rights reserved.
652 B. Deiminiat et al. / Sensors and Actuators B 238 (2017) 651659
simplicity, high sensitivity and selectivity, good stability, low cost
and fast response time.
Molecular imprinting, rst proposed by Wulff et al. [21] and
Vlatakis et al. [22], is a powerful technique for synthesizing
molecularly imprinted polymers (MIPs) with specic molecu-
lar recognition capacity [23,24]. The imprinting process involves
copolymerizing a functional monomer with a cross-linker in the
presence of a template molecule (print molecule). When the tem-
plate is removed, a molecular recognition site with appropriate size,
shape and chemical functionality is formed in the polymer matrix
to rebind the template. So far, MIPs have been extensively used in
chromatography [25], capillary electrochromatograp- hy [2628]
solid-phase extraction [29] and construction of chemical sensors
[3032]. Although the most of MIPs are prepared based on the bulk
polymerization of organic acrylate or acrylic type polymer, a main
drawback of this type imprinting is that the full potential of the MIP
as a specic recognition element is not exploited due to the poor
site accessibility for template molecules, slow interaction kinetics,
low afnity and heterogeneous nature of the binding sites [33]. To
overcome these problems, several methods for the preparation of
imprinting lms have been developed which the solgel imprinting
technique, is one of such progressions.
Solgel technology provides a simple way to produce a three
dimensional silicate network with high porosity, excellent physical
rigidity, chemical inertness and thermal stability [34]. Therefore,
the combination of the molecular imprinting with solgel tech-
nology is a promising way to construct electrochemical sensing
devices, but it suffers from some limitations including low electrical
conductivity, slow diffusion kinetics, long response time and low
sensitivity [3537]. Therefore, diffusion of the analytes across the
solgel MIP lm needs to be accelerated to obtain a quick response.
Furthermore, a proper design for the conduction of electrons from
the binding sites to the electrode surface is required to enhance
the sensitivity of this sensing system [38]. In order to improve the
electrical or electrochemical properties of the solgel MIP based
systems, the most effective way is the use of conducting polymers
and nanomaterials.
Conducting polymers, such as polypyrrole, polythiophene,
polyaniline and their derivatives have been used in many elds
of electrochemistry due to their good electrochemical characteris-
tics and their polymeric lm which can be easily prepared under
mild conditions using electrochemical methods. Electrosynthe-
sized polymers establish a powerful platform for the development
of biosensors and they permit easy localization of biomolecules on
the surface of the electrodes as well as the enhancement of the elec-
tron transfer between the active site of the enzymes and electrode.
Among the various conducting polymers, polypyrrole is one of the
most frequently used in design of chemical sensors and biosensors
due to its good stability and biocompatibility, signicant electri-
cal conductivity and facile polymerization by electrochemical or
chemical methods [39,40].
Carbon nanotubes (CNTs) have been considered as an important
class of nanomaterials with excellent mechanical, electrical and
chemical properties since they have been discovered [41]. These
unique properties make them extremely attractive for fabricating
electrochemical sensors because of the ability of CNTs to facilitate
the electron transfer reactions [42,43].
In the present work, we attempt to introduce a novel plat-
form to improve the electron transfer from solgel MIP layer to
the electrode surface via incorporation of conducting polymers
into the solgel networks and also using f-MWCNTs as a modied
layer before the formation of the imprinted lm. The combina-
tion of f-MWCNTs, conducting polymers and solgel technology,
led to enhance the selectivity and sensitivity of the electrochem-
ical sensor. Electrochemical impedance spectroscopy (EIS), cyclic
voltammetry (CV) and square wave voltammetry (SWV) were used
to investigate the electrochemical behavior of the constructed sen-
sor. The proposed sensor, exhibited a high selectivity towards the
tramadol molecules and it was successfully applied for tramadol
detection in real samples.
2. Experimental
2.1. Chemicals
Tetraethoxysilane (TEOS), phenyltriethoxysilane (PTEOS), tri-
uoroaceticacid (TFA) were purchased from Merck (Darmstadt,
Germany). Pyrrole (Merck) was puried by distillation and stored
in a dark bottle in a refrigerator before use. Lithium perchlorate
was purchased from Acros (New Jersey, USA). MWCNTs (>95%, OD
515 nm) were obtained from US research nanomaterials, Houston,
USA. Tramadol was supplied from Sajad Daru Shargh (Mashhad,
Iran). All of the solvents and the other salts used in this study,
were of analytical grade and were obtained from Merck (Darm-
stadt, Germany). All solutions were prepared with double-distilled
water. High purity nitrogen (99.999%, Sabalan Gas Company, Iran)
was used for deaeration of the solutions.
2.2. Instrumentation
The electrochemical measurements were carried out using an
Autolab PGSTAT 101 (Metrohm Autolab, Utrecht, The Netherlands)
managed by NOVA software. A conventional three-electrode cell
was used at room temperature. A glassy carbon electrode (with a
diameter of 2 mm, obtained from Azar Electrode Co., Urmia, Iran),
bare or modied according to the case, was used as the working
electrode. An Ag/AgCl electrode and a platinum wire were used as
the reference and auxiliary electrodes, respectively. Gill AC poten-
tioastate (ACM instruments) was applied for the electrochemical
impedance spectroscopic measurements. The pH of the solutions
was measured with Metrohm pH/mV-meter (model 827 pH Lab,
Swissmade). Morphological scanning electron microscope (SEM)
images were obtained using a LEO 1450VP SEM (Germany) at 20 kV.
The UVvis spectra were recorded by an Agilent 8453 UV/Vis spec-
trophotometer.
2.3. Preparation of polypyrrole@sol-gel MIP/f-MWCNTs/GC
electrode
Prior to use, the glassy carbon electrode (GCE) surface was care-
fully polished with a slurry of 0.5 m alumina. The polished surface
was then rinsed with double-distilled water and sonicated in a
1:1 mixture of waterethanol for 5 min. Then, a layer of MWCNTs
was coated on the surface of glassy carbon electrode to enhance
the accessible sites and electrical conductivity. At rst, carboxylic
functional group (COOH) was decorated on the surface of MWCNTs
to improve the dispersion and compatibility of MWCNTs. For this
purpose, 0.3 g of raw MWCNTs and 50 mL nitric acid were added
into a round-bottomed glass ask, and the MWCNTs mixture was
sonicated for 30 min in an ultrasonic bath. Next, it was reuxed
at 120 C and vigorous stirring rate for 24 h. After cooling to room
temperature, the mixture was ltered on a 0.22 m polycarbonate
membrane and washed with distilled water for several times until
the pH of the ltrate was neutral. The solid product was dried under
vacuum at 60 C for 12 h, to obtain carboxylic acid-functionalized
MWCNTs (f-MWCNTs). The f-MWCNTs/GCE was fabricated as is
described below: 5.0 mg of f-MWCNTs was dispersed in 2.5 mL
dimethylformamide (DMF) by ultrasonic agitation for 10 min to
give a homogeneous suspension, then, the pretreated GCE was
dipped into the resulting suspension for 45 min to fabricate f-
MWCNTs-layer (f-MWCNTs/GCE).
 B. Deiminiat et al. / Sensors and Actuators B 238 (2017) 651659
Fig. 1. The simplied sketch for the fabrication process of polypyrrole@sol-gel MIP/f-MWCNT/GCE.
The imprinted lm was then deposited on the assembled f-
MWCNTs layer by the solgel technology. The sol solution was
prepared according to the method developed by Rezaei and et al.
[44]. 75 L of PTEOS, 75 L of TEOS, 700 L of water, 1100 L of
ethanol and 10 L of TFA were added to tramadol (1.0 mM) in a vial
and the mixture was stirred for 2 h to obtain a homogeneous sol
at room temperature. Then, 50 L of pyrrole solution and 5.0 mg
of lithium perchlorate were added to the mixture. The resulting
solution was sonicated for 10 min. Then the cyclic voltammetry
between 0.8 V and +0.8 V (versus Ag/AgCl) for 10 cycles with a
scan rate of 50 mV s1 was applied in imprinted solgel solution
to fabricate the polypyrrole@sol-gel MIP/f-MWCNTs/GC electrode.
After that, the prepared polypyrrole@sol-gel MIP/f-MWCNTs/GCE
was dried for 2 h at room temperature. The most important step
in preparation of MIP, is the removal of the template molecules
from polymeric network. The doped tramadol was removed from
the imprinted lm by immersion the electrode in a mixture of
methanol and acetic acid (9:1, v/v) for 20 min under gentle stirring.
The complete removal of template is checked by UVvis analysis
at tramadol absorption peak, max = 271 nm [45]. For comparison,
a non-imprinted polymer (NIP) electrode was prepared under the
same procedure but in the absence of the TRA.
2.4. Electroanalytical measurements
Various electrochemical techniques including cyclic voltamme-
try (CV), electrochemical impedance spectroscopy (EIS) and square
wave voltammetry (SWV) were used to characterize the electro-
chemical properties of the imprinted sensor. In each measurement,
the sensor was rst incubated into a tramadol solution at pH 7.0
for 300 s. Then, electrochemical measurements were performed in
a phosphate buffer solution (pH 7.0) containing 0.1 mol L1 KCl and
5.0 mmol L1 K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] redox pair as a probe.
The cyclic voltammograms were recorded over a potential range
from 0.10 to 0.50 V with a scan rate of 50 mV s1 . EIS was per-
formed at an applied potential of 0.20 V, amplitude of 10 mV and
in a frequency range of 0.130000 Hz. The square wave voltam-
mograms (SWVs) were obtained under step potential of 1 mV,
amplitude of 20 mV and frequency of 15 Hz. All measurements were
carried out at room temperature.
2.5. Sample preparation
The pharmaceutical sample (100 mg tramadol tablet) was pur-
chased from a local drug store (Sajad Darou Shargh Pharmaceutical
653
Co., Mashhad, Iran). One tablet was accurately weighed, crushed
into powder and dissolved in 100 mL of distilled water with shaking
for 10 min in an ultrasonic bath. Then the solution was ltered and
the prepared solution was diluted with phosphate buffer solution
(pH 7.0) to obtain the required concentration for assay.
Drug-free human urine sample was obtained from a healthy vol-
unteer and stored at 4 C. Prior to measurement, the sample was
centrifuged for 10 min at 3500 rpm in order to remove the pre-
cipitated materials. The supernatant was diluted with phosphate
buffer solution (pH 7.0) and then spiked with different amounts of
tramadol. Standard addition method was applied for determination
of tramadol in the samples.
3. Results and discussion
3.1. Preparation and characterization of imprinted sensor
One of the essential requirements for successful imprinting of
solgel lm, is the presence of functional monomers in the solgel
matrix. The role of these monomers is to create specic binding
cavities inside the imprinted lm, which are complementary to the
template molecule in terms of the size, shape and functional groups
orientation, similar to the active sites of an enzyme. Fig. 1 shows the
stepwise construction process of imprinted electrode. F-MWCNTs
were used at the surface of the GCE to increase the surface area for
the formation of the MIP lm. Moreover, f-MWCNTs can improve
the electron conduction and sensitivity. Then, a thin uniform
imprinted lm composed of TEOS, PTEOS and pyrrole with tra-
madol as the template was coated on the f-MWCNTs/GCE surface.
TEOS was used as cross-linker to form a polymeric network around
the template through hydrogen bonds and ionic interactions, and
PTEOS was utilized as functional monomer for interaction with
the aromatic ring. Also, pyrrole was incorporated into the sol solu-
tion as an additional functional monomer to increase the stability
of the resultant MIP. The NH group of the pyrrole units are able
to form hydrogen bonds with the hydroxyl group of the tramadol
molecules. Tetraalkylorthosilicates monomers are hydrolyzed by
either acid or base catalyst. Acid catalyst is better to be used for the
design of sensing elements. Meanwhile the hydrolysis operates, the
condensation step of the hydrolyzed monomers starts. Completely
hydrolyzed monomers usually condense by elimination of water
while condensation between incompletely hydrolyzed monomers
occurs by the elimination of alcohol molecules. In order to obtain
the best imprinted solgel material with desirable physicochemical
properties, water content has to be adjusted before the synthesis of
654 B. Deiminiat et al. / Sensors and Actuators B 238 (2017) 651659

120 50
b
1 40
100 d
30 f
80 e
20
10
60 10 a
I(A)

I(A)
c
40 0
-10
20
-20
0
-30
-20 -40
-1 -0.5 0 0.5 1 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6
E(V) E(V)

Fig. 2. Cyclic voltammogram curves for electropolymerization of the imprinted
solgel on the GCE surface. Conditions: Scan rate 50 mV s1 , number of scans10,
potential range 0.8 to 0.8 V, tramadol concentration 1.0 mM.
Fig. 4. Cyclic voltammograms (CVs) of (a) bare GCE, (b) f-MWCNT/GCE, (c)
MIP/f-MWCNT/GCE, (d) MIP/f-MWCNT/GCE after removal of tramadol, (e) MIP/f-
MWCNT/GCE after 300 s incubating in 3.0 nM tramadol solution and (f) MIP/GCE
after removal of tramadol. Conditions: Potential scan range 0.1 V to +0.5 V, Scan
rate 50 mVs1 .

the sol solution. The ratio of water to alkyl orthosilicates monomers

was determined experimentally to be 4.5:1 (water:silane). This
ratio was found to be sufcient for complete hydrolysis of all
monomers before the start of condensation step which is necessary
to increase the incorporation of template molecules into the solgel
material before the growth of the polymeric networks around them
[46].
Cyclic voltammetric technique was employed to deposit a
uniform molecularly imprinted solgel layer on the surface of f-
MWCNTs/GC electrode. The cyclic voltammogram curves of the
polypyrrole@sol-gel MIP/f-MWCNTs/GC electrode at a scan rate of
50 mV s1 between 0.8 V and +0.8 V are shown in Fig. 2. The results
show that the response current decreases with increasing the cycle
number from the 1st to the 10th, indicating that the surface of the
electrode is covered continuously by a dense insulation layer of
molecularly imprinted polymer. In addition, no remarkable differ-
ences were observed in the cyclic voltammograms in the presence
and absence of tramadol, which reveals that tramadol does not have
any electrochemical behavior under the applied potential range
for electrodepostion, and the structure of the template was not
changed during the imprinting process. Scanning electron micro-
scope (SEM) was used to characterize the surface morphologies of
the modied electrodes, and the results are shown in Fig. 3. As is evi-
dent in Fig. 3B, a f-MWCNTs layer has been attached to the surface
of the glassy carbon electrode. Fig. 3C shows a dense and uniform
morphology for the polypyrrole@sol-gel MIP/f-MWCNT lm.
3.2. Electrochemical characterization of the polypyrrole@sol-gel
MIP/f-MWCNTs/GCE
CV and EIS were used to investigate the different constructed
electrodes to explain the effect of MWCNTs and MIP. Fig. 4 shows
the cyclic voltammograms of the bare GCE, f-MWCNTs modied

Fig. 3. Scanning electron microscope (SEM) images of (A) bare GCE, (B) f-MWCNT/GCE and (C) MIP/f-MWCNT/GCE.
 B. Deiminiat et al. / Sensors and Actuators B 238 (2017) 651659
4.5
4 700
3.5
3 500
Absorbance
2.5
a 10
2 300
1.5
1 100
0.5 b 0
0
-0.5
0 100 200 300 400 500
Wavelength (nm)
Fig. 5. The UVvis absorption spectra of: (a) MIPs lm with TRA, (b) MIPs lm
without the template.
GCE and polypyrrole@sol-gel MIP/f-MWCNTs/GCE. Compared to
the bare GCE (curve 4a), the peak current of the GCE which was
modied with f-MWCNTs increases and the redox peak separation
(Ep ) decreases (curve 4b). However, when the polypyrrole@sol-
gel MIP/f-MWCNTs/GC electrode with tramadol was applied as the
working electrode, the pair of the redox peaks declined notice-
ably in curve 4c. Since the polymer lm is coated on the surface
of the electrode, the molecules of [Fe(CN)6 ]3/4 cannot penetrate
through the layer of polymer and they cannot arrive at the elec-
trode surface for redox reaction and, therefore, the electron transfer
is blocked. After the template is removed from the MIP lm, the
current response of the sensor increases (curve 4d). It seems that
when the template is removed from the surface of the electrode,
the vacant recognition sites or binding cavities which are formed,
make the mass transfer and electronic transmission possible, and,
therefore, the [Fe(CN)6 ]3/4 redox couple, can pass through the
cavities due to their small size and reach the electrode surface. After
the polypyrrole@sol-gel MIP/f-MWCNT/GCE is incubated in 3.0 nM
tramadol solution for 300 s, the current response of the electrode
decreases (curve e). This result shows that some of the cavities
may be recombined with the tramadol molecules which results
in a less chance for the electron transfer of the redox probe on
the electrode surface. In addition, after the GCE was modied with
polypyrrole@sol-gel composite lm (without f-MWCNTs), the peak
current response was decreased (curve 4f). To resolve this problem,
the f-MWCNTs layer was introduced at the surface of the treated
GCE to increase the rate of electron transfer from solgel MIP layer
to the electrode surface.
The UVvis spectral analysis was carried out to illustrate the
effect of template removal method. Fig. 5a shows the UV- Vis spec-
trum of MIP lm which contains tramadol molecules. There is a
distinctive peak of the phenolic hydroxyl groups at 271 nm, which
after the removal of the template disappears.
Electrochemical impedance spectroscopy (EIS) is also an effec-
tive technique for probing the features of the modied electrodes.
The impedance spectra include a semicircle portion at higher fre-
quencies and a linear portion at lower frequencies. The semicircle
diameter corresponds to the electron-transfer resistance (Ret ), and
the linear part corresponds to the diffusion process. The stepwise
modication process of the sensor was characterized by EIS. Fig. 6
shows the Nyquist diagrams of the modied electrode at differ-
ent modication stages. As can be seen from this Figure, the Ret
is lower for f-MWCNTs/GCE than that for the bare GCE. The bare
GCE exhibits a semicircle part at high frequencies (Ret = 1323.4 )
and a linear part at low frequencies. After modication of GCE
with f-MWCNTs (curve 6b), the Ret decreases to 2.8427 , which is
an evidence for a faster electron transfer of [Fe(CN)6 ]3/4 on the
655
800 c
a 50
600 40
Z" (ohm)
30 b d f
Z" (ohm)
400 20
0
200 0 50 100
e
Z' (ohm)
0 500 1000 1500 2000 2500 3000 3500
Z' (ohm)
Fig. 6. Electrochemical impedance spectroscopy (EIS) of (a) bare GCE, (b) f-
MWCNT/GCE, (c) MIP/f-MWCNT/GCE, (d) MIP/f-MWCNT/GCE after removal of
tramadol, (e) MIP/f-MWCNT/GCE after 300 s incubating in 3.0 nM tramadol solution
and (f) MIP/GCE after removal of tramadol. Conditions: Potential 0.20 V, frequency
range of 0.130000 Hz and amplitude of 10 mV.
electrode surface. Subsequently, when the imprinted lm is elec-
trodeposited on to the modied electrode surface, the EIS shows an
increase in diameter (curve 6c), which indicates that the compact
imprinted lm acts as an additional barrier and the electron transfer
of [Fe(CN)6 ]3/4 is blocked. After removing the template from the
electrode surface, a small electron transfer resistance (Ret = 7.9 ) is
observed, because the cavities which are formed on the MIP lm can
facilitate the penetration of the probe through the imprinted layer
(curve 6d). Also, after incubating the polypyrrole@sol-gel MIP/f-
MWCNT GCE in 3.0 nM tramadol solution for 300 s, the electron
transfer resistance increases to Ret = 268.08 (curve 6e), which may
be due to the rebinding of the template into the imprinted cavities.
3.3. Optimization of experimental parameters
In order to fabricate an efcient imprinted sensor, the effect
of different inuencing factors such as scan cycles of electropoly-
merization process, applied potential interval, amount of the
monomers, template concentration, pH and the incubation time
was investigated. The change of [Fe(CN)6 ]3/4 peak current (ip )
was calculated by subtracting the current which resulted in the
presence of TRA from the current recorded in the absence of TRA
and it was used to optimize the experimental conditions.
The number of electropolymerization cycles and potential inter-
val are two important factors in the preparation of molecularly
imprinted polymer lm. The number of scan cycles during the
electropolymerization process can inuence the thickness of the
polymeric lm [47]. The thickness of the lm increases with
increasing the scan cycles of electropolymerization which affects
the sensitivity and linearity response of the electrochemical sensor.
The square wave voltammograms of polypyrrole@sol-gel MIP/f-
MWCNT/GCE at different scan cycles number were investigated.
As is evident in Fig. 7A, the current response increases with the
number of the scan cycles. However, more cycles lead to the forma-
tion of a thick MIP lm, and the template molecules cannot access
to the active site which located at the central area of the poly-
mer [48]. According to these results, the 10 cycles were used as
optimum number for the fabrication of the designed electrode. In
the electropolymerization step, different potential intervals were
examined and the best response was obtained when the potential
changed from 0.80 to +0.80 V.
The experimental results show that the composition and also
the thickness of the coating layer at the surface of the constructed
electrochemical sensor have important effect on the nal sensor
performance. The performance of the polypyrrole@sol-gel MIP/f-
MWCNT/GCE was examined by changing the amount of TEOS and
PTEOS in the range of 100300 L and the pyrrole concentration
656 B. Deiminiat et al. / Sensors and Actuators B 238 (2017) 651659

60
A
50

40
I(A)

30

20

10

0
0 2 4 6 8 10 12 14 16
Number of cycles
70
B
60

50

40
I(A)

Fig. 8. Selectivity of the imprinted and non-imprinted sensor for tramadol and
30 interferences.

20

10 The relationship between the current response and the rebind-

ing time was also studied. After removing the template from the
0
0 100 200 300 400 500 600 electrode surface, the imprinted electrode was incubated in a stir-
Time (s) ring 3.0 nM tramadol solution containing phosphate buffer solution
80
(pH 7.0) for various incubation times. As is shown in Fig. 7B, the
response current (i) increases with increasing the incubation time
C
70 up to 300 s and then it remains nearly constant. The results obtained
in this study, show that more tramadol molecules are accumu-
60 lated on the polypyrrole@sol-gel MIP/f-MWCNT/GC electrode with
increment of the accumulation time up to 300 s, but it seems that
I(A)

50 beyond this time, the accumulation of tramadol tends to be sat-

40
30
20
3 4 5 6 7 8
PH
Fig. 7. Optimization of factors affecting the performance of the MIP electrode, (A)
effect of the number cycles, (B) incubation time and (C) pH on the response current.
in the range of 5100 mM. The optimum amount was found to be
150 L of silane monomers (75 L of TEOS and 75 L of PTEOS)
and 25 mM pyrrole monomer that were chosen to obtain the best
performance. Below the optimum value of functional monomers
(TEOS, PTEOS, and pyrrole) the current decreases, which may be
due to lacking of enough imprinted sites and it seems that the
TRA molecules cannot be captured during the electropolymerisa-
tion process. On the other hand, when the imprinted polymer lm
is too thick due to high concentration of monomers, the tramadol
molecules which are located at the central area of the polymer
cannot be completely removed from the polymer network [47].
The quantity and quality of the binding cavities inside the
imprinted polymer is a direct function of template concentration.
Therefore, the amount of template in the electro- polymerization
step was studied and optimized. It was observed that the number of
recognition sites in the MIP lm decreases as the concentration of
the template molecules decreases. However, the excess amounts
of the template molecules can prohibit the formation of the MIP
lm because of increasing agglomerate and no bonded molecules.
As a result, the optimum template concentration was found to be
1.0 mM.
urated. Therefore, an accumulation time of 300 s was chosen for
further studies. The short response time of the electrochemical sen-
sor results from the porous structure of MIP lm using the solgel
technology.
It was found that the pH of solution is effective on tramadol
9 10 rebinding at the surface of the modied electrode. Therefore, the
pH of the solutions needs to be optimized in the incubation step in
order to facilitate the interaction of tramadol with the recognition
cavities. For this purpose, a series of 3.0 nM tramadol solution in the
pH range of 4.09.0 was tested with an incubation time of 300 s. As
is shown in Fig. 7C, the maximum current response is obtained at pH
7.0, which is selected as an optimum pH in the subsequent studies.
3.4. Selectivity of the constructed molecularly imprinted sensor
To investigate the selectivity of the prepared electrochemical
sensor, some of the substances commonly found with TRA in the
biological uids and pharmaceutical samples and often interfere
in the determination of TRA through conventional methods, such
as ascorbic acid, uric acid and citric acid were chosen as inter-
fering species with 10-fold excess of aforementioned substances.
As is evident in Fig. 8, the effect of the interfering species on
the electrochemical response to tramadol is negligible. The cur-
rent response of the MIP electrode for tramadol is signicantly
higher than that of the other interfering species present in solu-
tion. This can be explained by the size, conformation and also the
functional groups of the cavities that match tramadol molecules
in the imprinting lm. The selectivity can be also investigated by
calculating the imprinting factor (IF), which is dened as the ratio
of the iMIP to the iNIP . As shown in Fig. 8, the IF for tramadol
with polypyrrole@sol-gel MIP/f-MWCNT/GC sensor is 9.3. But in
the case of interference species it is in the range of 1.12.0 which
 B. Deiminiat et al. / Sensors and Actuators B 238 (2017) 651659 657

100 Table 2
Results of tramadol determination in real samples (n = 3).
90
Sample Added (nM) Detected (nM) Recovery RSD
80
Tablet 0.51 3.9
70
120 0.3 0.80 97 2.5
y = 1.4613x + 58.981
60 100 a 0.5 1.04 106 2.9
R = 0.9984
I(A)

80 0.7 1.23 103 4.1

50

I(A)
60
40 y = 24.759x + 12.713 40
R = 0.998 20
30
0 0.5
20 0 0.2 0.4
10 E (V)
0
0 5 10 15 20
Concentration (nM)
Fig. 9. Calibration curve of MIP sensor in different concentrations of tramadol
solutions. Error bars represent the standard deviations for three replicate mea-
surements. Inset is the Square wave voltammograms of polypyrrole@sol-gel
MIP/f-MWCNT/GCE before (a) and after incubation in 0.2 (b), 0.3 (c), 0.5 (d), 0.9 (e),
1.0 (f), 2.0 (g), 5.0 (h), 10.0 (i), 15.0 (j) and 20.0 (k) nM of tramadol solutions. Condi-
tions: Potential scan range 0.1 V to +0.50 V, Step potential 1 mV, Frequency15 Hz,
Amplitude, 20 mV in phosphate buffer pH 7.0 containing 0.1 M KCl and 1.0 mM
[Fe(CN)6 ]3/4 .
demonstrates a good selectivity of the constructed electrode for
tramadol in solutions.
3.5. Analytical performance of MIP electrode
The performance of the imprinted sensor for determination of
tramadol was investigated by square wave voltammetry technique
under optimized experimental conditions. After template removal
and background response measurements, MIP electrode was incu-
bated in different concentrations of TRA solution for 300 s with
stirring. The inset of Fig. 9 shows the decrease of the [Fe(CN)6 ]3/4
peak current with increasing the TRA concentration in solution.
Upon increasing the concentration of TRA, more imprinted sites
are rebound with TRA molecules at the surface of the sensor. As is
shown in Fig. 9, two linear ranges from 0.2 to 2.0 and 2.020.0 nM
for concentration of TRA are observed with the regression equation
of i = 24.759C + 12.713 (R2 = 0.9980) and i = 1.4613C + 58.981
(R2 = 0.9984), respectively, where C is the tramadol concentration
(nM) and i is the current difference (A). The limit of detection
(LOD) was calculated to be 0.03 nM based on 3Sb /S, where S and
Sb represent the slope of the calibration curve and the standard
deviation of the blank (n = 5), respectively.
The possible reason for the two linear concentration ranges in
calibration curve of TRA, is due to the existence of different kinds of
binding sites with different afnities. When the sensor is immersed
in TRA solution, the TRA molecules prefer to combine with the
high afnity binding sites. Upon increasing the TRA concentration,
the high afnity cavities are gradually occupied, and further bind-
ing takes place between the TRA molecules and the low afnity
sites. It seems that, it is difcult for TRA molecules to overcome
1.0 1.47 96 2.7
k
Urine Not detected
0.3 0.29 97 3.4
0.52 104 3.8
0.6 0.7 0.74 106 2.7
1.0 0.98 98 2.0
25
the transferring resistance and reach into the internal cavities. As a
result, it seems that the constructed sensor, demonstrates different
responses with increasing the TRA concentration in solution [49].
In order to evaluate the repeatability of the constructed MIP
sensor, 10 repeated measurements of 3.0 nM tramadol solution
were performed by the same electrode. After each experiment,
the sensor was washed with a mixture of methanol-acetic acid
(9/1 v/v) to remove the tramadol molecules from the cavities. The
relative standard deviation (RSD) was found to be 3.1% (n = 10).
Also, the reproducibility of the imprinted sensor was estimated
by measurement of 3.0 nM tramadol solution using ve fresh
electrodes prepared independently under identical experimental
conditions. The calculated RSD was about 4% (n = 5). According to
these experimental results, the reproducibility and repeatability
of the proposed sensor are acceptable. To ensure the stability, the
polypyrrole@sol-gel MIP/f-MWCNTs modied GC electrode was
stored at 4 C for two weeks. This electrode was tested every day for
two weeks and it was found that the response current decreases by
about 10% which demonstrates that the developed imprinted sen-
sor possesses a fair stability. The good stability of the MIP sensor
may be due to the formation of a stiff layer of imprinted polymer
on the surface of the modied electrode.
The results obtained in this work and some of the previous pro-
cedures for tramadol determination are summarized in Table 1.
The data in this Table, show that the present sensor has a lower
detection limit compared to the other electrochemical sensors,
indicating that the sensitivity of the electrochemical sensors, can
be improved by the MIP-modication.
In order to ascertain the applicability of the proposed method,
the developed imprinted sensor was used to determine the
tramadol concentration in tablet and urine samples, and the exper-
imental results are shown in Table 2. The samples were prepared
by the procedures which are described in the sample prepara-
tion section. A solution of each sample, which was diluted in the
concentration range of the calibration plot was used. The diluted
samples were spiked with appropriate concentrations of tramadol.
The standard addition method was employed for the measurement
of prepared samples. The tramadol content of the tablets which was
declared by the manufacturer, was 100 mg per tablet; the tramadol
content which was determined by the proposed sensor, was found

Table 1
Comparison of some characteristics of the different electrodes for determination of tramadol in solutions.

Method Linear range (mol L1 ) Detection limit (mol L1 ) Reference

D50wx2GNPGCPE 0.03445.5 0.011 [50]

GCE 1575 2.20 [16]
CNP/GCE 101000 5.0 [51]
MIP-MWCNT/CPE 0.0120 0.004 [52]
MIP-MWCNT/CPE 0.11000 0.5 [45]
Polypyrrole@Sol-Gel MIP/f-MWCNT/GCE 2 104 0.02 3 105 This work

CNP/GCE: Carbon nanoparticles modied glassy carbon electrode.

GCE: Glassy carbon electrode.
658 B. Deiminiat et al. / Sensors and Actuators B 238 (2017) 651659
to be: 101.9 mg per tablet (Table 2). As can be seen in Table 2, the
acceptable recoveries which obtained for all of the samples, were in
the range of 96.0%106.0% with RSDs of 2.04.1%, indicating that the
sensor response is not affected considerably by the sample matrix
and the proposed method can be efciently used for determination
of TRA in pharmaceutical and biological samples.
4. Conclusion
In this study, a novel, simple and low cost strategy was proposed
for the preparation of an electrochemical sensor for detection of
tramadol in pharmaceutical and biological samples. Under opti-
mized experimental conditions, the proposed electrode exhibited
a good performance in terms of sensitivity, selectivity, detection
limit, fast response, reproducibility and repeatability. The excellent
performance of the polypyrrole@sol-gelMIP/f-MWCNTs modied
electrode towards tramadol can be attributed to the f-MWCNTs
layer with high conductivity and large specic surface area and the
porous imprinted lm with numerous recognition sites. Addition-
ally, for real sample analysis, the perfect recovery of the imprinted
sensor for determination of tramadol, indicates that the matrix of
the real samples has no signicant interference and the imprinted
sensor is able to detect the tramadol in biological and commercial
pharmaceutical samples successfully.
Acknowledgment
The authors acknowledge Ferdowsi University of Mashhad,
Mashhad, Iran for generous nancial support to carry out this
research work (Grant No. 3.32159).
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Biographies
Behjat Deiminiat is a Ph.D. student of Analytical Chemistry at Ferdowsi University
of Mashhad, Mashhad, Iran. She received her M.Sc. degree in Analytical Chemistry
from the same university in 2010. Her research interests include the development of
electrochemical sensors based on molecularly imprinted polymers and aptasensors.
Gholam Hossein Rounaghi is a full distinguished professor of Chemistry at Ferdowsi
University of Mashhad, Mashhad, Iran. He received his B.Sc. degree in Chemistry
from Ferdowsi University of Mashhad, in 1970, his M.Sc. degree in Analytical Chem-
istry at the same university in 1973, and his PhD degree from Michigan State
University (MSU) in Analytical Chemistry in 1980. His research interests focus on the
study of complexation of macrocyclic ligands with metal cations in non-aqueous sol-
vents and development of electrochemical sensors for cations and pharmaceutical
compounds.
Mohammad Hossein Arbab-Zavar is a full distinguished professor of Chemistry at
Ferdowsi University of Mashhad, Mashhad, Iran. He was graduated in Chemistry
(B.Sc. degree) from Shahid Beheshti University, Tehran, (Iran) in 1968 and then he
received M.Sc. and Ph.D. in Analytical Chemistry from University of Southampton
(England) in 1978 and 1982, respectively. His research interests include elec-
trochemical hydride generation for measurement of some toxic elements and
development of electrochemical sensors and their application in electrochemical
analysis.