Biosensors and Bioelectronics 60 (2014) 77–83

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Biosensors and Bioelectronics

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

Caffeine electrochemical sensor using imprinted film as recognition

element based on polypyrrole, sol-gel, and gold nanoparticles
hybrid nanocomposite modified pencil graphite electrode
Behzad Rezaei n, Malihe Khalili Boroujeni, Ali A. Ensafi
Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran

art ic l e i nf o a b s t r a c t

Article history: In the present study, a novel sensitive and selective nanocomposite imprinted electrochemical sensor for
Received 18 January 2014 the indirect determination of caffeine has been prepared. The imprinted sensor was fabricated on the
Received in revised form surface of pencil graphite electrode (PGE) via one-step electropolymerization of the imprinted polymer
11 March 2014
composed of conductive polymer, sol-gel, gold nanoparticles (AuNPs), and caffeine. Due to such
Accepted 12 March 2014
Available online 13 April 2014
combination like the thin film of molecularly imprinted polymer (MIP) with specific binding sites, the
sensor responded quickly to caffeine. AuNPs were introduced for the enhancement of electrical response
Keywords: by facilitating charge transfer processes of [Fe(CN)6]3
/[Fe(CN)6]4
which was used as an electro-
Electrochemical sensor chemical active probe. The fabrication process of the sensor was characterized by cyclic voltammetry
Molecularly imprinted polymers
(CV) and electrochemical impedance spectroscopy (EIS). Several important parameters controlling the
Polypyrrole
performance of the sensor were investigated and optimized. The imprinted sensor has the advantages of
Sol-gel
Au nanoparticles high porous surface structure, inexpensive, disposable, excellent stability, good reproducibility and
Caffeine repeatability. The linear ranges of the MIP sensor were in the range from 2.0 to 50.0 and 50.0 to
1000.0 nmol L
1, with the limit of detection (LOD) of 0.9 nmol L
1 (S/N¼ 3). Furthermore, the proposed
method was successfully intended for the determination of caffeine in real samples (urine, plasma,
tablet, green tea, energy and soda drink).
& 2014 Elsevier B.V. All rights reserved.

1. Introduction
Caffeine (1, 3, 7-trimethylxanthine) is an active alkaloid and a
stimulant drug widely found in the human diet. We consume
caffeine daily in coffee, tea, chocolate, energy or soft drinks, and
in many painkillers and antimigraine drugs. Caffeine acts as a
central nervous system stimulant, cardiovascular system stimulant,
and alleviation of migraine. However, high amounts of caffeine in
the body may cause many undesired physical and mental condi-
tions (Gupta et al., 2013; Peri‐Okonny et al., 2005; Reissig et al.,
2009). Therefore, the investigation and determination of caffeine in
beverage, pharmaceutical, and biological samples is very important.
Various analytical methods have been reported for the determina-
tion of caffeine, including chromatography (Chen et al., 2010a;
Hadad et al., 2012; Rajabi Khorrami and Rashidpur, 2012), spectro-
metry (del Campo et al., 2010; Jafari et al., 2011), FT-Raman
(Armenta et al., 2005), and electrochemical sensors (Gupta et al.,
2013; Santos et al., 2012; Sun et al., 2011). Despite these techniques
n
Corresponding author. Tel.: þ 98 311 3913268; fax: þ98 311 3912350.
E-mail address: rezaei@cc.iut.ac.ir (B. Rezaei).
have high sensitivity and accuracy, some factors such as expensive
instrumentation, time consuming, highly skilled techniques and
complicated procedures restricted their application. Electrochemi-
cal methods, especially electrochemical molecularly imprinted
polymer (MIP) based sensors, have attracted more attention in
recent years due to their simplicity, high sensitivity and selectivity,
good stability, low cost instrumentation, fast response and real time
detection (Afkhami et al., 2013; Prasad et al., 2010; Xue et al., 2013).
The MIP technique has been demonstrated as a powerful techni-
que in designing and synthesizing some artificial receptor molecules
for the mimicking of biological functions usable in analytical chem-
istry. MIPs have been extensively utilized in various fields including
sensors, separations, sample pretreatment, and catalysis (Chen et al.,
2010b; Cirillo et al., 2011; Huang et al., 2011; Kirsch et al., 2009).
Traditional MIPs have encountered many limitations including
incomplete template removal, poor site accessibility, slow interaction
kinetics, heterogeneous nature of the binding sites, and irregularity
in the shape of materials (which relied upon the bulk polymerization
approach) (Liu et al., 2012; Zeng et al., 2013). To overcome these
problems, the use of surface MIPs was suggested (Jing et al., 2010; Lu
et al., 2007). In comparison with conventional methods for MIPs
preparation, electropolymerization offers a simple, reproducible,

http://dx.doi.org/10.1016/j.bios.2014.03.028
0956-5663/& 2014 Elsevier B.V. All rights reserved.
78 B. Rezaei et al. / Biosensors and Bioelectronics 60 (2014) 77–83
convenient and friendly to environment way for the deposition of
thin recognition film directly on the support surface of any size and
shape (Kan et al., 2012; Liu et al., 2012).
Conducting polymers comprise a group of compounds and
materials with very specific properties, which have permeated
many fields of electrochemical research. Polypyrrole (Ppy) and its
derivatives have become one of the most widely used conductive
polymers in the design of sensors and biosensors due to their
significant electrical conductivity, good biocompatibility, and its
stable films can easily be deposited electrochemically (Kan et al.,
2012; Mehdinia et al., 2013). The interaction between the con-
ductive polymers and the other constituents resulted in a novel
material that can be used for specific applications.
In addition to conductive polymers, the sol-gel technology has
also been applied to improve the performance of MIPs on the
sensor surface. The sol-gel technology provides a simple way to
prepare three dimensional silicate networks with desirable sorp-
tive properties, through efficient incorporation of organic compo-
nents into the inorganic polymeric structure. The sol-gel based
MIPs possess numerous significant advantages over conventional
MIPs, such as simple fabrication process, mild thermal reaction
condition, physical rigidity, chemical inertness of the matrix, and
matrix porosity due to highly cross-linked structure (Mao et al.,
2012; Yang et al., 2013). However, the electrical insulation property
of sol-gel based coatings is clearly an undesirable property and
poses severe limitations on the efficiency of the signal transduc-
tion process. Due to the electrically insulating layer of sol-gel MIP
based systems; a direct path for the conduction of electrons from
the binding sites to the electrode surface is required. In order to
overcome the limitations of rebinding capacity and conductivity,
the most effective way was the use of conducting nanomaterials.
Metal nanoparticles have attracted widespread attention in their
utilization for enhancing the sensitivity of the electrochemical
detection. Among all kinds of metal nanoparticles, gold nanopar-
ticles (AuNPs) have been extensively used in fabrication of
different kinds of sensors due to their unique structure and
fascinating properties such as high effective surface area, strong
adsorption ability, good biocompatibility and conductivity. The
nanocomposite of AuNPs doped MIPs can be easily constructed by
some strategies including direct electrostatic assembly, covalent
linking, polymer entrapment, co-mixing, and electrodeposition
methods (Guo and Dong, 2009; Li et al., 2013; Yu et al., 2012).
The incorporation of AuNPs, conductive polymers, and sol-gel
technology allows preparation of new nanocomposite that can
efficiently combine the advantages of all materials. The advantages
of using this nanocomposite MIP sensor compared to other sensors
(i.e. electrochemical sensors consisting of layer by layer modified
electrode) are ease of preparation (one-step electropolymeriza-
tion), low cost, more porous surface structure, higher recognition
capacity, higher functional groups, excellent selectivity and sensi-
tivity. In addition, it is possible to design the MIP film properties
and polarity through proper selection of the sol-gel precursor and
conductive polymer derivatives.
In the present paper, we propose a simple approach to improve the
conduction of electrons from sol-gel MIPs to the electrode surface with
the entrapment of conductive polymers and metal nanoparticles
within sol-gel networks. To the best of our knowledge, this is the first
work describing synthesis and use of a conducting polymer, sol-gel,
and AuNPs hybrid nanocomposite as a MIP based electrochemical
sensor (nanocomposite MIP/PGE). The experimental parameters
that affect the performance of the nanocomposite MIP sensor were
investigated and optimized. The nanocomposite MIP sensor was
evaluated to confirm its electrochemical properties (selectivity, con-
ductivity, stability, linearity, repeatability and reproducibility). Further-
more, the developed imprinted sensor was applied for the analysis of
the caffeine in real samples.
2. Experimental
2.1. Materials
Tetraethoxysilane (TEOS), phenyltriethoxysilane (PTEOS),
sodium dodecyl sulfate (SDS), trifluoroacetic acid (TFA), hydrogen
tetrachloroauratetrihydrate (HAuCl4d3H2O), and caffeine were
purchased from Merck (Darmstadt, Germany). All other solvents
and salts used in this study were of analytical grade and were
obtained from Merck (Darmstadt, Germany). Ethanol was pre-
pared from Bidestan Co. (Qazvin, Iran). Pyrrole (Merck, Darmstadt,
Germany) was distillated and stored in a dark bottle in a refrig-
erator before use.
Caffeine stock solution was prepared and stored at 4 1C prior to
use. More diluted working solutions used in further studies were
prepared daily by diluting different amounts of the intermediate
standard solution with phosphate buffer (pH 4.0) to the required
concentrations.
2.2. Instrumentation
The electrochemical measurements were performed with a
Potentiostat-Galvanostat μ-AutoLab (Echo Chemie, B.V., Nether-
lands, NOVA software). A three-electrode system consisted of an
Ag/AgCl reference electrode, a platinum rod auxiliary electrode,
and a nanocomposite MIP/PGE as the working electrode was used.
Autolab system (PGSTAT 12, Eco Chemie B.V., Utrecht, Nether-
lands) was applied for the electrochemical impedance spectro-
scopic measurements. The system was performed on a PC by GPES
and FRA 4.9 software.
A mechanical pencil was used as a holder for graphite leads
(Rotring, 2B, 0.7 mm diameter, Germany). Electrical contact with
the lead was obtained by soldering a metal wire to the metal part.
Metrohm pH meter (Model 827) with a glass electrode (Corning)
was used to adjust the solution pH. An atomic force microscope
(BRUKER, Germany) was used to investigate AuNPs electrodepos-
ited into the nanocomposite MIP. The scanning electron micro-
scopy images (SEM) were obtained using a Philips XL30 SEM
instrument.
2.3. Preparation of nanocomposite MIP/PGE
The surface of the pencil lead was first pretreated by applying
potential þ1.40 V for 300 s in 0.50 mol L
1 acetate buffer (pH 4.8)
containing 0.02 mol L
1 sodium chloride.
A typical procedure for preparing nanocomposite film is
described as follows. The sol solution was prepared using 75 mL
of PTEOS, 75 mL of TEOS, 700 mL of water, 1100 mL of ethanol, 10 mL
of TFA, and caffeine (2.5 mmol L
1) in a vial. The mixture was
stirred for 2 h at room temperature. Immediately following, 50 mL
of pyrrole solution (final concentration of 25.0 mmol L
1), 5.0 mg
of SDS (as the counter ion), and 50 mL of 0.01% HAuCl4 solution
(final concentration of 2.5  10
4%) were added to the mixture.
The resulting polymerization solution was sonicated for 10 min.
Then, the pretreated electrode was immersed into the polymer-
ization solution. The nanocomposite MIP film was prepared by
electrodeposition using CV in the potential range of
0.80 and
þ0.60 V (versus Ag/AgCl) during five cycles. After electrodeposi-
tion the modified electrode was dried at room temperature for 2 h.
The doped caffeine was extracted from the imprinted film by
immersion in acetic acid (50% V/V) solution with stirring magne-
tically for 20 min.
For the comparison purpose, a sol-gel/AuNPs nanocomposite
and a ppy/sol-gel/AuNPs layer-by-layer MIP electrode were fabri-
cated by the method given in Supplemental information. As a
reference electrode, the non-imprinted polymer (NIP) electrode
 B. Rezaei et al. / Biosensors and Bioelectronics 60 (2014) 77–83
was prepared under the same experimental conditions except that
no template molecule was used.
2.4. Electroanalytical measurements
The rebinding of caffeine on the modified sensor was per-
formed by incubating into the solution of caffeine in phosphate
buffer solution (pH 4.0) for 180 s. After that the electrochemical
measurements were performed in a phosphate buffer (pH 4.0)
containing 0.1 mol L
1 KCl and 5.0 mmol L
1 K3[Fe(CN)6]/K4
[Fe(CN)6] as probe solution.
CV and EIS were used for probing the electrochemical proper-
ties of the different modified electrodes. The cyclic voltammo-
grams were recorded between
0.10 and 0.50 V at a scan rate of
50 mV s
1. EIS was done at an applied potential of 0.20 V in a
frequency range of 0.005–105 Hz and at amplitude of 5 mV. The
square wave voltammograms (SWV) were obtained under fre-
quency of 15 Hz, amplitude of 20 mV and step potential of 1 mV.
SWV experiments were carried out in a phosphate buffer (pH 4.0)
containing 0.1 mol L
1 KCl and 1.0 mmol L
1 K3[Fe(CN)6]/K4[Fe
(CN)6] as probe solution due to its higher sensibility compared to
the CV technique. All electroanalytical measurements were per-
formed at room temperature.
2.5. Preparation of real samples
The energy drink (SYNERGY, Behnoush Co., Tehran, Iran), soft
drink (Canada Dry, Khoshgovar Co., Qazvin, Iran), and green tea
(Shahsavand Co., Mashhad, Iran) were purchased from the local
markets. The pharmaceutical sample (Novafen capsules from
Alhavi Pharmaceutical Co., Iran, 40 mg caffeine) was obtained
from the local drug store. Fresh human plasma sample was taken
from the Health Center of Isfahan University of Technology, and
human urine sample was collected from a healthy adult. The
preparation procedure is given in Supplemental information.
3. Results and discussion
3.1. Preparation and characterization of the imprinted sensor
The presence of interactions (i.e., noncovalent and/or covalent
interactions) between template and functional monomers in the
polymer matrix is a prerequisite for successful imprinting. The role
of these monomers is to form specific binding cavities comple-
mentary to the imprint species in terms of the shape and the
position of functional groups.
In this study, the incorporation of conductive polymers and sol-
gel technology allows preparation of new MIP that can efficiently
integrate the advantages of both. TEOS is a non-functionalized
silane monomer and alone cannot create a well-defined matrix
for imprinting. It was used as cross-linker to form a polymeric
Fig. 1. (A) AFM image of the nanocomposite MIP/PGE; (B) 3-D image (AFM) of the nanocomposite MIP/PGE.
79
network around the template molecule because of its rapid rate of
hydrolysis and condensation. Consequently, PTEOS was utilized as
functional monomer due to its phenyl group, which can interact
with the template via π–π interactions (Hu et al., 2012). Also,
pyrrole was incorporated into the sol solution to introduce addi-
tional functional group to the resultant MIP. Caffeine in aqueous
solution establishes equilibrium between CO
and CQO groups
(Santos et al., 2012), which allows electrostatic interaction with
the ppy film and also hydrogen bonding (Fig. S1).
CV was employed to electrodeposited nanocomposite MIP on the
modified PGE surface. No notable differences were observed in the
cyclic voltammogram with and without the present of caffeine,
which demonstrates that caffeine does not have any electrochemical
oxidation and reduction under the potential range of electrodeposi-
tion, and the structure of the caffeine was not electrochemically
changed during the imprinting process.
The surface morphological analysis of nanocomposite film was
investigated by atomic force microscope (AFM) and scanning electron
microscope (SEM). As shown in Fig. 1A, globular shaped AuNPs were
uniformly deposited on the electrode surface. According to the 3D
images in Fig. 1B, the modified electrode surface was very rough,
which increased the electrode specific surface area and the resulting
currents in voltammetric measurements. The average size of the
AuNPs determined by AFM supporting software was about 30 nm.
Also, the related SEM images are given in Supplemental information
(Fig. S2). Fig. S2A and B shows the SEM pictures of bare PGE and
pretreated PGE, respectively. The graphite layers were broken into
small pieces and they can be easily seen after the electrode was
pretreated with acetate buffer solution (Fig. S2B). Fig. S2C corresponds
to the sol-gel/AuNPs nanocomposite MIP film covered the entire
pretreated PGE surface, homogenously. As can be seen in Fig. S2D,
the pyrrole/AuNPs nanocomposite MIP film shows a densely packed
and globular surface morphology. On the surface of ppy/sol-gel/AuNPs
layer-by-layer MIP film (Fig. S2E), homogeneous and wrinkled surface
was observed, suggesting less compact and more porous structure
than pyrrole/AuNPs and sol-gel/AuNPs nanocomposite MIP film
(Fig. S2C and D). The ppy/sol-gel/AuNPs nanocomposite MIP film
(Fig. S2F) reveals different appearances, which suggest much rougher
surface, less compact and more porous structure than other MIP films
(Fig. S2C–E). The porous structure and rough surface could increase
the effective electrode surface area and improve the diffusion of
analytes into the polymer film.
3.2. Electrochemical characterization of the imprinted sensor
CV and EIS were valuable and convenient techniques to
monitor the electrochemical properties of the surface modified
electrodes. To characterize the stepwise fabrication of the MIP
sensor, CV was carried out in probe solution. Fig. 2A showed a
typical comparison of cyclic voltammograms of the bare PGE,
modified PGE, and nanocomposite MIP/PGE.
80 B. Rezaei et al. / Biosensors and Bioelectronics 60 (2014) 77–83
Fig. 2. (A) CVs of (a) bare PGE; (b) modified PGE; (c) MIP/PGE; (d) MIP/PGE after
removal of caffeine; (e) MIP/PGE after 180 s incubating in 0.5 μmol L
1 caffeine
solution and (f) MIP/PGE without AuNPs after removal of caffeine. Conditions:
Potential scan range, from
0.10 V to þ 0.50 V; Scan rate, 50 mV s
1. (B) EIS of
(a) bare PGE; (b) modified PGE; (c) MIP/PGE; (d) MIP/PGE after removal of caffeine;
(e) MIP/PGE after 180 s incubating in 0.5 μmol L
1 caffeine solution and (f) MIP/PGE
without AuNPs after removal of caffeine. Conditions: Potential: 0.20 V; frequency
range of 0.005–105 Hz and at amplitude of 5 mV.
At the bare PGE (curve a), the redox peaks of ferricyanide ions
were obtained with the peak-to-peak separation (ΔEp) of 190 mV.
As can be seen in curve b, after the electrode was modified with
acetate buffer solution, the peak current increased and the ΔEp
decreased to 141 mV. As shown in curve c, the current response of
the electrode after electropolymerization of MIPs declined
obviously. Since the polymer film coated on the electrode, the
molecules of [Fe(CN)6]3
/4
could not arrive at the surface of the
electrode and the electron transfer was blocked. After template
removal, the current response of the electrode increased appar-
ently (curve d). This could be explained that removal of the
template, leaving some channels and cavities for enhancing
the diffusion of [Fe(CN)6]3
/4
through the MIP and reached the
electrode surface more easily (ΔEp ¼ 110 mV). After the nanocom-
posite MIP/PGE was incubated in 0.5 mmol L
1 caffeine solution for
180 s, just a pair of weaker peaks (comparing with curve d)
detected (curve e), which reveals that some cavities were recom-
bined with caffeine, giving limited chances for the electron
transfer of the redox probe on the electrode surface.
After the modification of PGE with ppy/sol-gel composite film
(without AuNPs), due to a thin compact film of composite covered
on the surface of the electrode, the peak current was decreased
and the reversibility of electrochemical reaction became deterio-
rated (curve f). To overcome this problem the HAuCl4 solution was
dispersed in ppy/sol-gel solution and AuNPs entrapped within the
composite film during electrochemical deposition (curve d). The
presence of AuNPs caused a development in the electron
transfer rate.
EIS can also be used to investigate the features of the surface
modified electrode, in order to characterize the stepwise construc-
tion process of the sensor. The impedance spectra (presented in
the form of the Nyquist plot) include a semicircle portion and a
straight line. The semicircle part, which can be observed at higher
frequencies, corresponds to the electron transfer resistance. The
linear part at lower frequencies corresponds to the impedance of
the current because of the diffusion from the solution to the
interface. The semicircle diameter equals the electron transfer
resistance (Ret). Fig. 2B shows the impedance diagrams of the
modified electrode at different modification stages.
As shown in Fig. 2B (a), the bare PGE exhibited a semicircle part
at high frequencies and a linear part at low frequencies
(Ret ¼ 168 Ω). After PGE modification with acetate buffer solution
(curve b), the Ret decreased to 86 Ω, suggesting faster electron
transfer kinetics of [Fe(CN)6]3
/4
on the electrode surface. With
the electrodeposited of the nanocomposite, the resultant nano-
composite MIP/PGE displayed obvious increase in the interfacial
resistance (curve c). This indicated that the imprinted film formed
an additional barrier and the electron transfer of [Fe(CN)6]3
/4

was blocked.
As shown in curve d, a small electron transfer resistance
(Ret ¼65 Ω) was observed after removing the template. The reason
was that the formed cavities on the MIP could facilitate the penetra-
tion of the probe through the imprinted layer and make it easier for
electron transfer to take place. Because of the better conductivity of
AuNPs over PGE, the resistance of electrode modified with nanocom-
posite after removing the template was a little smaller than modified
PGE with acetate buffer solution. Furthermore, after immersing the
nanocomposite MIP/PGE in 0.5 mmol L
1 caffeine solution for 180 s,
the resistance increased to Ret ¼85 Ω (curve e), which would be
attributed to the rebinding of the template into the imprinted cavities
again and to the blocking of the arrival of the probe to the electrode
surface.
3.3. Optimization of experimental parameters
To obtain the best performance, the effects of various parameters
including electropolymerization cycles, applied potential interval, the
ratio of monomers and template, HAuCl4 percent, pH, and incubation
time were investigated by altering each variable in turn while
keeping the others constant. All the optimization experiments were
performed using CV. The change of [Fe(CN)6]3
/4
current (Δi) was
used to evaluate the optimum condition.
More discussion regarding the effect of various parameters on
the current response has been given in the Supplemental informa-
tion in the same section. The optimum conditions for the electro-
polymerization cycles (Fig. S3) and applied potential interval were
obtained as five cycles and a potential range from
0.80 to
þ0.60 V, respectively.
The optimum amount of sol-gel precursor, pyrrole monomer,
and HAuCl4 percentage were obtained as 150 mL, 25.0 mmol L
1,
and 2.5  10
4 percent, respectively. The results of amount of sol-
gel precursor, pyrrole monomer, and HAuCl4 percentage are
shown in Figs. S4–S6, respectively. The optimum template con-
centration was found to be 2.5 mmol L
1 (Fig. S7).
The results show that acetic acid (50% V/V) solution can remove
the template most quickly and completely, and the best extraction
 B. Rezaei et al. / Biosensors and Bioelectronics 60 (2014) 77–83
time was 20 min. As can be seen in Fig. S8, the incubation time of
180 s was selected in the subsequent studies.
The results (Fig. S9) showed that the maximum response was
observed at pH 4.0. This pH was also used for probe solution in the
determination step to prohibit leaching of the template.
3.4. Selectivity of the MIP sensor
The main purpose to prepare this nanocomposite MIP/PGE is to
improve the selectivity and sensitivity of the electrochemical
sensor. Subsequent removal of the template leaves binding sites
within the polymer possessing both shape and the correct orien-
tation of functional groups to allow selective rebinding of the
imprint species. However, when molecules that have similar
structures to the template molecule are present, these molecules
are also adsorbed on the surface of polymer and they can be
captured in the cavity sites of MIP. To evaluate the selectivity of the
prepared MIP sensor, theophylline, adenine, and guanine with
similar structure to caffeine were chosen as interference with an
equivalent concentration (0.5 mmol L
1). It can be seen from Fig. 3
that the current response of the imprinted sensor for caffeine was
higher than that of other interfering species. The excellent sensi-
tivity of the nanocomposite MIP/PGE was due to the cavities which
matched with the size, shape, and the functional group position of
the caffeine. Although the structures of the interferences are
relatively similar to caffeine, the imprinted cavities could not bind
them as tightly as caffeine.
In order to investigate the effect of the imprinted materials to
improve the selectivity of the sensor, the current response for
caffeine and its analogs (theophylline, adenine, and guanine) with
the nanocomposite MIP/PGE, sol-gel/AuNPs nanocomposite, ppy/
AuNPs nanocomposite, and layer-by-layer ppy/sol-gel/AuNPs MIP
and NIP sensors were compared. As shown in Fig. 3, the nano-
composite MIP electrode has higher adsorption capacity and
Fig. 3. Comparison selectivity of the nanocomposite MIP/PGE, sol-gel/AuNPs
nanocomposite, ppy/AuNPs nanocomposite, and ppy/sol-gel/AuNPs layer-by-layer
sensors response for caffeine and substances with structures analogous to that of
caffeine, selectivity expressed as imprinted factor (IF). Caffeine* was done at
presence of theophylline, adenine, and guanine. Ppy þsol-gelþ AuNPs* was nano-
composite MIP/PGE.
81
selectivity than other MIP electrodes. It can be attributed to the
more functional group of nanocomposite (through the incorpora-
tion of conductive polymers and sol-gel technology).
To further validate the selectivity of nanocomposite MIP/PGE,
another comparison was displayed in Fig. 3. The selectivity can be
evaluated intuitively by calculating the imprinting factor (IF),
which is defined as the ratio of the ΔiMIP to the ΔiNIP. As shown
in Fig. 3, the IF for caffeine with nanocomposite MIP/PGE, sol-gel/
AuNPs nanocomposite, ppy/AuNPs nanocomposite, and layer-
by-layer ppy/sol-gel/AuNPs MIP sensors are 12.7, 4.1, 1.1 and 5.4,
with RSDs 3.7%, 3.4%, 4.8% and 3.6%, respectively. Besides, the IF for
interference species with three different imprinted sensors is in
the range of 1.0–3.1, with RSDs in the range of 4.5–3.2%.
All results demonstrate that the nanocomposite MIP/PGE
sensor own higher sensitivity and selectivity toward caffeine than
two other sensors.
3.5. Analytical performance
The inset of Fig. 4 displays the SWV responses of the nano-
composite MIP electrode after incubation in different concentra-
tions of caffeine solution for 180 s. Under the optimum conditions,
SWV results of the MIP film were recorded in probe solution. Due
to the occupation of the cavity sites in the MIP film by caffeine
molecules, the peak current decreased as the concentration of
caffeine increases. As shown in Fig. 4, two linear ranges from 2.0 to
50.0 and 50.0 to 1000.0 nmol L
1 were observed with the regres-
sion equation of y¼0.309xþ 13.973 (R2 ¼ 0.9934) and y¼ 0.007x þ
28.986 (R2 ¼0.9969), respectively. The limit of detection (LOD) was
calculated to be 0.9 nmol L
1 based on 3sb/m, where m repre-
sented the slope of the calibration curve, and sb represented the
standard deviation of the blank (n ¼7).
By using acetic acid (50% V/V) for 5 min to elute caffeine
molecules, the MIP sensor was able to be reused for 12 repeated
analyses. The repeatability of one nanocomposite MIP sensor was
carried out for 0.1 mmol L
1 caffeine, and the calculated RSD was
3.7% (n ¼12). Besides, reproducibility is another important prop-
erty for the application of MIP sensors. To investigate the repro-
ducibility of the constructed MIP sensor, 0.1 mmol L
1 of caffeine
solution was determined using five fresh MIP sensors which
prepared independently under the same conditions. The RSD of
Fig. 4. Calibration curve of nanocomposite MIP sensor in different concentrations
of caffeine solutions. Inset is the SWV of nanocomposite MIP/PGE sensor before (a),
and after incubation in 2.0 (b), 5.0 (c), 10.0 (d), 20.0 (e), 30.0 (f), 50.0 (g), 100.0 (h),
200.0 (i), 300.0 (j), 500.0 (k), and 1000.0 nmol L
1 (l) of caffeine solution.
Conditions: Potential scan range, 0.0 V to þ 0.50 V; Step potential, 1 mV; Frequency,
15 Hz; Amplitude, 20 mV in phosphate buffer pH 4.0 containing 0.1 mol L
1KCl and
1.0 mmol L
1 [Fe(CN)6]3
/4
.
82 B. Rezaei et al. / Biosensors and Bioelectronics 60 (2014) 77–83

4.4% was observed. These values indicate that the reproducibility
and repeatability of the proposed MIP sensor are acceptable.
The storage stability of the MIP modified electrode was also
investigated. The prepared imprinted sensors were stored at 4 1C,
and the stability of the sensor was examined by measuring the
current response for 0.5 mmol L
1 of caffeine at regular intervals
(1 day) for a period of two weeks. After two weeks, the sensor
retained 92% of its initial response current, which suggested that
the developed caffeine imprinted sensor possessed good stability.
The good stability of the MIP sensor can be attributed to the stiff
layer of three dimensional nanocomposite MIP on the modified
electrode surface (Taheri et al., 2009).
Also, the comparison between the analytical characteristics of
the present nanocomposite MIP sensor and some previous reports
for the determination of caffeine is listed in Table 1. This reveals
that the present sensor shows superior sensitivity to all of the
other techniques.
3.6. Real sample analysis
The applicability of the proposed sensor was examined by
determining the caffeine concentration in urine, plasma, tablet,
green tea, energy and soft drink samples. The samples were
prepared by the procedures described in Section 2.5. Depending
on the concentration, each sample was diluted so as to move the
concentration within the range of detection limits. To each sample
solution was added three different concentrations of caffeine (5.0,
10.0, and 20.0 nmol L
1). The standard addition method was used
for the analysis of prepared samples. The recovery and precision
were determined for three replicate measurements for each
concentration. Caffeine declared content was 40 mg per tablet;
caffeine content was calculated to be 41.5 mg per tablet (according
to the detected amount in Table 2).
The results were shown in Table 2. Satisfactory recoveries for
all samples were obtained between 90% and 116% with RSDs of
1.1–6.6%. These results indicate that the complex matrix of the real
samples has no significant interference for the determination of
the caffeine using this nanocomposite MIP/PGE sensor.
4. Conclusion
In this study, a novel and simple strategy was proposed for the
preparation of an electrochemical sensor for caffeine determina-
tion based on one-step electropolymerization of nanocomposite
MIP at a modified pencil graphite electrode. Combination of
conductive polymer (ppy), sol-gel technology, and AuNPs allowed
the preparation of a novel electrochemical MIP sensor in a simple,
most convenient and reproducible way on a disposable electrode.
The excellent performance of the nanocomposite MIP/PGE sensor
can be ascribed to the porous imprinted composite film with
plentiful selective binding sites. The prepared sensor displayed

Table 1
Comparison of different electrochemical methods for determination of caffeine.

Electrode (method) Linear range (mmol L
1) LOD (mmol L
1) Reference

a
3
MWCNTs/GCE (SWV) 10–500 3.52  10 Gupta et al. (2013)
Nafion-Gr/GCEb (DPV) 0.4–40 and 40–600 0.12 Sun et al. (2011)
MIS/MWCNTs-VTMS/GCEc (DPV) 0.75–40 0.22 Santos et al. (2012)
Poly(AHNSA)/GCEd (SWV) 0.06–40 0.137 Amare and Admassie (2012)
Nanocomposite MIP/PGE 0.002–0.05 and 0.05–1 9  10
4 This work

a
Multiwall carbon nanotubes/glassy carbon electrode.
b
Nafion-graphene/glassy carbon electrode.
c
Molecularly imprinted siloxane/multiwall carbon nanotubes-vinyltrimethoxysilane/glassy carbon electrode.
d
Poly(4-amino-3-hydroxynaphthalene sulfonic acid)/glassy carbon electrode.

Table 2
Results of caffeine determination in real sample analysis (n¼3).

Sample Added (nmol L
1) Detected (nmol L
1) Recovery (%) RSD (%)

Soft drink – 22.9 7 1.5 – 6.6

5.0 28.17 1.7 104 6.0
10.0 33.271.9 103 5.7
20.0 42.7 7 1.9 99 4.4
Energy drink – 29.17 1.6 – 5.5
5.0 34.9 7 2.0 116 5.7
10.0 39.6 7 1.9 105 4.8
20.0 48.77 1.8 98 3.7
Green tea – 25.8 7 1.6 – 6.2
5.0 30.9 7 0.8 102 2.6
10.0 34.8 7 0.9 90 2.6
20.0 46.3 7 0.5 102.5 1.1
Tablet – 142.3 76.8 – 4.8
5.0 146.8 77.2 90 4.9
10.0 151.7 77.8 94 5.1
20.0 162.8 76.8 102.5 4.2
Urine – 19.3 71.0 – 5.2
5.0 24.971.4 112 5.6
10.0 29.6 7 1.4 103 4.7
20.0 397 1.5 98.5 3.8
Plasma – Not detected – –
5.0 5.5 7 0.2 110 3.6
10.0 10.8 70.5 108 4.6
20.0 19.5 70.6 97.5 3.0
 B. Rezaei et al. / Biosensors and Bioelectronics 60 (2014) 77–83
good selectivity, broad linearity, and excellent reproducibility and
repeatability. Moreover, this sensor had great potential application
in the real samples analysis without the interference of other
compounds with similar structure to that caffeine.
Acknowledgments
The authors acknowledge Iran National Science Foundation
(INSF), Isfahan University of Technology Research Council (IUT)
and Center of Excellency in Sensor and Green Chemistry of IUT for
supporting this work.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.bios.2014.03.028.
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