Author's Accepted Manuscript

Caffeine electrochemical sensor using im-

printed film as recognition element based on
polypyrrole, sol-gel, and gold nanoparticles
hybrid nanocomposite modified pencil gra-
phite electrode
Behzad Rezaei, Malihe Khalili Boroujeni, Ali A.
Ensafi

www.elsevier.com/locate/bios

PII: S0956-5663(14)00209-7
DOI: http://dx.doi.org/10.1016/j.bios.2014.03.028
Reference: BIOS6656

To appear in: Biosensors and Bioelectronics

Received date: 18 January 2014

Revised date: 11 March 2014
Accepted date: 12 March 2014

Cite this article as: Behzad Rezaei, Malihe Khalili Boroujeni, Ali A. Ensafi,
Caffeine electrochemical sensor using imprinted film as recognition element
based on polypyrrole, sol-gel, and gold nanoparticles hybrid nanocomposite
modified pencil graphite electrode, Biosensors and Bioelectronics, http://dx.doi.org/
10.1016/j.bios.2014.03.028

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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*, Malihe Khalili Boroujeni, Ali A. Ensafi

Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111,

Iran

*Correspondence: Behzad Rezaei, Department of Chemistry, Isfahan University of

Technology, Isfahan 84156-83111, Iran

Phone: +98 311 3913268. Fax: +98 311 3912350

E-mail: [email protected]
Abstract

In the present study, a novel sensitive and selective nanocomposite imprinted electrochemical

sensor for the indirect determination of caffeine has been prepared. The imprinted sensor was

fabricated on the surface of pencil graphite electrode (PGE) via one-step electropolymerization

of the imprinted polymer composed of conductive polymer, sol-gel, gold nanoparticles (AuNPs),

and caffeine. Due to such 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 by facilitating charge transfer processes of [Fe(CN)6]3-

/[Fe(CN)6]4- which was used as an electrochemical active probe. The fabrication process of the

sensor was characterized by cyclic voltammetry (CV) and electrochemical impedance

spectroscopy (EIS). Several important parameters controlling the performance of the sensor were

investigated and optimized. The imprinted sensor has the advantages of high porous surface

structure, inexpensive, disposable, excellent stability, good reproducibility and 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/N=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).

Keywords: Electrochemical sensor; Molecularly imprinted polymers; Polypyrrole; Sol-gel;

Au nanoparticles, Caffeine.
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 conditions (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 determination of caffeine, including

chromatography (Chen et al. 2010a; Hadad et al. 2012; Rajabi Khorrami and Rashidpur 2012),

spectrometry (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 have high sensitivity and accuracy, some factors such as expensive instrumentation,

time consuming, highly skilled techniques and complicated procedures restricted their

application. Electrochemical 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 technique in designing and

synthesizing some artificial receptor molecules for the mimicking of biological functions usable

in analytical chemistry. 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,

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 conductive 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 sorptive properties, through efficient

incorporation of organic components 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 transduction
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 nanoparticles (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 electropolymerization), low cost, more porous surface structure, higher recognition

capacity, higher functional groups, excellent selectivity and sensitivity. 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, conductivity, stability, linearity, repeatability and

reproducibility). Furthermore, 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 (HAuCl4.3H2O), 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 prepared from Bidestan Co. (Qazvin, Iran). Pyrrole (Merck, Darmstadt, Germany) was

distillated and stored in a dark bottle in a refrigerator before use.

Caffeine stock solution was prepared and stored at 4 ºC 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., Netherlands, 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, Netherlands) was applied for the electrochemical impedance

spectroscopic 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

electrodeposited into the nanocomposite MIP. The scanning electron microscopy 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 +1.40 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 µL of PTEOS, 75 µL of TEOS, 700 µL of water, 1100 µL of

ethanol, 10 µL 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 µL of pyrrole solution (final concentration of 25.0

mmol L-1), 5.0 mg of SDS (as the counter ion), and 50 µL 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 polymerization

solution. The nanocomposite MIP film was prepared by electrodeposition using CV in the

potential range of -0.80 and +0.60 V (versus Ag/AgCl) during five cycles. After
electrodeposition 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 magnetically for 20 min.

For the comparison purpose, a sol-gel/AuNPs nanocomposite and a ppy/sol-gel/AuNPs layer-

by-layer MIP electrode were fabricated by the method given in Supplemental information. As a

reference electrode, the non-imprinted polymer (NIP) electrode 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 performed 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 properties of the different modified

electrodes. The cyclic voltammograms 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

frequency 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 performed 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 complementary 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 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 additional

functional group to the resultant MIP. Caffeine in aqueous solution establishes equilibrium
between CO− and C=O groups (Santos et al. 2012), which allows electrostatic interaction with

the ppy film and also hydrogen bonding (Fig. S-1).

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 electrodeposition, 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. S-2). Fig. S-2A and B show 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. S-2B). Fig. S-2C corresponds to the sol-gel/AuNPs nanocomposite MIP film

covered the entire pretreated PGE surface, homogenously. As can be seen in Fig. S-2D, 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 S-2E),

homogeneous and wrinkled surface was observed, suggesting less compact and more porous

structure than pyrrole/AuNPs and sol-gel/AuNPs nanocomposite MIP film (Fig. S-2C and D).

The ppy/sol-gel/AuNPs nanocomposite MIP film (Fig. S-2F) reveals different appearance, which
suggests much rougher surface, less compact and more porous structure than other MIP films

(Fig. S-2C, D, and 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.

At the bare PGE (curve a), the redox peaks of ferricyanide ions were obtained with the peak-

to-peak separation (∆Ep) 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 ∆E p 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 apparently (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 (∆Ep=110 mV). After the nanocomposite MIP/PGE was incubated in 0.5 µmol 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 recombined 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 deteriorated (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 construction 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 =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

nanocomposite 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 =65 Ω) was observed after

removing the template. The reason was that the formed cavities on the MIP could facilitate the
penetration 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 nanocomposite 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 µmol L-1 caffeine solution for 180s, the resistance increased to Ret =85 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 (∆i) 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 information in the same section. The optimum conditions for the

electropolymerization cycles (Fig. S-3) and applied potential interval were obtained as five

cycles and a potential range from -0.80 to +0.60 V, respectively.

The optimum amount of sol-gel precursor, pyrrole monomer, and HAuCl4 percentage were

obtained as 150 µL, 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 were shown in Fig. S-4, Fig. S-5,

and Fig. S-6, respectively. The optimum template concentration was found to be 2.5 mmol L-1

(Fig. S-7).
 The results show that acetic acid (50% V/V) solution can remove the template most quickly

and completely, and the best extraction time was 20 min. As can be seen in Fig. S-8, the

incubation time of 180 s was selected in the subsequent studies.

The results (Fig. S-9) 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 orientation 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 µmol 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 sensitivity 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 nanocomposite MIP electrode has higher adsorption capacity and selectivity than other MIP

electrodes. It can be attributed to the more functional group of nanocomposite (through the

incorporation 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 ∆iMIP to the ∆iNIP. 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 nanocomposite MIP electrode after

incubation in different concentrations 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 regression equation of y=0.309x+13.973

(R2=0.9934) and y=0.007x+28.986 (R2=0.9969), respectively. The limit of detection (LOD) was
calculated to be 0.9 nmol L-1 based on 3sb/m, where m represented the slope of the calibration

curve, and sb represented the standard deviation of the blank (n=7).

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 µmol L-1 caffeine, and the calculated RSD was 3.7% (n=12). Besides,

reproducibility is another important property for the application of MIP sensors. To investigate

the reproducibility of the constructed MIP sensor, 0.1 µmol L-1 of caffeine solution was

determined using five fresh MIP sensors which prepared independently under the same

conditions. The RSD of 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°C, and the stability of the sensor was examined by measuring

the current response for 0.5 µmol 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 determination 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

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.

Acknowledgements\

The authors acknowledge Iran National Science Fundation (INSF), Isfahan University of

Technology Research Council (IUT) and Center of Excellency in Sensor and Green Chemistry of

IUT for supporting this work.

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

Electrode (method) Linear range LOD Reference

(µmol L-1) (µmol L-1)
MWCNTs/GCEa (SWV) 10-500 3.52×10-3 (Gupta et al.
2013)
Nafion-Gr/GCEb (DPV) 0.4-40 and 40-600 0.12 (Sun et al. 2011)
MIS/MWCNTs- 0.75-40 0.22 (Santos et al.
VTMS/GCEc (DPV) 2012)

Poly(AHNSA)/GCEd 0.06-40 0.137 (Amare and

(SWV) Admassie 2012)
Nanocomposite MIP/PGE 0.002-0.05 and 9×10-4 This work
0.05-1
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 (n=3).
Added Detected Recovery
Sample RSD %
(nmol L-1) (nmol L-1) (%)
Soft drink - 22.9 ± 1.5 - 6.6
5.0 28.1 ± 1.7 104 6.0
10.0 33.2 ± 1.9 103 5.7
20.0 42.7 ± 1.9 99 4.4
Energy drink - 29.1 ± 1.6 - 5.5
5.0 34.9 ± 2.0 116 5.7
10.0 39.6 ± 1.9 105 4.8
20.0 48.7 ± 1.8 98 3.7
Green tea - 25.8 ± 1.6 - 6.2
5.0 30.9 ± 0.8 102 2.6
10.0 34.8 ± 0.9 90 2.6
20.0 46.3 ± 0.5 102.5 1.1
Tablet - 142.3 ± 6.8 - 4.8
5.0 146.8 ± 7.2 90 4.9
10.0 151.7 ± 7.8 94 5.1
20.0 162.8 ± 6.8 102.5 4.2
Urine - 19.3 ± 1.0 - 5.2
5.0 24.9 ± 1.4 112 5.6
10.0 29.6 ± 1.4 103 4.7
20.0 39 ± 1.5 98.5 3.8
Plasma - Not detected - -
5.0 5.5 ± 0.2 110 3.6
10.0 10.8 ± 0.5 108 4.6
20.0 19.5 ± 0.6 97.5 3.0
Figure captions

Figure 1. (A) AFM image of the nanocomposite MIP/PGE; (B) 3-D image (AFM) of the

nanocomposite MIP/PGE.

Figure 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, -0.10 V to

+0.50 V; Scan rate, 50 mVs-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

Figure 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+sol-gel+AuNPs* was nanocomposite MIP/PGE.

Figure 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 (l) nmol L-1 of caffeine solution. Conditions: Potential scan range, 0.0 V to +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-.

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Highlights

• A novel nanocomposite MIP recognition electrochemical sensor has been developed.

• Au nanoparticles embedded in composite to improve electrical performance of sensor.

• It was made simple and fast for sensitive and selective determination of caffeine.

• The proposed sensor can be used to determine caffeine in different real samples.