Colloids and Surfaces B: Biointerfaces 89 (2012) 175–181

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Colloids and Surfaces B: Biointerfaces

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Electropolymerized molecular imprinting on gold nanoparticle-carbon nanotube

modified electrode for electrochemical detection of triazophos
Huaifen Li, Chenggen Xie ∗ , Shanqi Li, Ke Xu
Anhui Provincial Laboratory of Biomimetic Sensor and Detecting Technology, School of Materials and Chemical Engineering, West Anhui University, Lu’an, Anhui 237012, PR China

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

Article history: An electrochemical sensor for pesticide triazophos (TAP) was prepared by deposition of gold nanoparticles
Received 10 March 2011 (AuNPs) on carbon nanotubes (CNTs) modified glassy carbon (GC) electrode surface using a potentiostatic
Received in revised form 30 May 2011 method, followed by electropolymerizing of o-hydroxyphenol at the AuNP/CNT/GC electrode surface
Accepted 7 September 2011
in the presence of template triazophos via cyclic voltammetry. The electrochemical response of tri-
Available online 14 September 2011
azophos at the TAP-imprinted polyhydroxyphenol (PHP) modified AuNP/CNT/GC (PHP/AuNP/CNT/GC)
electrode was investigated by cyclic voltammetry. The cyclic voltammetric response of triazophos at
Keywords:
the TAP-imprinted PHP/AuNP/CNT/GC electrode was significantly higher than that at bare GC, CNT/GC,
Triazophos
Carbon nanotube
AuNP/CNT/GC, imprinted PHP/CNT/GC and non-imprinted PHP/AuNP/CNT/GC electrodes. The results
Electrochemical sensor indicated that the TAP-imprinted PHP/AuNP/CNT/GC electrode can effectively improve the reductive
Molecular imprinting properties of triazophos and eliminate interferences of other pesticides. In addition, the AuNPs can
strikingly amplify the electrochemical response of triazophos and improve the sensitivity to triazophos.
Finally, the electrochemical sensor was successfully applied to determination of triazophos in vegetable
samples with satisfactory results.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction cal/physical stability of the antibodies or enzymes prevents their

Triazophos (TAP), O,O-diethyl-O-(1-phenyl-1H-1,2,4-triazol-3-
yl) phosphorothiopate, has became one of the most important
pesticides for controlling insects, acarids, and some nematodes
in many agricultural crops of China since most high-toxic and
high-residual organophosphate pesticides, for example, methami-
dophos, parathion and methyl parathion, were banned for use on
crops by the Chinese government [1]. TAP is moderately toxic
to mammals, but highly toxic to fish and honeybees [2]. There-
fore, the residue of TAP in agricultural products and natural
environment is extremely harmful to human health and zoology.
Currently, gas chromatography and high-performance liquid chro-
matography have been well-established for the detection of TAP
residue in the agricultural and environmental samples [3–6]. How-
ever, these analysis techniques require expensive instrumentation,
tedious sample pretreatment and preparation, as well as highly
trained technical personnel. On the other hand, enzyme/antibody-
based immunoassay has also been developed for the detection of
organophosphate TAP by the readout of various electrochemical
or optical transducers [7–11]. Unfortunately, the poor chemi-
∗ Corresponding author. Tel.: +86 564 3305660; fax: +86 564 3305690.
E-mail address: cgxie@wxc.edu.cn (C. Xie).
use in harsh environments of acids or bases, organic solvents and
high temperatures. Accordingly, it still remains a great challenge
to develop a rapid, inexpensive but sensitive and selective method
for the detection of TAP residue.
Recently, numerous attempts have been made to replace biolog-
ical receptors with synthetic counterparts as a recognition element
in chemo/biosensors. Of various approaches, molecular imprint-
ing technique has become a powerful tool for the preparation of
polymeric materials that have the ability to specifically bind a
chemical species [12,13]. Molecular imprinting typically involves
the copolymerization of functional and cross-linking monomers
in the presence of template molecules. The removal of template
molecules from the polymer matrix generates the recognition sites
(cavities) complementary to the shape, size and functionality of
the template [14]. Because of mechanical/chemical stability, low
cost, and easy preparation, molecularly imprinted polymers (MIPs)
have widely been used as sensitive recognition elements in chemi-
cal/biological sensors for pesticide detection [15–18]. Typically, the
MIP-based sensors are fabricated by assembling MIP materials onto
the surface of transducer, and the analyte binding is transformed
into a measurable signal. Therefore, the efficiency of the MIP-based
sensors is largely dependent on the selectivity and sensitivity of the
used MIP materials to a target species [19]. However, traditional
imprinting techniques most often produce the polymer materials

0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2011.09.010
176 H. Li et al. / Colloids and Surfaces B: Biointerfaces 89 (2012) 175–181
exhibiting high selectivity but low binding capacity, poor site acces-
sibility, and slow binding kinetics because of most imprinted sites
to be embedded in high rigid polymer matrix interior [20]. There-
fore, controlling template molecules to locate at the surface or in
the proximity of materials’ surface is critical to create more effective
recognition sites and to improve sites accessibility [21]. To achieve
surface imprinting, the simplest method is imprinting molecules at
the surface of some solid to obtain the core–shell structural MIPs
[22–24]. Of various solid support materials, carbon nanotube (CNT)
is an excellent candidate as the supported material for preparation
of the core–shell structural MIPs because of its significant mechani-
cal strength, excellent electrical conductivity, high surface area and
good chemical stability [25,26].
Herein, we report an electropolymerized molecular imprint-
ing strategy for imprinting of TAP in the core–shell polymer
shells through the electropolymerization of o-hydroxyphenol at the
AuNP-modified CNTs surface. The prepared electrochemical sensor
was characterized by scanning electron microscopy (SEM), cyclic
voltammetry. As expected, the electrochemical sensor not only can
strikingly improve the sensitivity and selectivity of TAP analysis,
but also obtains good repeatability and thus can be potentially
exploited for the detection of pesticide residuals or other delete-
rious chemicals in environment.
2. Experimental
2.1. Chemicals and reagents
Carbon nanotubes (CNTs, the purity >95%, diameter 30–60 nm,
length 0.5–15 ␮m) was purchased from Shenzhen Nanoport Co.,
Ltd. (Shenzhen, China). TAP was purchased from Fluka. Other pes-
ticides were kindly provided by Anhui Huaxing Chemical Industry
Co., Ltd. Tetarachloroaurate (III) acid (HAuCl4 ), o-hydroxyphenol
(HP) and potassium ferricyanide (K3 Fe(CN)6 ) were obtained from
Shanghai Chemicals Ltd. All other reagents were commercially
available as analytical reagent grade and used without fur-
ther purification. Pesticide stock solutions of 1.0 × 10−2 M were
prepared in acetonitrile and stored at 4 ◦ C in refrigerator to
avoid exposure to light. Phosphate buffers solution (PBS, pH 7.0,
0.1 M) was prepared from KH2 PO4 and K2 HPO4 with 0.1 M KCl
solution. Double-distilled water was used throughout the exper-
iments.
2.2. Apparatus
Electrochemical measurements were performed using an elec-
trochemical analyzer LK98B II (Tianjin, China) connected to a
personal computer. A three-electrode configuration was employed,
consisting of a bare or imprinted modified glassy carbon (GC) elec-
trode (2 mm in diameter) serving as a working electrode, while an
Ag/AgCl/saturated KCl electrode and a platinum wire (1 mm diam-
eter) served as the reference electrode and the auxiliary electrode,
respectively.
2.3. Preprocessing of CNTs
CNTs were functionalized with carboxylic acid groups by meth-
ods that reported in the literature [27]. In brief, the commercial
CNTs of 0.1 g were added with 50 mL of concentrated nitric acid
(69% HNO3 ) in a reflux process at 70 ◦ C for 6 h. The resulting CNTs
were filtrated, washed repeatedly with the double-distilled water
until the filtrate became neutral, and finally dried under vacuum.
Then 5.0 mg of the acid processed CNTs were dispersed in 10 mL
of N,N-dimethylformamide (DMF) with the aid of ultrasonic agi-
tation to give a black suspension, which was used to prepare
CNTs-modified GC (CNT/GC) electrode.
2.4. Preparation of the AuNP/CNT/GC electrode
A bare GC electrode was polished carefully to a mirror-like
surface with 0.3 ∼ 0.05 ␮m alumina aqueous slurry and then suc-
cessively washed in an ultrasonic cleaner with the double-distilled
water and ethanol. Before the modification of CNTs, the bare GC
electrode was cyclic-potential scanned within the potential range
−0.2–0.6 V in 0.01 M K3 [Fe(CN)6 ] solution containing 0.1 M KCl as
the supporting electrolyte until a pair of rather well-defined redox
peaks were obtained. To prepare CNT/GC electrode, 10 ␮L of the
CNTs-DMF suspension (0.5 mg mL−1 ) was dropped onto the surface
of GC electrode and dried under an infrared lamp for 15 min. More-
over, gold nanoparticles (AuNPs) were electrochemically deposited
onto the surface of CNT/GC electrode to obtain AuNP/CNT/GC elec-
trode. Prior to the deposition of AuNPs, CNT/GC electrode was active
in 0.2 M H2 SO4 by cyclic voltammetric scan between −0.8 and
+1.3 V for 20 cycles [28]. Then the CNT/GC electrode was immersed
into the 0.2 g L−1 HAuCl4 solution and treated by the use of a con-
stant potential of −0.2 V for 100 s [29,30].
2.5. Preparation of the imprinted PHP/AuNP/CNT/GC electrode
The AuNP/CNT/GC electrode was immersed in the aqueous elec-
trolyte solution containing 0.1 mM TAP, 0.05 M o-hydroxyphenol,
and 0.1 M NaClO4 (pH 5.5). After deoxygenating the solution by
bubbling nitrogen gas for about 15 min, the electropolymeriza-
tion was performed by 15 consecutive cyclic scan in the potential
range of −0.6 and +1.2 V at a scan rate of 50 mV s−1 . After the
electropolymerization, the imprinted polyhydroxyphenol modi-
fied AuNP/CNT/GC electrode (PHP/AuNP/CNT/GC) was rinsed with
0.5 M H2 SO4 three times for 30 min each to remove the TAP tem-
plates. Then the imprinted PHP/AuNP/CNT/GC electrode was rinsed
with double-distilled water, ethanol and dried at nitrogen for fur-
ther use. At the same time, the non-imprinted PHP/AuNP/CNT/GC
electrode was prepared and treated in the same way except that
the template molecule was omitted from electropolymerization
stage. Meanwhile, the imprinted PHP/CNT/GC electrode was also
prepared under the same chemical conditions as the imprinted
PHP/AuNP/CNT/GC electrode, but only in the absence of AuNPs.
2.6. Measurement of electrochemical properties
The imprinted PHP/AuNP/CNT/GC electrode was dipped into a
stirring 0.1 M PBS (pH 7.0) solution containing the desired con-
centration of TAP for 15 min, washed with double-distilled water
carefully to remove the possible adsorptive substances on the elec-
trode surface, and then transferred to the electrochemical cell
containing 0.1 M PBS (pH 7.0) and 0.1 M KCl solution. Before elec-
trochemical measurements, the electrolyte solution was purged
with nitrogen for 15 min. The cyclic voltammograms were recorded
between −0.6 and +1.2 V at a scan rate of 100 mV s−1 . All electro-
chemical measurements were performed at room temperature.
3. Results and discussion
3.1. Preparation of the imprinted PHP/AuNP/CNT/GC electrode
Fig. 1 illustrates the preparation procedures of the imprinted
PHP/AuNP/CNT/GC electrode. The preparation procedures were
summarized as four steps: modification with CNTs on the sur-
face of GC electrode; electrodeposition of AuNPs on the surface of
CNT/GC electrode; electropolymerization of o-hydroxyphenol on
the surface of AuNP/CNT/GC electrode; removal of the imprinting
TAP molecules from the imprinted PHP matrix. In order to obtain
a uniform layer of CNTs onto GC electrode surface, 10 ␮L of the
CNTs-DMF suspension (0.5 mg mL−1 ) was first dropped onto the
 H. Li et al. / Colloids and Surfaces B: Biointerfaces 89 (2012) 175–181
Fig. 1. Schematic illustration of molecular imprinting at the surface of AuNP/CNT/GC electrode: (1) electrodeposition of AuNPs on the surface of CNT/GC electrode; (2)
electropolymerization of o-hydroxyphenol on the surface of AuNP/CNT/GC electrode; (3) removal/rebinding of TAP on the imprinted sites of the imprinted PHP/AuNP/CNT/GC
electrode.
surface of GC electrode and dried under an infrared lamp for 15 min.
Then, the deposition of AuNPs was performed on the CNT/GC elec-
trode surface in the 0.2 g L−1 HAuCl4 solution by using a constant
potential of −0.2 V. The deposition time was used to control the
amount of AuNPs on the CNT/GC electrode for obtaining an ade-
quate coverage of AuNPs on the CNT/GC electrode surface. The ideal
deposition time was determined by measuring the voltammetric
response of corresponding electrodes in 0.01 M K3 [Fe(CN)6 ] and
0.1 M KCl solution. The results showed that the redox peak current
of Fe(CN)6 3−/4− was the strongest with the first 100 s deposition and
then decreased slowly with deposition time. Although the longer
deposition time could increase the amount of AuNPs on the CNT/GC
electrode surface, aggregations of AuNPs on the CNT/GC electrode
surface would decrease the surface area of AuNPs, leading to the
decrease of redox peak currents of Fe(CN)6 3−/4− . Therefore, 100 s
of the deposition time was used to deposit AuNPs onto the CNT/GC
electrode surface.
Furthermore, electropolymerization of o-hydroxyphenol was
conducted onto the AuNP/CNT/GC electrode surface in 0.1 M
NaClO4 (pH 5.5) solution containing 0.05 M o-hydroxyphenol and
0.1 mM TAP by the scanning potential between −0.6 and +1.2 V for
15 consecutive cycles at a scan rate of 50 mV s−1 . Fig. 2 shows repre-
sentative cyclic voltammograms for electropolymerization process
of o-hydroxyphenol on the AuNP/CNT/GC electrode surface in the
1.5
1.0
Current (10 A)
-6
0.5
0.0
-0.5
-0.5 0.0 0.5 1.0
Potential (V)
Fig. 2. Cyclic voltammograms for the electropolymerization of 0.05 M o-
hydroxyphenol at the AuNP/CNT/GC electrode surface in 0.1 M NaClO4 (pH 5.5)
solution containing 0.1 mM TAP. Scan rate: 50 mV s−1 , sweep cycle: 15.
177
present of TAP. A strong oxidation peak at ∼1.0 V and a small reduc-
tion peak at ∼0 V were clearly observed on the first scan. Then, both
the oxidative and reduction peak decreased dramatically under
continuous cyclic scan. Finally, the reductive peak currents almost
approached to zero and the anodic peak current almost decreased
to about 70% after 15 cycle scan as indicated with the arrows in
Fig. 2. Moreover, the oxidation peak potential of o-hydroxyphenol
slightly shifted to more cathodic potentials from 1.0 to 0.83 V. The
results indicate that electropolymerization of the o-hydroxyphenol
monomer occurred at the electrode surface and PHP deposited onto
the surface of the AuNP/CNT/GC electrode. The decrease of the peak
currents seems to be related with the continual formation of PHP
that hinders o-hydroxyphenol monomer further access to the sur-
face of the AuNP/CNT/GC electrode.
In addition, similar cyclic voltammograms of the electropoly-
merization processes of o-hydroxyphenol on the AuNP/CNT/GC
electrode surface in the absence of TAP had also been observed.
Meanwhile, there is not any difference in the cyclic voltammo-
grams obtained in the presence/absence of TAP template, indicating
that TAP does not exhibit any electroactivity in the potential range
chosen for the polymerization and its structure was not electro-
chemically altered during electropolymerization.
In order to confirm whether PHP had been deposited
onto the AuNP/CNT/GC electrode surface, the morphologies of
AuNP/CNT/GC electrode before and after the electropolymeriza-
tion of o-hydroxyphenol were observed under scanning electron
microscope (SEM). From the SEM image of the AuNP/CNTs/GC in
Fig. 3(A), the obvious tube-shaped structure of nanotubes could
be distinguished one by one and the tips of nanotubes could be
clearly revealed as indicated with the arrows in Fig. 3(A). Fur-
thermore, their average diameter is approximately 50 nm, and the
surface in the side walls seems to be smooth. Interestingly, under
a high-magnification SEM of the AuNP/CNT/GC electrode, some
small light particles attaching to the nanotubes can be observed as
shown in the inset of Fig. 3(A), indicating that AuNPs had deposited
onto the CNTs surface. However, after electropolymerization of o-
hydroxyphenol was carried out on the surface of AuNP/CNT/GC
electrode, the CNTs could still be distinguished one by one, but the
diameters of the CNTs were slightly larger than those of CNTs in
AuNP/CNT/GC electrode, indicating that the polymer of PHP was
formed and wrapped around the surface of the CNTs. In addition,
a much rougher surface of the CNTs could be observed from the
SEM image of Fig. 3(B), which further supports the formation of
the PHP onto the CNTs surface. The highly selective occurrence
of the electropolymerization at the CNTs surface can be under-
stood from the imprinting synthesis strategies. In this work, CNTs
were first functionalized with carboxylic acid groups by treatment
with 69% HNO3 in a reflux process at 70 ◦ C for 6 h. Therefore, when
AuNP/CNT/GC electrode was immersed into electropolymerization
178 H. Li et al. / Colloids and Surfaces B: Biointerfaces 89 (2012) 175–181
Fig. 3. The typical SEM images of the CNTs (A) and the imprinted PHP/AuNP/CNT/GC
electrode (B) (inset is a high-magnification SEM image of AuNP/CNT/GC electrode).
solution, o-hydroxyphenol molecules could be adsorbed on the sur-
face of CNTs because of the strong hydrogen bond interactions
between the carboxylic group of CNTs and hydroxyl group of o-
hydroxyphenol molecules. The adsorbed o-hydroxyphenol on CNTs
surface can serve as an initial polymerizable monolayer to improve
the “wetting” of the surface by the polymer, which drives the selec-
tive occurrence of electropolymerization at the surface of CNTs.
On the other hand, CNTs could act as electronic wire to transfer
electrons between the o-hydroxyphenol and the electrode because
of their superior properties such as electron transfer promotion,
electrocatalytic capability and high surface-to-area ratio with high
electroactivity [31]. Therefore, during the electropolymerization
process of o-hydroxyphenol, CNTs can provide electrochemical
reaction sites, which drive the selective occurrence of electropoly-
merization at the surface of CNTs and results in PHP wrapped
around the CNTs surface.
In order to examine whether TAP molecules had been embed-
ded in the imprinted PHP matrix, cyclic voltammograms of
the imprinted PHP/AuNP/CNT/GC electrode before and after
the removal of the imprinted TAP molecules as well as the
non-imprinted PHP/AuNP/CNT/GC electrode were respectively
recorded in the N2 -saturated PBS (0.1 M, pH 7.0) containing 0.1 M
KCl. Fig. 4 shows a typical comparison of cyclic voltammograms
60
40
20
Current (10 A)
-6
0
a
c
-20
b
-40
-60
-0.5 0.0 0.5 1.0
Potential (V)
Fig. 4. Cyclic voltammograms of non-imprinted PHP/AuNP/CNT/GC electrode (a),
the imprinted PHP/AuNP/CNT/GC electrode before (b) and after (c) removal of the
imprinted TAP molecules in the N2 -saturated PBS (0.1 M, pH 7.0) containing 0.1 M
KCl as the supported electrolyte. Scan rate: 100 mV s−1 .
among the different electrodes. In the case of the non-imprinted
PHP/AuNP/CNT/GC electrode, no redox peak can be observed (Fig. 4,
curve a). However, a very sharp and well-defined reductive peak
at ∼0.45 V and a broad and small oxidation peak at ∼0.98 V
were clearly recorded with the imprinted PHP/AuNP/CNT/GC elec-
trode before the removal of the imprinting TAP molecules in the
potential range of −0.6 to 1.2 V (Fig. 4, curve b). Since the elec-
trochemical measurements were carried out in TAP-free solutions,
it implies that the oxidization–reduction peaks were entirely due
to the redox of TAP molecules which had been embedded into
the imprinted PHP matrix. From the imprinting strategies used
in this work, o-hydroxyphenol was selected as an electropoly-
meric functional monomer for non-covalently imprinting the TAP
molecules because of its strong non-covalent interaction with TAP
molecules. UV absorption spectra suggested that the formation of
hydrogen bonding interactions between the hydroxyl group of o-
hydroxyphenol and nitrogen/oxygen atom of TAP in the solution
of the electropolymerization precursors (data not shown). There-
fore, during the electropolymerization process, TAP molecules were
strongly absorbed to the electrode surface through hydrogen bond-
ing interactions, and then TAP molecules were embedded in the
imprinted PHP matrix to give rise to the recognition sites (cavities)
complementary to the molecular shape, size and functionality of
TAP molecules.
Furthermore, the TAP embedded in the imprinted PHP matrix
could be efficiently removed from the PHP matrix by rinsing with
0.5 M H2 SO4 three times for 30 min each. As shown in Fig. 4, curve
c, the characteristic oxidation–reduction peak of TAP molecules
disappeared completely after rinsing process and the cyclic voltam-
mogram became almost identical to that of the non-imprinted
PHP/AuNP/CNT/GC electrode (Fig. 4, curve a). Therefore, electro-
chemical polymerization of o-hydroxyphenol can give rise to the
recognition sites complementary to the molecular shape, size and
functionality of TAP molecules.
3.2. Electrochemical behavior of TAP at the imprinted
PHP/AuNP/CNT/GC electrode
The electrochemical response of the imprinted PHP/AuNP/CNT/
GC electrode to TAP was compared with the bare GC, CNT/GC,
 H. Li et al. / Colloids and Surfaces B: Biointerfaces 89 (2012) 175–181 179

30 0.43 V with modification of the imprinted PHP, which may result

from the catalytic activity of imprinted PHP matrix to the redox of
A TAP entering in the imprinted sites. With regard to the sensitivity
20 of determination of TAP, the reductive peak at 0.43 V was selected
for quantitative analysis because this peak was the most separated
and intense.
10 Moreover, the imprinted PHP/CNT/GC electrode exhibited a
Current (10 A)

well-defined reductive peak at ∼0.43 V and a broad oxidization

-6

peak at ∼0.95 V to 5.0 ␮M TAP (Fig. 5, curve e). With a compari-

0
a son with the imprinted PHP/AuNP/CNT/GC electrode, the reductive
peak current was decreased greatly by approximately 2.8-fold,
b indicating that the sensitivity of the MIP-based sensor could be
-10
c remarkably improved by the deposition of AuNPs onto the sur-
face of CNTs. A much higher sensitivity to TAP by the imprinted
-20 PHP/AuNP/CNT/GC electrode should be mainly attributed to two
advantages: (i) the AuNPs could enhance the electrical conduc-
tivity of MIP-based sensor and have good electrocatalytic activity
-30 towards the redox of TAP and (ii) the AuNPs could increase surface
-0.5 0.0 0.5 1.0 area of CNT/GC electrode and thus improve the amount of effective
Potential (V) imprinted sites on the sensor surface which would greatly improve
the enrichment of electrode to TAP species in solution.
Furthermore, the electrochemical response of the non-
B imprinted PHP/AuNP/CNT/GC electrode to TAP had also been
50
examined. Under the same conditions, there was no redox signal
of TAP observed at the non-imprinted PHP/AuNP/CNT/GC electrode
(Fig. 5, curve f). The high electrochemical response of the imprinted
PHP/AuNP/CNT/GC electrode might result from the imprinted cav-
Current (10 A)

0 ities in the PHP matrix and the functional groups on the cavities
-6

f produced by the template TAP molecules. Therefore, the imprinted

PHP matrix shows much higher binding properties to TAP than
non-imprinted ones.
e
-50 3.3. Optimization of conditions for the electrochemical sensor

It is well-known that the sensitivity of the MIP-based sensor

d is dictated by the amount of effective imprinted sites on the sen-
-100
-0.5 0.0 0.5 1.0
Potential (V)
Fig. 5. Cyclic voltammograms of the bare GC electrode (a), CNT/GC electrode (b),
AuNP/CNT/GC electrode (c), imprinted PHP/AuNP/CNT/GC electrode (d), imprinted
PHP CNT/GC electrode (e), and non-imprinted PHP/AuNP/CNT/GC electrode (f) in the
presence of 5.0 ␮M TAP in 0.1 M PBS (pH 7.0) and 0.1 M KCl. Scan rate: 100 mV s−1 .
AuNP/CNT/GC, non-imprinted PHP/AuNP/CNT/GC, and imprinted
PHP/CNT/GC electrode. Fig. 5 shows the cyclic voltammo-
grams of the bare GC, CNT/GC, AuNP/CNT/GC, non-imprinted
PHP/AuNP/CNT/GC, and imprinted PHP/CNT/GC electrode to
5.0 ␮M TAP. In the case of the bare GC electrode, no redox
peak can be observed (Fig. 5, curve a), suggesting that TAP is
non-electrochemically active on the bare GC electrode over this
potential range. However, a broad and small reductive peak of TAP
at ∼0.48 V was clearly observed with the CNT/GC electrode (Fig. 5,
curve b), and the reductive peak could be obviously increased
after the CNT/GC electrode was electrodeposited with the AuNPs
(Fig. 5, curve c). The results may be due to the large surface area
and good electrocatalytic activity of CNTs and AuNPs. Furthermore,
after the imprinted PHP were modified onto the AuNP/CNT/GC elec-
trode surface, a very strong and well-defined reductive peak at
∼0.43 V and a broad and small oxidization peak at ∼0.90 V were
clearly observed (Fig. 5, curve d). The reductive peak current of the
imprinted PHP/AuNP/CNT/GC electrode is about 10-fold compared
to that of the AuNP/CNT/GC electrode. Meanwhile, the reductive
peak potential shifted to more negative potentials from 0.48 to
sor surface. Although the amount of the imprinted sites increases
with the increase in the thickness of MIP layer, thick MIP layer
would lead to low binding capacity, poor site accessibility, and
slow binding kinetics. To control the moderate thickness of MIP
layer is a critical parameter for improving the sensitivity of MIP-
based sensor. The thickness of MIP layer can easily be adjusted
by controlling the number of cycles during electropolymeriza-
tion process. In order to determine the optimal MIP thickness,
the cyclic voltammetric response of 5.0 ␮M TAP at the imprinted
PHP/AuNP/CNT/GC electrode with different scanning cycles in
the electropolymerization process was respectively recorded. The
cyclic voltammetric response to TAP obviously rises and reaches
a maximum with the 15 cycles and then decreases with a further
increase in cycle number. Therefore, 15 cycles can obtain a suit-
able thickness of MIP layer to provide the highest sensitivity to TAP
analyte.
The accumulation step is usually a simple and effective way to
enhance the sensitivity of the MIP-based sensor [32]. After tem-
plate molecules were removed from the imprinted PHP matrix, the
imprinted PHP/AuNP/CNT/GC electrode was incubated in a stirring
0.1 M PBS (pH 7.0) containing 5.0 ␮M TAP for different incubation
time, the cyclic voltammetric responses were respectively recorded
in the N2 -saturated PBS (0.1 M, pH 7.0) containing 0.1 M KCl and
then reductive peak currents at 0.43 V were respectively recorded.
The results show that the reductive peak current increased rapidly
with the increase in the incubation time at the initial stage, and
a stable response was obtained after 15 min, suggesting that the
adsorption equilibrium was reached. Thus, the optimum incuba-
tion time should be 15 min for the electrochemical determination
of TAP.
180 H. Li et al. / Colloids and Surfaces B: Biointerfaces 89 (2012) 175–181
Table 1
Analytical results of triazophos (TAP) in the vegetable samples (n = 7).
Sample Content of TAP (␮M) TAP added (␮M)
Celery None 0.5
2.0
Lettuce None 0.5
2.0
Spinage None 0.5
2.0
Cabbage None 0.5
2.0
3.4. Selectivity and stability of the electrochemical sensor
The selectivity of the MIP-based sensor to TAP was eval-
uated by testing its cyclic voltammetric responses in the
presence of some possible interfering substances including
methyl parathion, methamidophos, chlorpyrifos, cartap, and 2,4-
dichlorophenoxy acetic acid, respectively. The selectivity of the
imprinted PHP/AuNP/CNT/GC electrode to TAP molecules was eval-
uated by calculating the peak current ratio (Is /I0 ), where Is and I0
were reductive peak currents of 5.0 ␮M TAP at 0.43 V in the present
and absence of interfering substances. It was found that 10-fold
excess of methyl parathion, methamidophos, chlorpyrifos, cartap,
and 2,4-dichlorophenoxy acetic acid over TAP hardly caused the
significant change in peak current of TAP, in which peak current
ratio (Is /I0 ) only slightly varied from 0.91 to 1.05. These results
indicate that the imprinted PHP/AuNP/CNT/GC electrode showed
higher recognition selectivity for TAP than for other pesticides. This
may be explained by the fact that the delicate recognition sites of
TAP molecules in the imprinted PHP matrix have the capability to
recognize target molecules through their size, shape and functional
group distribution. Therefore, application of the MIP as recognition
elements, combined with the electrochemical detection, may allow
the detection of TAP from a complex matrix without separation.
On the other hand, the reproducibility of the measurements
was evaluated by measuring the cyclic voltammetric responses of
5.0 ␮M TAP at the same imprinted PHP/AuNP/CNT/GC electrode.
The result indicates that the imprinted sensor shows excellent
reproducibility, with the relative standard deviation (RSD) for
seven successive determinations of being 2.3%. The stability of the
imprinted PHP/AuNP/CNT/GC electrode was investigated over a 30
day period by measuring the voltammetric responses to 5.0 ␮M TAP
with intermittent usage (every 3 days) and by storing under des-
iccated conditions at the room temperature when not in use. The
results showed that the voltammetric response of the imprinted
PHP/AuNP/CNT/GC electrode only decreased to ∼95% after storing
for 30 days. Furthermore, our detailed experiments reveal that the
voltammetric response of TAP at the imprinted PHP/AuNP/CNT/GC
electrode hardly changed after the imprinted electrode was used at
least 50 times with subsequent washing and measuring operations.
3.5. Analytical performance of the electrochemical sensor
Under the optimal experimental conditions described above,
the analytical performance of the electrochemical sensor to TAP
was examined. It showed that the reductive peak current at 0.43 V
was linearly proportional to the TAP concentration in the range
of 2.0 × 10−7 to 1.0 × 10−5 M. The linear regression equation was
I (␮A) = 17.1 + 16.4C (␮M), with a correlation coefficient of 0.9984.
The detection limit was estimated to be 9.3 × 10−8 M based on a
signal-to-noise of 3. However, with the imprinted PHP/CNT/GC as
a working electrode, the linear range, the detection limit and the
standard deviation for 1.0 × 10−5 M TAP were from 1.0 × 10−6 to
3.0 × 10−5 M, 8.2 × 10−7 M and 3.1% (n = 7), respectively. Therefore,
TAP found (␮M) Recovery (%) RSD (%)
0.46 ± 0.02 92 4.1
1.92 ± 0.07 96 3.5
0.47 ± 0.02 94 3.6
2.06 ± 0.06 103 2.8
0.53 ± 0.02 106 3.1
1.94 ± 0.05 97 2.3
0.49 ± 0.02 98 3.3
2.14 ± 0.06 107 2.9
the detection limit for TAP by the imprinted PHP/AuNP/CNT/GC
electrode is about 1 order of magnitude lower than that by the
imprinted PHP/CNT/GC electrode.
3.6. Analytical application of the electrochemical sensor
The established method was utilized for the determination
of TAP in vegetable samples. Vegetable samples (celery, lettuce,
spinage and cabbage) were obtained from the local markets. 100 g
of each sample were grinded and dispersed into 100 mL of double-
distilled water for 24 h, respectively. The samples were extracted
with 20 mL of ether and the extracts were evaporated to dryness.
Then, 2 mL of acetonitrile was added to the dry residue and diluted
to 100 mL with 0.1 M PBS (pH 7.0). In order to perform the recov-
ery test, a known amount of TAP standard solution was diluted to
suitable concentration with the extract solution of the vegetable
samples. The solutions obtained in this way were used as analyt-
ical solutions and determined using electrochemical sensor. The
analytical results are shown in Table 1. Obviously, the recoveries
from the vegetable samples were excellent, and varied from 92%
to 107%. Therefore, the method was applicable to detect TAP in
vegetable samples without the pretreatment of samples.
4. Conclusion
In this work, we have demonstrated a sensitive electrochemical
sensing protocol for the detection of organophosphate pesti-
cide TAP by an electropolymerized molecular imprinting strategy
for imprinting TAP in the electropolymerized PHP layers at the
AuNP/CNT/GC electrode surface. The cyclic voltammetric response
of TAP at the imprinted PHP/AuNP/CNT/GC electrode is about 10-
and 2.8-fold that of the AuNP/CNT/GC electrode and the imprinted
PHP/CNT/GC electrode, respectively. Moreover, the imprinted
PHP/AuNP/CNT/GC electrode also shows specific recognition selec-
tivity for TAP molecules over other pesticides. Therefore, this novel,
facile strategy reported herein can be further expected to fabricate
various electrochemical sensors for detecting the pesticide residu-
als and other environmentally deleterious chemicals.
Acknowledgment
This work was supported by the National Natural Science Foun-
dation of the People’s Republic of China (no. 20875070) and Science
and Technology Project of Anhui Province (no. 1106c0805007).
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