Sensors and Actuators B 208 (2015) 299306

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Sensors and Actuators B: Chemical

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

A new biomimetic sensor based on molecularly imprinted polymers

for highly sensitive and selective determination of hexazinone
herbicide
Maricely Janette Uria Toro, Luiz Diego Marestoni, Maria Del Pilar Taboada Sotomayor
Department of Analytical Chemistry, Institute of Chemistry, State University of So Paulo (UNESP), 14801-970 Araraquara, SP, Brazil

a r t i c l e

i n f o

Article history:
Received 17 April 2014
Received in revised form 18 October 2014
Accepted 8 November 2014
Available online 15 November 2014
Keywords:
1,3,5-Triazine
Hexazinone
Molecularly imprinted polymer
Carbon paste sensor
Molecular modeling

a b s t r a c t
A new selective sensor based on molecularly imprinted polymers (MIPs) was developed for the determination of hexazinone (HXZ) in environmental samples. MIPs were synthesized using a non-covalent
approach, and selection of the monomers employed in the polymerization reaction was carried out by
molecular modeling. Three functional monomers with high (2-vinylpyridine (MP17)) and intermediate
(methacrylic acid (MP12) and acrylamide (MP5)) energies of binding to the template (HXZ) were selected
for preparation of the MIPs, in order to conduct comparative studies and validate the theoretical data. For
sensor construction, carbon pastes were modied with each MIP or NIP (non-imprinted polymer), and
HXZ determination was performed using differential pulse adsorptive cathodic stripping voltammetry
(DPAdCSV). All parameters affecting the sensor response were optimized. In HCl at pH 2.5, the sensor
prepared with MP17 (5% w/w in the paste) showed a dynamic linear range between 1.9 1011 and
1.1 1010 mol L1 , and a detection limit of 2.6 1012 mol L1 , under the following conditions: accumulation time of 200 s at a potential of 0.5 V, scan rate of 50 mV s1 , pulse amplitude of 60 mV, and
pulse width of 50 ms. The sensor was selective in the presence of other similar compounds, and was
successfully applied to the analysis of HXZ in river water samples.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Herbicides that act as inhibitors of photosystem II are widely
used in agriculture, since they provide effective weed control at
low cost. This group accounts for about 30% of the total quantity
of herbicides currently used in agriculture. They encompass several classes of compounds, including triazines (atrazine, simazine,
and cyanazine), diazines (bentazone), triazinones (hexazinone
and metribuzin), phenylureas (diuron and tebuthiuron), phenols
(bromoxynil), and phenylamide. These harmful substances are
commonly found in surface and underground waters, as well as
in agricultural products, and their detection is important in terms
of health care and on economic grounds [1,2].
Identication and quantication of pesticides is routinely carried out by high performance liquid chromatography, with either
mass spectrometric or diode array detection [35], as well as
by gas chromatography with atomic emission or mass spectrometric detection [6]. Many of these methods are accurate and

Corresponding author.
E-mail address: mpilar@iq.unesp.br (M.D.P.T. Sotomayor).
http://dx.doi.org/10.1016/j.snb.2014.11.036
0925-4005/ 2014 Elsevier B.V. All rights reserved.

selective, but require relatively expensive instrumentation and

involve high consumption of reagents, which render them expensive or time consuming. Moreover, pesticides are usually found in
trace amounts in water, so that extraction or pre-concentration
steps must be performed prior to analysis [7].
Hexazinone (Chart 1) is the active compound of the herbicide
Velpar , which is used to control a wide variety of broadleaf weeds,
grasses, and woody plants in nurseries and forestry plantations. It
is also used in sugar cane and pineapple plantations, pasture, and
industrial sites [8].
Hexazinone is a soft base and is a pervasive ground water contaminant due to its water solubility [8]. In the literature, discussion
of the chemistry of the reduction reaction of substituted 1,3,5triazines in aqueous media is restricted to observations of reduction
waves and identication of the optimum conditions for their determination [9].
Molecularly imprinted polymers (MIPs) have been created to
mimic (to a certain degree) biological receptors. They possess
molecular recognition sites designed for a specic molecule. Polymer complexes are formed between the template and one or more
functional monomers in an appropriate solvent. Polymerization
then occurs in the presence of a cross-linker, and further removal

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Chart 1. Chemical structure of the pesticide hexazinone. The color correspondence

is as follows: white (H); gray (C); blue (N); red (O); pink (pair of free electrons from
N). (For interpretation of the references to color in this gure legend, the reader is
referred to the web version of this article.)

of the template yields the expected polymer [10]. In most cases, the
template is the analyte of interest. However, where the analyte may
be degraded during the polymerization process, due to its thermal
instability or photodegradability, a template molecule having similar chemical structure to the analyte but greater stability is used
in the synthesis of the MIP.
Molecular modeling and computational screening techniques
have been used to identify functional monomers capable of interacting with several different photosynthesis-inhibiting herbicides.
The process involved the design of a virtual library of molecular
models of functional monomers containing polymerizing residues
and residues able to interact with the template by means of electrostatic, hydrophobic, van der Waals, and dipoledipole interactions
[1].
Recent reports have described voltammetric sensors based on
carbon paste electrodes modied with MIPs for various analytes
such as parathion [11], para-nitrophenol [12], 2,4,6-trinitrotoluene
[13], and promethazine [14]. A computational approach was used to
design MIPs for cyanazine as template molecule [15], and the reduction process of the analyte was followed by its selective extraction
using a carbon paste electrode and stripping voltammetry (SV).
This work presents a new biomimetic sensor based on molecularly imprinted polymers for determination of the herbicide
hexazinone. The MIP containing recognition sites for hexazinone
was prepared with a functional monomer selected by means of
molecular modeling.
2. Materials and methods
2.1. Reagents and solutions
The pesticide standards (hexazinone, atrazine, s-atrazine,
desethyl atrazine, and metribuzin), functional monomers (FM)
(acrylamide (AA), 2-vinylpyridine (2-VP), methacrylic acid (MAA),
and 2,2-azobisisobutyronitrile (AIBN)), mineral oil, and graphite
powder (particle size < 150 m, 99.9% purity) were all acquired
from Sigma-Aldrich. Ethylene glycol dimethacrylate (EGDMA) was
obtained from Fluka. Hydrochloric acid and sodium hydroxide were
purchased from Synth. Other chemicals were of analytical grade
and were purchased from Merck. A 1.0 103 mol L1 stock solution of hexazinone was prepared in a mixture of distilled water
and N,N-dimethylformamide (50%, v/v). All buffer solutions were
prepared with deionized water (resistivity 18 M cm at 25 C)
obtained from a Milli-Q Direct-0.3 purication system.
2.2. Computational approach
The computer simulations employed HyperChem v. 8.0.5 software for modeling the molecules to be used in the preparations

of MIPs. The OpenEye program VIDA v. 3.0.0 was used to visualize the modeled molecules and check for possible errors in the
bonds between atoms. OpenEye Omega 2 was used to generate
different conformers of molecules, in order to identify possible
spatial hindrances. By default, 10 conformers of each pesticide
were generated, and three were randomly chosen for use in
the simulations. The OpenEye program Szybki v. 1.2.2 was used
to take the molecule to its lowest (and therefore most stable)
energy state. The Autolt v. 3.3.6.0 application was used to generate command lines (scripts) to automate repetitive processes
during the molecular modeling, hence eliminating human errors
during the procedures. Multiple Minima Hypersurfaces (MMH)
with MOPAC (Molecular Orbital Package) 2009 software was used
for the approach of the monomers to the molecule used as the
template (HXZ), calculating the energy released and the resulting
afnity.
The simulations were performed to evaluate the interaction
of hexazinone with twenty monomers contained in the simulation program (Table 1). The trials were performed in duplicate in
order to obtain reliable results. For each monomer, three different conformers were used in order to ensure accurate calculation
of the interaction energies between the different monomer conformations and the analyte. The duration of each simulation was
approximately 26 h.
2.3. Preparation of the molecularly imprinted polymers (MIPs)
The template (HXZ) molecule (0.1 mmol) was mixed for
10 min with the selected functional monomer (0.4 mmol) in a
10.0 mL screw capped glass vial, followed by addition of 30 mL of
dichloromethane as polymerization solvent. Subsequently, EGDMA
(2 mmol) and the initiator (AIBN, 0.2 mmol) were added to the rst
solution. Dissolved oxygen was removed from the solution by purging with high purity nitrogen for 10 min. Finally, the test tube was
sealed under the nitrogen atmosphere and placed in a water bath at
60 C for 24 h. The polymer obtained was broken up, mechanically
ground, and sieved to 2038 m particle size. The template (HXZ)
molecule was extracted from the polymers with a methanol/acetic
acid mixture (90:10, v/v) in a Soxhlet extraction system for two
days. A reference non-imprinted polymer (NIP) was prepared using
the same procedure, but in the absence of the template (HXZ)
molecule.
Three functional monomers were used in MIP preparation,
based on the theoretical data. These were 2-vinylpyridine (MP17),
methacrylic acid (MP12), and acrylamide (MP5).
For comparative purposes, two other MIPs were also synthesized, using 2-VP monomers with the metribuzim (MTZ) and
atrazine (ATZ) molecules as templates.
2.4. Characterization and adsorption experiment
The morphology of the products was characterized by scanning
electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The BrunauerEmmettTeller (BET) method was
used to obtain the pore size distributions and surface areas of the
washed polymers.
The adsorption capacities of the MIPs for hexazinone were
studied by obtaining UV absorbance spectra at 242 nm. For
this, 5 mg of polymer were added to a 10 mL ask containing
5.0 mL of hexazinone solution (at concentrations of 2200 mg L1 ).
After being shaken for 12 h at room temperature, the samples
were centrifuged and ltered. The free hexazinone concentration
in the ltrate was determined by HPLC [16]. The adsorption of hexazinone by the NIPs was measured in a similar
manner.

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301

Table 1
Sensors based on MIPs and their analytical characteristics in quantication of triazines and other pesticides.
Analyte

Atrazine

Cyanazine

Desmetryn

Parathion
Propazine
Hexazinone

Sensor construction

Linear response range

(mol L1 )

Detection limit (mol L1 )

Ref.

Electropolymerization of thiophene, EDOT, and AAT

onto a Pt electrode
Direct polymerization by UV irradiation of the
atrazine-imprinted polymer composed of MAA and
EGDMA on the surface of a gold electrode modied
with allyl mercaptan and 1-butanethiol
Conductimetric sensors prepared with molecularly
imprinted polymer membranes obtained by
copolymerization of MAA and TEGDMA
MIP prepared by bulk procedure and modifying an Ag
electrode with 5 L of a solution prepared with MIP,
THF, and PVC. The measurements were carried out
with a thickness-shear mode (TSM) acoustic sensor
MIP prepared by bulk procedure and modifying a
carbon paste using a cathodic stripping voltammetric
method
Grafting polymerization to obtain thin lms of MIP on
the surface of polypropylene membranes on
hydrophobized gold electrodes and UV irradiation
Casting deposition of 4 L of a mixture of MIP-PEI/SiO2
and chitosan onto the surface of a GCE
MIP prepared by bulk procedure and modifying a
carbon paste using cathodic stripping voltammetry
MIP prepared by bulk procedure and modifying a
carbon paste by cathodic stripping voltammetry

Data not shown

1.0 107

[19]

1 106 to 1 105

Data not shown

[20]

Data not shown

5.0 109

[21]

Data not shown

2.0 106

[22]

5.0 109 to 1.0 106

3.2 109

[15]

Data not shown

Data not shown

[23]

5.15 108 to 5.15 105

(0.01515 mg kg1 )
1.0 108 to 1.0 106 and
1.055.0 106
1.9 1011 to 1.1 1010

1.03 108 (0.003 mg kg1 )

[24]

1 10

[25]

2.6 1012

This work

EDOT: 3,4-ethylenedioxythiophene; AAT: acetic acid thiophene; MAA: methacrylic acid; EGDMA: ethylene glycol dimethacrylate; TEGDMA: tri(ethylene glycol) dimethacrylate; THF: tetrahydrofuran; PVC: polyvinylchloride; PEI: polyethyleneimine; SiO2 : silica gel; GCE: glassy carbon electrode.

2.5. Analytical system for the determination of hexazinone

Electrochemical measurements were carried out using a micropotentiostat (Autolab) consisting of a conventional three-electrode
electrochemical cell containing a commercial Ag/AgCl,KClsat reference electrode (Analion), a platinum wire coil as the counter
electrode, and the modied carbon paste as the working electrode.
The electroanalytical techniques used were cyclic voltammetry for
characterization of the proposed sensor, and differential pulse with
cathodic stripping voltammetry for the quantitative analysis of
hexazinone.
2.6. Construction of the biomimetic sensor
The unmodied carbon paste was prepared by thoroughly mixing graphite and parafn oil, in a ratio of 70:30 (%, w/w). The
paste obtained was then inserted into the cavity (3 mm i.d., 1 mm
depth) of the working electrode, where a Pt disk was used to
provide the electrical contact. The modied carbon paste was prepared by mixing different percentages of graphite powder, parafn
oil, and the MIP for hexazinone (or its corresponding NIP reference polymer). This mixture was homogenized in a mortar for
at least 10 min, and then packed into the same working electrode described above. The electrode surface was polished with
sulte paper to ensure a reproducible working surface. The electrochemical behavior of hexazinone at these different electrodes was
studied using the cyclic voltammetry technique. The best results
were obtained with a 66.7:28.5:4.8 (w/w) ratio of graphite powder/parafn oil/MIP17. This optimized electrode composition was
then used for the voltammetric determination of hexazinone.
2.7. General analytical procedure
A 10.0 mL volume of HCl at pH 2.5 was introduced into the
electrochemical cell, and the solution was bubbled with anhydrous nitrogen for 10 min. An accumulation potential of 0.5 V was
applied to the electrode for 200 s, while the solution was stirred.

The stirring was then stopped, and the solution was allowed to rest
for 10 s before recording differential pulse voltammograms in the
potential range from 0.6 to 1.6 V. Subsequently, an appropriate
volume of sample solution was added to the voltammetry cell, the
solution was bubbled with anhydrous nitrogen for 3 min, and the
voltammogram was recorded after 200 s accumulation at 0.5 V.
2.8. Study of sensor selectivity
The selectivity of the sensor was evaluated for atrazine,
s-atrazine, desethyl atrazine, and metribuzin, as well as for hexazinone. The concentrations of all the pesticides used in the
measurement cell were 100 pmol L1 .
2.9. Sensor application using environmental samples
Water samples were collected from the Tiet River in the central
region of the State of So Paulo (Brazil). After collection, the samples
were enriched with hexazinone at a concentration of 109 mol L1 ,
and were then analyzed using the proposed biomimetic method
in order to calculate the recoveries. The nal concentration in the
measurement cell was around 1011 mol L1 .
3. Results
3.1. Computer simulations and monomer interactions
The values obtained in the computer simulations (Fig. 1) can
be interpreted as the energy released on approach between each
monomer and hexazinone. The greater the energy released (negative value), the higher is the afnity between them, and the
resulting polymer should be more selective. It can be observed
from the results in Fig. 1 that the monomers most suitable for
hexazinone were (in order) methylene succinic acid (MP11) and 2vinylpyridine (MP17). Monomers with intermediate energy were
acrylamide (MP5) and methacrylic acid (MP12), and monomers

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-140

simulations, with a higher current being observed for the sensor

prepared with MIP17 using 2-VP as the functional monomer. The
only discrepancy in these results was observed for MIP5 and MIP12,
for which the experimental sensor response was inverted, with a
higher current for MIP5 and a lower current for MIP12. The subsequent experiments were therefore carried out using MIP17.

-120

EB / kJ mol

-1

-100
-80

3.2. Characterization of MIP17

-60

The use of FTIR spectroscopy to evaluate polymeric materials provides information about the extent of incorporation of
monomers into the polymeric network. The FTIR spectra of EGDMA

-40

M
P1
M 7
P1
M 1
P1
3
M
P
M 2
P1
6
M
P7
M
P9
M
P
M 5
P1
M 0
P1
M 8
P1
M 4
P2
M 0
P1
M 2
P1
M 5
P1
9
M
P8
M
P1
M
P3
M
P4
M
P6

-20

(A)

100

Transmitance (%)

Functional monomers
Fig. 1. Calculated interaction energies (EB /kJ mol1 ) for hexazinonemonomer complexes.

MIP17
MIP5
MIP12

-6

9x10

80
60

C=C
1640

40
C=O
1700

20

EGDMA
0

-6

6x10

|i| / A

4000

3000

2000

1000

-1

Wavenumber (cm )
-6

3x10

(B)

-1.6

-1.4

-1.2

-1.0

E vs Ag|AgCl / V
Fig. 2. Differential pulse cathodic stripping voltammetry analyses of 250 nmol L1
hexazinone using sensors constructed with MIP17, MIP5, and MIP12. Conditions: pH
2.5 (HCl); Eac = 0.5 V; tac = 200 s; scan rate = 50 mV s1 ; pulse amplitude = 100 mV;
pulse width = 50 ms.

Transmitance (%)

100

1500
60

40

MIP-2-VP

1731

4000

3000

2000

1000

-1

Wavenumber (cm )

(C)

100

Transmitance (%)

with low interaction energy were acrolein (MP6), ethyl ester

imidazole-4-acrylic acid (MP3), and acrylic acid (MP4).
It is important to emphasize that the computational simulation
was carried out in vacuum, which is a medium completely different to the experimental situation. However, previous work by
our research group has shown that computer simulation helps in
selection of the best functional monomer to obtain highly selective
MIPs suitable for use in different applications (such as adsorbent
materials for solid phase extraction). In the present case, the graphs
(Figs. 1 and 2) showed that the theoretical results were in agreement with the experimental behavior of the sensor.
The structure of hexazinone (Chart 1) includes the presence of
nitrogen atoms with free electrons, so hexazinone can be considered a soft base with preference for molecules that present acidic
and neutral characteristics. In light of this, the accuracy of the
theoretical calculations was evaluated by preparation (using precipitation polymerization) of three MIPs for hexazinone, using MP5,
MP12, and MP17 (2-VP) as functional monomers. The MIPs were
used to prepare modied carbon paste electrodes, and these sensors were applied to quantify HXZ using the differential pulse with
cathodic stripping voltammetric technique. It can be seen from
Fig. 2 that the experimental results agreed with the theoretical

2940

80

2940
80
1500
60
NIP-2-VP
4000

1731
3000

2000

1000

-1

Wavenumber (cm )
Fig. 3. FTIR spectra of (A) EGDMA, (B) MIP17 prepared with 2-vinylpyridine (2-VP),
and (C) NIP17 prepared with 2-VP.

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303

Q =

(C0 C)V
M

(1)

where C0 is the initial hexazinone concentration (mg L1 ), C is the

hexazinone concentration after adsorption, V is the volume of the

60

MIP
NIP

-1

and the imprinted (MIP17) and non-imprinted (NIP17) polymers

are shown in Fig. 3AC, respectively.
The infrared spectrum of the EGDMA crosslinking agent (Fig. 3A)
showed a characteristic band in the region of 1700 cm1 , corresponding to axial deformation stretching of the C O ester group,
while bands in the range 16701640 cm1 corresponded to stretching of the C C (vinyl groups) present in EGDMA. A characteristic
band of the amide group could be seen at 1731 cm1 (Fig. 3B and
C), and a band at 2960 cm1 was characteristic of carbon bonded to a
primary or secondary amine, indicating formation of the polymer.
The characteristic bands of the monomers used in the synthesis,
which could be seen in the spectra of the MIP and NIP polymers, were those of EGDMA and the 2-VP monomer at around
1700 cm1 , corresponding to C O stretching of the ester group,
and at 1550 cm1 , due to axial deformation of the ester. In addition,
there was an absence of bands at 16701640 cm1 corresponding
to stretching of the vinyl groups of the monomer. For all the polymers, these disappeared (Fig. 3B and C) due to the breaking of C C
bonds in the polymerization process, giving rise to single bonds.
Additionally, the infrared spectra of MIP17 and NIP17 (Fig. 3B and
C) were almost identical, reecting the presence of the same chemical groups and suggesting that the hexazinone had been removed
in the MIP washing step.
SEM micrographs of MIP and NIP, synthesized in bulk with 2-VP
(MP17), are shown in Fig. 4A and B, respectively. These analyses
revealed the presence of particles of irregular shapes and sizes,
with diameters of approximately 50 nm or more. Unfortunately,
higher magnication images were unable to improve differentiation between the MIP and the NIP. However, it could be clearly
observed that MIP17 was more porous than NIP17, which presented a smoother and relatively uniform appearance, with fewer
cavities. This difference in porosity of these polymeric materials
was conrmed by N2 adsorption/desorption experiments using
the BrunauerEmmettTeller (BET) model. Specic surfaces were
683.53 m2 g1 (MIP17) and 632.03 m2 g1 (NIP17), and pore sizes
were 30.74 nm (MIP17) and 26.75 nm (NIP17). These results indicated a real difference between these materials.
The efciency of the MIP, relative to the NIP, for the recognition
of HXZ was studied in afnity experiments carried out for 12 h at
room temperature. The adsorption capacity (Q, mg g1 ) was calculated based on the difference in the hexazinone concentration
before and after adsorption by the polymer, using a constant volume of methanol and a precise weight of the MIP or NIP, according
to Eq. (1):

Adsorption capacity, Q ( mg g )

Fig. 4. SEM micrographs (at 50,000 magnication) of (A) MIP17 and (B) NIP17 synthesized by the bulk method.

50
40
30
20
10
0
0

20

40

60

80

100 120 140 160

-1

CHXZ (mg L )
Fig. 5. HXZ adsorption proles of the MIP17 and NIP17 polymers.

solution of hexazinone in methanol (mL), and M is the weight of the

MIP or the NIP (g). As shown in Fig. 5, the amount of adsorbed hexazinone was greater for the MIP, compared to the NIP, suggesting
that the imprinted polymer provided better molecular recognition,
due to the creation (in the MIP) of nanocavities with size, shape,
and functionality that enabled selective interaction with the analyte. This enabled the use of this imprinted polymer to construct
highly selective biomimetic sensors for HXZ.
3.3. Electrochemical behavior of hexazinone on the biomimetic
sensor
The rst investigation of the reduction of hexazinone was
described by Polak and Volke [17]. The initial reduction step was
attributed to a two-electron process, based on comparison with
the reduction of 6-azauracils. The hexazinone molecule contains
an electroactive C C C N moiety, which would result in formation
of a species bearing two reducible centers. In other work, Priman
and Zuman [8] developed a method for hexazinone detection using
adsorption of the analyte by lignin in a slightly acidic medium.
Priman and Zuman [9] attributed the two-electron reduction to
the protonated form of the azomethine moiety in hexazinone. The
reduction of hexazinone was studied at different pH, and a decrease
in current was observed at pH > 2.5.
Given the fact that triazines are electrochemically active, evaluation was made of the effect of varying the pH in the range from
1 to >4. The peak height decreased as the pH increased, and no
current was detected at pH > 4. The best results were obtained at
pH around 2.5, so solutions with this pH value were used for the
preconcentration and electrochemical determination of HXZ.

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M.J.U. Toro et al. / Sensors and Actuators B 208 (2015) 299306

MIPCPE
NIPCPE
CPE

-6

6x10

-5

4x10

-6

-4x10

|i| / A

i/A

Fig. 6. Schematic representation of the sensor response.

-5

-8x10

CPE
NIPCPE
MIPCPE

-4

-1x10

-1.6

-1.4

-1.2

-6

2x10

-1.0

E vs Ag|AgCl / V
Fig. 7. Cyclic voltammograms obtained for 100 nmol L1 HXZ using MIPCPE, NIPCPE,
and CPE. Measurements carried out in HCl (pH 2.5) at a scan rate of 50 mV s1 .

Fig. 6 presents a schematic mechanism describing the sensor

response, based on the experimental evidence and the ndings of
Priman and Zuman [8,9]. In the rst step, an accumulation potential
(0.5 V) was used to preconcentrate HXZ within the MIP cavities
of the paste, after which the applied potential resulted in quantitative reduction of HXZ (in an acidic medium) and its return to the
solution.
The electroreduction of hexazinone in HCl at pH 2.5 was investigated for a plain carbon paste electrode (CPE) and electrodes
modied with the NIP (NIPCPE) and the MIP (MIPCPE), using cyclic
voltammetry (Fig. 7). As expected, only the MIPCPE sensor presented an irreversible reduction prole in the presence of HXZ.
This response indicated the suitability of the MIP as a recognition
element in the construction of a sensor for the determination of
hexazinone.
The ability to quantify hexazinone with the MIPCPE was evaluated using differential pulse with cathodic stripping voltammetry

-1.6

-1.4

-1.2

-1.0

E vs Ag|AgCl / V
Fig. 8. Differential pulse cathodic stripping voltammetry analysis of 100 pmol L1
hexazinone using MIPCPE, NIPCPE, and CPE. Conditions: pH 2.5 (HCl); accumulation
potential (Eac ) = 0.5 V; accumulation time (tac ) = 200 s; v = 60 mV s1 ; pulse amplitude = 50 mV; pulse width = 40 ms.

(DPCSV), with the MIPCPE, NIPCPE, and CPE electrodes immersed

in a solution containing hexazinone at a concentration of
100 nmol L1 . The results (Fig. 8) showed that under identical conditions, the signal provided by the proposed sensor (MIPCPE) was
higher than for the NIPCPE or the CPE. This indicated that the
MIP electrode provided greater adsorption of hexazinone from the
aqueous solution, due to the presence of selective nanocavities and
greater porosity, compared to the NIP.
After optimization of the paste composition, the best sensitivity was achieved using a graphite powder/parafn oil/MIP17 mass
ratio (w/w) of 66.7:28.5:4.8.
Further evaluation of the efciency of the proposed sensor
was performed by measuring the response to HXZ obtained for
sensors constructed using MIPs synthesized with other templates
(metribuzin and atrazine), under the same conditions used to

M.J.U. Toro et al. / Sensors and Actuators B 208 (2015) 299306

(A)

the stability of MIPs enables simple and highly reproducible preparation of pastes, so that determinations can be carried out with the
same batch of sensors. Good repeatability can be achieved in both
sensor construction and subsequent measurements.

-6

6x10

(f)

-6

|i| / A

4x10

305

3.5. Study of selectivity

-6

2x10

The selectivity of the proposed biomimetic sensor was evaluated

by testing its response to atrazine, s-atrazine, desethyl atrazine,
metribuzin, and hexazinone. With the exception of hexazinone, the
sensor showed no response to these pesticides, using either cyclic
voltammetry or differential pulse with cathodic stripping voltammetry. This demonstrated that the proposed biomimetic sensor was
highly selective for the detection and quantication of hexazinone.

(a)

0
-1.5

-1.2

-0.9

-0.6

E vs Ag|AgCl / V

(B)

3.6. Analysis of environmental samples (water)

-6

ip / A

6.0x10

The proposed sensor (MIPCPE) was used with differential pulse

with cathodic stripping voltammetry to evaluate the recovery of
hexazinone from real water samples. An average recovery of 95.8%
(n = 3) was obtained for samples of river water enriched with
6.0 nmol L1 of hexazinone. This level of recovery at nanomolar
concentrations illustrates the efciency of the proposed sensor.

-6

4.0x10

-6

2.0x10

-8

ip = -0.9x10 + 56817[HXZ]

4. Conclusions

R = 0.9949
-11

4.0x10

-11

8.0x10

-10

1.2x10

-1

[HXZ] / mol L

Fig. 9. (A) Typical voltammograms for DPCSV analysis of HXZ using the proposed biomimetic sensor under previously optimized conditions. Conditions: pH
2.5 (HCl); tac = 200 s; Eac = 0.5 V; scan rate = 50 mV s1 ; pulse amplitude = 100 mV;
pulse width = 50 ms. [HXZ]: (a) 1.90 1011 mol L1 ; (b) 3.85 1011 mol L1 ;
(c) 5.66 1011 mol L1 ; (d) 7.41 1011 mol L1 ; (e) 9.09 1011 mol L1 ; (f)
1.10 1010 mol L1 . (B) Corresponding analytical curve with error bars (for triplicates).

prepare the hexazinone MIP. The results (Supplementary Data,

Figures S1 and S2) conrmed the effectiveness of the MIP and
sensor prepared with the hexazinone template.
3.4. Analytical characterization
Differential pulse with cathodic stripping voltammetry was
employed, using an accumulation potential (Eac ) of 0.5 V and a
subsequent stripping step from 0.6 to 1.6 V. The voltammetric
responses of the MIPCPE are shown in Fig. 9A. With 200 s accumulation (tac ), the proposed sensor exhibited a working concentration
range of 1.9 1011 to 1.1 1010 mol L1 and a sensitivity of
56,817 A L mol1 (Fig. 9B). The limits of detection (LOD) and quantication (LOQ) were 2.6 1012 mol L1 and 8.7 1012 mol L1 ,
respectively [18], which indicates the high sensitivity of the
proposed method. The repeatability of the measurements was evaluated from the slopes of ve calibration curves obtained using
different pastes, and the resulting RSD value was 3.7%.
The literature describes only a few sensors based on MIPs for
the detection of triazines and other pesticides, which are listed in
Table 1. It can be seen that in all cases, the concentration ranges for
pesticide quantication, as well as the LODs, were inferior, compared to the values obtained with the sensor proposed here. The
new sensor therefore offers considerable potential for use in pesticide measurements.
It is important to emphasize that the use of sensors based on
modied carbon paste allows the possibility of renewing the sensor
surface by polishing after each set of measurements, or changing
the paste after a predetermined period of operation. Additionally,

Molecular modeling can be used to identify functional

monomers that are capable of producing effective imprinted
polymers. These computer calculations are an effective design technique and can be used for the rapid development and optimization
of polymers that act as synthetic receptors. It is important to note
that monomer prediction does not only depend on the ionization
constants of the monomers, or acidbasic interactions, and that
other parameters are included in the theoretical prediction. Here,
the 2-VP monomer, which simulation predicted to be superior to
two other monomers, enabled the production of a more effective
MIP for hexazinone (MIP17).
The MIP17 polymer was employed as the recognizer in a modied carbon paste biomimetic sensor used in association with the
DPCSV technique. The sensor was highly sensitive, enabling detection of HXZ at concentrations in the region of 1010 mol L1 . In
addition, the sensor was highly selective for this herbicide, unlike
other enzyme-based biosensors where the response relies on nonselective enzyme inhibition. The ndings indicate that the proposed
sensor could be used for the detection of HXZ in different types of
samples, including food and environmental matrices.
Acknowledgements
The authors gratefully acknowledge nancial support from
FAPESP (Proc. 2011/12296-6) and CNPq (Proc. 400459/2012-4).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.snb.2014.11.036.
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Biographies
Maricely Janette Uria Toro studied Chemistry at the National University of San
Marcos (Lima, Peru), and received her PhD in Chemistry at UNESP (Brazil) in 2014.
She received an MSc degree at the Federal University of Par (UFPA, Brazil) in 1999.
She currently lectures at UFPA. Her area of expertise includes molecularly imprinted
polymers and biosensors.
Luiz Diego Marestoni studied Physics at Londrina State University (UEL, Brazil)
from 2001 to 2004, when he obtained his degree in Physics. In February 2007,
he obtained an MSc degree in Applied Nuclear Physics at UEL, and in June 2014
received a PhD in Analytical Chemistry at the Chemistry Institute of UNESP. He
currently lectures at the Federal Institute of Paran, in Telmaco Borba, Paran
(Brazil). His research mainly involves the development of molecularly imprinted
polymers.
Maria del Pilar Taboada Sotomayor studied Chemistry at the Universidad Nacional
de Ingenieria (Lima, Peru) from 1987 to 1992, when she obtained her BSc degree in
Chemistry. In 1996, she obtained her MSc degree in Analytical Chemistry at UNICAMP (Brazil), and in March 2000 received her PhD in Sciences at UNICAMP. She
currently lectures at the Institute of Chemistry of UNESP, in Araraquara, So Paulo
State (Brazil). Her research concerns the development of electrochemical and optical biomimetic sensors based on biomimetic catalysts and molecularly imprinted
polymers.