Abstract widely used benzimidazolic IV class systemic

Abstract

Poly
(metacryloylhydrazide) was chemically synthesized using hydrazine hydrate and
poly methyl methacrylate through a simple reaction. The synthesized material
was employed for fabrication of modified carbon paste electrode (CPE). The modified electrode was used as a novel impedimetric
sensor for the determination of an important pesticide, Carbendazim. The
performance of poly (metacryloyihydrazide) toward Carbendazim was examined
using electrochemical impedance spectroscopy. Binding of Carbendazim to the
polymer on the surface of the electrode changes the impedance of the
electrode/electrolyte interface which can be monitored as signal for the
selective and sensitive determination of Carbendazim. Under
optimized experimental conditions, the proposed modified electrode shows a
linear response range from 40 to 240 ppb Carbendazim with a detection limit of
14 ppb (based on 3sb). Furthermore, the fabricated electrode was
successfully applied to determine Carbendazim in Carbendazim-added artificial
samples.

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Keywords: Electrochemical
Impedance Spectroscopy; Ploy (Methyl methacrylate); Methacryloyl Hydrazide;
Carbendazim; CPE.

 

Introduction

For many years
now, pesticides have been extensively employed to increase agriculture products
in order to supply human food. Carbendazim (methyl 1H-benzodimidazol-2-yl
carbamate) (CBZ) is a widely used benzimidazolic IV class systemic fungicide on
plants, seeds or soils that can be absorbed by plants through the roots, leaves
and seeds. It is stable for long time and degrades slowly so can be accumulated
in the human body through consumption of infected drinking water, vegetables
and tree crops and fruits. Several reports have been published regarding the
toxic and carcinogenic effects of CBZ in human and animals.1 Hence, it is necessary to develop reliable
analytical methods to monitor and determine CBZ residues in water and
foodstuffs. Various quantitative analytical methods mainly based on
chromatography technique have been established for determination of CBZ which
is expensive and time consumption2,.3.
UV-vis spectrophotometry, spectrofluorometry and Raman spectroscopy have also been
used for this purpose.4-6 On the other
hand electrochemical techniques such as differential pulse voltammetry (DPV) in
combination with modified electrodes have been applied to determine the
residues of the CBZ in real samples.7-12  Great attention devoted to the
electroanalytical techniques is due to their high sensitivity and excellent selectivity,
simple instrumentation, low cost, and miniaturization capability that are
accountable for these techniques. Several articles in literature report the use
of modified electrodes recognizing pesticides selectively along with the
electrochemical techniques for quantified assay of toxic materials. For example
an ionic liquid modified CPE was prepared and introduced for determination of CBZ
using DPV by Ya et al.13 The
proposed modified electrode was applied for the determination of CBZ in
sugarcane samples.  Petroni et al.
designed a commercial screen-printed carbon electrode modified with
multi-walled carbon nanotubes for square-wave voltammetric determination of the
fungicide CBZ in the presence of an anionic surfactant.14
They showed that the use of the anionic surfactant sodium dodecyl sulphate
provided a significant improvement on analytical sensitivity of the
measurement. The developed electrode was employed for determination of CBZ in a
spiked orange juice sample. An electrochemically reduced nitrogen-doped
graphene oxide-modified glassy carbon electrode (ERNGO/GCE) was developed for
the determination of CBZ in food samples by Ya et al.15 The ERNGO/GCE was used to measure the
concentration of CBZ in Dendrobium candidum samples. For pre-preparation, the
samples was treated in ethanol and then centrifuged.  The resulting ethanol solution was evaporated
to dryness using a mild nitrogen stream, and the residue was reconstituted in
1.0 ml of ethanol. Recently a neat CPE (CPE) was used for the DPV
quantification of CBZ in water and orange juice samples by Arruda et al.9 An oxidation peak was observed at high over
potential of 0.88 V vs. Ag/AgCl/Cl- reference electrode in
citric acid-phosphate buffer solution adjusted at pH=5.0.  The simultaneous determination of two
pesticides, namely isoproturon and CBZ, by single drop analysis using a disposable
graphene based screen-printed electrochemical sensor with square wave stripping
voltammetry was developed by Noyrod et.al.8
The objective of the work was no requirement to use a conventional cell because
the low-cost graphene-based sensor was used as an electrochemical cell with
three integrated electrodes. Also only a single 60 µL drop of the sample
solution is required to be dispensed onto the surface of the sensor for each
stripping voltammetric measurement. In all above mentioned works, measurements
have been conducted on the non-selective recognizing surfaces.  

Carboxylic group functionalized poly (3,4-ethylenedioxythiophene) (PC4-EDOT-COOH)
electro synthesized in aqueous micro emulsion system exhibited good
electrochemical recognition towards CBZ and was employed for the
electrochemical detection of CBZ by differential pulse adsorptive stripping
voltammetry.16. An oxidation peak
was observed at high over potential of 0.87 V vs. Hg/Hg2Cl2/Cl-
reference electrode in buffer solution adjusted at pH=7.0. Moreover the
electrode is stable for less than one week and the modifying polymer should be
removed and synthesized again to achieve new and fresh electrode. Recently Dai
et al. reported an impedimetric determination of formaldehyde using a glassy
carbon surface covered by poly metacryloylhydrazide/carbon nanotube (PMAH/CNT)
electro spun nanofibers as recognizing agent.17
The condensation reaction of -NH2 functional group on the PMAH
attached to the electrode surface with carbonyl group of target molecules
(formaldehyde) and formation of an imine (C=N) bond (due to the removing a H2O
molecule) leads to the change of impedance of the electrode/electrolyte
interface. This change in impedance was quantitatively related to the
concentration of the target molecules.17
In the present study we introduce a new modified CPE to recognize CBZ
sensitively and selectively at trace levels in real samples. PMAH is
synthesized based on the method reported by Gong et al. with slight
modification.17 Then the polymer
is well mixed with carbon powder and a mineral oil to achieve a conductive
paste. As it is well known, CPEs can be easily prepared and achievement to the
new surface and refreshing the electrode is very simple only by polishing the
surface of the electrode on a suitable polishing material. They are also usable
for long time applications. The high efficient condensation reaction of -NH2
functional group on the PMAH inside the electrode with carbonyl group of CBZ in
test solution and elimination of a methanol molecule (instead of water) leads
to the change in impedance of electrode/electrolyte interface which quantitatively
is correlated to the concentration of CBZ in solution (Scheme
1). Because, methoxy is an excellent leaving group in CBZ (compared to
hydrogen in formaldehyde), the condensation reaction occurs efficiently.  Here we utilize electrochemical impedance
spectroscopy (EIS) technique that comprising all features associated with
electrochemical techniques mentioned above and also it can be well used to the
detection of interfacial binding events. 

Scheme
1. Mechanism
of reaction between poly methacryloyl hydrazide and CBZ.

Experimental

Chemicals

Polymethylmethacrylate
(average molecular weight Mw ~350,000) and CBZ standard were
purchased from Sigma-Aldrich. Other chemical used were
of analytical grade and purchased from Merck. Doubly
distilled water was used for preparation of all solutions.

Instruments and methods

All
electrochemical measurements were made using an Autolab general purpose
electrochemical potentiostat/galvanostat system PG302N and Autolab frequency
response analyser system (AUT20. FRA2- AUTOLAB, Eco
Chemie, B.V., Netherlands). Typical impedimetric measurements were
conducted in a conventional electrochemical cell housing three-electrode which
employed PMAH modified CPE as the working electrode, an Ag/AgCl electrode as
the reference and a Pt rod as the counter electrode. The solution was containing
10 mL 0.1 mol l?1 KCl and 0.005 M Fe(CN)63-/4-
. The working CPE electrode was immersed in 0.1 M phosphate buffer solution (pH
7.0) containing certain amounts of CBZ for reaction time of 60 s before each
impedimetric test. The frequency range used for the electrochemical impedance
spectroscopy (EIS) measurement ranged from 0.1 Hz to 100 KHz with an
alternating amplitude of 10 mV super imposed onto the formal potential of
Fe(CN)63-/4- (0.25 V vs. Ag/AgCl reference electrode).

Synthesis of
ploy-methacryloylhydrazide (PMAH)

PMAH was
synthesized based on the method was reported by Die et.al. with some
modification (Scheme 2).17 1.0 g PMMA was dissolved in 10 ml
dichloromethane and added dropwise to the equimolar solution of hydrazine hydrate.
During the addition, the mixture was stirred moderately at 35 °C. A white solid
crystalline material was extracted by addition of excess amounts of ethanol and
dried at room temperature for subsequent uses.   

 

Scheme
2. Synthesis
of poly metacryloyl hydrazide.

 

Preparation of CPE

Conventional CPE
(CPE) was prepared by thoroughly mixing 0.10 g graphite powder and three drop
paraffin oil until a uniform wetted paste was obtained. The PMAH modified CPEs
were prepared by hand mixing 0.10 g of graphite powder and 0.020, 0.025, 0.033
and 0.05 g of PMAH and three drop paraffin oil. The resulting pastes were
firmly packed into a cylindrical Teflon tube (3.0 mm diameter) fitted with a
copper piston, which serves as an inner electrical contact. The surface of the
electrodes was polished on a glossy paper prior to each electrochemical use.

 

Results and
Discussion

Fig.
1 compares
the IR spectra of the pristine PMMA and synthesized PMAH. Appearance of a
two-branch absorption band at 3350-3520 cm-1 characteristic of the
NH2 functional group can be considered as a strong evidence of
successful synthesis of PMAH through simple reaction of PMMA and hydrazine
hydrate. Moreover, absorption bands corresponding to the bending vibration of
N-H and stretching of C-N are observable at 1618 and 1238 cm-1 respectively

Fig. 1

The
electrochemical impedance spectroscopic characterization of the neat CPE and
its composites with various amounts of PMAH were conducted using Fe(CN)6?3/?4 as redox
probe. Impedance spectra (Nyquist plots) for each electrode were obtained after
immersing the electrode in phosphate buffer blank solution (pH=7.0) and
phosphate buffer solution containing 80 ppb CBZ for 60 s. The resulted Nyquist plots
show a semicircle with diameter corresponds to the charge-transfer resistance (Rct)
and straight line corresponds to the Warburg impedance (Zw). The changes in
charge transfer resistance due to the reaction of CBZ with PMAH at the surface
of the electrodes were taken into account according to the following
calculation:

   ?R/R0=(R-R0)/R0

where R0
and R are charge transfer resistances at the surface of each electrode immersed
in phosphate buffer blank solution (pH=7.0) 
and solution containing target molecules respectively.

Fig 2 shows
Nyquist response of neat CPE (a and b) and composite electrode
including 33.3 wt% of PMAH (c and d). 
Comparison of plot a and b reveals that the Rct for neat
CPE remain constant when the electrode is immersed in solution containing
target molecules. However a great change of Rct occurs when the modified
electrode is immersed in solution containing 80 ppb CBZ target molecules (plots
c and d). This great change is attributed to the combination of CBZ molecules
with active sites of the PMAH on the surface of the electrode. Comparison of
plot a and c indicates that the presence of PMAH in composite
electrode increases the charge transfer resistance.

Fig. 2

Fig
3 (inset) shows
Nyquist response of the electrodes containing 0.0, 20.0, 25.0, 33.3, and 50.0
wt% of PMAH.  Comparison of these plots
shows that with increasing the content of the PMAH, semicircle diameters
(charge transfer resistance) increases because of the more combination of
target molecules (CBZ) with active sites of the PMAH on the surface of the
electrode. Fig. 3 shows the changes in
analytical signal (?R/R0) vs. content of PMAH in composite
electrodes. As it is observable, ?R/R0 increases rapidly with
increasing the amount of PMAH up to 33.3 wt% and then the rate of increase is
slowed. Then CPE electrode modified with 33.3% PMAH was selected and used for
further optimization process.

Fig. 3

The effect of pH of buffer solutions on the reaction of CBZ with
PMAH on the surface of the modified electrode was studied, and the relative
charge transfer resistance (?R/R0) as a function of pH ranging from 3.0
to 10.0 in the presence of 80 ppb CBZ are shown in Fig.
4. The
corresponding Nyquist plots are illustrated as inset. As shown in Fig.
4, charge transfer resistance of the modified electrode increased gradually
with the increasing the pH value until it attained the maximum at pH 7.0, and
then decreased rapidly when pH increased further. At pHs lower than 7.0, NH2
functional groups on the PMAH react with hydronium ions in acidic buffer
solution and are converted to NH3+ with little
nucleophilic properties that are not able to react with CBZ target molecules.
More over in this range of pH CBZ is also converted into soluble salt. With
increasing the pH of buffer solution to the values higher than 7.0 CBZ
molecules degrade slowly.16,18
resulted in the rapid decrease in response function. Therefore, pH of 7.0 was
chosen for the impedimetric determination of CBZ.

Fig.
4

To achieve a highly sensitive detection of CBZ, immersing time of
the PMAH modified electrode in sample solution (buffered at pH 7.0) has been
optimized. Immersion time can influence on the transport of the CBZ to the
electrode surface and its more reaction with PMAH immobilized on the surface of
the electrode. Fig. 5 shows the Nyquist
plots (inset) and corresponding analytical signal against reaction time for
PMAH modified electrode dipped in CBZ molecules for various immersion times.
The enhancement of Rct value indicates that the amount of CBZ reacting in the
recognition sites of the PMAH modified electrode increased with the increase of
the immersion time. It shows that with increasing the immersion time up to 120
s the analytical response function increases rapidly and then with more
increasing the immersion time the rate of increase of ?R/R0
decreases and finally reaches the platform after 120 s.  Therefore, the 120 s immersion time was
employed in subsequent experiments.

Fig. 5

The
effects of some potential interfering species on the determination of 80 ppb
CBZ were examined. The tolerance limit was taken as the maximum concentration
of the foreign substances that caused an approximately ±5% relative error in
the determination. The results revealed that more than 100-fold all examined
species indicated in Table 1 did not affect
the determination of CBZ.

Table 1

In
the determination step of CBZ, after the PMAH modified electrode was immersed
in buffer solution containing various amounts of target molecules for 60 s
Nyquist plots were recorded in 5 mM Fe(CN)63?/Fe(CN)64?
containing 0.1 M KCl. Calibration studies include the successively dipping the
electrode into CBZ solutions with a surface regeneration step, in which the
electrode surface was polished on a glassy paper to achieve a smooth surface
after each impedance run. The values of relative change of electron transfer
resistance were plotted as a function of CBZ concentration (Fig. 6). A linear relationship between the CBZ
concentration and charge transfer value was obtained over the concentration
range from 0.0 to 240 ppb CBZ. The linearity of this method was described by
the equations ?R/R0% = 1.048 C (ppb) – 3.0714, R2= 0.997.
The limit of detection (LOD) was found to be 14 ppb CBZ using equation LOD = 3sb/m,
where sb is the standard deviation of the blank response and m is
the slope of the calibration plot.

Fig. 6

To
demonstrate the feasibility of the electrode, the PMAH modified CPE was used
for the detection of CBZ in well water, commercial formulation and wheat
samples under optimized conditions by the standard addition method. Results are
measure of the precision and accuracy of the method for analysis of the CBZ in
real samples are listed in Table 2. The
recoveries range were 92.5-112.5%, 95.0-104.0 and 95.0-104.0% in well water,
commercial formulation and Wheat samples respectively, indicating that the
prepared sensor could be employed for practical application without sample purification
step. Moreover, satisfactory results revealed that the proposed method was
accurate, suitable and efficient for the trace level detection of CBZ in
practical samples.

Table 2

Table 3 compares the response
characteristics for the determination of CBZ at various modified electrodes
reported in recent studies. As can be seen from Table 3, the PMAH/CPE exhibited
high sensitivity, low detection limit, and wide linearity toward CBZ.

Table 3

Conclusion

In
summary, we successfully fabricated a modified CPE for impedimetric
determination of carbendazim by introducing methacryloyl hydrazide in carbon
paste. The high reactivity of the carbonyl hydrazide functional group toward
esters such as carbendazim was employed to change the electrode/electrolyte
interface charge transfer resistance.19
The electrochemical impedance spectroscopy was employed for the first time in
monitoring the electrode/electrolyte charge transfer resistance resulted from
the reaction taking place between carbendazim molecules and binding sites on
the surface of the poly methacryloyl hydrazide modified CPE. The
electrode was successfully applied for the determination of carbendazim in
spiked well water, wheat and commercial formulation samples with high precision
and recovery. The proposed method offered linearity in a concentration range
from 40 to 240 ppb with detection limit of 14 ppb.