Materials Science and Engineering C 32 (2012) 1001–1004

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Materials Science and Engineering C

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Short communication

Determination of chlorpyriphos in broccoli using a voltammetric acetylcholinesterasesensor based on carbon nanostructure–chitosan composite material

Ion Ion, Alina C. Ion ⁎University Politehnica of Bucharest, Department of Analytical Chemistry, Gh. Polizu street no. 1-7, Bucharest 011061, Romania

⁎ Corresponding author. Tel./fax: +40 212319492.E-mail address: ac_ion@yahoo.com (A.C. Ion).

0928-4931/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.msec.2012.01.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 June 2011Received in revised form 24 November 2011Accepted 10 January 2012Available online 16 January 2012

Keywords:Organophosphate pesticidesEnzymeAmperometric biosensor

The widespread use of organophosphate pesticides (OPPs) in agriculture leads to residue accumulation in theenvironment, which is dangerous to human health and disrupts the ecological balance. This study reports re-sults obtained by a standard chromatographic method (high performance liquid chromatography, HPLC) andan amperometric method for chlorpyriphos (CPF) determination in broccoli. Reversed-phase HPLC with UV–VIS detection was used for the separation, and the mobile phase was acetonitrile–water (75:25 v/v). The elec-trochemical experiments were performed in phosphate buffer solutions at pH=7.4, with an incubation timeof 10 min. The response of the sensor was a linear function of CPF concentration from 10−10 to 10−7 Mwith acorresponding equation: y=0.1467x+1.4472 (R2=0.9959) and a detection limit of 1.58×10−10 M. Thebiosensor methodology was used to analyze CPF directly in broccoli that had been previously spiked withCPF solution.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

The widespread use of organophosphate pesticides (OPPs) leadsto residue accumulation in the agricultural environment. OPPs arewidely used in agriculture because of their ability to modify acetyl-cholinesterase (AChE) [1]. The analytical methods used in pesticidequantification consist of gas chromatography with electron capturedetection (ECD), mass spectrometric (MS) [2] detectors and highperformance liquid chromatography (HPLC) with UV detection [3].Due to their selectivity, response and size for on-field analysis [4],methods based on the inhibition of the AChE enzyme by OPPsare convenient and cheaper alternative tools for OPP quantitativeanalysis. The decrease of the biosensor response is correlated withthe amount of OPP present in samples after an incubation time. Thematrix is usually water, but recently, applications for fruit and vegeta-ble samples have been reported in the literature [5–7].

Carbon-based nanomaterials [8], especially graphene [9–11], haveshown new advantages in electrochemistry due to the remarkableelectrochemical properties of these nanomaterials [12–14].

Compared with other analytical techniques, especially chromato-graphic techniques, [15] enzyme-based electrochemical biosensorswith carbon nanomaterials [16,17] exhibit good selectivity [18], sen-sitivity [19] and rapid response [20].

This work presents the application of a biosensor [21] previouslystudied by our group based on carbon nanostructures–chitosancomposite material in spiked vegetable samples (broccoli) and the

rights reserved.

comparison of the results obtained by our group with the resultsobtained by HPLC with UV detection for CPF analysis.

2. Experimental

2.1. Reagents

The organic solvents acetonitrile and ethyl acetate were HPLCgrade purchased from Sigma-Aldrich (Laborchemikalien GmbH D-30918 Seelze).

Acetylthiocholine iodide (ATCI) and glutaraldehyde (25%) werepurchased from Sigma-Aldrich (United Kingdom). Chitosan (95%),bovine serum albumin (BSA) and other reagents were analytical re-agent grade. Aqueous solutions were prepared with double-distilledwater. Phosphate buffer was prepared using KH2PO4 and K2HPO4

salts purchased from Proanalysis Merck KGaA (Darmstadt, Germany).A 10 mg/mL solution of acetylcholinesterase (AChE) enzyme from bo-vine erythrocyte (Type Xll-S, 833.3 mg solid, activity 0.3 unit/mg,solid, SIGMA) was prepared in 0.01 M potassium phosphate buffer,pH 7.4. Exfoliated graphite nanoplatelets (xGnP) were purchasedfrom XGScience, Michigan, USA. Chloropyriphos pestanal® referencematerial (o,o-diethyl-o-(3,5,6-trichloro-2-pyridylphosphorothioate))was purchased from Sigma-Aldrich (Laborchemikalien GmbH D-30918 Seelze).

2.2. Broccoli samples

The vegetables were grown at Romanian farms, in exper-imental fields. The matured broccoli-bearing plants treated with the

0.0E+00

1.0E-06

2.0E-06

3.0E-06

4.0E-06

5.0E-06

6.0E-06

0 0.1 0.2 0.3 0.4 0.5 0.6

C, mM

i, A

y = 2.02E-05x + 4.85E-070.0E+00

1.0E-06

2.0E-06

0 0.02 0.04 0.06 0.08C, mM

i, A

y = 5.2E-06x + 1.7E-06

0.0E+00

2.0E-06

4.0E-06

6.0E-06

0.1 0.3 0.5C, mM

i, A

Fig. 1. Calibration plot of the voltammetric response of AChE–chitosan modified-GCEElectrode towards ATCI6.

-1.70E-05

-1.30E-05

-9.00E-06

-5.00E-06

-1.00E-06

3.00E-06

7.00E-06

1.10E-05

-1.5 -1 -0.5 0 0.5 1 1.5

E, V

i, A

1 2 3 4

Fig. 2. Cyclic voltammograms obtained from the AChE–chitosan modified-GCE withxGnPs in 0.01 M phosphate buffer pH 7.4 (1) containing 0.250 mM ATCI (1) addedand 10−10 M CPF (2); 10−9 M CPF (3); 10−8 M CPF (4); scan rate 100 mV s−1.

1002 I. Ion, A.C. Ion / Materials Science and Engineering C 32 (2012) 1001–1004

recommended doses of chlorpyriphos (CPF) were collected duringsummer (August, 2010).

3. Instrumentation

Electrochemical measurements were performed with an AutolabPGSTAT 30 potentiostat/galvanostat (EcoChemie), equipped withthe VA 663 stand (Metrohm) nd using a single-compartment, three-electrode cell at room temperature. A 2 mm diameter glassy carbonGC disk electrode (Metrohm) was used as the working electrode, asaturated silver–silver chloride electrode was the reference, and agraphite rod (Metrohm) was the auxiliary. Before electrochemicaltests were performed, the surface of the working electrode waspolished to a mirror finish with 0.3 μm alumina powder and rinsedwith double-distilled water. All solutions used for the electrochemicalmeasurements were bubbled with Ar for 10 min, and an Ar flow wasmaintained over the solution during the experiment.

The chromatographic measurements were performed using highperformance liquid chromatography (HPLC) on a Waters 2487 chro-matograph with dual absorbance detectors.

3.1. Biosensor preparation, electrochemical and chromatographicmeasurements

Chitosan stock solution (0.5% w/v) was prepared by dissolvingchitosan flakes in a 2 M aqueous solution of acetic acid. Three acids(HCl, HCOOH and CH3COOH) were tested. Among these three acids,the best dispersion of chitosan was obtained in CH3COOH, and theworst dispersion in the solution containing HCl, probably because ofthe large number of NH4

+ groups in the chitosan molecule.A volumeof 5 μL of 25% glutaraldehydewasmixedwith 1 mL 0.5%w/

v chitosan solution to form cross-linked chitosan with free CHO groups.Pretreated xGnP (1 mg)were added to 1 mL of themixture and sonicat-ed to obtain a homogeneous suspension. The distribution of the xGnPsheets in the chitosan matrix is influenced by their dispersion state inwater. A good and stable dispersion was obtained in the aqueous 2 Macetic acid solution, even after two weeks, using an ultrasonic disper-sion. Better dispersion of the xGnP in CH3COOH can be attributed to hy-drogen bonding of the COOH groups of the two acids with the oxygen-containing groups on the xGnP surface. Four microliters of this mixturewas applied to the glassy carbon electrode surface and kept at roomtemperature for 2 h. The modified electrode obtained was treated with6 μL AChE solution (300 mU, containing 5 g/L BSA tomaintain the stabil-ity of AChE) and kept at room temperature for 1 h for covalent linkage ofthe AChE to the electrode surface. The film contains cross-linked chito-san, exfoliated graphite nanoplatelets and BSA. The sensor was thenwashed with phosphate buffer (pH 7.4) to remove the excess of AChE.

Phosphate buffer (pH 7.4) was added to the electrochemical cell,the desired volume of ATCI was added, and voltammograms wererecorded from 0.3 V to 1.25 V.

Chromatographic measurements were obtained using separatecalibration stock solutions, and the measurements were performedin triplicate. The mobile phase consisted of a mixture of acetonitrile:water 75:25 (v/v) with a flow rate of 1 mL min−1. The wavelengthof the dual absorbance detector was fixed at 230 nm, and the injec-tion volume was of 20 μL.

3.2. Extraction procedure before chromatographic analysis

3.2.1. ExtractionParts of each broccoli sample (10 g) were chopped, blended and

kept in the freezer in polyethylene bags at temperatures below−10 °C. The samples were fortified with 10−8 to 10−7 mol L−1 CPFin water and extracted with ethyl acetate (30 mL) for 10 min. Theethyl acetate extract was filtered, evaporated to dryness, re-dissolvedin 2 mL of acetonitrile and analyzed by high performance liquid

chromatography (HPLC) with UV/Vis detection and with the biosensorby immersion for an incubation time of 10 min.

3.3. Determination of CPF in broccoli from the farm

Two pieces of 10 g each of the broccoli plant were cut and choppedwith a small hole inside. One was spiked with 0.5 mL of the CPF solu-tion with a concentration between 10−8 to 10−7 mol L−1 in phos-phate buffer solution. The other was treated with only the 0.5 mL ofthe phosphate buffer solution. Both solutions were gently mixedwith the chopped juiced pulp for 10 min. After this time, the biosensorwas introduced into the juiced pulp of both pieces for an incubationtime of 10 min. The biosensor was then introduced into the electro-chemical cell for CPF measurements as described in the experimentalsection. From the inhibition current, the recovered concentration wascalculated.

3.4. Validation of the electrochemical method using the proposedbiosensor

Linearity of the method was proved by running the final extractsof the broccoli samples in triplicate at two spiking concentrations.

y = 0.1467x + 1.4472

R2 = 0.99592.40

2.60

2.80

3.00

6 8 10 12

-log CCPF, M

i,µA

Fig. 3. Calibration curve obtained from the AChE–chitosan modified GCE with xGnPs in0.01 M phosphate buffer containing 0.250 mM ATCl, pH=7.4 for different concentra-tions of chloropyriphos (10−10 to 10−7 mol L−1), T=35 °C.

Table 2Concentrations of CPF in the broccoli samples analyzed by HPLC and in the field usingthe AChE biosensor (n=5).

Sample Chlorpyriphos, M, HPLC Chlorpyriphos, M,AChE biosensor

1 6.2×10−8 5.7×10−8

2 5.7×10−8 4.2×10−8

3 3.7×10−8 2.8×10−8

4 bdl nd5 bdl nd

bdl — below detection limit of the method.

1003I. Ion, A.C. Ion / Materials Science and Engineering C 32 (2012) 1001–1004

The limit of detection (LOD) for each pesticide was defined as thevalue between the common mean and three times the standard devi-ation of the blank signal. The limit of quantification (LOQ) was de-fined as the common mean and ten times the standard deviation ofthe blank signal [22]. Recoveries were estimated by comparing the re-sponses of the biosensor before and after adding broccoli samples ofknown concentrations to the CPF standard solution.

4. Results and discussion

Detection of cholinesterase activity and measurements of the in-hibitory action of organophosphate pesticides are based on the vol-tammetric determination of the concentration of thiocholine, whichis a product of the enzymatic hydrolysis of the substrate.

The oxidation of thiocholine proceeded via the one-electronmechanism shown below, perhaps with a subsequent dimerizationof the generated radical that is: [23]

CH3COSCH2CH2Nþ

CH3ð Þ3→AChE=H2O HSCH2CH2 Nþ

CH3ð Þ3 þ CH3COO−

þHþ2HSCH2CH2 Nþ

CH3ð Þ3→voltage−2e−

CH3ð Þ3 NþCH2CH2

−S−S−CH2CH2 Nþ

CH3ð Þ3 þ 2Hþ

By increasing the ATCI concentration, the voltammetric responseof the enzyme electrode increased. The response of the sensor was alinear function of ATCI concentration in two segments, one segmentwas from 0.005 to 0.039 mM with the corresponding equation:ip(A)=2.26×10−5 E+4.39×10−7 (R2=0.992) and the second seg-ment from 0.064 mM to 0.258 mM with the corresponding equation:ip(A)=6.80×10−6 E+1.30×10−6 (R2=1.000) (Fig. 1).

Fig. 2 shows the voltammetric behavior of the AChE–chitosanmodified-GCE sensor after treatment with chloropyrifos (CPF), an or-ganophosphate pesticide. After incubation of the biosensor in10−10 M CPF aqueous solution in phosphate buffer (pH 7.4) for10 min, the signal obtained in the cyclic voltammogram was muchlower at the same concentration of ATCI (0.250 mM). The peak cur-rent was observed to decrease as the concentration of CPF increased.This voltammetric response to ATCI can be used to monitor AChE in-hibitors and provides a method to study the effect of organophos-phate pesticides on the AChE enzyme.

Table 1Percentage of recovery in broccoli analysis at two levels of CPF concentrations.

Method Added, M Recovered, M Percentage, %

AChE biosensor 1×10−7 1×10−7 100HPLC 1×10−7 7.55×10−8 75AChE biosensor 1×10−8 1×10−8 100HPLC 1×10−8 0.8×10−8 80

The improved performance of these biosensors with the additionof chloropyrifos can be attributed to their high surface area. The de-velopment of biosensors with good characteristics is possible becauseof the nanoscale thickness of the graphene nanoplatelets (Fig. 3).

4.1. Recovery after the extraction procedures

The recovery measurements were performed at varying CPF con-centrations of 10−8 to 10−7 mol L−1 at 35 °C because, during sum-mer, the temperatures at the farms are elevated far above 35 °C. Theincubation time was 10 min, and the analyses were repeated threetimes for each sample. Matrix effects were not observed in the bio-sensor analysis. In Table 1, the recovery values for CPF in broccolisamples for two concentrations of CPF are presented.

From the recoveries, the success of the biosensor voltammetricmethod in recovering the added concentrations in the linear range ofthe CPF concentration is demonstrated. The biosensor can be used di-rectly at the farm for fast analysis.

Table 2 presents the correlation between the analysis using the bio-sensor and the HPLCmethod. The values correlate well. Each value rep-resents the mean of three replicates. Each replicate was injected twice.The samples revealedmedium amounts of CPF one day after treatment.After 2 weeks, the concentrations of CPF in all samples were below thedetection limit using the proposed biosensor. The fast sample prepara-tion and the low detection limit represent important advantages for de-veloping new environmental and agricultural sensing techniques.

The regression results are based on three replicates at five concen-trations in the range 10−7–10−10 M; the response was linear with avalue of the correlation coefficient of 0.9994. Repeatability and repro-ducibility were calculated on the basis of 5 replicates in the sameday with relative standard deviations (RSDs) of 20%. Recovery exper-iments were carried out in triplicate at two fortification levels of 10−8

and 10−7 M, by adding known concentrations of CPF standard direct-ly into broccoli chopped pulp. The samples were analyzed accordingto the proposed electrochemical method (Table 3).

Chloropyriphosmay decompose in vegetables producing chloropyr-iphos-oxon and 3,5,6-trichloro-2-pyridinol degraded to 3,5,6-trichloro-2-methoxypyridine and carbon dioxide, but these compoundswere notobserved in chromatographic measurements.

5. Conclusions

The results demonstrate that xGnPs can be used as a convenientand cost-effective alternative in the fabrication of sensitive and fast

Table 3Characteristic parameters of the validated method for CPF analysis.

Parameter Value

Regression equation y=0.0147x+0.144Correlation coefficient, R2 0.9994Linearity range, M Till 10−10 MLOD, M 1.5×10−10

Recovery and precision (n=5) 95 (+5)

1004 I. Ion, A.C. Ion / Materials Science and Engineering C 32 (2012) 1001–1004

ATCI biosensors is presented. The addition of the xGnPs enhanced theredox peak current in the ATCI solution in comparison to the AChEbiosensor without xGnPs, and the sensor exhibited excellent sensitiv-ity and stability. A simple method for the production of AChE biosen-sors for organophosphate pesticides has been established, and thebiosensor that was prepared is used in broccoli analysis directly atthe farm. The results were compared with the results obtained fromHPLC analysis. The HPLC measurements including the extractionstep did not give satisfactory recovery values because of the pre-treatment steps involving organic solvents and pre-concentration.

The direct use of the proposed biosensor can be very useful in anintegrated form for in situ monitoring of OPPs in cultures of vegeta-bles and fruits.

Acknowledgments

The results described in this work have been partially funded bythe Romanian Ministry of Education and Research, through PNCD IIprojects BIOMICROTECH and POPMED (2008–2011).

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